技术资料

OBJECTIVE: The S100A9 and S100A8 proteins are highly expressed by neutrophils and monocytes and are part of a group of damage-associated molecular pattern molecules that trigger inflammatory responses. Sera and synovial fluids of patients with rheumatoid arthritis (RA) contain high concentrations of S100A8/A9 that correlate with disease activity.backslashnbackslashnMETHODS: In this study,we investigated the importance of S100A9 in RA by using neutralizing antibodies in a murine lipopolysaccharide-synchronized collagen-induced arthritis model. We also used an in vitro model of stimulation of human immune cells to decipher the role played by S100A9 in leukocyte migration and pro-inflammatory cytokine secretion.backslashnbackslashnRESULTS: Treatment with anti-S100A9 antibodies improved the clinical score by 50%,diminished immune cell infiltration,reduced inflammatory cytokines,both in serum and in the joints,and preserved bone/collagen integrity. Stimulation of neutrophils with S100A9 protein led to the enhancement of neutrophil transendothelial migration. S100A9 protein also induced the secretion by monocytes of proinflammatory cytokines like TNFα,IL-1β and IL-6,and of chemokines like MIP-1α and MCP-1.backslashnbackslashnCONCLUSION: The effects of anti-S100A9 treatment are likely direct consequences of inhibiting the S100A9-mediated promotion of neutrophil transmigration and secretion of pro-inflammatory cytokines from monocytes. Collectively,our results show that treatment with anti-S100A9 may inhibit amplification of the immune response and help preserve tissue integrity. Therefore,S100A9 is a promising potential therapeutic target for inflammatory diseases like rheumatoid arthritis for which alternative therapeutic strategies are needed. View Publication

The 2009 H1N1 influenza pandemic was a major international public health crisis which caused considerable morbidity and mortality worldwide. The goal of this study was to produce anti-H1 monoclonal antibodies (MAbs) for improving diagnostic immunological assays and to develop potential immunotherapeutics. Nine MAbs were produced after immunizing mice with recombinant hemagglutinin (HA) protein from A/California/06/09. Two spleenocyte myeloma fusions yielded 1588 hybridoma cultures. After screening the hybridoma culture supernatants for antibody reactivity to rHA,nine clones were selected for further characterization. Cross-reactivity studies of the anti-rHA antibodies against a panel of influenza viruses (H1-H16) revealed eight out of nine MAbs were specific to the pandemic H1 subtype,except for MAb F256G2sc1 which also cross-reacted with H5 subtype virus. All MAbs were of the IgG1κ isotype,except F256G2sc1 which was IgG2aκ. The anti-rHA MAbs had binding affinities to rHA that ranged from a K(D) (disassociation constant) of 1.34×10(-9)M (F255G7sc1) to the weakest affinity of 4.60×10(-8)M (F255G4sc1). Interestingly,in a plaque reduction neutralization assay,all MAbs except F255G3sc1 demonstrated neutralizing ability. Furthermore,all MAbs except F255G3sc1 and F255G9sc1 exhibited anti-hemagglutinin activity against pandemic H1N1 viruses,but not against classical North American swine influenza viruses of the same subtype. Immunofluorescence assay (IFA) demonstrated that all MAbs except F255G1sc1 and F255G3sc1 were able to detect 2009 pandemic H1N1 (2009) virus- infected MDCK cells. The MAbs were also evaluated for potential use in competitive ELISA (cELISA),and with the exception of F255G3sc1,all MAbs showed competitive activity with serum collected from pigs infected with pandemic H1N1 virus (2009). The developed MAbs have demonstrated utility as immunodiagnostic and research reagents,and their neutralizing capabilities also hold potential for designing antiviral drugs against pandemic influenza. View Publication

Deriving lung progenitors from patient-specific pluripotent cells is a key step in producing differentiated lung epithelium for disease modeling and transplantation. By mimicking the signaling events that occur during mouse lung development,we generated murine lung progenitors in a series of discrete steps. Definitive endoderm derived from mouse embryonic stem cells (ESCs) was converted into foregut endoderm,then into replicating Nkx2.1+ lung endoderm,and finally into multipotent embryonic lung progenitor and airway progenitor cells. We demonstrated that precisely-timed BMP,FGF,and WNT signaling are required for NKX2.1 induction. Mouse ESC-derived Nkx2.1+ progenitor cells formed respiratory epithelium (tracheospheres) when transplanted subcutaneously into mice. We then adapted this strategy to produce disease-specific lung progenitor cells from human Cystic Fibrosis induced pluripotent stem cells (iPSCs),creating a platform for dissecting human lung disease. These disease-specific human lung progenitors formed respiratory epithelium when subcutaneously engrafted into immunodeficient mice. View Publication

Induced pluripotent stem (iPS) cells are generated from somatic cells by the forced expression of a defined set of pluripotency-associated transcription factors. Human iPS cells can be propagated indefinitely,while maintaining the capacity to differentiate into all cell types in the body except for extra-embryonic tissues. This technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large amounts of disease-specific cells for biomedical research. Despite their great potential,the long reprogramming process (up to 1. month) remains one of the most significant challenges facing standard virus-mediated methodology. In this study,we report the accelerated generation of human iPS cells from adipose-derived stem (ADS) cells,using a new combination of chemical inhibitors under a setting of physiological hypoxia in conjunction with retroviral transduction of Oct4,Sox2,Klf4,and L-Myc. Under optimized conditions,we observed human embryonic stem (ES)-like cells as early as 6. days after the initial retroviral transduction. This was followed by the emergence of fully reprogrammed cells bearing Tra-1-81-positive and DsRed transgene-silencing properties on day 10. The resulting cell lines resembled human ES cells in many respects including proliferation rate,morphology,pluripotency-associated markers,global gene expression patterns,genome-wide DNA methylation states,and the ability to differentiate into all three of the germ layers,both in vitro and in vivo. Our method,when combined with chemical inhibitors under conditions of physiological hypoxia,offers a powerful tool for rapidly generating bona fide human iPS cells and facilitates the application of iPS cell technology to biomedical research. textcopyright 2011 Elsevier Inc. View Publication

Symptomatic dengue virus infection ranges in disease severity from an influenza-like illness to life-threatening shock. One model of the mechanism underlying severe disease proposes that weakly neutralizing,dengue serotype cross-reactive antibodies induced during a primary infection facilitate virus entry into Fc receptor-bearing cells during a subsequent secondary infection,increasing viral replication and the release of cytokines and vasoactive mediators,culminating in shock. This process has been termed antibody-dependent enhancement of infection and has significantly hindered vaccine development. Much of our understanding of this process has come from studies using mouse monoclonal antibodies (MAbs); however,antibody responses in mice typically exhibit less complexity than those in humans. A better understanding of the humoral immune response to natural dengue virus infection in humans is sorely needed. Using a high-efficiency human hybridoma technology,we isolated 37 hybridomas secreting human MAbs to dengue viruses from 12 subjects years or even decades following primary or secondary infection. The majority of the human antibodies recovered were broadly cross-reactive,directed against either envelope or premembrane proteins,and capable of enhancement of infection in vitro; few exhibited serotype-specific binding or potent neutralizing activity. Memory B cells encoding enhancing antibodies predominated in the circulation,even two or more decades following infection. Mapping the epitopes and activity of naturally occurring dengue antibodies should prove valuable in determining whether the enhancing and neutralizing activity of antibodies can be separated. Such principles could be used in the rational design of vaccines that enhance the induction of neutralizing antibodies,while lowering the risk of dengue shock syndrome. View Publication

Tools permitting the isolation of live pancreatic cell subsets for culture and/or molecular analysis are limited. To address this,we developed a collection of monoclonal antibodies with selective surface labeling of endocrine and exocrine pancreatic cell types. Cell type labeling specificity and cell surface reactivity were validated on mouse pancreatic sections and by gene expression analysis of cells isolated using FACS. Five antibodies which marked populations of particular interest were used to isolate and study viable populations of purified pancreatic ducts,acinar cells,and subsets of acinar cells from whole pancreatic tissue or of alpha or beta cells from isolated mouse islets. Gene expression analysis showed the presence of known endocrine markers in alpha and beta cell populations and revealed that TTR and DPPIV are primarily expressed in alpha cells whereas DGKB and GPM6A have a beta cell specific expression profile. View Publication

Trypanosoma congolense is an African trypanosome that causes serious disease in cattle in Sub-Saharan Africa. The four major life cycle stages of T. congolense can be grown in vitro,which has led to the identification of several cell-surface molecules expressed on the parasite during its transit through the tsetse vector. One of these,glutamic acid/alanine-rich protein (GARP),is the first expressed on procyclic forms in the tsetse midgut and is of particular interest because it replaces the major surface coat molecule of bloodstream forms,the variant surface glycoprotein (VSG) that protects the parasite membrane,and is involved in antigenic variation. Unlike VSG,however,the function of GARP is not known,which necessarily limits our understanding of parasite survival in the tsetse. Toward establishing the function of GARP,we report its three-dimensional structure solved by iodide phasing to a resolution of 1.65 Å. An extended helical bundle structure displays an unexpected and significant degree of homology to the core structure of VSG,the only other major surface molecule of trypanosomes to be structurally characterized. Immunofluorescence microscopy and immunoaffinity-tandem mass spectrometry were used in conjunction with monoclonal antibodies to map both non-surface-disposed and surface epitopes. Collectively,these studies enabled us to derive a model describing the orientation and assembly of GARP on the surface of trypanosomes. The data presented here suggest the possible structure-function relationships involved in replacement of the bloodstream form VSG by GARP as trypanosomes differentiate in the tsetse vector after a blood meal. View Publication

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia in the United States and globally. Despite the availability of pneumococcal capsular polysaccharide (PPS) and protein conjugate-based vaccines,the prevalence of antibiotic-resistant pneumococcal strains,serotype (ST) replacement in nonconjugate vaccine strains,and uncertainty as to whether the PPS vaccine that is used in adults protects against pneumonia emphasize the need for continued efforts to understand the nature of protective PPS antibody responses. In this study,we generated mouse monoclonal antibodies (MAbs) to a conjugate consisting of the PPS of serotype 8 (PPS8) S. pneumoniae and tetanus toxoid. Thirteen MAbs,including four IgMs that bound to PPS8 and phosphorylcholine (PC) and five IgMs and four IgG1s that bound to PPS8 but not PC,were produced,and their nucleotide sequences,epitope and fine specificity,and efficacy against lethal challenge with ST8 S. pneumoniae were determined. MAbs that bound to PPS8 exhibited gene use that was distinct from that exhibited by MAbs that bound to PC. Only PPS8-binding MAbs that did not bind PC were protective in mice. All 13 MAbs used germ line variable-region heavy (V(H)) and light (V(L)) chain genes,with no evidence of somatic hypermutation. Our data reveal a relationship between PPS specificity and V(H) gene use and MAb efficacy in mice. These findings provide insight into the relationship between antibody molecular structure and function and hold promise for the development of novel surrogates for pneumococcal vaccine efficacy. View Publication

PURPOSE: In vitro sensitivity to the proapoptotic receptor agonists dulanermin (rhApo2L/TRAIL) and drozitumab (DR5-agonist antibody) is strongly predicted by the expression of the O-glycosylation enzymes GALNT14 in non-small cell lung cancer (NSCLC) cell lines (among others) and of FUT3/6 in colorectal cancer (CRC) cell lines. We developed immunohistochemistry (IHC) assays that measure GALNT14 and FUT3/6 levels in archival formalin-fixed,paraffin-embedded human tumor tissue to determine marker prevalence in NSCLC and CRC tissue and to enable the future examination of these markers in clinical trials. EXPERIMENTAL DESIGN: GALNT14 or FUT3/6 ELISA-positive hybridoma clones were screened through IHC on cell pellets with known mRNA levels. The specificity of staining was examined in cell lines,normal tissue,and tumor tissue. RESULTS: GALNT14 and FUT3/6 IHC exhibited a golgi staining pattern and correlated with GALNT14 and FUT3/6 (but not GALNT2 and FUT4) mRNA expression levels in cell lines and normal tissues,suggesting specificity. GALNT14 and FUT3/6 H-scores were significantly higher in cell lines sensitive to dulanermin (P = 0.01 and P = 0.0004,respectively) and drozitumab (P = 0.03 and P textless 0.0001,respectively) versus resistant cell lines. GALNT14 and FUT3/6 H-scores varied widely,with approximately 45% of NSCLC samples exhibiting weak to moderate GALNT14 staining (H-score of at least 25) and 70% of CRC samples exhibiting moderate to strong FUT3/6 staining (H-score of at least 125). CONCLUSIONS: GALNT14 and FUT3/6 expression can be assessed in human tumors using sensitive and specific IHC assays. Both assays are being deployed in ongoing clinical trials of dulanermin and drozitumab to assess potential utility for patient selection. View Publication

Antiangiogenic effects of the proteasome inhibitor bortezomib were analyzed on tumor xenografts in vivo. Bortezomib strongly inhibited angiogenesis and vascularization in the chicken chorioallantoic membrane. Bortezomib's inhibitory effects on chorioallantoic membrane vascularization were abrogated in the presence of distinct tumor xenografts,thanks to a soluble factor secreted by tumor cells. Through size-exclusion and ion-exchange chromatography as well as mass spectroscopy,we identified GRP-78,a chaperone protein of the unfolded protein response,as being responsible for bortezomib resistance. Indeed,a variety of bortezomib-resistant solid tumor cell lines (PC-3,HRT-18),but not myeloma cell lines (U266,OPM-2),were able to secrete high amounts of GRP-78. Recombinant GRP-78 conferred bortezomib resistance to endothelial cells and OPM-2 myeloma cells. Knockdown of GRP78 gene expression in tumor cells and immunodepletion of GRP-78 protein from tumor cell supernatants restored bortezomib sensitivity. GRP-78 did not bind or complex bortezomib but induced prosurvival signals by phosphorylation of extracellular signal-related kinase and inhibited p53-mediated expression of proapoptotic Bok and Noxa proteins in endothelial cells. From our data,we conclude that distinct solid tumor cells are able to secrete GRP-78 into the tumor microenvironment,thus demonstrating a hitherto unknown mechanism of resistance to bortezomib. View Publication

The anthrax protective antigen (PA) is the receptor-binding subunit common to lethal toxin (LT) and edema toxin (ET),which are responsible for the high mortality rates associated with inhalational Bacillus anthracis infection. Although recombinant PA (rPA) is likely to be an important constituent of any future anthrax vaccine,evaluation of the efficacies of the various candidate rPA vaccines is currently difficult,because the specific B-cell epitopes involved in toxin neutralization have not been completely defined. In this study,we describe the identification and characterization of two murine monoclonal immunoglobulin G1 antibodies (MAbs),1-F1 and 2-B12,which recognize distinct linear neutralizing epitopes on domain 4 of PA. 1-F1 recognized a 12-mer peptide corresponding to residues 692 to 703; this epitope maps to a region of domain 4 known to interact with the anthrax toxin receptor CMG-2 and within a conformation-dependent epitope recognized by the well-characterized neutralizing MAb 14B7. As expected,1-F1 blocked PA's ability to associate with CMG-2 in an in vitro solid-phase binding assay,and it protected murine macrophage cells from intoxication with LT. 2-B12 recognized a 12-mer peptide corresponding to residues 716 to 727,an epitope located immediately adjacent to the core 14B7 binding site and a stretch of amino acids not previously identified as a target of neutralizing antibodies. 2-B12 was as effective as 1-F1 in neutralizing LT in vitro,although it only partially inhibited PA binding to its receptor. Mice passively administered 1-F1 or 2-B12 were partially protected against a lethal challenge with LT. These results advance our fundamental understanding of the mechanisms by which antibodies neutralize anthrax toxin and may have future application in the evaluation of candidate rPA vaccines. View Publication

Serotonergic axons from the raphe nuclei in the brainstem project to every region of the brain,where they make connections through their extensive terminal arborizations. This serotonergic innervation contributes to various normal behaviors and psychiatric disorders. The protocadherin-alpha (Pcdha) family of clustered protocadherins consists of 14 cadherin-related molecules generated from a single gene cluster. We found that the Pcdhas were strongly expressed in the serotonergic neurons. To elucidate their roles,we examined serotonergic fibers in a mouse mutant (Pcdha(Delta CR/Delta CR)) lacking the Pcdha cytoplasmic region-encoding exons,which are common to the gene cluster. In the first week after birth,the distribution pattern of serotonergic fibers in Pcdha(Delta CR/Delta CR) mice was similar to wild-type,but by 3 weeks of age,when the serotonergic axonal termini complete their arborizations,the distribution of the projections was abnormal. In some target regions,notably the globus pallidus and substantia nigra,the normally even distribution of serotonin axonal terminals was,in the mutants,dense at the periphery of each region,but sparse in the center. In the stratum lacunosum-molecular of the hippocampus,the mutants showed denser serotonergic innervation than in wild-type,and in the dentate gyrus of the hippocampus and the caudate-putamen,the innervation was sparser. Together,the abnormalities suggested that Pcdha proteins are important in the late-stage maturation of serotonergic projections. Further examination of alternatively spliced exons encoding the cytoplasmic tail showed that the A-type (but not the B-type) cytoplasmic tail was essential for the normal development of serotonergic projections. View Publication