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1.
Class II major histocompatibility complex (MHC) molecules are cell surface glycoproteins that bind and present immunogenic peptides to T cells. Intracellularly, class II molecules associate with a polypeptide referred to as the invariant (Ii) chain. Ii is proteolytically degraded and dissociates from the class II complex prior to cell surface expression of the mature class II alpha beta heterodimer. Using human fibroblasts transfected with HLA-DR1 and Ii cDNAs, we now demonstrate that truncation of the cytoplasmic domain of Ii results in the failure of Ii to dissociate from the alpha beta Ii complex and leads to stable expression of class II alpha beta Ii complexes on the cell surface. Furthermore, biochemical analysis and peptide presentation assays demonstrated that transfectants with stable surface alpha beta Ii complexes expressed very few free alpha beta heterodimers at the surface and were very inefficient in their ability to present immunogenic peptides to T cells. These results support the hypothesis that the cytoplasmic domain of Ii is responsible for endosomal targeting of alpha beta Ii and directly demonstrate that association with Ii interferes with the antigen presentation function of class II molecules. 相似文献
2.
B Y Wei J M Buerstedde M Bell C Chase A Nilson A Browne L Pease D J McKean 《Journal of immunology (Baltimore, Md. : 1950)》1991,146(7):2358-2366
To evaluate the potential functional role of the alpha- and beta-chain N-linked oligosaccharides we used site-directed mutagenesis to construct class II Ak alpha and Ak beta genes that encode polypeptides with altered N-linked oligosaccharide acceptor sites in the N-terminal domain of both polypeptides. The alpha 1 domain acceptor site at positions 82 to 84 was eliminated by substituting Gln for Asn at position 82. The beta 1 domain acceptor site at positions 19 to 21 was deleted by substituting Gln for Asn at position 19 or Ala for Thr at position 21. The mutant genes (Ak alpha* or Ak beta*) were transfected either individually (mutants T.19, T.21, and T.82) or together (mutant T.82-21) into class II cell surface negative B lymphoma cell lines. Quantitative immunofluorescence with a panel of Ak beta- or Ak alpha- reactive mAb demonstrated that although the oligosaccharide-deleted Ak alpha Ak beta molecules were serologically wild type, the Ad alpha serologic epitope defined by mAb K24-199 was eliminated in both the T.19 and T.21 Ak beta* Ad alpha molecules. Cloned cell lines expressing the T.19 or T.21 Ak beta* Ak alpha molecules exhibited limited functional Ag presentation defects. Cells expressing the T.82 Ak alpha* Ak beta molecules exhibited defects in Ag presentation function to nine of the ten T hybridomas tested. Surprisingly, cells expressing the mutant T.82-21 class II molecule stimulated a response that was equal to the wild-type response from three of the nine T hybrids and a response that was significantly greater than that of wild-type cells from five of nine T hybridomas. These functional and serological analyses also indicate that some of the observed Ag presentation defects may be due to altered secondary structure caused by either deletion of the oligosaccharide or the amino acid substitution used to delete the N-linked oligosaccharide acceptor site. 相似文献
3.
A Ben-Nun E Choi K R McIntyre S A Leeman D J McKean J G Seidman L H Glimcher 《Journal of immunology (Baltimore, Md. : 1950)》1985,135(2):1456-1464
A20.2J B lymphoma cells have been co-transfected with the A alpha b, A beta b or with the A alpha b, A beta bm12 and neomycin resistance genes. The transfected cell lines constitutively express the I-Ab or I-Abm12 class II molecules at a level comparable with that of the endogenous I-Ad antigen. The I-Ab antigens expressed on three independently transfected B cell clones (A20.Ab.1, A20.Ab.2, and A20.Ab.3) are serologically and functionally indistinguishable from the I-Ab molecules expressed by control H-2bxd B hybridoma cells (LB cells). These transfected cell lines were potent I region-restricted antigen-presenting cells to a large panel of antigen-specific, autoreactive and alloreactive T cell hybridomas, as well as normal T cell clones. There were not significant differences in the efficiency of antigen presentation by the Ia molecules encoded by the transfected, as compared with the endogenous, I-A genes. The expression of a functional I-Ab antigen on the surface of cells transfected with A beta bm12 and A alpha b genes is consistent with previous work that implicated the A beta-chain alone in the bm 12 mutation. Furthermore, because the transfected A20.Ab and A20.Abm12 cells display the serologic and functional properties of normal spleen cells from the wild-type and mutant mouse strains, respectively, it is clear that class II genes do not undergo unexpected and unpredictable alterations after transfection in this system. This system permits us to investigate the structural requirements for interactions between class II major histocompatibility complex antigens, a foreign antigen, and the T cell receptor by in vitro site-directed mutagenesis coupled with DNA-mediated gene transfer. 相似文献
4.
Analysis of the molecular specificities of anti-class II monoclonal antibodies by using L cell transfectants expressing HLA class II molecules 总被引:4,自引:0,他引:4
E P Klohe R Watts M Bahl C Alber W Y Yu R Anderson J Silver P K Gregersen R W Karr 《Journal of immunology (Baltimore, Md. : 1950)》1988,141(6):2158-2164
Expressible HLA class II alpha- and beta-chain cDNA were used for DNA-mediated gene transfer to produce L cell transfectants expressing single types of human class II molecules. Cloned transfectants expressing nine different class II molecules were isolated: DR alpha: DR1 beta I, DR alpha: DR4 beta I, DR alpha: DR5 beta I, DR alpha: DR5 beta III (DRw52), DR alpha: DR7 beta I, DR alpha: DR4/7 beta IV (DRw53), DQ7 alpha: DQw2 beta, DQ7 alpha: DQw3 beta, and DPw4 alpha: DPw4 beta. These class II-expressing transfectants were used to analyze by flow cytometry the molecular specificities of 20 anti-class II mAb. These analyes indicate that some mAb are more broadly reactive than was previously thought based on immunochemical studies. In contrast, the narrow molecular specificities of other anti-class II mAb were confirmed by this approach. Transfectants expressing human class II molecules should be valuable reagents for studies of B cell and T cell defined epitopes on these molecules. 相似文献
5.
N K Puri I D Walker M R Brandon 《Journal of immunology (Baltimore, Md. : 1950)》1987,139(9):2996-3002
Four monoclonal antibodies, SBU.II 28-1, 37-68, 38-27, and 42-20, each recognizing a distinct, non-overlapping subset of sheep class II molecules, were used to purify class II molecules from a single sheep. Four class II alpha subunits designated 28-1 alpha, 37-68 alpha, 42-20 alpha, and 38-27 alpha and five class II beta subunits designated 28-1 beta, 37-68 beta 1, 37-68 beta 2, 42-20 beta, and 38-27 beta were compared by N-terminal sequence analyses. Two distinct alpha subunits were identified; the 28-1 alpha, 37-68 alpha, and 42-20 alpha subunits all had identical N-terminal amino acids sequences, which exhibited about 75% homology with HLA-DR alpha and mouse E alpha polypeptides. In contrast, the 38-27 alpha sequence exhibited about 80% sequence homology with HLA-DQ alpha and mouse A alpha polypeptides. In general, sheep beta subunits displayed insufficient sequence homology to enable correlation with human beta-chain sequences; however, the 38-27 beta-chain sequence showed homology with the HLA-DQ beta sequence. The conserved sequence surrounding the site for N-linked glycosylation within human/mouse beta polypeptides (residues 19 to 21) was not present in sheep beta sequences and in contrast with the beta-chains of mouse and man, sheep beta polypeptides contained between 1 and 3 positionally variable cysteine residues (residues 13 to 15 inclusive). Individual sheep beta subunits exhibited extensive sequence heterogeneity and each consisted of a unique population of beta polypeptide species. At least 16 different beta polypeptide sequences were identified from a single sheep and the existence of no fewer than nine non-allelic beta genes was inferred from the sequence data. We have previously provided evidence suggesting that the sheep has multiple major histocompatibility complex class II alpha and beta genes related to those of all three HLA-D subregions. The present results suggest that a number of these genes encode HLA-DQ-like heterodimers and that a sheep DR-like alpha gene product is shared with the products of a large and heterogeneous sheep beta gene family. 相似文献
6.
Integrin alpha v beta 5 selectively promotes adenovirus mediated cell membrane permeabilization 总被引:22,自引:0,他引:22
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《The Journal of cell biology》1994,127(1):257-264
Human adenovirus type 2 (Ad2) enters host cells by receptor-mediated endocytosis, an event mediated by the virus penton base binding to cell surface integrins alpha v beta 3 and alpha v beta 5. While both alpha v integrins promote virus internalization, alpha v beta 5 is involved in the subsequent event of membrane permeabilization. Cells transfected with the beta 5 or beta 3 subunit, expressing either alpha v beta 5 and alpha v beta 3, respectively, were capable of supporting Ad2 infection to varying degrees. In this case, cells expressing alpha v beta 5 were significantly more susceptible to Ad2-induced membrane permeabilization, as well as to Ad2 infection, than cells expressing alpha v beta 3. Adenovirus-mediated gene delivery was also more efficient in cells expressing alpha v beta 5. These results suggest that the interaction of alpha v beta 5 with Ad2 penton base facilitates the subsequent step of virus penetration into the cell. These studies provide evidence for the involvement of a cellular receptor in virus- mediated membrane permeabilization and suggest a novel biological role for integrin alpha v beta 5 in the infectious pathway of a human adenovirus. 相似文献
7.
SB subregion of the human major histocompatibility complex: gene organization, allelic polymorphism and expression in transformed cells 总被引:7,自引:0,他引:7
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K Okada H L Prentice J M Boss D J Levy D Kappes T Spies R Raghupathy R A Mengler C Auffray J L Strominger 《The EMBO journal》1985,4(3):739-748
The SB region of the human major histocompatibility complex (MHC) has been cloned from cosmid and lambda phage libraries made from the human B-lymphoblastoid cell line Priess (DR4/4, DC4/4, SB3/4). Two alpha genes and two beta genes are encoded in the 100 kb long SB region in the order SB alpha-SB beta-SX alpha-SX beta. The SB alpha and SB beta genes encode the alpha and beta subunits of the SB subset of class II MHC molecules. Both the SX alpha and the SX beta genes are pseudogenes in the haplotype examined. From the isolated clones, the two haplotypes of the Priess cell line, SB3 and SB4, are distinguished by nucleotide sequencing and blot hybridization analyses. Restriction site polymorphisms between the SB3 and SB4 clones were observed only in relatively small regions of the SB beta and SX beta genes. A mouse macrophage cell line was transfected with one of the cosmid clones containing both SB alpha and SB beta genes. Expression of the alpha and beta genes was detected by fluorescene-activated cell sorting (FACS) and two-dimensional gel electrophoresis using SB-specific monoclonal antibodies. 相似文献
8.
Association with BiP and aggregation of class II MHC molecules synthesized in the absence of invariant chain. 总被引:2,自引:1,他引:1
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C Bonnerot M S Marks P Cosson E J Robertson E K Bikoff R N Germain J S Bonifacino 《The EMBO journal》1994,13(4):934-944
Class II molecules of the major histocompatibility complex (MHC) are composed of two polymorphic glycoprotein chains (alpha and beta), that associate in the ER with a third, non-polymorphic glycoprotein known as the invariant chain (Ii). We have examined the relationship between the intracellular transport and physico-chemical characteristics of various combinations of murine alpha, beta and Ii chains. Biochemical and morphological analyses of transfected fibroblasts expressing class II MHC chains show that both unassembled alpha and beta chains, as well as a large fraction of alpha+beta complexes synthesized in the absence of Ii chain, are retained in the ER in association with the immunoglobulin heavy chain binding protein, BiP. Analyses by sedimentation velocity on sucrose gradients show that most incompletely assembled class II MHC species exist as high molecular weight aggregates in both transfected fibroblasts and spleen cells from mice carrying a disruption of the Ii chain gene. This is in contrast to the sedimentation properties of alpha beta Ii complexes from normal mice, which migrate as discrete, stoichiometric complexes of M(r) approximately 200,000-300,000. These observations suggest that assembly with the Ii chain prevents accumulation of aggregated alpha and beta chains in the ER, which might relate to the known ability of the Ii chain to promote exit of class II MHC molecules from the ER. 相似文献
9.
10.
K. T. BALLINGALL H. WRIGHT J. REDMOND B. M. DUTIA J. HOPKINS J. LANG† E. V. DEVERSON‡ J. C. HOWARD‡ N. PURI§ D. HAIG 《Animal genetics》1992,23(4):347-359
Previous work made use of nucleic acid probes corresponding to different subtypes of the class II regions of the human and murine major histocompatibility complex (MHC) to isolate seven different alpha and 24 different beta genes of the ovine MHC from two cosmid libraries. In an attempt to identify pairs of alpha and beta genes capable of cell surface expression, all permutations of alpha and beta genes were in turn transfected into mouse L-cells. Two pairs of alpha and beta genes co-expressed and stable ovine MHC class II L-cell lines were developed. The expressed alpha genes had previously been defined as DR-alpha homologues (DRA) by differential Southern hybridization to human subtype specific class II probes. The expressed ovine beta genes were also assigned as ovine DR-beta homologues (DRB) on the basis of their sequence having a higher degree of similarity with human DRB than any other subtype. A total of eight out of 23 anti-sheep class II specific monoclonal antibodies were typed OLA-DR specific by FACScan analysis using the L-cell lines. 相似文献
11.
J McCluskey T Munitz L Boyd R N Germain J E Coligan A Singer D H Margulies 《Journal of immunology (Baltimore, Md. : 1950)》1988,140(6):2081-2089
To evaluate the relationship between structure and function of the alpha 1 and beta 1 domains of class II MHC molecules, a strategy has been developed whereby isolated alpha 1 or beta 1 domains can be expressed on the surface of transfected mouse L cells as a part of a hybrid class II/class I molecule. The construction and expression of a chimeric class II/class I gene encoding the first two exons of A kappa alpha linked to the C2, TM and cytoplasmic exons of H-2Dd (A kappa alpha 1/DdC2) are described. High levels of A kappa alpha 1/DdC2 protein were detected on transfected L cells with a DdC2-specific mAb. An anti-Ia xenoantiserum specifically bound to A kappa alpha 1/DdC2-transfected L cells, although 12 different mAb reactive against I-A kappa alpha and an anti-Ia kappa alloantiserum did not bind these cells. However, alloreactive Ia kappa-specific CTL lines were able specifically to lyse cells expressing the hybrid A kappa alpha 1/DdC2 molecule. This indicates that the isolated A kappa alpha 1 domain preserves some of the structure of the native molecule and demonstrates the recognition by T cells of domain-specific A alpha allodeterminants. 相似文献
12.
Class II major histocompatibility complex genes of the sheep 总被引:2,自引:0,他引:2
E. V. DEVERSON H. WRIGHT S. WATSON† ‡ K. BALLINGALL† N. HUSKISSON§ A. G. DIAMOND§ ¶ J. C. HOWARD 《Animal genetics》1991,22(3):211-225
The class II genes of the sheep major histocompatibility complex (MHC) have been cloned from two unrelated heterozygous sheep into cosmid vectors. By restriction mapping and hybridization with a number of class II probes of human and mouse origin, the cloned genetic material has been assigned to seven distinct alpha genes, 10 distinct beta genes and 14 beta-related sequences. It was difficult to identify homologues of specific HLA class II genes because of a tendency for the ovine genes to cross-hybridize between HLA probes representing different loci. Such cross-hybridization was especially marked among the beta genes. While DQ and DR homologues have been tentatively identified by several criteria, no genes corresponding to DP have been identified. Cosmids containing class II alpha and beta genes have been transfected into mouse LTK- cells, and surface expression of a sheep class II molecule has been obtained. 相似文献
13.
When human T cell receptor for antigen (TCR) alpha chain V-genes were compared pair-wise, the numbers of nucleotide differences
showed a characteristic distribution; most were in the range of 100 to 200 differences out of a total of about 300 bases.
The same distribution was observed for mouse TCR alpha chains. Even more interesting was that comparing human alpha chains
and mouse alpha chains gave essentially the same nucleotide difference pattern. It is inferred from the large number of differences
and from the nonspecificity of trans-species (human and mouse) nucleotide sequence differences of TCR V-genes that TCR alpha
chains probably diverged early during evolution. The same feature was also observed for human and mouse TCR beta chains, although
the alpha and beta chain V-genes were distinct. This evolutionary preservation could be of vital importance to the fidelity
of the complicated trimolecular interactions among TCR alpha and beta chains, the processed peptide, and the major histocompatibility
complex (MHC) class I or II molecules.
Received: 22 January 1996 / Accepted: 9 September 1996 相似文献
14.
Cells from a mouse B lymphoma were transfected by DQ alpha and DQ beta genes derived from a DR4 haplotype. Quantitatively, the resulting expression of human class II molecules was similar to that of human B lymphoblastoid cell lines. Qualitatively, the transformant class II molecules differed from normal class II molecules in their carbohydrate moiety. As for their antigenic specificity, they were shown to carry two determinants previously identified on DQ molecules controlled by DR4 haplotypes, i. e., DQw3 and DCHON. The transformant molecules did not carry a third DR4-associated specificity, DC5 (equivalent to TA10), and must possess a structure allelic to DC5. However, no corresponding alloantigenic specificity was detected by a screening of relevant alloantisera. 相似文献
15.
The HLA-DQ region contains highly polymorphic alpha and beta loci, so that a diverse set of cis- and trans-associated class II alpha/beta dimers are potentially generated in heterozygous individuals. To evaluate the extent of this predicted diversity, DQ2 beta or DQ3.2 beta cDNA were introduced into a panel of homozygous B cell lines that expressed different DQ alpha alleles. Restricted patterns of alpha/beta pairing were observed in which DQ2 beta and DQ3 beta molecules were unable to pair efficiently with DQ1 alpha chains. This pairing anomaly may contribute to altered class II phenotypes in heterozygous individuals, and is reflected in the absence of either DQ1 alpha, DQ2 beta or DQ1 alpha, DQ3 beta haplotypes in the known human gene pool. 相似文献
16.
Adenovirus type 5 fiber knob binds to MHC class I alpha2 domain at the surface of human epithelial and B lymphoblastoid cells. 总被引:1,自引:1,他引:0
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Adenovirus serotype 5 (Ad5) fiber receptor was investigated using reverse antibody biopanning of a phage-displayed hexapeptide library, and virus-neutralizing monoclonal antibodies (mAbs 1D6.3 and 7A2.7) raised against recombinant Ad5 fiber knob. Both mAbs inhibited attachment of Ad5 to HeLa cells. Mimotopes of 1D6.3 showed homology with the C-terminal segment of the alpha2 domain of the heavy chain of human MHC class I molecules (MHC-I alpha2), and mimotopes of 7A2.7 were consensus to human fibronectin type III (FNIII) modules. In vitro, GST-fused MHC-I alpha2- and FNIII-derived oligopeptides interacted with recombinant fibers in a subgroup-specific manner. In vivo, the MHC-I alpha2 synthetic icosapeptide RAIVGFRVQWLRRYFVNGSR showed a net neutralization effect on Ad5 in HeLa cells, whereas the FNIII icosapeptide RHILWTPANTPAMGYLARVS significantly increased Ad5 binding to HeLa cells. Daudi cells, which lack surface expression of HLA class I molecules, showed a weak capacity for Ad5 binding. In beta2-microglobulin-transfected Daudi cells, Ad5 attachment and permissivity were restored to HeLa cell levels, with 4000 receptors per cell and a binding constant of 1.4x10(10)/M. The results suggested that the conserved region of MHC-I alpha2-domain including Trp167 represents a high affinity receptor for Ad5 fiber knob, whereas ubiquitous FNIII modules would serve as auxiliary receptors. 相似文献
17.
Gene transfer of H-2 class II genes: antigen presentation by mouse fibroblast and hamster B-cell lines 总被引:13,自引:0,他引:13
B Malissen M P Price J M Goverman M McMillan J White J Kappler P Marrack A Pierres M Pierres L Hood 《Cell》1984,36(2):319-327
We have transferred the mouse Ak alpha and Ak beta genes, which encode the class II I-Ak molecule, into mouse L-cell fibroblasts and hamster B cells. I-Ak molecules are expressed on the surface of both cell types. The L-cell and hamster B-cell I-Ak molecules appear normal by serological analyses and two-dimensional gel electrophoresis. Furthermore, the I-Ak molecules on L cells can act as targets for the allogenic T-cell killing of the transformed L cells. The I-Ak molecules in both mouse fibroblasts and hamster B cells can present certain antigens to T-cell helper hybridomas. Thus only class II molecules are required to convert the nonantigen-presenting cell. Accordingly, it will be possible to dissect the structure-function relationships existing between Ia molecules, foreign antigen, and T-cell receptor molecules by in vitro site-directed mutagenesis and gene transfer. 相似文献
18.
S Karaki M Tanabe H Nakauchi M Takiguchi 《Journal of immunology (Baltimore, Md. : 1950)》1992,149(5):1613-1618
It is known that the alpha-chain of CD8 binds to a negatively charged loop composed of residues 223 to 229 on MHC class I Ag and that binding of CD8 alpha enhances Ag recognition of T cells. We have recently shown that the mouse CD8 alpha homodimer does not bind to either the HLA class I alpha 3 domain or a mutant of H-2Kb Ag containing a substitution of glutamine for methionine at residue 224, which brings this residue toward the human consensus. Here we report a complementary study of the CD8 beta-chain. The functional role of the CD8 beta-chain was analyzed by using four T cell hybridoma lines expressing mouse CD8 alpha and transfected with the mouse CD8 beta gene. As compared with the lines expressing only CD8 alpha, allorecognition of the chimeric H-2Kb Ag that contains the HLA class I alpha 3 domain was enhanced in lines expressing both CD8 alpha and -beta. This enhancement was blocked by either anti-CD8 mAb or anti-HLA class I alpha 3 domain mAb. In addition, we show that CD8 alpha beta binds the H-2Kb mutant Ag at residue 224. These results suggest that the beta-chain allows the CD8 alpha beta heterodimer to recognize the chimeric H-2Kb Ag. A model for the role of the beta-chain is presented. 相似文献
19.
H Wada S Ohno K Kubo C Taya S Tsuji S Yonehara K Suzuki 《Biochemical and biophysical research communications》1989,165(1):533-538
By the use of cloned cDNAs for protein kinase C isozymes alpha, beta I, beta II, gamma, and those for novel protein kinase C, epsilon and zeta, the expression of the corresponding mRNA species was examined in various mouse tissues, human lymphoid cell lines, and mouse cell lines of neuronal origin. In adult brain, mRNAs for all the isozymes of PKC family are expressed. However, the expression of these mRNA species in brain is low at birth. A similar pattern of expression was also observed for beta I/beta II mRNAs in spleen. These expression patterns are in clear contrast to that for beta I/beta II mRNAs in thymus where the mRNAs are expressed at birth and the levels of expression decrease with age. Human lymphoid cell lines express large amounts of PKC beta mRNAs in addition to PKC alpha. Further, nPKC epsilon mRNA is expressed in some of these cell lines. On the other hand, all the mouse cell lines of neuronal origin tested express nPKC epsilon and zeta in addition to PKC alpha. In a mouse neuroblast cell line, Neuro 2a, down modulation of mRNAs for both PKC alpha and nPKC epsilon was observed in association with in vitro differentiation. 相似文献
20.
K Imanishi H Igarashi T Uchiyama 《Journal of immunology (Baltimore, Md. : 1950)》1990,145(10):3170-3176
We investigated the mechanisms of murine T cell activation by streptococcal pyrogenic exotoxin type A (SPE A), focusing on the role of MHC class II molecules on accessory cells (AC) and V beta usage in alpha beta TCR of SPE A-reactive T cells in comparison with staphylococcal enterotoxin B-reactive T cells. L cells transfected with I-Ab genes functioned as effective AC for SPE A-induced responses by C57BL/6 T cells, proliferation, and IL-2 production, but control L cells were not effective AC. Anti-I-Ab mAb inhibited the SPE A-induced responses. Staphylococcal enterotoxin B-induced C57BL/6 T cell blasts were composed of cells bearing V beta 3, members of the V beta 8 family, and V beta 11. Most of the SPE A-induced T cell blasts (about 80%) bore V beta 8.2. mAb reactive to V beta 8.2 markedly inhibited SPE A-induced T cell responses. Apparently, SPE A activates mainly T cells bearing V beta 8.2 in physical association with MHC class II molecules expressed on AC. We also discuss the pathogenic activities of SPE A in relation to toxic shock syndrome. 相似文献