Comparative analysis of the fate of donor dendritic cells and B cells and their influence on alloreactive T cell responses under tacrolimus immunosuppression

We have shown that tacrolimus (TAC)-induced liver allograft acceptance is associated with migration and persistence of donor B cells and dendritic cells (DC). To clarify whether these MHC class II+ leukocytes have favorable roles in inducing tolerance, we analyzed recipient T cell reactions after allogeneic B or DC infusion. LEW rat B cells localized exclusively in BN host B cell follicles without any direct contact with host T cells. While few donor DC migrated to T cell areas and marginal zones, they were captured by host APC, suggesting that allogeneic MHC class II+ cells may induce immune reactions via the indirect pathway. Although DC-infused non-immunosuppressed recipients showed enhanced ex vivo anti-donor responses, persistent in vitro donor-specific hyporeactivity was seen equally with donor DC or B cell infusion under TAC. The results indicate that donor MHC class II+ APC are capable of regulating recipient immune reactions under TAC. Possible involvement of the indirect pathway of allorecognition is discussed.     

Antibody producing human-human hybridomas. I. Technical aspects

Technical aspects of generation of antibody-secreting human-human hybridomas are evaluated as based on 100 human-human fusions with a human B-lymphoma cell line (RH-L4) or the SKO-007 myeloma cell line as malignant fusion partners, and compared with similar fusion conditions in the mouse hybridoma system. The yield of hybrids was significantly lower when normal peripheral blood lymphocytes were used as fusion partners as compared with spleen lymphocytes, but could be substantially improved by increasing the amount of mitotic active B-lymphocytes by mitogen stimulation of the lymphocytes, preferably in HAT medium, prior to fusion. Furthermore, human hybrids grew slower and had a higher degree of chromosomal instability than usually observed in the mouse hybridoma system. Thus, out of 72 fusions, only 3 stable hybrids with antibody production against a predefined antigen were established. The importance of improved sources of human B-lymphocytes for human-human hybridoma production is discussed and methods of obtaining such improvement suggested.     

Antigen presentation by proliferating thymic macrophages to A (T,G)-A-L specific T cell line in an H-2 restricted manner

Long-term cultures of murine homogeneous populations of Ia-bearing thymus-derived murine macrophages were tested for their ability to present antigen to a (T,G)-A-L specific IL-2-dependent continuous T cell line. Thymus-derived macrophages, with and without pretreatment for Ia induction, triggered efficiently antigen-specific T cell proliferation in an MHC restricted way. This experimental system, consisting of two normal proliferating homogeneous populations of macrophages and antigen specific T cells, provides an ideal tool for studying the mechanism of antigen presentation to T cells and for elucidation of the role of macrophages in T-B cell collaboration for antibody production.     

Monoclonal antibodies to rabbit lymphoid cells: Preparation and characterization of a T-cell-specific antibody

Spleen cells of mice immunized with rabbit spleen and mesenteric lymph node (MLN) cells were fused with mutant mouse myeloma cells. Twenty-six clones which react with rabbit lymphoid cells were obtained. By membrane immunofluorescence, as analysed visually and by the fluorescence-activated cell sorter, one of these clones, 9AE10, produced an antibody that reacted with nearly all thymocytes (> 90%) and with from 46 to 78% of spleen, MLN and peripheral blood lymphocytes. Double-membrane immunofluorescence with the 9AE10 monoclonal antibody (MAb) and anti-Ig showed that 9AE10⁺ and Ig⁺ cells of spleen, MLN and peripheral blood were distinct and non-overlapping populations. Thus, the 9AE10 MAb is a T-cell-specific antibody.The 9AE10 MAb also reacted with most brain cells and with approximately 30% of bone marrow cells. Biochemical analysis of the antigen recognized by the 9AE10 MAb indicated that the antigen is a glycoprotein with an apparent molecular weight of 25,000. These data indicate that the 9AE10 MAb may be directed against a Thy-1-like antigen.     

A simplified new method for HLA-DR typing using the TM1 monoclonal antibody

A new modification of an HLA-DR typing technique is described which makes DR typing as rapid and simple as routine HLA-A,B,C typing. In this new method, designated the TM1 technique, carboxyfluoresceindiacetate labeled peripheral blood lymphocytes are added directly to DR typing trays. The T cells are then lysed by addition of TM1, a pan-T cytotoxic IgM monoclonal antibody, and residual B-cell reactivity with cytotoxic DR alloantibodies is read as in routine fluorochromasia microlymphocytotoxicity. HLA-DR typing by the TM1 technique compares favorably to typing by methods using B cells enriched by sheep red blood cell rosetting or by Degalan bead columns. The TM1 technique also works well with cells that have been cryopreserved as well as with cells that have been separated from whole blood drawn as much as 3 days earlier. Finally, because TM1 is so effective in lysing normal T lymphocytes, this antibody may prove useful in functional in vitro and in vivo studies requiring T-cell depletion.     

A new assay for rapid measurement of MIF levels by 3H-labelled cells in liquid scintillation counting vials: Statistical implications for the measurement of migration inhibition

A new quantitative assay for migration inhibitory factor (MIF) employs ³H-labelled cultured mouse or human lymphoid cells migrating from capillary tubes. Capillaries filled with labelled cells are placed in liquid scintillation counting vials, along with the MIF- containing sample and are removed at the end of a five-hour incubation period. The residual, labelled cells which have migrated out of the tubes are solubilized and counted in a liquid scintillation counter. While cultured lymphoblast cells are routinely used in the assay, the method was checked against mouse and guinea pig peritoneal exudate cells in both the labelled cell technique and the conventional chamber assay. The assay is technically simple to perform and a useful tool for laboratory research purposes because of the short span of time needed to obtain the results. These advantages indicate a potential for automation and use of this assay in a clinical immunology laboratory. Statistical analysis of data from both assays demonstrated that the relative variation among replicates is lower in the labelled cell assay. The new assay also measured a significant difference between controls and MIF-containing samples when the migration index (MI) was greater than 80%. Criteria for significant inhibition of migration are discussed in regard to the use of analysis variance (ANOVA) and other statistical procedures, and the inadequacy of a single measure, such as the MI, is discussed.     

Recognition of HLA-B27 and related antigen by a monoclonal antibody

A monoclonal antibody that binds specifically to HLA-B27, B7, and B22 is described. Binding to B27 appeared to be slightly stronger than to B7 and stronger than to B22 in an indirect binding assay, but no difference in B7 and B27 binding could be detected by Scatchard analysis. No distinction could be made between B27 on cells from normal and from ankylosing spondylitis patients in any assay system. The antibody, which was not cytotoxic, blocked complement-dependent cytolysis mediated by human HLA typing sera specific for B7 and B27. Competitive binding studies with other monoclonal antibodies showed that ME1 could block the binding of antibodies that recognized different antigenic sites on HLA. ME1 did not bind to Klebsiella pneumoniae. This reagent will be useful in further analysis of the relationship between B27 and ankylosing spondylitis.     

Denbinobin induces apoptosis in human lung adenocarcinoma cells via Akt inactivation, Bad activation, and mitochondrial dysfunction

Increasing evidence demonstrated that denbinobin, isolated from Ephemerantha lonchophylla, exert cytotoxic effects in cancer cells. The purpose of this study was to investigate whether denbinobin induces apoptosis and the apoptotic mechanism of denbinobin in human lung adenocarcinoma cells (A549). Denbinobin (1-20microM) caused cell death in a concentration-dependent manner. Flow cytometric analysis and annexin V labeling demonstrated that denbinobin increased the percentage of apoptotic cells. A549 cells treated with denbinobin showed typical characteristics of apoptosis including morphological changes and DNA fragmentation. Denbinobin induced caspase 3 activation, and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor, prevented denbinobin-induced cell death. Denbinobin induced the loss of the mitochondrial membrane potential and the release of mitochondrial apoptotic proteins including cytochrome c, second mitochondria derived activator of caspase (Smac), and apoptosis-inducing factor (AIF). In addition, denbinobin-induced Bad activation was accompanied by the dissociation of Bad with 14-3-3 and the association of Bad with Bcl-xL. Furthermore, denbinobin induced Akt inactivation in a time-dependent manner. Transfection of A549 cells with both wild-type and constitutively active Akt significantly suppressed denbinobin-induced Bad activation and cell apoptosis. These results suggest that Akt inactivation, followed by Bad activation, mitochondrial dysfunction, caspase 3 activation, and AIF release, contributes to denbinobin-induced cell apoptosis.     

Arsenic trioxide in environmentally and clinically relevant concentrations interacts with calcium homeostasis and induces cell type specific cell death in tumor and non-tumor cells

While arsenic compounds are known as environmental toxicants (especially in drinking water) and as carcinogens, some arsenic compounds, like arsenic trioxide (As2O3), are clinically used in humans to treat some forms of cancer (e.g. leukemia). Although arsenic compounds have been studied intensively, their interactions with living cells are still not fully elucidated. We have previously proposed that modulation of intracellular calcium ([Ca2+]i) homeostasis induced by As2O3 could be an important mechanism to induce cytotoxicity. Here we demonstrate, using human cell models (neuroblastoma (SY-5Y) or embryonic kidney cells (HEK)) and confocal microscopy in combination with the calcium sensitive dye fluo 4-AM, that As2O3 interferes with calcium signaling at low (environmentally and clinically relevant concentrations of 100 pM to 1 microM). Within this concentration range, As2O3 had cell type specific cytotoxic effects, with neuroblastoma cells being more sensitive to As2O3 than HEK 293. In addition, by staining with Hoechst 33347 and counting micronucleated cells as well as apoptotic nuclei, As2O3 was found to increase the rate of apoptosis and DNA damage, which was also cell type specific. These results indicate that the As2O3-induced cell death could be triggered or mediated by [Ca2+]i signals and suggest that low concentrations of As2O3 are able to interfere with specific physiological processes in diverse cell models.     

Case studies putting the decision-making framework for the grouping and testing of nanomaterials (DF4nanoGrouping) into practice

Case studies covering carbonaceous nanomaterials, metal oxide and metal sulphate nanomaterials, amorphous silica and organic pigments were performed to assess the Decision-making framework for the grouping and testing of nanomaterials (DF4nanoGrouping). The usefulness of the DF4nanoGrouping for nanomaterial hazard assessment was confirmed. In two tiers that rely exclusively on non-animal test methods followed by a third tier, if necessary, in which data from rat short-term inhalation studies are evaluated, nanomaterials are assigned to one of four main groups (MGs). The DF4nanoGrouping proved efficient in sorting out nanomaterials that could undergo hazard assessment without further testing. These are soluble nanomaterials (MG1) whose further hazard assessment should rely on read-across to the dissolved materials, high aspect-ratio nanomaterials (MG2) which could be assessed according to their potential fibre toxicity and passive nanomaterials (MG3) that only elicit effects under pulmonary overload conditions. Thereby, the DF4nanoGrouping allows identifying active nanomaterials (MG4) that merit in-depth investigations, and it provides a solid rationale for their sub-grouping to specify the further information needs. Finally, the evaluated case study materials may be used as source nanomaterials in future read-across applications. Overall, the DF4nanoGrouping is a hazard assessment strategy that strictly uses animals as a last resort.