An inhibitor of cytotoxic functions produced by CD8+CD57+ T lymphocytes from patients suffering from AIDS and immunosuppressed bone marrow recipients

An inhibitor of the cytotoxic functions (ICF) mediated by human immunodeficiency virus (HIV)- or HLA-specific cytotoxic T lymphocytes, natural killer and lymphokine-activated killer (LAK) cells is secreted by CD8⁺CD57^? T lymphocytes, a subset expanded during infection with HIV and after bone marrow transplantation. We previously showed an apparent molecular mass of 20–30 kDa for this soluble glycosylated concanavalin A-binding inhibitor which is distinct from known cytokines. Here, we report a characterization of the mechanism of action of this CD8⁺CD57⁺ ICF. We show that the ICF-induced inhibition of LAK cell cytolytic activity is transient, with a spontaneous recovery of cytolytic potential after 18 h. When testing interactions of ICF with a large set of cytokines we found that the ICF-mediated inhibition of cytotoxic functions is antagonized by two cytokines: recombinant interleukin (rIL)-4 and recombinant interferon (rIFN)-γ. Finally, we show that ICF acts at the level of cytolytic effector cells, where it induces a significant increase of cyclic AMP (cAMP) level. In contrast, no modification of either cell surface antigen expression or of target/effector cell conjugate formation could be evidenced. Addition of rIL-4 and rIFN-γ reverses such an increase of cAMP levels and in parallel restores the cytolytic activity. Altogether, these data demonstrate that the glycoprotein ICF produced by CD8⁺CD57⁺ cells (1) inhibits cell-mediated cytotoxicity by sensitizing cytolytic effector cells to the cAMP pathway, and (2) is part of a cytokine network controlling cell-mediated cytotoxic functions.     

In vitro stimulation by prostate extracts of rat ventral prostate stromal and epithelial cell division

We examined the effect of prostate cell extracts on the replication of highly enriched rat ventral prostate stromal and epithelial cells cultured in RPMI-1640. Extracts from normal rat prostates completely inhibited cell division in both fractions, while a 10% (v/v) extract of Dunning R-3327G adenocarcinoma inhibited replication of stromal cells but permitted that of epithelial cells. The cytotoxic effect of prostate extracts was dialyzable, heat stable, unaffected by proteases, soluble in acid/alcohol, and insoluble in chloroform. These properties are consistent with those of polyamines incubated in the presence of fetal calf serum. Dialyzed extracts from Dunning adenocarcinomas, and from human benign hypertrophic and carcinomatous prostates stimulated rat prostate stromal and epithelial cell division, in keeping with other reports of "growth factors" in prostate tissue. This mitogenic activity was stable to temperature (70 degrees C, 4 hours), and marginally if at all affected by exposure to trypsin or to pronase coupled to Sephadex. Short-term culture of separated prostate cells should provide a useful assay system for detecting putative autocrine or paracrine stromal and/or epithelial cell growth factors and identifying suspected homo- or heterotypic cellular interactions in normal and diseased prostates.     

Macromolecular DNA-damage in murine and human leukemic and lymphoid cells after in vitro exposure to ASTA Z 7557 (INN mafosfamide)

Summary Cyclophosphamide (CPA) is widely used against leukemic and lymphoproliferative diseases, but in vitro studies on response to this agent so far have been limited to instable derivatives with poor galenic properties. ASTA Z 7557 is a newly synthesized activated cyclophosphamide that circumvents the need for hepatic activation and has good stability. The critical cytotoxic lesions after exposure to bifunctional alkylating agents presumably are DNA interstrand crosslinks (ISC). We have, therefore, examined the formation and apparent removal of ISC after in vitro treatment with ASTA Z 7557 by use of the highly sensitive alkaline elution technique. Survival of murine L1210 cells was determined after 1 hour in vitro exposure with a D 37 value of 5.7 g/ml (from the initial shoulder part of the survival curve) and a Do value of 1.5 g/ml (from the exponential part of the curve). Previous labelling of L1210 cells by ¹²⁵IUdR simplified the alkaline elution procedure but there was some cytotoxicity of the radiochemical itself with a reduction of cloning efficiency from 77% to 61 %. The maximum of ISC was observed at 6 h after initiation of treatment with much of the damage apparently removed at 24 h. The simultaneous presence of DNA single strand breaks (SSB), however, confounds the analysis of DNA damage at 24 h and early cytolysis and unaided death of human lymphocytes often preclude the analysis of macromolecular damage at this time. Human peripheral blood cells isolated from patients with leukemic or lymphoproliferative diseases showed a remarkable heterogeneity with regard to the formation of ISC at 3 h. Thus, analysis of macromolecular damage may become an additional prognostic factor for response to CPA beyond the morphologic classification of these diseases.     

Pediatric intestinal transplantation

Advancements in donor management, organ preservation and operative techniques, as well as immunosuppressive therapies, have provided children with intestinal failure and its complications a chance not only for enteral autonomy but also long-term survival through intestinal transplantation (ITx). First described in the 1960’s, experience has grown in managing these complex patients both pre- and post-transplant. The goals of this review are to provide a brief history of intestinal transplantation and intestinal rehabilitation in pediatric patients, followed by focused discussions of the indications for ITx, induction and maintenance immunosuppression therapies, common post-operative complications, and outcomes/quality of life post-transplant.     

Extracellular signal-regulated kinase (ERK) activation by the pre-T cell receptor in developing thymocytes in vivo

The first checkpoint in T cell development occurs between the CD4(-)CD8(-) and CD4(+)CD8(+) stages and is associated with formation of the pre-T cell receptor (TCR). The signaling mechanisms that drive this progression remain largely unknown. Here, we show that extracellular signal-regulated kinases (ERKs)-1/2 are activated upon engagement of the pre-TCR. Using a novel experimental system, we demonstrate that expression of the pre-TCR by developing thymocytes induces ERK-1/2 activation within the thymus. In addition, the activation of this pre-TCR signaling cascade is mediated through Lck. These findings directly link pre-TCR complex formation with specific downstream signaling components in vivo.     

Interactions of Phosphodiester and Phosphorothioate Oligonucleotides with Intestinal Epithelial Caco-2 Cells

Purpose. Oral bioavailability for antisense oligonucleotides has recently been reported but the mechanistic details are not known. The proposed oral delivery of nucleic acids will, therefore, require an understanding of the membrane binding interactions, cell uptake and transport of oligonucleotides across the human gastro-intestinal epithelium. In this initial study, we report on the cell-surface interactions of oligonucleotides with human intestinal cells. Methods. We have used the Caco-2 cell line as an in vitro model of the human intestinal epithelium to investigate the membrane binding interactions of 20-mer phosphodiester (PO) and phosphorothioate (PS) oligonucleotides. Results. The cellular association of both an internally [³H]-labelled and a 5end [³²P]-labelled PS oligonucleotide (3.0% at 0.4 µM extracellular concentration) was similar and was an order of magnitude greater than that of the 5end [³²P]-labelled PO oligonucleotide (0.2%) after 15 minutes incubation in these intestinal cells. The cellular association of PS was highly saturable with association being reduced to 0.9% at 5 µM whereas that of PO was less susceptible to competition (0.2% at 5 µM, 0.1% at 200 µM). Differential temperature-dependence was demonstrated; PS interactions were temperature-independent whereas the cellular association of PO decreased by 75% from 37°C to 17°C. Cell association of oligonucleotides was length and pH-dependent. A decrease in pH from 7.2 to 5.0 resulted in a 2- to 3-fold increase in cell-association for both backbone types. This enhanced association was not due to changes in lipophilicity as the octanol:aqueous buffer distribution coefficients remained constant over this pH range. The ability of NaCl washes to remove surface-bound PS oligonucleotides in a concentration-dependent manner suggests their binding may involve ionic interactions at the cell surface. Cell-surface washing with the proteolytic enzyme, Pronase^®, removed approximately 50% of the cell-associated oligonucleotide for both backbone types. Conclusions. Binding to surface proteins seems a major pathway for binding and internalization for both oligonucleotide chemistries and appear consistent with receptor (binding protein)-mediated endocytosis. Whether this binding protein-mediated entry of oligonucleotides can result in efficient transepithelial transport, however, requires further study.     

γ—干扰素对喉癌细胞株HEP—2增殖活性的影响

用单克隆抗体Ｋｉ－６７（抗ＰＣＮＡ），以链霉菌素－生物素技术（ＬＳＡＢ）检测喉癌细胞株ＨＥＰ－２增殖细胞核抗原（ＰＣＮＡ）表达。人重组γ－干扰素（ｒｈｕ－ＩＦＮ－γ）抑制ＰＣＮＡ表达（即抗增殖活性）强弱与其剂量有关；雌激素对ｒｈｕ－ＩＦＮ－γ抗增殖作用无影响。提示：ｒｈｕ－ＩＦＮ－γ对喉癌细胞株ＨＥＰ－２有抗增殖作用，因而在喉癌的治疗中具有应用价值的。     

Platelet proteins as autoantibody targets in idiopathic thrombocytopenic purpura

Idiopathic thrombocytopenic purpura (ITP), caused by autoantibodies directed against certain platelet antigens, is the most common entity of the immune thrombocytopenias. ITP is an acquired disorder and can affect both children and adults. However, the clinical syndromes of ITP are distinct between children and adults. Childhood (acute) ITP characteristically is acute in onset, occurs within 1-2 weeks of an infection, usually of viral origin, resolves spontaneously within 6 months. Adult (chronic) ITP has an insidious onset and rarely resolves spontaneously. Over the last decade considerable new information has accumulated as to the pathophysiological mechanisms of immune thrombocytopenias. In addition, most of the knowledge on this disorder has been obtained from studies of adult patients with chronic ITP. The present work gives an updated overview of the platelet autoantigens and the molecular immunological reactions in ITP.     

Application of a statistical dynamic model investigating the short-term cellular kinetics induced by riddelliine, a hepatic endothelial carcinogen.

In recent studies, riddelliine, a pyrrolizidine alkaloid, was found to increase rates of replication and apoptosis and induce hemangiosarcoma in the liver of rats and mice. To analyze DNA replication and apoptosis data taken from the same animals, we have developed a predictive mathematical model for describing BrdU labeling and apoptotic processes. The model allows the incorporation of simple diurnal patterns in cellular kinetics and is applied to data on hepatocytes and endothelial cells taken from riddelliine exposed rats. Predictions from the model were used with multivariable nonlinear regression techniques to estimate replication and apoptotic rate constants for both cell types and all treatment groups. Hypothesis tests were used with the predicted rates to separate the competing effects of riddelliine on replication and apoptosis of hepatocytes and endothelial cells as well as compare replication rates between cell types. That estimated replication rates were found to be significantly higher for endothelial cells supports the supposition of induction of hemangiosarcoma by riddelliine in the liver.