PDZ Peptide of the ZO-1 Protein Significantly Increases UTP-Induced MUC8 Anti-Inflammatory Mucin Overproduction in Human Airway Epithelial Cells.

Mucus hyperproduction and hypersecretion are observed often in respiratory diseases. MUC8 is a glycoprotein synthesized by epithelial cells and generally expressed in the respiratory track. However, the physiological mechanism by which extracellular nucleotides induce MUC8 gene expression in human airway epithelial cells is unclear. Here, we show that UTP could induce MUC8 gene expression through P2Y2-PLCß3-Ca2+ activation. Because the full-length cDNA sequence of MUC8 has not been identified, a specific siRNA-MUC8 was designed based on the partial cDNA sequence of MUC8. siRNA-MUC8 significantly increased TNF-α production and decreased IL-1Ra production, suggesting that MUC8 may downregulate UTP/P2Y2-induced airway inflammation. Interestingly, the PDZ peptide of ZO-1 protein strongly abolished UTP-induced TNF-α production and increased IL-1Ra production and MUC8 gene expression. In addition, the PDZ peptide dramatically increased the levels of UTP-induced ZO proteins and TEER (trans-epithelial electrical resistance). These results show that the anti-inflammatory mucin MUC8 may contribute to homeostasis, and the PDZ peptide can be a novel therapeutic candidate for UTP-induced airway inflammation.

Change of inspired oxygen concentration in low flow anesthesia.

BACKGROUND: There are several advantages of low flow anesthesia including safety, economics, and eco-friendliness. However, oxygen concentration of fresh gas flow and inspired gas are large different in low flow anesthesia. This is a hurdle to access to low flow anesthesia. In this study, we aimed to investigate the change in inhaled oxygen concentration in low flow anesthesia using oxygen and medical air. METHODS: A total of 60 patients scheduled for elective surgery with an American Society of Anesthesiologist physical status I or II were enrolled and randomly allocated into two groups. Group H: Fresh gas flow rate (FGF) 4 L/min (FiO2 0.5). Group L: FGF 1 L/min (FiO2 0.5). FGF was applied 4 L/min in initial phase (10 min) after intubation. After initial phase FGF was adjusted according to groups. FGF continued at the end of surgery. Oxygen and inhalation anesthetic gas concentration were recorded for 180 min at 15 min interval. RESULTS: The inspired oxygen concentration decreased by 5.5% during the first 15 min in the group L. Inspired oxygen decreased by 1.5% during next 15 min. Inspired oxygen decreased by 1.4% for 30 to 60 min. The inspired oxygen of group L is 35.4 ± 4.0% in 180 min. The group H had little difference in inspired oxygen concentration over time and decreased by 1.8% for 180 min. CONCLUSIONS: The inspired oxygen concentration is maintained at 30% or more for 180 min in patients under 90 kg. Despite some technical difficulties, low flow anesthesia may be considered.

Distinct roles of transforming growth factor-ß signaling and transforming growth factor-ß receptor inhibitor SB431542 in the regulation of p21 expression.

Transforming growth factor-ß (TGF-ß) has both tumor suppressive and oncogenic activities. Autocrine TGF-ß signaling supports tumor survival and growth in certain types of cancer, and the TGF-ß signaling pathway is a potential therapeutic target for these types of cancer. TGF-ß induces p21 expression, and p21 is considered as an oncogene as well as a tumor suppressor, due to its anti-apoptotic activity. Thus, we hypothesized that autocrine TGF-ß signaling maintains the expression of p21 at levels that can support cell growth. To verify this hypothesis, we sought to examine p21 expression and cell growth in various cancer cells following the inhibition of autocrine TGF-ß signaling using siRNAs targeting TGF-ß signaling components and SB431542, a TGF-ß receptor inhibitor. Results from the present study show that p21 expression and cell growth were reduced by knockdown of TGF-ß signaling components using siRNA in MDA-MB231 and A549 cells. Cell growth was also reduced in p21 siRNA-transfected cells. Downregulation of p21 expression induced cellular senescence in MDA-MB231 cells but did not induce apoptosis in both cells. These data suggest that autocrine TGF-ß signaling is required to sustain p21 levels for positive regulation of cell cycle. On the other hand, treatment with SB431542 up-regulated p21 expression while inhibiting cell growth. The TGF-ß signaling pathway was not associated with the SB431542-mediated induction of p21 expression. Specificity protein 1 (Sp1) was downregulated by treatment with SB431542, and p21 expression was increased by Sp1 knockdown. These findings suggest that downregulation of Sp1 expression is responsible for SB43154-induced p21 expression.

The ADAM15 ectodomain is shed from secretory exosomes.

We demonstrated previously that a disintegrin and metalloproteinase 15 (ADAM15) is released into the extracellular space as an exosomal component, and that ADAM15-rich exosomes have tumor suppressive functions. However, the suppressive mechanism of ADAM15-rich exosomes remains unclear. In this study, we show that the ADAM15 ectodomain is cleaved from released exosomes. This shedding process of the ADAM15 ectodomain was dramatically enhanced in conditioned ovarian cancer cell medium. Proteolytic cleavage was completely blocked by phenylmethylsulfonyl fluoride, indicating that a serine protease is responsible for exosomal ADAM15 shedding. Experimental evidence indicates that the ADAM15 ectodomain itself has comparable functions with those of ADAM15-rich exosomes, which effectively inhibit vitronectininduced cancer cell migration and activation of the MEK/extracellular regulated kinase signaling pathway. We present a tumor suppressive mechanism for ADAM15 exosomes and provide insight into the functional significance of exosomes that generate tumor-inhibitory factors.

Therapeutic effect of a TM4SF5-specific monoclonal antibody against colon cancer in a mouse model.

Transmembrane 4 superfamily member 5 protein (TM4SF5) is presumed to serve as a molecular target to prevent or treat hepatocellular carcinoma (HCC) and colon cancer in a mouse model. Previously, we reported the efficacy of anti-cancer peptide vaccine targeting TM4SF5. In addition, we reported an anti-proliferative effect of anti-TM4SF5 monoclonal antibody in HCC. Here, we investigated expression of TM4SF5 in 45 primary colon cancer tissues. Almost all of the colon cancer tissues expressed TM4SF5 based on immunohistochemistry using anti-TM4SF5 monoclonal antibody. The treatment of human colon cancer cells with anti-TM4SF5 antibody reduced growth of TM4SF5 expressing cells and enhanced expression of E-cadherin and ß-catenin. Using mouse colon cancer models, we then evaluated the in vivo anti-cancer effect of anti-TM4SF5 antibody. Injection of the antibody significantly reduced growth of tumors priorly established by subcutaneous injection of human colon cancer cells HT-29 in a xenograft setting. We obtained similar results with mouse colon cancer cell line CT-26 in an allograft setting. Therefore, we suggest that the TM4SF5-specific monoclonal antibody has a therapeutic effect against colon cancer.

Plasminogen activator inhibitor-1 regulates infiltration of macrophages into melanoma via phosphorylation of FAK-Tyr9²5.

Tumor-infiltrating macrophages are potential candidates for cancer immunotherapy. However, the detailed molecular mechanism underlying macrophage infiltration into tumors is poorly understood. Based on our previous finding that plasminogen activator inhibitor-1 (PAI-1) enhances vitronectin-dependent migration of macrophages, we investigated the potential role of PAI-1 in macrophage invasion into melanoma. Experimental evidence obtained from spheroid confrontation assay clearly showed that PAI-1 overexpression significantly enhanced the invasion of RAW 264.7 cells into B16F10 melanoma. We further demonstrated that PAI-1 induces phosphorylation of focal adhesion kinase (FAK) at Tyr(925), which, in turn, mediated the invasion of macrophages into the melanoma. This work further illustrates that low-density lipoprotein receptor related-protein 1 (LRP1) is essential for PAI-1-mediated FAK phosphorylation and macrophage invasion into melanoma. In conclusion, our study demonstrates a novel role of PAI-1 in macrophage invasion into melanoma and provides insights into the underlying molecular mechanism.

Monoclonal antibody targeting of the cell surface molecule TM4SF5 inhibits the growth of hepatocellular carcinoma.

The cell surface transmembrane receptor TM4SF5 has been implicated in hepatocellular carcinoma (HCC), but its candidacy as a therapeutic target has not been evaluated. Building on findings that immunization with a peptide vaccine targeting human TM4SF5 can exert prophylactic and therapeutic effects in a murine model of HCC, we developed a monoclonal antibody to characterize expression of TM4SF5 in HCC and to target its function there as an anticancer strategy. We found that the antibody modulated cell signaling in HCC cells in vitro, reducing cell motility, modulating E-cadherin expression, altering p27(kip1) localization, and increasing RhoA activity. Using a mouse xenograft model of human HCC, we documented the in vivo efficacy of the antibody, which suppressed tumor growth in either tumor prevention or treatment designs. Our work offers a preclinical proof of concept for TM4SF5 as a promising target for antibody therapeutics to treat HCC. Cancer Res; 74(14); 3844-56. ©2014 AACR.

The LRP1-independent mechanism of PAI-1-induced migration in CpG-ODN activated macrophages.

CpG-oligodeoxynucleotides (CpG-ODNs) induces plasminogen activator inhibitor type-1 (PAI-1) expression in macrophages, leading to enhanced migration through vitronectin. However, the precise role of low-density lipoprotein receptor-related protein 1 (LRP1) in PAI-1 induced migration of macrophages in the inflammatory environment is not known. In this study, we elucidated a novel mechanism describing the altered role of LRP1 in macrophage migration depending on the activation state of the cells. Experimental evidence clearly shows that the blocking of LRP1 function inhibited the PAI-induced migration of resting RAW 264.7 cells through vitronectin but exerted a pro-migratory effect on CpG-ODN-activated cells. We also demonstrate that CpG-ODN downregulates the protein and mRNA levels of LRP1 both in vivo and in vitro, a function that depends on the NF-κB signaling pathway, resulting in reduced internalization of PAI-1. This work illustrates the distinct mechanism that PAI-1-induced migration of CpG-ODN-activated cells through vitronectin depends on the interaction of PAI-1 with vitronectin but not LRP1 unlike in the resting cells, where the migration is LRP1 dependent and vitronectin independent. In conclusion, our experimental results demonstrate the altered function of LRP1 in the migration of resting and activated macrophages in the context of microenvironmental extracellular matrix components.

Exosomes derived from human macrophages suppress endothelial cell migration by controlling integrin trafficking.

Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial for the regulation of cellular functions. Exosomes, nano-sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage-derived exosomes promote internalization of integrin ß1 in primary HUVECs. The internalized integrin ß1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage-derived exosomes stimulate trafficking of internalized integrin ß1 to lysosomal compartments with a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin ß1 is enhanced by the exosomes, and exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage-derived exosomes were also shown to effectively suppress collagen-induced activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin ß1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking.

Therapeutic effect of a TM4SF5-specific peptide vaccine against colon cancer in a mouse model.

Molecular-targeted therapy has gained attention because of its high efficacy and weak side effects. Previously, we confirmed that transmembrane 4 superfamily member 5 protein (TM4SF5) can serve as a molecular target to prevent or treat hepatocellular carcinoma (HCC). We recently extended the application of the peptide vaccine, composed of CpG-DNA, liposome complex, and TM4SF5 peptide, to prevent colon cancer in a mouse model. Here, we first implanted mice with mouse colon cancer cells and then checked therapeutic effects of the vaccine against tumor growth. Immunization with the peptide vaccine resulted in robust production of TM4SF5-specific antibodies, alleviated tumor growth, and reduced survival rate of the tumor-bearing mice. We also found that serum levels of VEGF were markedly reduced in the mice immunized with the peptide vaccine. Therefore, we suggest that the TM4SF5-specific peptide vaccine has a therapeutic effect against colon cancer in a mouse model.

Prophylactic effect of a peptide vaccine targeting TM4SF5 against colon cancer in a mouse model.

Expression of transmembrane 4 superfamily member 5 protein (TM4SF5) was implicated in hepatocellular carcinoma (HCC) and colon cancer. Previously, we have shown that immunization with TM4SF5 peptide-CpG-DNA-liposome complex induces production of TM4SF5-specific antibodies and protects mice from HCC progression in an allograft model. Here, we confirmed expression of TM4SF5 in the mouse colon cancer cell line CT-26 and found that anti-TM4SF5 antibody inhibits growth of CT-26 cells. We then immunized mice with TM4SF5 peptide-CpG-DNA-liposome complex and transplanted CT-26 cells to investigate the vaccination effects. Robust production of TM4SF5-specific antibodies was induced by challenge with CT-26 cells and the tumor growth was significantly suppressed in the immunized mice. The peptide vaccine targeting TM4SF5 consequently showed a prophylactic effect against colon cancer development in a mouse model. These results suggest that the peptide vaccine can be potentially applied in humans to treat colon cancer.

Matrix metalloproteinase 9 (MMP-9)-dependent processing of ßig-h3 protein regulates cell migration, invasion, and adhesion.

Cell migration is critically involved in inflammation, cancer, and development. In this study, transforming growth factor-ß-induced protein (ßig-h3) was identified as a substrate of matrix metalloproteinase-9 (MMP-9) by site-directed mutagenesis. ßig-h3 has two cleavage sites with the consensus sequence Pro-Xaa-Xaa-Hy-(Ser/Thr) (Hy is a hydrophobic amino acid) (PGSFT beginning at amino acid 135 and PPMGT beginning at amino acid 501). Using recombinant human ßig-h3 and MMP-9, ßig-h3 from ßig-h3-transfected HEK293F cells, and MMP-9 from MMP-9-transfected HEK293F cells, human macrophages, and neutrophils, we found that MMP-9 proteolytically cleaves ßig-h3. Cleavage leads to the loss of its adhesive property and its release from extracellular matrix proteins, collagen IV, and fibronectin. Spheroids formed by increased cell-cell interactions were observed in ßig-h3-transfected HEK293F cells but not in vehicle-transfected HEK293F cells. In human glioma U87MG cells, MMP-9 constitutive overexpression resulted in endogenous ßig-h3 cleavage. ßig-h3 cleavage by MMP-9 led to increased cell invasion, and ßig-h3 knockdown also resulted in increased cell invasion. The ßig-h3 fragment cleaved by MMP-9 could bind to the surface of macrophages, and it may play a role as a peptide chemoattractant by inducing macrophage migration via focal adhesion kinase/Src-mediated signal activation. Thus, intact ßig-h3 is responsible for cell migration inhibition, cell-cell contact, and cell-extracellular matrix interaction. Experimental evidence indicates that MMP-9-cleaved ßig-h3 plays a role in MMP-9-mediated tumor cell and macrophage migration.

Dimerization of matrix metalloproteinase-2 (MMP-2): functional implication in MMP-2 activation.

Matrix metalloproteinase-2 (MMP-2) functions in diverse biological processes through the degradation of extracellular and non-extracellular matrix molecules. Because of its potential for tissue damage, there are several ways to regulate MMP-2 activity, including gene expression, compartmentalization, zymogen activation, and enzyme inactivation by extracellular inhibitors. Enzyme regulation through zymogen activation is important for the regulation of MMP-2 activity. In our previous studies, we showed that thrombin directly cleaved the propeptide of MMP-2 at specific sites for enzyme activation. We also demonstrated that heparan sulfate was required for thrombin-mediated activation of pro-MMP-2 by binding to thrombin, presumably through conformational changes at the active site of the enzyme. This suggests a regulatory mechanism for thrombin-mediated activation of pro-MMP-2. In this study, we found that MMP-2 formed a reduction-sensitive homodimer in a controlled manner and that Ca(2+) ion was essential for homodimerization of MMP-2. Homodimerization was not associated with protein kinase C-mediated phosphorylation of MMP-2. MMP-2 formed a homodimer through an intermolecular disulfide bond between Cys(102) and the neighboring Cys(102). Homodimerization of MMP-2 enhanced thrombin-mediated activation of pro-MMP-2. Moreover, the MMP-2 homodimer could cleave a small peptide substrate without removal of the propeptide. Taken together, our experimental data suggest a novel regulatory mechanism for pro-MMP-2 activation that is modulated through homodimerization of MMP-2.

Exosome release of ADAM15 and the functional implications of human macrophage-derived ADAM15 exosomes.

A disintegrin and metalloproteinase 15 (ADAM15), the only ADAM protein containing an Arg-Gly-Asp (RGD) motif in its disintegrin-like domain, is a widely expressed membrane protein that is involved in tumor progression and suppression. However, the underlying mechanism of ADAM15-mediated tumor suppression is not clearly understood. This study demonstrates that ADAM15 is released as an exosomal component, and ADAM15 exosomes exert tumor suppressive activities. We found that exosomal ADAM15 release is stimulated by phorbol 12-myristate 13-acetate, a typical protein kinase C activator, in various tumor cell types, and this results in a corresponding decrease in plasma membrane-associated ADAM15. Exosomes rich in ADAM15 display enhanced binding affinity for integrin αvß3 in an RGD-dependent manner and suppress vitronectin- and fibronectin-induced cell adhesion, growth, and migration, as well as in vivo tumor growth. Exosomal ADAM15 is released from human macrophages, and macrophage-derived ADAM15 exosomes have tumor inhibitory effects. This work suggests a primary role of ADAM15 for exosome-mediated tumor suppression, as well as functional significance of exosomal ADAM protein in antitumor immunity.

Prevention and therapy of hepatocellular carcinoma by vaccination with TM4SF5 epitope-CpG-DNA-liposome complex without carriers.

Although peptide vaccines have been actively studied in various animal models, their efficacy in treatment is limited. To improve the efficacy of peptide vaccines, we previously formulated an efficacious peptide vaccine without carriers using the natural phosphodiester bond CpG-DNA and a special liposome complex (Lipoplex(O)). Here, we show that immunization of mice with a complex consisting of peptide and Lipoplex(O) without carriers significantly induces peptide-specific IgG2a production in a CD4(+) cells- and Th1 differentiation-dependent manner. The transmembrane 4 superfamily member 5 protein (TM4SF5) has gained attention as a target for hepatocellular carcinoma (HCC) therapy because it induces uncontrolled growth of human HCC cells via the loss of contact inhibition. Monoclonal antibodies specific to an epitope of human TM4SF5 (hTM4SF5R2-3) can recognize native mouse TM4SF5 and induce functional effects on mouse cancer cells. Pre-immunization with a complex of the hTM4SF5R2-3 epitope and Lipoplex(O) had prophylactic effects against tumor formation by HCC cells implanted in an mouse tumor model. Furthermore, therapeutic effects were revealed regarding the growth of HCC when the vaccine was injected into mice after tumor formation. These results suggest that our improved peptide vaccine technology provides a novel prophylaxis measure as well as therapy for HCC patients with TM4SF5-positive tumors.

Novel regulatory mechanism and functional implication of plasminogen activator inhibitor-1 (PAI-1) expression in CpG-ODN-stimulated macrophages.

Macrophages are activated by recognizing bacterial DNA and CpG-oligodeoxynucleotides (CpG-ODNs) through Toll-like receptor-9 (TLR-9). Plasminogen activator inhibitor-1 (PAI-1) has been shown to be an important factor in inflammation-induced macrophage migration which is essential for defense functions. The aim of this study was to demonstrate the molecular mechanism associated with the regulation of PAI-1 expression and its biological significance in CpG-ODN-stimulated mouse macrophages. Our results clearly show that PAI-1 expression in macrophages was highly up-regulated by CpG-ODN-stimulation in vitro and in vivo. The TLR-9-mediated stimulation of PAI-1 expression was independent of the NF-κB pathway and involved the synergistic activation of Sp1 and Elk-1 by the MEK1/2-ERK and JNK signaling pathways. The elevated PAI-1 expression resulted in significantly enhanced transmigration of RAW264.7 cells through vitronectin but not through fibronectin. We suggest that CpG-ODN plays a role in regulating macrophage migration by stimulating the expression of PAI-1, and the migration is modulated depending on the microenvironmental extracellular matrix components.

Predictors of difficult intubation defined by the intubation difficulty scale (IDS): predictive value of 7 airway assessment factors.

BACKGROUND: The intubation difficulty scale (IDS) has been used as a validated difficulty score to define difficult intubation (DI). The purpose of this study is to identify airway assessment factors and total airway score (TAS) for predicting DI defined by the IDS. METHODS: There were 305 ASA physical status 1-2 patients, aged 19-70 years, who underwent elective surgery with endotracheal intubation. During the pre-anesthetic visit, we evaluated patients by 7 preoperative airway assessment factors, including the following: Mallampati classification, thyromental distance, head & neck movement, body mass index (BMI), buck teeth, inter-incisor gap, and upper lip bite test (ULBT). After endotracheal intubation, patients were divided into 2 groups based on their IDS score estimated with 7 variables: normal (IDS < 5) and DI (IDS ≥ 5) groups. The incidence of TAS (> 6) and high score of each airway assessment factor was compared in two groups: odds ratio, confidence interval (CI) of 95%, with a significant P value ≤ 0.05. RESULTS: The odds ratio of TAS (> 6), ULBT (class III), head & neck movement (< 90°), inter-incisor gap (< 4 cm), BMI (≥ 25 kg/m(2)) and Mallampati classification (≥ class III) were respectively 13.57 (95% CI = 2.99-61.54, P < 0.05), 12.48 (95% CI = 2.50-62.21, P < 0.05), 3.11 (95% CI = 0.87-11.13), 2.32 (95% CI = 0.75-7.19), 2.22 (95% CI = 0.81-6.06), and 1.22 (95% CI = 0.38-3.89). CONCLUSIONS: We suggest that TAS (> 6) and ULBT (class III) are the most useful factors predicting DI.

Application of pulsed radiofrequency currents to rat dorsal root ganglia modulates nerve injury-induced tactile allodynia.

BACKGROUND: Application of pulsed radiofrequency (PRF) currents to the dorsal root ganglia (DRG) has been reported to produce relief from certain pain states without causing thermal ablation. In this study, we examined the direct correlation between PRF application to DRG associated with spinal nerve injury and reversal of injury-induced behavioral hypersensitivity in a rat neuropathic pain model. METHODS: Neuropathic lesioning was performed via left L5 spinal nerve ligation on male adult Sprague-Dawley rats. Once the injured rats had developed tactile allodynia, one group was then assigned to PRF treatment of the L5 DRG and another group was assigned to the sham treatment to the DRG. Behavioral testing was performed on both the control and treated paws using the von Frey filament test before the surgery and at indicated days. The resulting data were analyzed using a linear mixed model to assess the overall difference between the treatment groups and the overall difference among the study days. Cohen's d statistic was computed from paired difference-from-baseline scores for each of the 14 study days after treatment and these measures of effect size were then used to descriptively compare the recovery patterns over time for each study group. RESULTS: Spinal nerve injury resulted in the development of behavioral hypersensitivity to von Frey filament stimulation (allodynia) in the hindpaw of the left (injury) side. Mixed linear modeling showed a significant difference between the treatment groups (P = 0.0079) and a significant change of paw withdrawal threshold means over time (P = 0.0006) for all 12 animals. Evaluation of Cohen's d (effect size) revealed that the PRF-treated animals exhibited better recovery and recorded larger effect sizes than the sham-treated animals on 10 of the 14 post-PRF treatment days and exhibited moderate-to-strong effects posttreatment at days 8 to 10 and at and beyond day 32. CONCLUSIONS: Findings from this study support that PRF of the DRG causes reversal of nerve injury (spinal nerve ligation)-induced tactile allodynia in rats. This allodynia reversal indicates that nonablative PRF acting via modulation of the DRG can speed recovery in nerve injury-induced pain.

Production of antibodies with peptide-CpG-DNA-liposome complex without carriers.

BACKGROUND: The screening of peptide-based epitopes has been studied extensively for the purpose of developing therapeutic antibodies and prophylactic vaccines that can be potentially useful for treating cancer and infectious diseases such as influenza virus, malaria, hepatitis B, and HIV. To improve the efficacy of antibody production by epitope-based immunization, researchers evaluated liposomes as a means of delivering vaccines; they also formulated adjuvants such as flagella and CpG-DNA to enhance the magnitude of immune responses. Here, we provide a potent method for peptide-based epitope screening and antibody production without conventional carriers. RESULTS: We present that a particular form of natural phosphodiester bond CpG-DNA encapsulated in a specific liposome complex (Lipoplex(O)) induces potent immunomodulatory activity in humans as well as in mice. Additionally, Lipoplex(O) enhances the production of IgG2a specific to antigenic protein in mice. Most importantly, immunization of mice with several peptides co-encapsulated with Lipoplex(O) without carriers significantly induces each peptide-specific IgG2a production in a TLR9-dependent manner. A peptide-specific monoclonal antibody produced against hepatocellular carcinoma-associated antigen has functional effects on the cancer cells. CONCLUSIONS: Our overall results show that Lipoplex(O) is a potent adjuvant and that complexes of peptide and Lipoplex(O) are extremely useful for B cell epitope screening and antibody production without carriers. Therefore, our strategy may be promptly used for the development of therapeutic antibodies by rapid screening of potent B cell epitopes.

Thrombin-dependent MMP-2 activity is regulated by heparan sulfate.

Like most metalloproteases, matrix metalloprotease 2 (MMP-2) is synthesized as a zymogen. MMP-2 propeptide plays a role in inhibition of catalytic activity through a cysteine-zinc ion pairing, disruption of which results in full enzyme activation. A variety of proteases have been shown to be involved in the activation of pro-MMP-2, including metalloproteases and serine proteases. In the previous study we showed that MMP-2 activation occurred via specific cleavages of the propeptide by thrombin followed by intermolecular autoproteolytic processing for full enzymatic activity. Thrombin also degraded MMP-2, but this degradation was reduced greatly under cell-associated conditions with a concomitant increase in activation, prompting us to elucidate the molecular mechanisms underlying thrombin-mediated MMP-2 activation. In the present study we demonstrate that heparan sulfate is essential for thrombin-mediated activation of pro-MMP-2. Binding of heparan sulfate to thrombin is primarily responsible for this activation process, presumably through conformational changes at the active site. Furthermore, interaction of MMP-2 with exosites 1 and 2 of thrombin is crucial for thrombin-mediated MMP-2 degradation, and inhibition of this interaction by heparan sulfate or hirudin fragment results in a decrease in MMP-2 degradation. Finally, we demonstrated interaction between exosite 1 and hemopexin-like domain of MMP-2, suggesting a regulatory role of hemopexin-like domain in MMP-2 degradation. Taken together, our experimental data suggest a novel regulatory mechanism of thrombin-dependent MMP-2 enzymatic activity by heparan sulfate proteoglycans.