Multi-parameter prediction of HLA class Ⅰ restricted cytotoxic T lymphocyte epitopes for human proliferating cell nuclear antigen

Objective To predict the HLA class Ⅰ restricted cytotoxic T lymphocyte epitopes for human proliferating cell nuclear antigen (PCNA). Methods By using 3 epitope prediction databases which including MHC binder ( BIMAS and SYFPEITHI databases) and proteasome cutting site ( PAProC databases), the epitopes of PCNA were predicted by analyzing several parameters and methods. Results After comprehensive analysis,we have obtained two possible HLA-A0201 restricted CTL epitopes SMSAD-VPLV (228-236, score 447 633. 595 ) and LINEACWDI ( 22-28, score 127 966.779);two HLA-A24 re-stricted CTL epitopos DYEMKLMDL ( 113-121, score 563 994.9 ) and EFARICRDL ( 143-151, score 40 540.7);two HLA-A1101 restricted CTL epitopes LTSMSKILK (72-80, score 1334. 2680 ) and DVPLV-VEYK (232-240,score 736.9236). Conclusion The multi-parameter epitope prediction method is feasi-ble for cytotoxic T lymphocyte epitope prediction.     

Massive Immune Hemolysis Caused by Anti-D After Dual Kidney Transplantation

Massive immune hemolysis due to passenger lymphocyte-derived anti-D has not been reported in renal transplantation. A 50-year-old (B-positive) male received a dual deceased-donor kidney transplant (B-negative) for diabetic renal failure. Two weeks post-transplant, the patient developed severe hemolytic anemia. The donor anti-D titer was 1:8. The recipient anti-D titer (zero pre-transplant) increased from 1:4 to 1:16 over 4 days. Rapid hemolysis caused severe anemia, minimum Hb = 4.2 g/dL, while selectively lysing the patient's autologous red cells during this time. The hemolytic anemia did not impair the allografts and subsided without monoclonal B-cell pharmacotherapy or apheresis. The anti-D titer decreased to barely detectable levels at four months and had cleared when checked 2 years post-transplant. Transfusion support subsided after two months. If complications of anemia can be avoided, the deleterious effects of hemolysis may be well tolerated by renal allografts using antigen negative transfusion alone.     

神经肽Y对免疫细胞活性的影响

神经肽Y（NPY）是广泛分布于中枢神经系统和外周神经各部位的神经肽类物质。本实验观察NPY在体外对几种免疫细胞活性的直接作用。结果表明，NPY对小鼠T淋巴细胞丝裂原反应性和NK细胞的杀伤活性均无明显影响（P＞0．05），对巨噬细胞分泌溶菌酶有明显抑制作用（P＜0．05）；而对B淋巴细胞丝裂原反应性则有明显的促进作用（P＜0．05）。上述结果提示，NPY对部分免疫细胞功能的影响因细胞种类而异。     

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Actions on αT3-1 Gonadotrophs Show Desensitization

PACAP is a hypothalamic hypophysiotropic factor that acts upon a number of pituitary cells, including gonadotrophs. In the gonadotroph-derived αT3-1 cell line, PACAP acts via PVR1 receptors to stimulate adenylyl cyclase and phosphoinositidase C. PACAP-stimulated cAMP accumulation is inhibited by protein kinase C-activating phorbol esters in these cells and the current work was undertaken primarily to establish whether it is also subject to homologous regulation. In acute experiments, PACAP27-stimulated cAMP accumulation (intracellular plus extracellular) was measured (in the presence of phosphodiesterase inhibitor) both in intact cells and in cell membranes. The peptide increased cAMP accumulation, but initial rates of PACAP27-stimulated cAMP accumulation were reduced to between 10 and 50% within 10 min of stimulation in both cells and membranes. The initial rate of forskolin-stimulated cAMP accumulation was maintained in membranes but not in intact cells (although the deviation from linearity was less pronounced than with PACAP27). Thus, rapid homologous desensitization to PACAP27 occurs in intact αT3-1 cells, but is not entirely receptor specific. Rapid homologous desensitization of PACAP27-stimulated cAMP accumulation also occurred in the presence of a protein kinase C activating phorbol ester, which inhibited cAMP accumulation without altering the kinetics of the PACAP27 effect. Brief pre-treatment (3 min) with PACAP27 also reduced the ability of PACAP27, but not gonadotrophin-releasing hormone, to cause a spike-type elevation of cytosolic Ca²⁺ concentration (a consequence of phosphoinositidase C activation). In chronic desensitization studies, pre-treatment for 6 h with PACAP27 caused a dose-dependent (IC₅₀ approximately 10 nM) reduction of PACAP-stimulated cAMP accumulation and down regulated cell surface PVR1 receptors (to approximately 50%). Thus, it appears that PACAP27-stimulated (PVR-1 receptor mediated) adenylyl cyclase undergoes rapid homologous desensitization in αT3-1 cells, which is paralleled by homologous desensitization of PACAP27-stimulated phosphoinositidase C activity and involves mechanisms distinct from those underlying heterologous desensitization by phorbol esters. Chronic desensitization of PACAP-stimulated cAMP accumulation and down-regulation of cell surface PVR-1 receptors also occurs in these cells although the receptor loss may not entirely explain the observed desensitization.     

In vivo induction of CD4+ T cell responses by antigens covalently linked to synthetic microspheres does not require adjuvant

Macrophages, dendritic cells or B lymphocytes have been shownto play a major role in the presentation of soluble antigensto CD4⁺ T cells. In contrast, the capacity of these cells topresent particulate antigens such as bacterial or parasiticantigens to T cells remains controversial. To investigate thisquestion, well defined particulate antigens were prepared bycovalent linkage of proteins or peptides to 1 µm in diametersynthetic microspheres. The T cell immunogenicity of such particulateantigens was analyzed in vitro and in vivo. In vitro, a solubleprotein such as hen egg lysozyme (HEL) coupled to beads stimulateda strong proliferative T cell response of lymph node cells fromHEL-primed mice or of specific T cell hybridomas. HEL coupledto beads was presented to the specific T cell hybridomas bysplenocytes or by peritoneal macrophages, but not by lymphomaB cells. Immunization of mice with several different proteinantigens or with a synthetic peptide covalently linked to beadsinduced strong CD4⁺ T cell responses in the absence of adjuvant.The strong in vivo immunogenicity of proteins coupled to beadsdid not result from a non-specific adjuvant effect of beadssince covalent linkage of the antigen to beads was strictlyrequired to induce T cell responses in the absence of adjuvant.In vivo treatment by carrageenan showed that macrophages arerequired for the in vivo stimulation of T cell responses bythese particulate antigens. Thus, these results demonstratedthe role of phagocytic cells, especially macrophages, for invivo presentation of particulate antigens. These particulateantigens represent an interesting approach for the developmentof new vaccines, and for the in vivo analysis of the role ofvarious antigen presenting cells in T cell activation and differentiation.     

Development of intestinal epithelial cell lines using a transgenic mouse

The epithelial cells of the colonic mucosa of the animal have proved impossible to culture using standard tissue culture techniques. Immortalization of adult colonic epithelial cells has been unsuccessful due to the lack of DNA synthesis in these cells once they are isolated from the tissue. Recently an unique transgenic mouse bearing a temperature sensitive mutant of the known immortalizing gene, SV40 large T has become available. The advantage of this mouse is that the SV40 large T gene is expressed in every cell. Active immortalizing protein is produced in each cell at the permissive temperature. We have used colonic mucosa from these mice to initiate cultures of epithelial cells from the colon of adult mice. The cells grow readily at the permissive temperature but die within 7 days at the non-permissive temperature. The methods used to develop these cultures are described.     

Antisperm antibodies and lymphocyte subsets in semen—not a simple relationship

The significance of white blood cells in the ejaculate remains a matter of controversy. Several authors have suggested that such cells are important in the modulation of an antisperm antibody response, i.e. a predominance of suppressor/cytotoxic to helper/inducer T cells may prevent the development of antisperm antibodies. In order to examine this relationship further we have documented the white blood cell types, with emphasis on the T-lymphocyte populations, in the ejaculates of men from infertile couples with and without antisperm antibodies; the latter group was divided further into two groups--vasovasostomized men and idiopathic men. All seven of the men without antisperm antibodies had a predominance of suppressor/cytotoxic T cells to helper/inducer T cells in the ejaculate. However, only in some of the men with antibodies was there a predominance of T-helper/inducer cells. It is clear that the relationship between antisperm antibodies and seminal leucocytes is therefore not as straightforward as has been proposed.     

Interactions between peripheral blood CD8 T lymphocytes and intestinal epithelial cells (iEC)

Intestinal intraepithelial lymphocytes (iIEL) are primarily CD8 cells and most of them have a CD28^? phenotype, the phenotype of effector cytotoxic T cells. We asked whether the predominance of CD8⁺ CD28^? T cells in the gut may result from peripheral blood T cells preferentially migrating to the iIEL compartment and adhering to iEC. Compared with CD4 cells, adhesion of resting CD8⁺ T cells to iEC cell lines was significantly higher. Adhesion could be blocked with a MoAb to gp180, a molecule expressed on iEC which is known to interact with CD8/lck. No significant difference in the level of adhesion was observed between CD8⁺ CD28⁺ and CD8⁺ CD28^? T cells. Thus CD8 cells may preferentially migrate to the iIEL compartment, but loss of CD28 expression could occur in situ after migration. Consistent with this hypothesis, the CD8⁺ CD28^? cells became enriched after co-culturing T cells with iEC cell lines and primary iEC. Induction of the CD8⁺ CD28^? phenotype in cord blood and adult T cells was observed in co-cultures with iEC and also with mitogens and superantigens. In the latter case, CD28 down-modulation was seen specifically in the Vβ subset targeted by the superantigen, indicating that loss of CD28 expression is a direct result of T cell receptor (TCR)-mediated stimulation. The combined results suggest that CD8⁺ CD28^? T cells are antigen experienced T cells, and that they may have a survival advantage in the presence of gut epithelial cells in vitro. This may contribute to the predominance of CD8⁺ CD28^? T cells in the iIEL compartment.     

小鼠胸腺和脾脏细胞的体外转化培养

建立了小鼠脾脏、胸腺细胞体外转化微量培养系统,确定了最佳实验条件.点蜡法充CO_2,简便易行,效果好.RPMI-1640培养基内补充胎牛血清,其浓度确定为在脾细胞培养为10%,在胸腺细胞培养为20%.脾细胞培养最适细胞密度为2～5×10~6/ml,Con A最适量为2～4μg/培养;胸腺细胞培养细胞最适密度为1×10~7/ml,Con A最适量为2～4μg/培养.两种已知免疫活性的药物在脾细胞培养体系有明确肯定的反应.