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BACKGROUND Identification of species-specific trypanosome molecules is important for laboratory- and field-based research into epidemiology and disease diagnosis. Although Trypanosoma congolense is the most important trypanosome pathogen of cattle in Africa,no species-specific molecules found in infective bloodstream forms (BSF) of the parasites have been identified,thus limiting development of diagnostic tests. METHODS Immuno-mass spectrometric methods were used to identify a protein that is recognized by a T. congolense-specific monoclonal antibody (mAb) Tc6/42.6.4. The identified molecule was expressed as a recombinant protein in E. coli and was tested in several immunoassays for its ability to interact with the mAb. The three dimensional structure of the protein was modeled and compared to crystal- and NMR-structures of the homologous proteins from T. cruzi and T. brucei respectively,in order to examine structural differences leading to the different immunoreactivity of the T. congolense molecule. Enzyme-linked immunosorbent assays (ELISA) were used to measure antibodies produced by trypanosome-infected African cattle in order to assess the potential for use of T. congolense calflagin in a serodiagnostic assay. RESULTS The antigen recognized by the T. congolense-specific mAb Tc6/42.6.4 was identified as a flagellar calcium-binding protein,calflagin. The recombinant molecule showed immunoreactivity with the T. congolense-specific mAb confirming that it is the cognate antigen. Immunofluorescence experiments revealed that Ca2+ modulated the localization of the calflagin molecule in trypanosomes. Structural modelling and comparison with calflagin homologues from other trypanosomatids revealed four non-conserved regions on the surface of the T. congolense molecule that due to differences in surface chemistry and structural topography may form species-specific epitopes. ELISAs using the recombinant calflagin as antigen to detect antibodies in trypanosome-infected cattle showed that the majority of cattle had antibody responses. Area under the Receiver-Operating Characteristic (ROC) curves,associated with host IgG and IgM,were calculated to be 0.623 and 0.709 respectively,indicating a positive correlation between trypanosome infection and the presence of anti-calflagin antibodies. CONCLUSIONS While calflagin is conserved among different species of African trypanosomes,our results show that T. congolense calflagin possesses unique epitopes that differentiate this protein from homologues in other trypanosome species. MAb Tc6/42.6.4 has clear utility as a laboratory tool for identifying T. congolense. T. congolense calflagin has potential as a serodiagnostic antigen and should be explored further for its utility in antigen-detection assays for diagnosis of cattle infections. View Publication

It has been proposed that the ancestral fungus was mating competent and homothallic. However,many mating-competent fungi were initially classified as asexual because their mating capacity was hidden behind layers of regulation. For efficient in vitro mating,the essentially obligate diploid ascomycete pathogen Candida albicans has to change its mating type locus from heterozygous MTL a /α to homozygous MTL a / a or MTL α/α and then undergo an environmentally controlled epigenetic switch to the mating-competent opaque form. These requirements greatly reduce the potential for C. albicans mating. Deletion of the Yci1 domain gene OFR1 bypasses the need for C. albicans cells to change the mating type locus from heterozygous to homozygous prior to switching to the opaque form and mating and allows homothallic mating of MTL heterozygous strains. This bypass is carbon source dependent and does not occur when cells are grown on glucose. Transcriptional profiling of ofr1 mutant cells shows that in addition to regulating cell type and mating circuitry,Ofr1 is needed for proper regulation of histone and chitin biosynthesis gene expression. It appears that OFR1 is a key regulator in C. albicans and functions in part to maintain the cryptic mating phenotype of the pathogen. View Publication

The EphA2 receptor tyrosine kinase is overexpressed in many different types of human cancers where it functions as a powerful oncoprotein. Dramatic changes in the subcellular localization and function of EphA2 have also been linked with cancer,and in particular,unstable cancer cell-cell contacts prevent EphA2 from stably binding its ligand on the surface of adjoining cells. This change is important in light of evidence that ligand binding causes EphA2 to transmit signals that negatively regulate tumor cell growth and invasiveness and also induce EphA2 degradation. On the basis of these properties,we have begun to target EphA2 on tumor cells using agonistic antibodies,which mimic the consequences of ligand binding. In our present study,we show that a subset of agonistic EphA2 antibodies selectively bind epitopes on malignant cells,which are not available on nontransformed epithelial cells. We also show that such epitopes arise from differential cell-cell adhesions and that the stable intercellular junctions of nontransformed epithelial cells occlude the binding site for ligand,as well as this subset of EphA2 antibodies. Finally,we demonstrate that antibody targeting of EphA2 decreases tumor cell growth as measured using xenograft tumor models and found that the mechanism of antibody action relates to EphA2 protein degradation in vivo. Taken together,these results suggest new opportunities for therapeutic targeting of the large number of different cancers that express EphA2 in a manner that could minimize potential toxicities to normal cells. View Publication

Therapeutic mAbs that target tumor-associated Ags on the surface of malignant cells have proven to be an effective and specific option for the treatment of certain cancers. However,many of these protein markers of carcinogenesis are not expressed on the cells' surface. Instead these tumor-associated Ags are processed into peptides that are presented at the cell surface,in the context of MHC class I molecules,where they become targets for T cells. To tap this vast source of tumor Ags,we generated a murine IgG2a mAb,3.2G1,endowed with TCR-like binding specificity for peptide-HLA-A*0201 (HLA-A2) complex and designated this class of Ab as TCR mimics (TCRm). The 3.2G1 TCRm recognizes the GVL peptide (GVLPALPQV) from human chorionic gonadotropin beta presented by the peptide-HLA-A*0201 complex. When used in immunofluorescent staining reactions using GVL peptide-loaded T2 cells,the 3.2G1 TCRm specifically stained the cells in a peptide and Ab concentration-dependent manner. Staining intensity correlated with the extent of cell lysis by complement-dependent cytotoxicity (CDC),and a peptide concentration-dependent threshold level existed for the CDC reaction. Staining of human tumor lines demonstrated that 3.2G1 TCRm was able to recognize endogenously processed peptide and that the breast cancer cell line MDA-MB-231 highly expressed the target epitope. The 3.2G1 TCRm-mediated CDC and Ab-dependent cellular cytotoxicity of a human breast carcinoma line in vitro and inhibited in vivo tumor implantation and growth in nude mice. These results provide validation for the development of novel TCRm therapeutic reagents that specifically target and kill tumors via recognition and binding to MHC-peptide epitopes. View Publication

The extended substrate specificity of granzyme B (GrB) was used to identify substrates among the chaperone superfamily. This approach identified Hsp90 and Bag1-L as novel GrB substrates,and an additional GrB cleavage site was identified in the Hsc70/Hsp70-Interacting Protein,Hip. Hsp90,Bag1L,and Hip were validated as GrB substrates in vitro,and mutational analysis confirmed the additional cleavage site in Hip. Because the role of Hip in apoptosis is unknown,its proteolysis by GrB was used as a basis to test whether it has anti-apoptotic activity. Previous work on Hip was limited to in vitro characterization; therefore,it was important to demonstrate Hip cleavage in a physiological context and to show its relevance to natural killer (NK) cell-mediated death. Hip is cleaved at both GrB cleavage sites during NK-mediated cell death in a caspase-independent manner,and its cleavage is due solely to GrB and not other granule components. Furthermore,Hip is not cleaved upon stimulation of the Fas receptor in the Jurkat T-cell line,suggesting that Hip is a substrate unique to GrB. RNA interference-mediated reduction of Hip within the K562 cell line rendered the cells more susceptible to NK cell-mediated lysis,indicating that proteolysis by GrB of Hip contributes to death induction. The small effect of RNA interference-mediated Hip deficiency on cytotoxicity is in agreement with the inherent redundancy of NK cell-mediated cell death. The identification of additional members of the chaperone superfamily as GrB substrates and the validation of Hip as an anti-apoptotic protein contribute to understanding the interplay between stress response and apoptosis. 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

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

It has been 50 years since cellular senescence was first described in human diploid fibroblasts (HDFs),yet its mechanism as well as its physiological and clinical implications are still not fully appreciated. Recent progress suggests that cellular senescence is a collective phenotype,composed of complex networks of effector programs. The balance and quality within the effector network varies depending on the cell type,the nature of the stress as well as the context. Therefore,understanding each of these effectors in the context of the whole network will be necessary in order to fully understand senescence as a whole. Furthermore,searching for new effector programs of senescence will help to define this heterogeneous and complex phenotype according to cellular contexts. View Publication

We identify the tumor necrosis factor receptor superfamily 25 (TNFRSF25)/TNFSF15 pair as critical trigger for allergic lung inflammation,which is a cardinal feature of asthma. TNFRSF25 (TNFR25) signals are required to exert T helper cell 2 (Th2) effector function in Th2-polarized CD4 cells and co-stimulate interleukin (IL)-13 production by glycosphingolipid-activated NKT cells. In vivo,antibody blockade of TNFSF15 (TL1A),which is the ligand for TNFR25,inhibits lung inflammation and production of Th2 cytokines such as IL-13,even when administered days after airway antigen exposure. Similarly,blockade of TNFR25 by a dominant-negative (DN) transgene,DN TNFR25,confers resistance to lung inflammation in mice. Allergic lung inflammation-resistant,NKT-deficient mice become susceptible upon adoptive transfer of wild-type NKT cells,but not after transfer of DN TNFR25 transgenic NKT cells. The TNFR25/TL1A pair appears to provide an early signal for Th2 cytokine production in the lung,and therefore may be a drug target in attempts to attenuate lung inflammation in asthmatics. View Publication