技术资料

BACKGROUND: We previously identified a novel serine protease inhibitor (serpin),megsin,which is predominantly expressed in the kidney. Megsin expression is up-regulated in human and experimental renal diseases associated with mesangial proliferation and expansion,suggesting that urinary megsin may be a novel diagnostic marker for some renal diseases. METHODS: We established a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for megsin and measured urinary megsin of patients with various renal diseases. RESULTS: Megsin ELISA specifically detected megsin but not other serpins. The detection limit was 0.04 ng/ml,which allowed detection of urinary megsin in 3.6% of healthy individuals. The antigenic epitope in the urine detected by the ELISA was confirmed as megsin protein by time-of-flight mass spectrometry. Among patients with rapidly progressive glomerulonephritis (n = 18),55.6% were urinary megsin-positive,while 24.1% in IgA nephropathy (n = 112) and 15.1% in chronic non-IgA glomerulonephritis (n = 245) were urinary megsin-positive,respectively. Among patients with chronic renal failure due to unknown causes (n = 74),18.9% were positive for urinary megsin. In diabetic patients with or without nephropathy (n = 1073),12.3% were urinary megsin-positive,while positivity of urinary megsin in patients with non-renal diseases (n = 768) was equivalent (3.3%) to that of healthy individuals. Of note,when urinary megsin-positive patients with diabetic nephropathy (n = 71) were classified into four stages by their proteinuria and estimated glomerular filtration rate,urinary megsin excretion increased as the stage progressed up to stage 3A,suggesting correlation of that with mesangial expansion level. Urinary megsin decreased in the advanced stage,probably reflecting development of glomerulosclerosis. CONCLUSION: We established a high-sensitive megsin ELISA,which detects urinary megsin in some patients with renal diseases and in only a few healthy subjects. Megsin ELISA may be a novel diagnostic tool for renal diseases. 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

The trypsin-like serine protease marapsin is a member of the large protease gene cluster at human chromosome 16p13.3,which also contains the structurally related proteases testisin,tryptase epsilon,tryptase gamma,and EOS. To gain insight into the biological functions of marapsin,we undertook a detailed gene expression analysis. It showed that marapsin expression was restricted to tissues containing stratified squamous epithelia and was absent or only weakly expressed in all other tissues,including the pancreas. Marapsin was constitutively expressed in nonkeratinizing stratified squamous epithelia of human esophagus,tonsil,cervix,larynx,and cornea. In the keratinizing stratified squamous epidermis of skin,however,its expression was induced only during epidermal hyperproliferation,such as in psoriasis and in murine wound healing. In fact,marapsin was the second most strongly up-regulated protease in psoriatic lesions,where expression was localized to the upper region of the hyperplastic epidermis. Similarly,in the hyperproliferative epithelium of regenerating murine skin wounds,marapsin localized to the suprabasal layers,where keratinocytes undergo squamous differentiation. The transient up-regulation of marapsin,which closely correlated with re-epithelialization,was virtually absent in a genetic mouse model of delayed wound closure. These results suggested a function during the process of re-epithelialization. Furthermore,in reconstituted human epidermis,a model system of epidermal differentiation,members of the IL-20 subfamily of cytokines,such as IL-22,induced marapsin expression. Consistent with a physiologic role in marapsin regulation,IL-22 was also strongly expressed in re-epithelializing skin wounds. Marapsin's restricted expression,localization,and cytokine-inducible expression suggest a role in the terminal differentiation of keratinocytes in hyperproliferating squamous epithelia. View Publication

An immunochromatographic assay (Campy-ICA) using a newly generated single monoclonal antibody against a 15-kDa cell surface protein of Campylobacter jejuni was developed. When cell suspensions of 86 C. jejuni strains and 27 Campylobacter coli strains were treated with a commercially available bacterial protein extraction reagent and the resulting extracts were tested with the Campy-ICA,they all yielded positive results. The minimum detectable limits for the C. jejuni strains ranged from 1.8 x 10(4) to 8.2 x 10(5) CFU/ml of cell suspension,and those for the C. coli strains ranged from 1.4 x 10(5) to 4.6 x 10(6) CFU/ml of cell suspension. All 26 non-Campylobacter species tested yielded negative results with the Campy-ICA. To evaluate the ability of the Campy-ICA to detect C. jejuni and C. coli in human stool specimens,suspensions of 222 stool specimens from patients with acute gastroenteritis were treated with the bacterial protein extraction reagent,and the resulting extracts were tested with the Campy-ICA. The Campy-ICA results showed a sensitivity of 84.8% (28 of 33 specimens) and a specificity of 100% (189 of 189 specimens) compared to the results of isolation of C. jejuni and C. coli from the stool specimens by a bacterial culture test. The Campy-ICA was simple to perform and was able to detect Campylobacter antigen in a fecal extract within 15 min. These results suggest that Campy-ICA testing of fecal extracts may be useful as a simple and rapid adjunct to stool culture for detecting C. jejuni and C. coli in human stool specimens. View Publication

A method employing Percoll gradient centrifugation was developed to purify Kudoa thyrsites spores from somatic muscle tissue of Atlantic salmon Salmo salar. Highly purified spores were then used to immunize inbred BALB/c mice for derivation of hybridomas secreting Kudoa-specific monoclonal antibodies (mAbs). Analysis of mAbs by immunofluorescence microscopy and flow cytometry showed that several were specific for antigens on the surface of K. thyrsites spores whereas other mAbs reacted with polar capsules or with polar filaments of spores of K. thyrsites,K. paniformis and K. crumena. Immunoblots on spore lysates using the surface-binding mAbs showed a broad band of 46 to textgreater 220 kDa,whereas mAbs specific for antigens of polar capsules and polar filaments detected sharper bands of various molecular masses,depending on the Kudoa species. The dominant epitope of the K. thyrsites spore surface antigen was shown to be carbohydrate as determined by its sensitivity to treatment with anhydrous trifluoromethane sulfonic acid and by its resistance to treatment with Proteinase K. Immunofluorescence microscopy using the K. thyrsites-specific mAbs on isolated,intact,permeabilized plasmodia and on thin sections of somatic muscle tissue containing plasmodia revealed intense labeling of spores both within the spore-producing plasmodia and in the flesh of infected Atlantic salmon. As few as 100 spores were detected by immunoblotting,indicating that these mAbs have potential for use in developing a field-based diagnostic test. View Publication

In Treponema denticola,a ribbon-like structure of cytoplasmic filaments spans the cytoplasm at all stages of the cell division process. Insertional inactivation was used as a first step to determine the function of the cytoplasmic filaments. A suicide plasmid was constructed that contained part of cfpA and a nonpolar erythromycin resistance cassette (ermF and ermAM) inserted near the beginning of the gene. The plasmid was electroporated into T. denticola,and double- crossover recombinants which had the chromosomal copy of cfpA insertionally inactivated were selected. Immunoblotting and electron microscopy confirmed the lack of cytoplasmic filaments. The mutant was further analyzed by dark-field microscopy to determine cell morphology and by the binding of two fluorescent dyes to DNA to assess the distribution of cellular nucleic acids. The cytoplasmic filament protein-deficient mutant exhibited pleiotropic defects,including highly condensed chromosomal DNA,compared to the homogeneous distribution of the DNA throughout the cytoplasm in a wild-type cell. Moreover,chains of cells are formed by the cytoplasmic filament- deficient mutant,and those cells show reduced spreading in agarose,which may be due to the abnormal cell length. The chains of cells and the highly condensed chromosomal DNA suggest that the cytoplasmic filaments may be involved in chromosome structure,segregation,or the cell division process in Treponema. View Publication

We have generated rat monoclonal antibodies specific for the mouse eotaxin receptor,C-C chemokine receptor 3 (CCR3). Several anti-CCR3 mAbs proved to be useful for in vivo depletion of CCR3-expressing cells and immunofluorescent staining. In vivo CCR3 mAbs of the IgG2b isotype substantially depleted blood eosinophil levels in Nippostrongyus brasiliensis-infected mice. Repeated anti-CCR3 mAb treatment in these mice significantly reduced tissue eosinophilia in the lung tissue and bronchoalveolar lavage fluid. Flow cytometry revealed that mCCR3 was expressed on eosinophils but not on stem cells,dendritic cells,or cells from the thymus,lymph node,or spleen of normal mice. Unlike human Th2 cells,mouse Th2 cells did not express detectable levels of CCR3 nor did they give a measurable response to eotaxin. None of the mAbs were antagonists or agonists of CCR3 calcium mobilization. To our knowledge,the antibodies described here are the first mAbs reported to be specific for mouse eosinophils and to be readily applicable for the detection,isolation,and in vivo depletion of eosinophils. View Publication

Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here,we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains,both lacking canonical export signals,are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGs-named 'cyclonals'-effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation. View Publication

Helicobacter pylori causes cellular vacuolation in host cells,a cytotoxic event attributed to vacuolating cytotoxin (VacA) and the presence of permeant weak bases such as ammonia. We report here the role of γ-glutamyl transpeptidase (GGT),a constitutively expressed secretory enzyme of H. pylori,in potentiating VacA-dependent vacuolation formation in H. pylori-infected AGS and primary gastric cells. The enhancement is brought about by GGT hydrolysing glutamine present in the extracellular medium,thereby releasing ammonia which accentuates the VacA-induced vacuolation. The events of vacuolation in H. pylori wild type (WT)- and Δggt-infected AGS cells were first captured and visualized by real-time phase-contrast microscopy where WT was observed to induce more vacuoles than Δggt. By using semi-quantitative neutral red uptake assay,we next showed that Δggt induced significantly less vacuolation in AGS and primary gastric epithelial cells as compared to the parental strain (Ptextless0.05) indicating that GGT potentiates the vacuolating effect of VacA. Notably,vacuolation induced by WT was significantly reduced in the absence of GGT substrate,glutamine (Ptextless0.05) or in the presence of a competitive GGT inhibitor,serine-borate complex. Furthermore,the vacuolating ability of Δggt was markedly restored when co-incubated with purified recombinant GGT (rGGT),although rGGT itself did not induce vacuolation independently. Similarly,the addition of exogenous ammonium chloride as a source of ammonia also rescued the ability of Δggt to induce vacuolation. Additionally,we also show that monoclonal antibodies against GGT effectively inhibited GGT activity and successfully suppressed H. pylori-induced vacuolation. Collectively,our results clearly demonstrate that generation of ammonia by GGT through glutamine hydrolysis is responsible for enhancing VacA-dependent vacuolation. Our findings provide a new perspective on GGT as an important virulence factor and a promising target in the management of H. pylori-associated gastric diseases. View Publication

Murine hybridomas producing neutralizing mAbs specific to the pandemic influenza virus A/California/07/2009 haemagglutinin (HA) were isolated. These antibodies recognized at least two different but overlapping new epitopes that were conserved in the HA of most Spanish pandemic isolates. However,one of these isolates (A/Extremadura/RR6530/2010) lacked reactivity with the mAbs and carried two unique mutations in the HA head (S88Y and K136N) that were required simultaneously to eliminate reactivity with the murine antibodies. This unusual requirement directly illustrates the phenomenon of enhanced antigenic change proposed previously for the accumulation of simultaneous amino acid substitutions at antigenic sites of the influenza A virus HA during virus evolution (Shih et al.,Proc Natl Acad Sci USA,104,6283-6288,2007). The changes found in the A/Extremadura/RR6530/2010 HA were not found in escape mutants selected in vitro with one of the mAbs,which contained instead nearby single amino acid changes in the HA head. Thus,either single or double point mutations may similarly alter epitopes of the new antigenic site identified in this work in the 2009 H1N1 pandemic virus HA. Moreover,this site is relevant for the human antibody response,as shown by competition of mAbs and human post-infection sera for virus binding. The results are discussed in the context of the HA antigenic structure and challenges posed for identification of sequence changes with possible antigenic impact during virus surveillance. 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

PURPOSE: The PRL-3 mRNA is consistently elevated in metastatic samples derived from colorectal cancers. We sought to generate a specific PRL-3 monoclonal antibody (mAb) that might serve as a potential diagnostic marker for colorectal cancer metastasis. EXPERIMENTAL DESIGN: PRL-3 is one of three members (PRL-1,PRL-2,and PRL-3) in a unique protein-tyrosine phosphatase family. Because the three PRLs are 76% to 87% identical in their amino acid sequences,it poses a great challenge to obtain mAbs that are specific for respective phosphatase of regenerating liver (PRL) but not for the other two in the family. We screened over 1,400 hybridoma clones to generate mAbs specific to each PRL member. RESULTS: We obtained two hybridoma clones specifically against PRL-3 and another two clones specifically against PRL-1. These antibodies had been evaluated by several critical tests to show their own specificities and applications. Most importantly,the PRL-3 mAbs were assessed on 282 human colorectal tissue samples (121 normal,17 adenomas,and 144 adenocarcinomas). PRL-3 protein was detected in 11% of adenocarcinoma samples. The PRL-3- and PRL-1-specific mAbs were further examined on 204 human multiple cancer tissues. The differential expressions of PRL-3 and PRL-1 confirmed the mAbs' specificity. CONCLUSIONS: Using several approaches,we show that PRL-3- or PRL-1-specific mAbs react only to their respective antigen. The expression of PRL-3 in textgreater10% of primary colorectal cancer samples indicates that PRL-3 may prime the metastatic process. These mAbs will be useful as markers in clinical diagnosis for assessing tumor aggressiveness. View Publication