A8, an anti-uPA agonistic antibody, promotes metastasis of cancer cells via ERK pathway.

Urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR) are expressed in many tumors and have been reported to be correlated to protein expression and poor prognosis in malignant tumors. In a previous study, we reported on the selection of human single-chain variable fragment (scFv) A8 specific to uPA from phage-displayed human naïve scFv library. In this study, scFv A8 was converted to minibody form and evaluated for its functional ability on the uPA system involved in cellular signaling and cancer cell metastasis. A8 minibody increased enzyme activity of uPA and enhanced the migration and invasion of HT1080 colon cancer cells in a dose-dependent manner. A8 increased ERK phosphorylation, and enhanced migration was blocked by U0126, but not by LY0294002, SB2203580, and SP600125. A8 minibody also enhanced migration of MDA-MB231 by mediated expressing surface uPA, but not that of MCF-7 non-expressing surface uPA. Taken together, the A8 anti-uPA antibody is a uPA agonistic antibody, enhancing migration and invasion of cancer cells that express uPA via activation of ERK pathway.

JNK1 and 2 play a negative role in reprogramming to pluripotent stem cells by suppressing Klf4 activity.

Embryonic stem (ES) cells are pluripotent cells with the capacity for unlimited self-renewal or differentiation. Inhibition of MAPK pathways enhances mouse ES cell pluripotency characteristics. Compared to wildtype ES cells, jnk2(-/-) ES cells displayed a much higher growth rate. To determine whether JNKs are required for stem cell self-renewal or differentiation, we performed a phosphorylation kinase array assay to compare mouse ES cells under LIF+ or LIF- culture conditions. The data showed that activation of JNKs was induced by LIF withdrawal. We also found that JNK1 or 2 phosphorylated Klf4 at threonines 224 and 225. Activation of JNK signaling and phosphorylation of Klf4 inhibited Klf4 transcription and transactivation activity. Importantly, jnk1(-/-) and jnk2(-/-) murine embryonic fibroblasts (MEFs) exhibited a significantly greater potency in the ability to increase the number of iPS colonies compared with jnk wildtype MEFs. Overall, our results demonstrated that JNK1 and 2 play a negative role in reprogramming to pluripotent stem cells by suppressing Klf4 activity.

Regulation of inflammatory responses and fibroblast-like synoviocyte apoptosis by calcineurin-binding protein 1 in mice with collagen-induced arthritis.

OBJECTIVE: Calcineurin-binding protein 1 (CABIN-1) regulates calcineurin phosphatase activity as well as the activation, apoptosis, and inflammatory responses of fibroblast-like synoviocytes (FLS), which actively participate in the chronic inflammatory responses in rheumatoid arthritis (RA). However, the mechanism of action of CABIN-1 in FLS apoptosis is not clear. This study was undertaken to define the regulatory role of CABIN-1 in FLS from mice with collagen-induced arthritis (CIA). METHODS: Transgenic mice overexpressing human CABIN-1 in joint tissue under the control of a type II collagen promoter were generated. Expression of human CABIN-1 (hCABIN-1) in joints and FLS was determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. The expression of cytokines, matrix metalloproteinases (MMPs), and apoptosis-related genes in FLS was determined by enzyme-linked immunosorbent assay, gelatin zymography, and RT-PCR, respectively. Joints were stained with hematoxylin and eosin and with tartrate-resistant acid phosphatase for histologic analysis. RESULTS: Human CABIN-1-transgenic mice with CIA had less severe arthritis than wild-type mice with CIA, as assessed according to hind paw thickness and histologic features. The milder arthritis was accompanied by significantly enhanced apoptosis in transgenic mice, evidenced by a significantly greater number of TUNEL-positive cells in synovial tissue. Expression of inflammatory cytokines and MMPs in the transgenic mice with CIA was reduced, and they exhibited decreased Akt activation and increased expression of p53, caspase 3, caspase 9, and Bax. CONCLUSION: Our findings demonstrate that hCABIN-1 plays a critical role in promoting apoptosis of FLS and in attenuating inflammation and cartilage and bone destruction in RA. These results help elucidate the pathogenic mechanisms of RA and suggest that CABIN-1 is a potential target for treatment of this disease.

A human monoclonal antibody scFv to urokinase plasminogen activator.

A human monoclonal antibody can be a good method for tumor diagnosis and treatment. This study is aimed at the generation of human antibody fragments against urokinase plasminogen activator (uPA) known to be related to tumor metastasis using the naive human antibody phage display library. Three clones--A2, A8, and E4--were selected from 1 x 10(10) sized human naïve antibody phage library using BIAcore rescue and screen. Clone A8 was finally selected by flow cytometry against Hep3 and HT1080, uPA overexpressing tumor cell lines. A8 clone consisted of 324 bp lambda and 402 bp heavy chains. The affinity (K(D)) of purified A8 antibody fragments was 1.44 x 10(-8) M(-1). The antibody fragment was reacted with HT1080 in a dose-dependent manner but not reacted with LS513 normal fibroblast. In this study, uPA specific human monoclonal antibody fragment A8 was made with BIAcore selection. Selected A8 was bound specifically to uPA expressed on the tumor cell surface. Further study for the application of A8 antibody clones will be needed.

Cyanoacrylate for colonic anastomosis; is it safe?

BACKGROUND: This experimental study evaluated the effectiveness and safety of using cyanoacrylate adhesive for sutureless colonic anastomosis and as a protective seal to prevent leakage. METHODS: Sixty male Sprague-Dawley rats (300 +/- 10 g, 9 weeks old) were divided into three groups: in group I, the anastomosis was sutured in a single layer with 5-0 polypropylene; in group II, the anastomosis was fixed using N-butyl-2-cyanoacrylate (Histoacryl(R)); and in group III, the anastomosis was sutured and then sealed with N-butyl-2-cyanoacrylate. The rats were sacrificed on postoperative day 7. The anastomoses among the three groups were compared by measuring wound infection, anastomotic leakage, anastomotic stricture, adhesion formation, anastomotic bursting pressure, and histological appearance. RESULTS: No anastomotic leakage was observed in any group. Anastomotic stricture was significantly more extensive in groups II and III (p < 0.001). Bursting pressure was significantly lower in groups II and III (168 +/- 58, 45 +/- 21, and 60 +/- 38 mmHg for groups I to III, respectively, p < 0.001). The severity of inflammatory reactions was significantly greater and collagen deposition was significantly lower in groups II and III (p < 0.05). CONCLUSIONS: N-butyl-2-cyanoacrylate could be a useful method for sutureless colonic anastomosis based on the absence of anastomotic leakage, but it may impede healing of the colonic anastomosis. In addition, when used to seal sutured colonic anastomoses, cyanoacrylate may have a negative influence on anastomotic healing. The clinical use of N-butyl-2-cyanoacrylate in colonic anastomosis does not appear to be acceptable and safer anastomotic methods or alternative forms of cyanoacrylate should be developed.

Modified technique for endoscopic endonasal reduction of medial orbital wall fracture using a resorbable panel.

PURPOSE: To describe a modified technique using resorbable panel for endoscopic endonasal reduction of medial orbital wall fracture. METHODS: A prospective, small interventional case series involving 2 patients with medial orbital wall fractures that were treated with the modified technique. Postoperatively, patients were evaluated for visual acuity, enophthalmos, extraocular motility, and diplopia. RESULT: Both patients recovered completely without any residual eye symptoms or complications, and postoperative CT showed a completely resolved medial orbital wall. CONCLUSION: The modified technique for endoscopic endonasal reduction using a resorbable panel was effective for the reconstruction of orbital wall fractures, without complications. The modified technique may be expected to have advantages over the conventional endoscopic approach using a Silastic sheet and Merocel because it does not require long-term nasal packing, and the resorbable panel supports the orbital contents longer.