Group 3 innate lymphoid cells produce the growth factor HB-EGF to protect the intestine from TNF-mediated inflammation.

Tumor necrosis factor (TNF) drives chronic inflammation and cell death in the intestine, and blocking TNF is a therapeutic approach in inflammatory bowel disease (IBD). Despite this knowledge, the pathways that protect the intestine from TNF are incompletely understood. Here we demonstrate that group 3 innate lymphoid cells (ILC3s) protect the intestinal epithelium from TNF-induced cell death. This occurs independent of interleukin-22 (IL-22), and we identify that ILC3s are a dominant source of heparin-binding epidermal growth factor-like growth factor (HB-EGF). ILC3s produce HB-EGF in response to prostaglandin E2 (PGE2) and engagement of the EP2 receptor. Mice lacking ILC3-derived HB-EGF exhibit increased susceptibility to TNF-mediated epithelial cell death and experimental intestinal inflammation. Finally, human ILC3s produce HB-EGF and are reduced from the inflamed intestine. These results define an essential role for ILC3-derived HB-EGF in protecting the intestine from TNF and indicate that disruption of this pathway contributes to IBD.

ErbB1 and ErbB4 generate opposing signals regulating mesenchymal cell proliferation during valvulogenesis.

Heparin-binding EGF-like growth factor (HB-EGF) plays an indispensable role in suppression of cell proliferation during mouse valvulogenesis. However, ligands of the EGF receptor (EGFR/ErbB1), including HB-EGF, are generally considered as growth-promoting factors, as shown in cancers. HB-EGF binds to and activates ErbB1 and ErbB4. We investigated the role of ErbB receptors in valvulogenesis in vivo using ErbB1- and ErbB4-deficient mice, and an ex vivo model of endocardial cushion explants. We show that HB-EGF suppresses valve mesenchymal cell proliferation through a heterodimer of ErbB1 and ErbB4, and an ErbB1 ligand (or ligands) promotes cell proliferation through a homodimer of ErbB1. Moreover, a rescue experiment with cleavable or uncleavable isoforms of ErbB4 in ERBB4-null cells indicates that the cleavable JM-A, but not the uncleavable JM-B, splice variant of ErbB4 rescues the defect of the null cells. These data suggest that the cytoplasmic intracellular domain of ErbB4, rather than the membrane-anchored tyrosine kinase, achieves this suppression. Our study demonstrates that opposing signals generated by different ErbB dimer combinations function in the same cardiac cushion mesenchymal cells for proper cardiac valve formation.

Anti-HB-EGF Antibody-Mediated Delivery of siRNA to Atherosclerotic Lesions in Mice.

For atherosclerotic cardiovascular diseases (ACD), gene therapy may be a potential therapeutic strategy; however, lack of effective and safe methods for gene delivery to atherosclerotic plaques have limited its potential therapeutic applications. To overcome this limitation, we developed a novel antibody-based gene delivery system (anti-HB-EGF/NA vector) by chemically crosslinking antibodies against human heparin-binding epidermal growth factor-like growth factor (HB-EGF). It has been shown to be excessively expressed in human atherosclerotic plaques and NeutrAvidin (NA) for conjugating biotinylated siRNA. Immunofluorescence staining and quantitative flow cytometry analysis using human HB-EGF-expressing cells showed both antibody-mediated selective cellular targeting and efficient intracellular delivery of conjugated biotin-fluorescence. Moreover, we demonstrated antibody-mediated significant and selective gene knockdown via conjugation with anti-HB-EGF/NA vector and biotinylated siRNA (anti-HB-EGF/NA/b-siRNA) in vitro. Furthermore, using high fat-fed human HB-EGF knock-in and apolipoprotein E-knockout (Hbegf hz/hz; Apoe-/-) mice, we demonstrated that the anti-HB-EGF/NA vector, conjugating biotin-fluorescence, increasingly accumulated within the atherosclerotic plaques of the ascending aorta in which human HB-EGF expression levels were highly elevated. Moreover, in response to a single intravenous injection of anti-HB-EGF/NA/b-siRNA in a dose-dependent manner, qPCR analysis of laser-dissected atherosclerotic plaques of the ascending aorta showed significant knockdown of the reporter gene expression. These results suggest that the anti-HB-EGF antibody-mediated siRNA delivery could be a promising delivery system for gene therapy of ACD.

BK-UM in patients with recurrent ovarian cancer or peritoneal cancer: a first-in-human phase-I study.

BACKGROUND: BK-UM (CRM197) is a mutant form of diphtheria toxin and a specific inhibitor of heparin-binding epidermal growth factor-like growth factor (HB-EGF). We assessed the safety, pharmacokinetics, recommended dose, and efficacy of BK-UM in patients with recurrent ovarian cancer (OC) or peritoneal cancer (PC), and measured HB-EGF levels in serum and abdominal fluid after BK-UM administration. METHODS: Eleven patients with advanced or recurrent OC or PC were enrolled and treated with BK-UM via the intraperitoneal route. The dose was escalated (1.0, 2.0, 3.3, and 5.0 mg/m2) using a 3 + 3 design. RESULTS: Eight of 11 patients completed treatment. No dose-limiting toxicity (DLT) was experienced at dose levels 1 (1.0 mg/m2) and 2 (2.0 mg/m2). Grade 3 transient hypotension as an adverse event (defined as a DLT in the present study) was observed in two of four patients at dose level 3 (3.3 mg/m2). Treatment with BK-UM was associated with decreases in HB-EGF levels in serum and abdominal fluid in seven of 11 patients and five of eight patients, respectively. Clinical outcomes included a partial response in one patient, stable disease in five patients, and progressive disease in five patients. CONCLUSIONS: BK-UM was well tolerated at doses of 1.0 and 2.0 mg/m2, with evidence for clinical efficacy in patients with recurrent OC or PC. A dose of 2.0 mg/m2 BK-UM is recommended for subsequent clinical trials. TRIAL REGISTRATION: This trial was prospectively performed as an investigator-initiated clinical trial. The trial numbers are UMIN000001002 and UMIN000001001, with registration dates of 1/30/2008 and 2/4/2008, respectively. UMIN000001001 was registered as a trial for the continuous administration of BK-UM after UMIN000001002 .

Tetraspanin is required for generation of reactive oxygen species by the dual oxidase system in Caenorhabditis elegans.

Reactive oxygen species (ROS) are toxic but essential molecules responsible for host defense and cellular signaling. Conserved NADPH oxidase (NOX) family enzymes direct the regulated production of ROS. Hydrogen peroxide (H(2)O(2)) generated by dual oxidases (DUOXs), a member of the NOX family, is crucial for innate mucosal immunity. In addition, H(2)O(2) is required for cellular signaling mediated by protein modifications, such as the thyroid hormone biosynthetic pathway in mammals. In contrast to other NOX isozymes, the regulatory mechanisms of DUOX activity are less understood. Using Caenorhabditis elegans as a model, we demonstrate that the tetraspanin protein is required for induction of the DUOX signaling pathway in conjunction with the dual oxidase maturation factor (DUOXA). In the current study, we show that genetic mutation of DUOX (bli-3), DUOXA (doxa-1), and peroxidase (mlt-7) in C. elegans causes the same defects as a tetraspanin tsp-15 mutant, represented by exoskeletal deficiencies due to the failure of tyrosine cross-linking of collagen. The deficiency in the tsp-15 mutant was restored by co-expression of bli-3 and doxa-1, indicating the involvement of tsp-15 in the generation of ROS. H(2)O(2) generation by BLI-3 was completely dependent on TSP-15 when reconstituted in mammalian cells. We also demonstrated that TSP-15, BLI-3, and DOXA-1 form complexes in vitro and in vivo. Cell-fusion-based analysis suggested that association with TSP-15 at the cell surface is crucial for BLI-3 activation to release H(2)O(2). This study provides the first evidence for an essential role of tetraspanin in ROS generation.

Metastasis suppressor tetraspanin CD82/KAI1 regulates ubiquitylation of epidermal growth factor receptor.

Ligand-induced ubiquitylation of EGF receptor (EGFR) is an important regulatory mechanism that controls endocytic trafficking of the receptor and its signaling potential. Here we report that tetraspanin CD82/KAI1 specifically suppresses ubiquitylation of EGFR after stimulation with heparin-binding EGF or amphiregulin and alters the rate of recruitment of the activated receptor to EEA1-positive endosomes. The suppressive effect of CD82 is dependent on the heparin-binding domain of the ligand. Deletion of the C-terminal cytoplasmic domain of CD82 (CD82ΔC mutant) inhibits endocytic trafficking of the tetraspanin and compromises its activity toward heparin-binding EGF-activated EGFR. Reduced ubiquitylation of EGFR is accompanied by PKC-dependent increase in serine phosphorylation of c-Cbl in cells expressing elevated levels of CD82. Furthermore, phosphorylation of threonine 654 (PKC phosphorylation site) in the juxtamembrane domain of the receptor is considerably increased in CD82-expressing cells. These results describe previously unsuspected links between tetraspanin proteins and ubiquitylation of their molecular partners (e.g., EGFR). Our data identify CD82 as a new regulator of c-Cbl, which discriminatively controls the activity of this E3 ubiquitin ligase toward heparin-binding ligand-EGFR pairs. Taken together, these observations provide an important new insight into the modulatory role of CD82 in endocytic trafficking of EGF receptor.

Antibody-modified lipid nanoparticles for selective delivery of siRNA to tumors expressing membrane-anchored form of HB-EGF.

An Fab' antibody against heparin-binding epidermal growth factor-like growth factor (HB-EGF) was applied to achieve advanced tumor-targeted delivery of siRNA. Lipid nanoparticles (LNP) encapsulating siRNA (LNP-siRNA) were prepared, pegylated, and surface modified with Fab' fragments of anti-HB-EGF antibody (αHB-EGF LNP-siRNA). αHB-EGF LNP-siRNA showed high-binding affinity to recombinant human HB-EGF in a Biacore assay. In addition, αHB-EGF LNP-siRNA selectively associated with cells expressing HB-EGF in vitro. Confocal microscopic images showed that siRNA formulated in αHB-EGF LNP-siRNA was efficiently internalized into MDA-MB-231 human breast cancer cells, on which HB-EGF is highly expressed. In addition, siRNA encapsulated in αHB-EGF LNP induced obvious suppression of both target mRNA and protein levels in MDA-MB-231 cells. These results indicate that αHB-EGF LNP have excellent potential to deliver siRNA to target cancer cells, resulting in effective gene silencing.

Conditional loss of heparin-binding EGF-like growth factor results in enhanced liver fibrosis after bile duct ligation in mice.

Our aims were to evaluate the involvement of heparin-binding EGF-like growth factor (HB-EGF) in liver fibrogenesis of humans and mice and to elucidate the effect of HB-EGF deficiency on cholestatic liver fibrosis using conditional HB-EGF knockout (KO) mice. We first demonstrated that gene expression of HB-EGF had a positive significant correlation with that of collagen in human fibrotic livers, and was increased in bile duct ligation (BDL)-induced fibrotic livers in mouse. We then generated conditional HB-EGF knockout (KO) mice using the interferon inducible Mx-1 promoter driven Cre recombinase transgene and wild type (WT) and KO mice were subjected to BDL. After BDL, KO mice exhibited enhanced liver fibrosis with increased expression of collagen, compared with WT mice. Finally, we used mouse hepatic stellate cells (HSCs) to examine the role of HB-EGF in the activation of these cells and showed that HB-EGF antagonized TGF-ß-induced gene expression of collagen in mouse primary HSCs. Interestingly, HB-EGF did not prevent the TGF-ß-induced nuclear accumulation of Smad3, but did lead to stabilization of the Smad transcriptional co-repressor TG-interacting factor. In conclusion, our data suggest a possible protective role of HB-EGF in cholestatic liver fibrosis.

HB-EGF function in cardiac valve development requires interaction with heparan sulfate proteoglycans.

HB-EGF, a member of the EGF family of growth factors, plays an important role in cardiac valve development by suppressing mesenchymal cell proliferation. Here, we show that HB-EGF must interact with heparan sulfate proteoglycans (HSPGs) to properly function in this process. In developing valves, HB-EGF is synthesized in endocardial cells but accumulates in the mesenchyme by interacting with HSPGs. Disrupting the interaction between HB-EGF and HSPGs in an ex vivo model of endocardial cushion explants resulted in increased mesenchymal cell proliferation. Moreover, homozygous knock-in mice (HB(Delta)(hb/)(Delta)(hb)) expressing a mutant HB-EGF that cannot bind to HSPGs developed enlarged cardiac valves with hyperproliferation of mesenchymal cells; this resulted in a phenotype that resembled that of Hbegf-null mice. Interestingly, although Hbegf-null mice had abnormal heart chambers and lung alveoli, HB(Delta)(hb/)(Delta)(hb) mice did not exhibit these defects. These results indicate that interactions with HSPGs are essential for the function of HB-EGF, especially in cardiac valve development, in which HB-EGF suppresses mesenchymal cell proliferation.

Conditional knockout of heparin-binding epidermal growth factor-like growth factor in the liver accelerates carbon tetrachloride-induced liver injury in mice.

AIM: We previously demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is induced in response to several liver injuries. Because the HB-EGF knockout (KO) mice die in utero or immediately after birth due to cardiac defects, the loss of function study in vivo is limited. Here, we generated liver-specific HB-EGF conditional knockout mice using the interferon-inducible Mx-1 promoter driven cre recombinase transgene and investigated its role during acute liver injury. METHODS: We induced acute liver injury by a single i.p. injection of carbon tetrachloride (CCl4 ) in HB-EGF KO mice and wild-type mice and liver damage was assessed by biochemical and immunohistochemical analysis. We also used AML12 mouse hepatocyte cell lines to examine the molecular mechanism of HB-EGF-dependent anti-apoptosis and wound-healing process of the liver in vitro. RESULTS: HB-EGF KO mice exhibited a significant increase of alanine aminotransferase level and also showed a significant increase in the number of apoptotic hepatocytes assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling staining at 24 h after CCl4 injection. We also demonstrated that HB-EGF treatment inhibited tumor necrosis factor-α-induced apoptosis of AML12 mouse hepatocytes and promoted the wound-healing response of these cells. CONCLUSION: This study showed that HB-EGF plays a protective role during acute liver injury.

Proteolytic activation of heparin-binding EGF-like growth factor by membrane-type matrix metalloproteinase-1 in ovarian carcinoma cells.

Increased expression of heparin-binding EGF-like growth factor (HB-EGF) and membrane-type matrix metalloproteinase-1 (MT1-MMP) is frequently associated with various types of malignant tumor. HB EGF-like growth factor has been reported to promote the malignant progression of ovarian carcinoma. Based on this finding, inhibition of HB-EGF activity with CRM197 is now under phase I clinical evaluation. On the other hand, MT1-MMP expressed in ovarian carcinoma cells is thought to promote invasion and growth of tumor cells by degrading the extracellular matrix. However, we recently demonstrated that co-expression of MT1-MMP and HB-EGF in gastric carcinoma cells leads to cleavage of HB-EGF within its N-terminal heparin-binding region, converting it into a potent heparin-independent growth factor. In this study, we evaluated the importance of regulation of HB-EGF by MT1-MMP in clinical samples of ovarian carcinoma. We detected co-expression of HB-EGF and MT1-MMP in clear cell ovarian carcinoma tissues, particularly at the invasion front and in tumor cells that had disseminated into the ascites, whereas HB-EGF alone was expressed in non-invasive borderline ovarian tumor tissue. Furthermore, a soluble HB-EGF fragment that corresponds to that processed by MT1-MMP was detected in malignant ascites obtained from patients with metastatic ovarian carcinoma. Ovarian carcinoma cells that express MT1-MMP and HB-EGF exhibited enhanced cell growth in a 3D-collagen matrix and anchorage-independent growth in suspension. These results indicate that MT1-MMP co-expressed with HB-EGF in ovarian carcinoma cells potentiates the activity of HB-EGF to promote invasive tumor growth and spreading in vivo.

Integrin signal masks growth-promotion activity of HB-EGF in monolayer cell cultures.

The extracellular environment and tissue architecture contribute to proper cell function and growth control. Cells growing in monolayers on standard polystyrene tissue culture plates differ in their shape, growth rate and response to external stimuli, compared with cells growing in vivo. Here, we showed that the EGFR (epidermal growth factor receptor) ligand heparin-binding EGF-like growth factor (HB-EGF) strongly stimulated cell growth in nude mice, but not in cells cultured in vitro. We explored the effects of HB-EGF on cell growth under various cell culture conditions and found that growth promotion by HB-EGF was needed in three-dimensional (3D) or two-dimensional (2D) culture systems in which cell-matrix adhesion was reduced. Under such conditions, cell growth was extremely suppressed in the absence of HB-EGF, but markedly potentiated in the presence of HB-EGF. When the integrin signal was reduced using antibodies or knockout of either integrin beta1 or focal adhesion kinase (FAK), cells showed HB-EGF-dependent growth. We also showed that EGF, transforming growth factor-alpha (TGFalpha) or ligands of other receptor tyrosine kinases (RTKs) stimulated cell growth in 3D culture, but not in tissue culture plates. These results indicate that the integrin signal was sufficient to support cell growth in 2D tissue culture plates without addition of the growth factor, whereas stimulation by growth factors was clearly demonstrated in culture systems in which integrin signals were attenuated.

The fusing ability of sperm is bestowed by CD9-containing vesicles released from eggs in mice.

Membrane fusion is an essential step in the encounter of two nuclei from sex cells-sperm and egg-in fertilization. However, aside from the involvement of two molecules, CD9 and Izumo, the mechanism of fusion remains unclear. Here, we show that sperm-egg fusion is mediated by vesicles containing CD9 that are released from the egg and interact with sperm. We demonstrate that the CD9(-/-) eggs, which have a defective sperm-fusing ability, have impaired release of CD9-containing vesicles. We investigate the fusion-facilitating activity of CD9-containing vesicles by examining the fusion of sperm to CD9(-/-) eggs with the aid of exogenous CD9-containing vesicles. Moreover, we show, by examining the fusion of sperm to CD9(-/-) eggs, that hamster eggs have a similar fusing ability as mouse eggs. The CD9-containing vesicle release from unfertilized eggs provides insight into the mechanism required for fusion with sperm.

Spread of genetically similar noroviruses in Bangkok, Thailand, through symptomatic and asymptomatic individuals.

Norovirus infection is a major cause of acute gastroenteritis, although some infected individuals are asymptomatic. GII.4 is the predominant genotype worldwide and, since 2000, has been the most prevalent in patients in Thailand with acute gastroenteritis. We screened stool samples for norovirus in 786 patients with acute gastroenteritis who were admitted to a hospital in Bangkok from 2017 to early 2019 and detected it in 136 specimens (17.3%). Eight and 124 specimens were positive for the GI and GII genogroups, respectively, and the remaining 4 specimens were double-positive. Nine genotypes (GI.3, GI.5, GII.2, GII.3, GII.4, GII.6, GII.8, GII.13, and GII.17) were identified from 140 strains, and 72 strains (51.4%) were GII.4. We had previously conducted a one-year survey of norovirus infection in residents of a community in Bangkok from May 2018 to April 2019 and found that a substantial portion of the residents were infected asymptomatically. The 9 genotypes identified in the patients were also commonly identified in the community residents. To investigate the relationship between noroviruses identified in the acute gastroenteritis patients and those identified in the community residents, phylogenetic tree analysis was conducted. Of the 9 genotypes, 8 showed similarities in both their genomic sequences and their deduced amino acid sequences. In addition, strain replacement of GI.3 was observed in both the patients and the community residents within the overlapping period. These results suggested that norovirus spreads efficiently to the community by simultaneously causing symptomatic and asymptomatic infections.

Humanized gene replacement in mice reveals the contribution of cancer stroma-derived HB-EGF to tumor growth.

Tumor progression is a complex process that involves the interaction of cancer cells with the cancer-surrounding stromal cells. The cancer stroma influences the cancer cell growth and metastatic potential. The EGF family growth factor HB-EGF is synthesized in cancer cells and plays pivotal roles in oncogenic transformation and tumor progression, but the contribution of HB-EGF expressed in tumor stromal cells to tumor growth remains unclear. In the present study, we found that HB-EGF was expressed in host-derived cancer stromal cells in xenograft and allograft mouse tumor models. CRM197 is a specific inhibitor of human HB-EGF that has no effect on mouse HB-EGF. To elucidate whether host-derived stromal HB-EGF contributes to tumor growth, we generated knock-in mice expressing a CRM197-inhibitable humanized mutant form of HB-EGF. Administration of CRM197 to humanized knock-in mice that were bearing tumors derived from human or mouse cancer cells revealed that inhibition of host-derived stromal HB-EGF by CRM197 significantly reduced tumor growth. These results suggest that HB-EGF in the cancer-associated stroma plays a significant role for tumor growth, and that the HB-EGF derived from the stroma, as well as that expressed by cancer cells, is a potential target for cancer therapy. The present results also suggest that the humanized HB-EGF knock-in mice could be utilized for pathophysiological studies of HB-EGF as well as the development of therapeutic strategies targeting HB-EGF.

Maternal heparin-binding-EGF deficiency limits pregnancy success in mice.

An intimate discourse between the blastocyst and uterus is essential for successful implantation. However, the molecular basis of this interaction is not clearly understood. Exploiting genomic Hbegf mutant mice, we show here that maternal deficiency of heparin-binding EGF-like growth factor (HB-EGF) defers on-time implantation, leading to compromised pregnancy outcome. We also demonstrate that amphiregulin, but not epiregulin, partially compensates for the loss of HB-EGF during implantation. In search of the mechanism of this compensation, we found that reduced preimplantation estrogen secretion from ovarian HB-EGF deficiency is a cause of sustained expression of uterine amphiregulin before the initiation of implantation. To explore the significance specifically of uterine HB-EGF in implantation, we examined this event in mice with conditional deletion of uterine HB-EGF and found that this specific loss of HB-EGF in the uterus still defers on-time implantation without altering preimplantation ovarian estrogen secretion. The observation of normal induction of uterine amphiregulin surrounding the blastocyst at the time of attachment in these conditional mutant mice suggests a compensatory role of amphiregulin for uterine loss of HB-EGF, preventing complete failure of pregnancy. Our study provides genetic evidence that HB-EGF is critical for normal implantation. This finding has high clinical relevance, because HB-EGF signaling is known to be important for human implantation.

Norovirus transmission mediated by asymptomatic family members in households.

The transmission of human norovirus excreted from infected persons occasionally causes sporadic infections and outbreaks. Both symptomatic patients and asymptomatic carriers have been reported to contribute to norovirus transmission, but little is known about the magnitude of the contribution of asymptomatic carriers. We carried out a 1-year survey of residents of a district of Bangkok, Thailand to determine the percentage of norovirus transmissions originating from asymptomatic individuals. We screened 38 individuals recruited from 16 families from May 2018 to April 2019 for GI and GII genotypes. Norovirus was detected every month, and 101 of 716 stool samples (14.1%) from individuals with no symptoms of acute gastroenteritis were norovirus-positive. The average infection frequency was 2.4 times per person per year. Fourteen genotypes were identified from the positive samples, with GII.4 being detected most frequently. Notably, 89.1% of the norovirus-positive samples were provided by individuals with no diarrhea episode. Similar to cases of symptomatic infections in Thailand, asymptomatic infections were observed most frequently in December. We detected 4 cases of NV infection caused by household transmission, and 3 of the 4 transmissions originated from asymptomatic individuals. We also identified a case in which norovirus derived from an asymptomatic individual caused diarrhea in a family member. These results suggest that asymptomatic individuals play a substantial role in both the maintenance and spreading of norovirus in a community through household transmission.

Identification of GII.14[P7] norovirus and its genomic mutations from a case of long-term infection in a post-symptomatic individual.

Norovirus is a leading cause of acute gastroenteritis worldwide. Norovirus shedding typically lasts one week to one month after the onset of diarrhea in immunocompetent hosts. The occurrence of mutations in the genome during infection has contributed to the evolution of norovirus. It has been suggested that genomic mutations in the P2-domain of capsid protein VP1, the major antigenic site for virus clearance, are involved in the evasion of host immunity and prolonged shedding of norovirus. In our previous study, we found a case of long-term shedding of GII.14 norovirus in a post-symptomatic immunocompetent individual that lasted about three months. In this study, we characterized the genomic sequence of the GII.14 strain to gain insight into the context of long-term shedding. By sequencing a 4.8 kb region of the genome corresponding to half of ORF1 and the entire ORF2 and ORF3, which encode several non-structural proteins and the structural proteins VP1 and VP2, the GII.14 strain was found to be classified as recombinant GII.14[P7]. Six point-mutations occurred during the three-month period of infection in a time-dependent manner in the genomic regions encoding RNA-dependent RNA polymerase, VP1, and VP2. Three of the six mutations were sense mutations, but no amino acid substitution was identified in the P2-domain of VP1. These results suggest that there is a mechanism by which long-term shedding of norovirus occurs in immunocompetent individuals independent of P2-domain mutations.

Synergistic anti-tumor effect of paclitaxel with CRM197, an inhibitor of HB-EGF, in ovarian cancer.

Heparin-binding EGF-like growth factor (HB-EGF) plays a pivotal role in tumor growth and clinical outcomes in patients with ovarian cancer, leading to the validation of HB-EGF as a target for ovarian cancer therapy. In this study, we investigated the anti-tumor effects of paclitaxel, as an anti-cancer agent, and CRM197, as a specific inhibitor off HB-EGF, in ovarian cancer. Paclitaxel induced transient ERK activation and sustained activation of JNK and p38 MAPK through the ectodomain shedding of HB-EGF in SKOV3 cells. In addition, the overexpression of HB-EGF in paclitaxel-treated SKOV3 cells resulted in modulation of paclitaxel-evoked MAPK signaling, including marked activation of ERK and Akt, and minimized activation of JNK and p38 MAPK, indicating that HB-EGF is involved in drug sensitivity through the balance of anti-apoptotic and pro-apoptotic signals induced by paclitaxel. The combination of paclitaxel with CRM197 had an inhibitory effect on cell proliferation and enhanced apoptosis via the inhibition of ERK and Akt activation and the stimulation of p38 and JNK activation. More prominently, the administration of paclitaxel with CRM197 resulted in synergistic anti-tumor effects in SKOV3 cells and in SKOV3 cells overexpressing HB-EGF in xenografted mice. Accordingly, inhibitory agents against HB-EGF, such as CRM197, represent possible chemotherapeutic and chemosensitizing agents for ovarian cancer.

Tetraspanins CD9 and CD81 function to prevent the fusion of mononuclear phagocytes.

Tetraspanins CD9 and CD81 facilitate the fusion between gametes, myoblasts, or virus-infected cells. Here, we investigated the role of these tetraspanins in the fusion of mononuclear phagocytes. Expression of CD9 and CD81 and their complex formation with integrins were up-regulated when blood monocytes were cultured under normal conditions. Under fusogenic conditions in the presence of Con A, CD9 and CD81 up-regulation was inhibited, and their complex formation with integrins was down-regulated. Anti-CD9 and -CD81 antibodies, which were previously shown to inhibit the fusion of gametes, myoblasts, and virus-infected cells, unexpectedly promoted the fusion of monocytes and alveolar macrophages. However, these effects were not due to altered cell adhesion, aggregation, or cytokine production. When stimulated in vitro or in vivo, alveolar macrophages and bone marrow cells of CD9- and CD81-null mice formed larger numbers of multinucleated cells than those of wild-type mice. Finally, CD9/CD81 double-null mice spontaneously developed multinucleated giant cells in the lung and showed enhanced osteoclastogenesis in the bone. These results suggest that CD9 and CD81 coordinately prevent the fusion of mononuclear phagocytes.