益生菌抑制大肠埃希菌K1株黏附与侵袭的实验研究

目的评价三联活菌片中的益生菌对大肠埃希菌K1株在肠上皮黏附和侵袭的抑制作用。方法将大肠埃希菌K1株与Lovo细胞共孵育,采用黏附性抑制和竞争性排除方法,检测大肠埃希菌K1株黏附侵袭于Lovo细胞的数量,并采用流式细胞术分析益生菌对大肠埃希菌K1株诱导的细胞凋亡的抑制作用。将Sprague Dawley乳鼠随机分为实验组（益生菌和致病菌）与对照组（致病菌）。应用活菌计数法,对各组乳鼠肠道和血液中大肠埃希菌K1株进行定量检测,观察益生菌对大肠埃希菌K1株血行转移的拮抗作用。结果益生菌在体外能显著抑制大肠埃希菌K1株黏附侵袭于Lovo细胞（P〈0.01）,其抑制作用呈剂量依赖性;益生菌能显著降低大肠埃希菌K1株诱导的细胞凋亡（P〈0.01）;益生菌能显著抑制大肠埃希菌K1株的生长与血行转移,患菌血症的乳鼠数量显著降低（P〈0.01）。结论三联活菌片中益生菌能显著抑制大肠埃希菌K1株对肠上皮的黏附损伤和血行转移。     

Expression of LFA-1 by a lymphoblastoid cell line from a patient with monosomy 21: effects on intercellular adhesion.

Monosomy 21 (M21) is a rare aneuploid condition which in certain cases leads to reduced levels of chromosome 21 gene products. We have prepared an Epstein-Barr virus lymphoblastoid cell-line (LCL) from patient with M21 who has immunological abnormalities, and analysed the expression of lymphocyte function-associated antigen-1 (LFA-1). This heterodimeric leucocyte integrin consists of CD11a (alpha) subunits non-covalently associated with CD18 (beta) subunits coded, respectively, by genes on chromosomes 16 and 21. To determine whether monosomy 21 results in decreased expression of LFA-1, monoclonal antibodies were used to compare the expression of CD11a and CD18 on the M21 LCL with LCL from trisomy 21 (Down's syndrome, T21), normal controls and a possible case of leucocyte adhesion deficiency. In addition, phorbol-ester-induced homotypic adhesion, an LFA-1-mediated effect, was compared in these LCLs. The results are consistent with a gene dosage mediated reduction of LFA-1 expression by the M21 LCL.     

Identification of monoclonal antibodies reactive with the rat homolog of ICAM-1, and evidence for a differential involvement of ICAM-1 in the adherence of resting versus activated lymphocytes to high endothelial cells

We have produced four mAbs reactive with the rat homolog of ICAM-1 (intercellular adhesion molecule-1), and presented evidence to indicate that the ICAM-1 molecule plays a differential role in the binding between high endothelial cells and lymphocytes, depending on the state of lymphocyte activation. The conclusion that the four antibodies (1A29, 6A22, 10A25, 10A56) specifically recognize the rat ICAM-1 homolog was based on: (i) their ability to inhibit homotypic aggregation of PHA-blasts; (ii) SDS-PAGE analysis of the antigen recognized; (iii) antigen distribution as assessed by immunoperoxidase staining of frozen sections; and (iv) cytokine-induced up-regulation of the antigen. Involvement of the ICAM-1 molecule in lymphocyte binding to high endothelial cells, the vital step in lymphocyte extravasation in lymph nodes, was examined by the use of one of the newly developed antibodies, and it was found that binding of activated but not resting lymphocytes to cultured high endothelial cells was significantly blocked by the anti-ICAM-1, implying that ICAM-1 has differential involvement in the adherence of lymphocytes to high endothelial cells and that it may play an important role in dissemination of memory lymphocytes to systemic circulation by facilitating or strengthening the binding to the specialized postcapillary high endothelial venules (HEV).     

Inhibition of Basal and Stimulated Release of Endothelin-1 from Guinea Pig Tracheal Epithelial Cells in Culture by Beta 2-adrenoceptor Agonists and Cyclic AMP Enhancers

The effects of cyclic AMP-related compounds and beta adrenoceptor agonists on the basal and lipopolysaccharide (LPS)-stimulated release of endothelin-1 (ET-1) from guinea-pig tracheal epithelial cells (GPTEpCs) in culture were studied. Forskolin (a potent activator of adenylyl cyclase), 8-bromo-cyclic AMP (a cyclic AMP analogue), salbutamol and salmeterol (two beta 2-adrenoceptor agonists), were used to increase cyclic AMP levels. Cultured GPTEpCs released ET-1 continuously over a 24 h incubation period. The values reached 1,938 ± 122 pg/mg of total cell proteins after 24 h. LPS (10 μg/ml) significantly stimulated the release of ET-1 by 1.6- to 1.8-fold, up to 1,262 ± 56 pg/mg total cell proteins after an 8 h incubation period. Compound 8-bromo-cyclic AMP (10⁻⁵, 10⁻⁴ and 10⁻³ M) reduced the basal release of ET-1 from GPTEpCs by up to 31% (P < 0.01) and the LPS stimulated release by up to 42% (P < 0.05), after an 8 h incubation period. Forskolin (10⁻⁶, 10⁻⁵ and 10⁻⁴ M) also inhibited the basal release of ET-1 by up to 28% (P < 0.05) and LPS-stimulated release of ET-1 by up to 50% (P < 0.05), after an 8 h incubation period. At the concentration of 10⁻⁵ M, forskolin increased cyclic AMP levels in GPTEpCs by 17-fold (P < 0.001) in the medium, 15 min after the beginning of the incubation. Salbutamol (10⁻⁸ to 10⁻⁶ M) had no effect on the basal production and release of ET-1 after 8 h. Conversely, this short acting beta 2-adrenoceptor agonist significantly reduced LPS-mediated increase of ET-1 production by up to 55% (P < 0.05) after an 8 h incubation period. Salmeterol (10⁻⁹ M to 10⁻⁵ M) inhibited basal and LPS-stimulated production and release of ET-1 after an 8 h incubation period (between 44 and 51%, P < 0.01). Both salbutamol and salmeterol (10⁻⁶ M) increase cyclic AMP levels by five- and twofold, respectively (P < 0.05). In summary, these observations indicate that beta 2-adrenoceptor agonists or cyclic AMP enhancers can modulate both basal and more markedly, the enhanced production of ET-1 from LPS-activated guinea pig airway EpCs. In addition, these compounds increase cyclic AMP levels in the cells. It is suggested that there is a correlation between cyclic AMP increase and inhibition of ET-1 release by guinea pig airway EpCs. Since ET-1 production was shown to be elevated in asthmatic subjects and in patients suffering from other inflammatory lung disorders, the inhibition of its production by beta adrenoceptor agonists, such as salbutamol and salmeterol, could be added to their therapeutical benefits.     

Inhibition of HIV-1-mediated syncytium formation and virus replication by the lipophosphoglycan from Leishmania donovani is due to an effect on early events in the virus life cycle

Previous findings have indicated that the major surface molecule of Leishmania, lipophosphoglycan (LPG), could abrogate HIV-1-induced syncytium formation and virus replication. In the present work, we were interested in characterizing this inhibitory process. Data from a new luciferase-based semiquantitative assay for syncytium formation, relying on the coincubation of a T-cell line containing an HIV-1 LTR-driven luciferase construct with a cell line chronically infected with HIV-1, confirmed that LPG was indeed a strong inhibitor of HIV-1-dependent syncytium formation and that this inhibition was dose-dependent. As determined by flow cytometric analyses, this inhibition was not apparently due to downregulation of CD4, CXCR4 or LFA-1, three distinct surface glycoproteins known to be important in HIV-1 mediated syncytium formation. Furthermore, LPG did not seem to affect signal transduction pathways in T cells as judged by measurement of HIV-1 LTR-driven reporter gene activity upon treatment with different stimuli. However, pretreatment of either of the cell lines used in the assay with LPG led to a significant decrease of virus-mediated syncytium formation, which was further accentuated when both cell lines were pretreated. LPG inhibition of HIV-1 replication was next assessed. When measuring either infection with luciferase-encoding recombinant HIV-1 particles or multinucleated giant cell formation following an acute virus infection, we again observed that LPG was efficient at blocking HIV-1 replication. Specific assays probing different steps of viral entry demonstrated that attachment was not hindered by LPG but that viral entry was modulated, suggesting that LPG targets a postbinding step. Hence, incorporation of LPG into a target cell membrane could influence its fluidity and diminish both the virus-cell and cell-to-cell fusion processes initiated by HIV-1.     

自体角朊细胞引起Th1向Th2极化抑制同种异体免疫应答

我们已有的工作揭示 ,在混合皮肤移植的体外模型MELC中 ,自体角朊细胞可通过间接递呈同种异体抗原诱导免疫抑制。本文在此基础上 ,进一步分析其中Th1和Th2相关细胞因子含量的变化。结果发现 :(1)向MELC引入自体角朊细胞后约 32h ,原同种异体应答中出现的IFN γ和IL 2分泌格局开始让位于IL 4和IL 10 ,6 4h后IFN γ和IL 2完全消失 ,表明发生了淋巴细胞Th1向Th2亚群的极化 ;(2 )单抗封闭试验表明 ,IL 10在其中起关键作用 ;(3)经自体和异体角朊细胞调变过的淋巴细胞对MELR的作用相反 ,前者抑制 ,后者促进。这是首次从T亚群相互作用上探索并揭示混合皮肤移植引起局部免疫耐受的机制。     

TcR-α/β CD4CD8 T Cells in Humans with the Autoimmune Lymphoproliferative Syndrome Express a Novel CD45 Isoform That Is Analogous to Murine B220 and Represents a Marker of Altered O-Glycan Biosynthesis

Autoimmune lymphoproliferative syndrome (ALPS), caused by inherited defects in apoptosis secondary to mutations in genes encoding Fas/CD95/APO-1 and Fas ligand (Fasl)/CD95L, is characterized by nonmalignant lymphadenopathy and splenomegaly, increased T cell receptor α/β⁺ CD4⁻CD8⁻ T cells (α/β⁺ double-negative T cells [α/β⁺-DNT cells]), autoimmunity, hypergammaglobulinemia, and cytokine abnormalities. The α/β⁺-DNT cells are immunophenotypically and functionally similar to α/β⁺-DNT cells that accumulate in lpr and gld mice, which bear genetic mutations in Fas and FasL. In these mice, α/β⁺-DNT cells express the B-cell-specific CD45R isoform B220. We show that α/β⁺-DNT cells of ALPS patients, with either Fas or FasL mutations, also express B220. In addition, also similar to LPR/gLD mice, they have an unusual population of B220-positive CD4⁺ T cells. B220 expression, together with our finding of characteristic lectin binding profiles, demonstrates that cell surface O-linked glycoproteins have undergone specific modifications, which may have consequences for lymphocyte trafficking, cell–cell interactions, and access to alternative apoptosis pathways.     

Virion-bound ICAM-1 and activated LFA-1: a combination of factors conferring resistance to neutralization by sera from human immunodeficiency virus type 1-infected individuals independently of the disease status and phase

The role of the supplementary interaction between virion-bound host ICAM-1 and LFA-1 on target cells in sensitivity to neutralization of human immunodeficiency virus type 1 (HIV-1) is poorly studied. Serum samples from four long-term nonprogressors (LTNPs) and sequential sera from one progressor were used to assess neutralization sensitivity of isogenic ICAM-1-negative and ICAM-1-bearing HIV-1(NL4-3), a prototype of T-cell-line-adapted viruses. We found that virus neutralization sensitivity to the studied sera is not modified by the additional interaction between virally embedded ICAM-1 and LFA-1 under an inactive state. However, expression on the target cell surface of an activated LFA-1 form renders ICAM-1-bearing virus particles, but not viruses devoid of ICAM-1, more refractory to neutralization by sera from three out of four LTNPs and all sequential sera from the person who has experienced a progression of the HIV-1-associated disease. Although no conclusive correlation could be drawn between virus susceptibility to neutralization and the disease status or stages of HIV-1 infection, these findings demonstrate that other nonspecific virus-cell interactions mediated by virion-anchored host proteins and their normal cognate ligands on target cells represent factors that can affect the mechanism of HIV-1 neutralization.