An experimental model for canine visceral leishmaniasis

Seven mixed-breed dogs were challenged with either promastigotes or amastigotes of Leishmania donovani infantum strains recently isolated from naturally infected dogs. Different routes and numbers of parasites were utilized and each dog was monitored for at least 1 year post-infection. Anti-parasite specific antibody levels were measured by enzyme-linked immunosorbence, immunofluorescence, crossed-immune electrophoresis and Western blotting on crude antigen. Western blotting on two pure parasite proteins, dp72 and gp70-2, was also done. Mitogenic and antigen-specific stimulation of peripheral blood lymphocytes was monitored; and the haematological, clinical and parasitological parameters measured. Dogs challenged with amastigotes exhibited a more pronounced humoral response to leishmanial antigens. Only in one case was strong antigen-specific proliferation detected. Clinical signs of disease, including hypergammaglobulinaemia, enlarged lymph nodes and the presence of parasites, were also more apparent in the dogs challenged with amastigotes. None of the seven dogs died. Serum antibodies to leishmanial antigens were apparent between 1.5 to 3 months following challenge and correlated with the appearance of enlarged lymph nodes, hypergammaglobulinaemia and the presence of parasites in tissue biopsies. Serum antibodies remained chronically high in these dogs throughout the period of the study. Only one dog (1/3) challenged intravenously with promastigotes and the dog challenged intradermally with amastigotes produced transient antibody responses to leishmanial antigen.     

小儿白血病瘤细胞P—糖蛋白测定方法及应用

本文介绍免疫组化法检测小儿白血病瘤细胞（Ｐ－ｇｐ）。检测３０例小儿白血病细胞膜Ｐ－ｇｐ，观察其原发耐药性。３０例中有８例ｐ－ｇｐ阳性，总阳性率２６．６７％，其中ＡＮＬＬ单项阳性率３３．３３％，ＡＬＬ为２０％。本组病例耐约性最高的是ＣＭＬ，其次是ＡＮＬＬ，ＡＬＬ组最低。同时发现ＭＤＲ阳性者复发率高，ＭＤＲ阴性者复发率低，多因素分析表明ＭＤＥＲ对初诊白血病患者预后及抗癌药物选择有重要意义。目的临床评价ＭＤＲ采用检测ｍｄｒ１ｍＲＮＡ或Ｆ－ｇｐ，本文认为采用简便、快速的免疫组化法检测Ｐ－ｇｐ在临床上具有较大的实用价值。     

结核分枝杆菌Ag85B/GST融合蛋白表达载体构建及在大肠杆菌中的表达

目的 构建结核分枝杆菌Ag85B／GSF融合蛋白表达质粒，并在大肠杆菌(E．coli)中诱导表达。方法 以质粒pUC18／Ag85B为模板，用PCR法扩增结核杆菌Ag85B基因，扩增的Ag85B上游含有EcoR Ⅰ酶切位点，下游含有SalⅠ酶切位点；将Ag85B基因定向插入质粒pGEX-4T-1中，构建原核表达质粒pGEX-Ag85B并转化E．coli B121，筛选阳性重组子，限制性内切酶切鉴定和DNA序列测定，异丙基硫代半乳糖苷(IPTG)诱导表达，SDS-PAGE和Westem blot鉴定结果。结果 成功构建原核表达质粒pGEX-Ag85B并表达出Mr约56000大小的Ag85B／GSY融合蛋白，表达量占总菌体的30％。结论 为进一步进行纯化Ag85B蛋白和研究其在膀胱肿瘤治疗中的作用奠定了基础。     

Rectal Absorption Enhancement of Des-Enkephalin-γ-Endorphin (DEγE) by Medium-Chain Glycerides and EDTA in Conscious Rats

The stability of the neuroleptic peptide des-enkephalin--endorphin (DEE; Org 5878) in the rectal lumen and the rectal bioavailability of DEE were investigated in conscious rats. Furthermore, the influence of peptidase inhibition, peptidase saturation, and absorption enhancement on DEE bio-availability were evaluated. Na₂EDTA (0.25%, w/v) prolonged the degradation half-life of DEE in the ligated colon from 33 ± 7 to 93 ± 45 min. Without adjuvant, tritium-labeled DEE was absorbed from the rat rectum to a very low extent (0–4%). After administration of an excess of unlabeled DEE or with Na₂EDTA, comparable results were obtained. The medium-chain glyceride preparation MGK markedly enhanced the rectal DEE bioavailability, up to 8–20%, which was further increased to 10–44% by coadministration of Na₂EDTA. No substantial influence of varying the rectal delivery rate was observed. The results suggest that absorption enhancement and enzyme inhibition both are essential for effective increase of rectal peptide bioavailability.     

Inhibition of HIV-1 by modification of a host membrane protease

While it is clear that CD4 Is the receptor for the gp120 envelopeprotein of HIV-1, substantial evidence suggests that other hostcell proteins are required for successful membrane fusion. Studieswere initiated to examine the potential for a protein receptorwhich has an elastase-like character to participate in fusionof HIV-1 with permissive host cells. A synthetic elastase inhibitorwas shown to significantly reduce HIV-1 infectivity when presentduring, but not after, the initial contact between virus andcells. A human T cell elastase-like membrane component was purifiedand shown to be lipid-associated. By competitive Inhibition,the purified protein was shown to bind gp160 within the HIV-1fusion domain. The binding parameters of whole T cell membraneextract, with a hydrophobic pentapeptide representative of thefusion domain, suggested an elastase-like protein is the single,secondary T cell receptor for HIV-1 (K = 1x10³ M^–1). Thepentapeptide interacted with porcine and human (epithelial andpolymorphonuclear leukocyte), but not murine, elastase isoforms,suggesting its participation In the permissiveness of host cellsto infection.     

ATL and HTLV-I:In vivo cell growth of ATL cells

The mechanism of leukemogenesis or neoplastic cell growth in adult T cell leukemia (ATL) still remains unclear, although Tax of human T cell leukemia/lymphoma virus type I (HTLV-I), the etiologic virus, has been reported to affect the expression of various cellular genes which encode molecules involved in cell growth or cell death. We have studied the cell growth of HTLV-I-infected human T cells in severe combined immunodeficiency (SCID) mice and found that fresh leukemic cells or cell lines derived from leukemic cell clones but not HTLV-I-infected cell lines of nonleukemic cell origin showed tumorigenicity, and neither HTLV-I nor IL-2 expression was needed for cell growthin vivo, indicating that accumulating changes in addition to the initial events induced by HTLV-I infection were required for the development of ATL. The interaction between ATL cells and vascular endothelial cells appears to be one of the important factors which determine the pattern of organ infiltration by leukemic cells. E-selectin and its ligand are one of the major cell adhesion pathways between ATL cells and human umbilical vein endothelial cells (HUVEC). Another pathway that had not been identified was studied using newly developed monoclonal antibodies capable of blocking cell adhesion. The molecules which directly mediate adhesion between ATL cells and HUVEC were determined to be OX40 and gp34, a member of the tumor necrosis factor receptor (TNF-R) family and TNF family, respectively. The OX40/gp34 system may play a key role in the trafficking and homing of not only ATL cells but also activated normal T cells.     

Identification of a novel gp100/pMel17 peptide presented by HLA-A*6801 and recognized on human melanoma by cytolytic T cell clones

Melanoma-associated peptides recognized by cytolytic T lymphocytes (CTL) in the context of several histocompatibility leukocyte antigens (HLA) are required for the development of specific immunotherapies. Using a transient transfection assay into COS-7 cells, we identified the gp100/pMel17 melanosomal protein as the shared antigen recognized by three independent CD8+ CTL clones in HLA-A*6801-restricted fashion. This finding was confirmed by the correlation between lack of gp100/pMel17 protein in a number of HLA-A*6801-positive melanomas and their resistance to lysis/cytokine production by the specific effectors. The gp100/pMel17 antigenic epitope was identified based on recognition of subfragments and on a computer-based prediction algorithm. Among a panel of gp100/pMel17-derived synthetic peptides only the 10-mer HTMEVTVYHR (gp100/pMel17182-191) induced tumor necrosis factor (TNF) release by CTL clones when pulsed on suitable target cells whereas both the 10-mer and the shorter 9-mer gp100/pMel17183-191 sensitized the same antigen-pulsed cells to lysis. In conclusion, the identification of the HTMEVTVYHR peptide will extend to HLA-A*6801 melanoma patients the possibility to exploit gp100/pMel17 melanosomal protein for experimental and clinical studies.     

gp49B1 suppresses stem cell factor-induced mast cell activation-secretion and attendant inflammation in vivo

We report that gp49B1, a mast cell membrane receptor with two immunoreceptor tyrosine-based inhibitory motifs (ITIM), constitutively inhibits mast cell activation-secretion induced by stem cell factor (SCF), a tissue-derived cytokine that also regulates mast cell development. The intradermal injection of SCF into the ears of gp49B1 null (gp49B(-/-)) mice elicited approximately 4- and 2.5-fold more degranulating mast cells and tissue swelling caused by edema, respectively, than in gp49B(+/+) mice. SCF did not induce tissue swelling in mast cell-deficient mice, and the responsiveness of gp49B(-/-) mice to mast cell-associated amine and lipid mediators was unaltered. When gp49B(+/+) and gp49B(-/-) mice were pretreated with antagonists of the amines, SCF-induced tissue swelling was reduced by >90% and 60%, respectively, and it was reduced by >90% in both genotypes when a cysteinyl leukotriene receptor antagonist was also provided. Hence, the dominant contribution of secretory granule amines to SCF-induced tissue swelling is the result of gp49B1-mediated inhibition of the production of cysteinyl leukotrienes by mast cells. Our findings also provide the first example of an ITIM-bearing receptor that constitutively suppresses inflammation generated in vivo independently of the adaptive immune response by a receptor that signals through intrinsic tyrosine kinase activity rather than immunoreceptor tyrosine-based activation motifs.     

结核分枝杆菌联合DNA疫苗初免-BCG加强的免疫效果观察

目的:研究并比较结核分枝杆菌免疫保护性抗原DNA(Ag85A和ESAT-6)疫苗联合免疫,BCG免疫以及联合DNA疫苗初免-BCG加强免疫等不同的免疫策略,诱导免疫应答效果观察.方法:健康雌性BALB/c小鼠24只,随机分成PBS 阴性对照组,DNA初免-BCG异源加强组,DNA(Ag85A和ESAT-6)初免DNA同源加强组和BCG阳性对照组,共进行3次免疫,初免2次,最后1次加强,间隔2周1次.PBS组3次均注射PBS 溶液;DNA/BCG组以质粒DNA免疫2次,最后1次以BCG加强免疫;DNA/DNA组3次均以质粒DNA进行免疫;BCG组则注射PBS溶液2次后以BCG免疫.末次免疫后4、6、8周分别分离血清测定总IgG水平,同时分离小鼠脾细胞,体外经TB-PPD刺激后进行淋巴细胞增殖实验(XTT法)并测定脾细胞培养上清中IFN-γ和IL-4水平.结果:DNA/BCG、DNA/DNA、BCG组体外经TB-PPD刺激后均检测到特异性IgG抗体产生,3组平均效价为1:120、1:160、1:80,DNA/DNA组的抗体效价高于另外2组;小鼠脾细胞体外经TB-PPD刺激后,均能产生特异性淋巴细胞增殖并诱生较强的IFN-γ反应,其中DNA/BCG组IFN-γ的分泌水平高于DNA/DNA组和BCG组(P<0.05).结论:联合DNA疫苗初免-BCG加强的免疫策略能在小鼠体内诱导较强的特异性细胞免疫反应,产生高水平的IFN-γ.     

结核分枝杆菌Ag85B基因疫苗免疫保护作用的初步研究

目的:研究编码结核分枝杆菌分泌蛋白Ag85B的基因疫苗pTB30m和pTB30s对免疫动物的保护作用。方法:以基因疫苗pTB30m和pTB30s肌注免疫BALB/c小鼠。免疫完成6 wk后,用5×105 CFU的MTB H37Rv毒株经小鼠尾静脉攻击感染。同时用尼龙毛柱分离基因免疫BALB/c小鼠的T细胞,并以5×106 T细胞/只小鼠过继免疫正常BALB/c小鼠,立即用105 CFU的MTB毒株经小鼠尾静脉攻击感染。4 wk后分别计数脾脏中的细菌负荷。结果:与生理盐水对照组相比较,pTB30m及pTB30s质粒免疫组BALB/c小鼠脾脏中的细菌负荷均减少,分别为0.645(log10 CFU,P<0.01)和0.839(log10CFU,P<0.001);而空质粒对照组小鼠脾脏中的细菌负荷减少较少。经质粒pTB30m和pTB30s免疫的BALB/c小鼠的T细胞,过继免疫的正常BALB/c小鼠,对攻击感染的MTB H37Rv毒株在脾脏中的增殖具有部分抑制作用。结论:pTB30s免疫的BALB/c小鼠,对MTB H37 Rv毒株攻击的保护作用优于pTB30m质粒免疫,有望进一步用于结核病的防治研究。