Identification of a naturally processed cyclin D1 T-helper epitope by a novel combination of HLA class II targeting and differential mass spectrometry

T-helper (Th) cells play an important role in orchestrating the effector function of CTL in anti-tumor immunity. However, only a limited number of Th cell epitopes has been characterized. Here we describe a novel approach for identifying naturally processed and presented peptides derived from chosen antigens. This method combines a transfection step of antigen-presenting cells with a vector encoding a fusion protein between the Ii chain and the antigen of interest, elution of the HLA-bound peptides and identification of the antigen-derived peptides by mass spectrometric comparison to the non-transfected cells. In vitro-stimulated Th cells against the identified peptide of interest specifically recognize transfectants overexpressing the cognate antigen. Using this approach, we were able to identify the HLA-DR4-restricted Th cell epitope NPPSMVAAGSVVAAV derived from cyclin D1, which is frequently overexpressed in tumors. This method will help in identifying peptide candidates for vaccination studies for tumor immunotherapy.     

Emergence in Cx knockout mice of a diverse cytotoxic T lymphocyte repertoire that recognizes a single peptide from the immunoglobulin constant x light chain region

Allotype- or idiotype-specific CD4⁺ T cells have been reported to recognize immunoglobulin (Ig) peptides presented by class II molecules. In contrast, few data are available concerning the generation of Ig peptide-specific CD8⁺ T cells. We have therefore investigated whether T-depleted spleen cells from Ig x light chain-expressing 129/Sv mice (129x^+/+) could induce, in Cx knockout mice (129 x^?/?), the generation of Ig constant x light chain region (Cx)-specific cytotoxic T lymphocytes (CTL). The determination of TCRβ chain expressed by nine CTL clones, together with the use of a library of overlapping peptides spanning the whole Cx sequence, show that the B cells from x^+/+ mice are able to elicit in Cx knockout mice, the emergence of a diverse CTL repertoire that recognizes one single Cx peptide presented by the H-2K^b class I molecule. In addition, these data support the notion that B cells are able to process and present on their class I molecules, peptides generated from their own x light chains.     

The rational design of TAP inhibitors using peptide substrate modifications and peptidomimetics

The major histocompatibility complex (MHC)-encoded transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the lumen of the endoplasmic reticulum. This step precedes the binding of peptides to MHC class I molecules and is essential for cell surface expression of the MHC class I/peptide complex. TAP has a broad sequence specificity and a preference for peptides of around 9 amino acids. To synthesize inhibitors for TAP, we studied various alterations of the peptide substrate. The results indicate that TAP is stereospecific and that peptide bonds engineered into isosteric structures can improve translocation of the peptide. Furthermore, TAP is able to translocate peptides with large side chains that correspond to a peptide of ～ 21 amino acids in extended conformation. Peptides with longer side chains compete for the peptide binding site of TAP but fail to be translocated. Therefore, they represent the first rationally designed inhibitors of TAP.     

心包积液中几种多肽类物质的观察

目的 :为探讨C型利钠多肽 (CNP)、神经肽Y(NPY)、降钙素基因相关肽 (CGRP)在心包积液时的变化及其作用。方法 :用放射免疫分析测定心包积液患者的血液和心包积液中的CNP、NPY、CGRP水平。并与正常对照组的血液中的水平进行比较。结果 :血液和心包积液中的CNP显著高于对照组 (p <0 0 1) ,血液中NPY和CGRP则与对照组无显著性差异 (p >0 0 5 ) ,心包积液中NPY水平显著低于血液中 (p <0 0 5 ) ,心包积液中的CGRP水平则显著高于血液中 (p <0 0 1)。心包积液的量与心包积液中CNP、CGRP呈正相关 (r=0 71,p <0 0 1,r=0 5 7,p <0 0 1)与NPY呈负相关 (r=- 0 6 7,p <0 0 1) ,心包积液中的CNP、NPY和CGRP与其血液中的浓度间无显著相关性 (p >0 0 5 )。结论 :心包积液中有一定量的CNP、NPY和CGRP ,很可能由心包分泌。心包积液患者体内的CNP、NPY和CGRP的改变可能对心包积液的病理生理状况起到重要的调节作用。     

蝎蜂毒肽对大鼠纤溶系统作用初探

本研究采用大鼠肢体血管灌流和整体给药两种模型，观察蝎蜂毒（ＳＢＰ）对血管理灌流液内纤溶酶原激活物（ＰＡ）活性、血浆优球蛋白纤溶性（ＥＦＡ）和纤溶酶（ＰＬ）活性的影响。结果说明，ＳＢＰ有明显激活纤溶系统作用；其机制可能涉及血管内皮细胞释放ＰＡ活性增加，进一步促使纤溶酶原活化为ＰＬ增多的途径。     

A comparative study of the proteolytic enzymes of Trypanosoma brucei, T. equiperdum, T. evansi, T. vivax, Leishmania tarentolae and Crithidia fasciculata

Four types of proteolytic activity were detected in the bloodstream form of each of the four Trypanosoma species: (i) HPAase, active on hide powder azure and detected on polyacrylamide gels containing denatured haemoglobin; (ii) AZCase, active on azocasein; (iii) type 1, active on the chromogenic peptide N-benzoyl-L-prolyl-L-phenylalanyl-L-arginine p-nitroanilide in the presence of dithiothreitol, and (iv) type 2, active against several nitroanilide derivatives in the absence of dithiothreitol. Studies of the pH optimum, dithiothreitol requirement and inhibitor sensitivities of the proteolytic activities suggested that: (a) HPAase and type 1 activities could be due to the same enzymes, probably a family of cysteine proteinases; (b) AZCase had some characteristics of a cysteine proteinase, but was not identical to HPAase, and (c) type 2 activity could be due to a serine proteinase. Procyclic T. brucei contained relatively low cysteine proteinase activities (HPAase, AZCase and type 1) but high type 2 activity. Their proteolytic enzymes thus were apparently more similar to those in Crithidia fasciculata and Leishmania tarentolae promastigotes than those in T. brucei bloodstream forms.     

Differential effects of interleukin-4 in peptide induced autoimmunity

BALB/c mice immunized with multimeric DWEYSVWLSN develop IgG1 anti-DNA antibodies and glomerular immunoglobulin deposits, leading us to investigate the role of IL-4 in this model of antigen induced lupus. Splenocytes from DWEYSVWLSN immunized mice secreted IL-4 but not gamma-interferon. Following peptide immunization, IgG1 anti-peptide and anti-DNA antibodies were significantly higher in IL-4 wild type mice, while IgM and IgG3 anti-DNA levels were significantly higher in IL-4 knockout mice. Titers of IgG anti-laminin and anti-histone, but not anti-Sm/RNP and anti-cardiolipin antibodies, were significantly higher in the IL-4 wild type group. Glomerular immunoglobulin deposition was substantially decreased in IL-4 knockout mice. We conclude that while IL-4 does not materially affect the generation of some autoantibody responses associated with peptide induced autoimmunity, IL-4 deficiency inhibits kidney immunoglobulin deposition. The effect of IL-4 on humoral autoimmunity in lupus is complex, and is dependent on genetic background, the antigenic trigger and stage of disease.     

Antigenic and immunogenic mimicry of the HER2/neu oncoprotein by phage-displayed peptides

To recover peptides that antigenically and immunogenically mimic the p185^HER2 oncoprotein, we selected the phage-peptide libraries pVIII-9aa and pVIII-9aa. Cys using murine monoclonal antibodies (mAb) MGr2 and MGr6, directed against two distinct epitopes of the p185^HER2 extracellular domain. Phagedisplayed peptides containing consensus amino acid motifs were recovered and shown to compete specifically for mAb binding on tumor cells that overexpress p185^HER2. The deduced amino acid sequence of the peptides suggests that both epitopes defined by the mAb on p185^HER2 are discontinuous and that hydrophobic interactions are involved in binding with the mAb. A phage clone displaying the GPLDSLFAQ peptide elicited a specific immune response against the p185^HER2 in BALB/c mice, demonstrating that this phage-displayed peptide represents an immunological equivalent of the MGr2 epitope on p185^HER2 and might be used as a substitute for this oncoprotein in in vitro and in vivo immunological studies.     

人类免疫缺陷病毒包膜糖蛋白V3区对细胞结合能力的研究

目的：研究人类免疫缺陷病毒（ＨＩＶ）不同亲嗜株包膜糖蛋白Ｖ３区结合于靶细胞的能力。方法：合成来源于不同嗜性ＨＩＶ－１Ｖ３区的生物素标记和非标记的多肽。采用流式细胞计数分析生物素化的 Ｖ３多肽对细胞的结合能力以及细胞表面的结合配体。结果：ＨＩＶ－１Ｘ４　亲嗜株ＩＩＩＢＶ３区能结合于多种细胞的表面，包括辅助受体ＣＸＣＲ４；竞争实验结果显示蛋白酶抑制剂能抑制该结合。Ｒ５亲嗜株 ＡＤＡ Ｖ３区只极微弱地结合于外周血单核细胞和表达ＣＣＲ５ 的人星形胶质细胞表面。结论：不同嗜性ＨＩＶ－１Ｖ３区结合于细胞表面的能力不同从亲嗜株Ｖ３区直接结合于细胞表面并被其自身所增强，其靶分子至少包括辅助受体 ＣＸＣＲ４和蛋白酶分子；而Ｒ５亲嗜株 ＡＤＡ Ｖ３区则不结合于 ＣＣＲ５和蛋白酶。     

Carcinoid Tumor of the Middle Ear Containing Serotonin and Multiple Peptide Hormones A Case Report and Review of the Pathology Literature

A 69-year old man complaining of longstanding hearing loss and mild otorrhea was found to have a mass obliterating the external auditory canal and polypous tympanic mucosa with accompanying absence of the tympanic membrane and ossicular chain. Tumors excised from the external auditory canal and tympanum showed histologic features essentially characteristic of a carcinoid tumor: a ribbon or festoon arrangement of tumor cells, formation of anastomosing cords and glandular spaces, presence of numerous argyrophilic as well as argentaffin secretory granules within many of the tumor cells, and ultrastructur-al evidence of neurosecretory granules in the tumor cell cytoplasm. Immunohistochemically, the tumor was found to contain not only neuronal marker substances such as neuron-specific enolase, S 100 protein and chromogranin A, but also serotonin and multiple peptide hormones such as pancreatic polypeptide, glucagon, cholecystokinin and leucine-enkephalin. A review of the pathology of 17 previous cases of carcinoid of the middle ear suggested that this type of carcinoid may have a variegated hormone profile among carcinoids of foregut origin, and hormonally may resemble ileal carcinoid arising from the midgut, although their histogenetic origins may differ, because of frequent production of serotonin. Acta Pathol Jpn 42: 614–620, 1992.