A novel in vivo follicular dendritic cell-dependent iccosome-mediated mechanism for delivery of antigen to antigen-processing cells

Recent scanning electron microscopic studies on isolated follicular dendritic cells (FDC) showed that dendrites of certain FDC were "beaded," i.e., consisting of a series of interconnected immune complex coated bodies (termed "iccosomes," measuring 0.3 to 0.7 micron diameter). In vitro these iccosomes detach from one another with ease. The major objectives herein were to establish whether these structures can be detected in sections and whether iccosomes serve to disseminate antigen in vivo. Beginning at day 1, the time point used for isolating beaded FDC, the popliteal lymph nodes of immune C3H mice were studied with light and transmission electron microscopy for 2 wk (i.e., at days 1, 3, 5, 8, and 14) after hind footpad injection of the histochemically detectable antigen, horseradish peroxidase (HRP). Iccosomes (0.25 to 0.38 micron diameter), contoured by a peroxidase (PO)-positive coat of HRP-anti-HRP complexes, were first detected by transmission electron microscopy at day 1 adjacent to cell bodies of certain FDC. Within their limiting membrane they contained flocculent material that was PO positive. At day 3 by light microscopy, germinal centers were seen enlarged and the antigen-retaining reticulum, composed of antigen-bearing FDC, appeared diffuse. This coincided with the transmission electron microscopic visualization of a dispersed state of iccosomes among the follicular lymphocytes. At that time iccosomes were seen attached to the surface of lymphocytes via PO-positive immune complexes and were surrounded by microvillous processes of these cells. Germinal center lymphocytes and tingible body macrophages both responded to contact with iccosomes by endocytosis. Antigen-containing tingible body macrophage were most conspicuous by light microscopy at day 5, when transmission electron microscopy showed that the majority of germinal center lymphocytes contained endocytosed HRP in secondary lysosome-like granules associated with the Golgi apparatus. The number of dispersed iccosomes was markedly reduced by day 5. In controls injected with HSA, a PO-negative antigen, lymphocytes and tingible body macrophages were PO-negative. The presence of antigen in both cell types was confirmed through the use of a gold-conjugated antigen (goat IgG). Simultaneous immunoperoxidase labeling of the same tissues with anti-Ia showed the gold conjugate containing B cells to be Ia+. Antigen-positive B cells and tingible body macrophages were greatly reduced in numbers by day 14, suggesting the intracellular fragmentation of the antigen.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evolution of the ATP-binding-cassette transmembrane transporters of vertebrates

The ATP-binding-cassette transmembrane transporters (ABC transporters) known from vertebrates belong to four major subfamilies: (1) the P- glycoproteins (Pgp); (2) the cystic fibrosis transmembrane conductance regulators (CFTR); (3) the Tap proteins encoded with the major histocompatibility complex of mammals; and (4) the peroxisomal membrane proteins. Both Pgp and CFTR have a structure suggesting a past internal gene duplication; a phylogenetic analysis indicated that these duplications occurred independently, while an independent tandem gene duplication occurred in the case of the Tap family. Both the Pgp and Tap proteins show evidence of relationship to bacterial ABC transporters lacking internal duplication, and both are significantly more closely related to the HlyB and MsbA families of transporters from purple bacteria than they are to ABC transporters from nonpurple bacteria. The simplest hypothesis to explain this observation is that eukaryotic Pgp and Tap genes are descended from a mitochondrial gene or genes that were subsequently translocated to the nuclear genome. The Pgp genes of eukaryotes are characterized by a remarkable degree of convergent evolution between the ATP-binding cassettes of their N- terminal and C-terminal halves, whereas no such convergence is seen between the two halves of CFTR genes or between the duplicated Tap genes. Exon 13 of the CFTR gene, which encodes a putative regulatory domain not found in other ABC transporters apart from CFTR, showed high levels of both synonymous and nonsynonymous difference in comparisons among different mammalian species, suggesting that this region is a mutational hot spot.      

Evidence for the presence of β-lactamase inStreptomyces glaucescens and its inhibition by sodium clavulanate

Streptomyces glaucescens is shown to possess -lactamase activity which is inhibitable by clavulanate. This is important in regard to its use as a cloning host for enzymes of \-lactam biosynthesis.     

Tissue localization and retention of antigen in relation to the immune response

Antigen persists for months or even years in lymphoid tissues of immune animals and this antigen is believed to participate in the induction and maintenance of B-cell memory as well as in the maintenance of serum antibody levels. In the present report we describe the phenomenon of antigen localization and long-term retention on mouse follicular dendritic cells (FDCs). The antigens used were injected in the hind footpads of immune mice and the popliteal lymph nodes were the lymphoid organs generally studied. In addition to presenting the morphological features of mouse FDCs, we report the results of a study of the mechanism of antigen migration from the site of initial localization in the lymph node subcapsular sinus to the regions of follicular retention in the cortex. The migration was followed by light and electron microscopy. The results support the concepts that immune complexes are trapped in the subcapsular sinus and are transported by a group of nonphagocytic cells to follicular regions. The mechanism of transport may involve either migration of pre-FDCs with a concomitant maturation into FDCs, or cell-to-cell transport utilizing dendritic cell processes and membrane fluidity; or a combination of the two mechanisms may be in operation.     

In vivo obtained antigen presented by germinal center B cells to T cells in vitro

Shortly after secondary immunization germinal center (GC) B cells obtain antigen from follicular dendritic cells (FDC) in the form of immune complexes. This antigen appears to be degraded by the GC B cells and may be processed for presentation to T cells. The present study was undertaken to determine whether GC B cells can process and present antigen obtained from FDC in vivo to appropriate T cells in vitro. GC B cells were isolated from immune mice with the use of Percoll density separation followed by a panning procedure which utilizes the ability of the plant lectin, peanut agglutinin (PNA), to selectively bind to GC B cells. The enriched GC B cells were approximately 80% highly positive for PNA, 97% positive for Ia and surface IgM, but less than 0.01% positive for Thy-1.2 or esterase. In some experiments, this population was further purified to near 100% highly PNA-positive cells with the use of fluoresceinated PNA and a fluorescence-activated cell sorter. Cell sorting analysis indicated that the antigen (125I-labeled ovalbumin (OVA)) was restricted to the highly PNA-positive cell fraction. The capacity of these highly PNA-positive B cells to present antigen was assessed by monitoring interleukin 2 (IL-2) production by the OVA-specific T cell hybridoma, 3DO-54.8. GC B cells obtained from mice 3 wk or more after secondary immunization did not elicit IL-2 production in the absence of added OVA. However, GC B cells isolated as early as 1 day and for over 1 wk after a challenge with OVA, were able to stimulate high levels of IL-2 production, in the absence of adding OVA to the cell cultures. This response was maximal on day 5 and corresponded precisely with the kinetics of the ultrastructural studies which document the uptake of antigen by GC B cells in vivo. The FDC-derived antigen was remarkably immunogenic when compared with exogenous antigen. These findings demonstrated that antigen obtained in vivo by GC B cells could be processed and presented to T cells. In vivo, GC B cells may induce the T cell help needed for the germinal center reaction, generate B memory cells, and help induce the high titers of antibody associated with the secondary antibody response.     

Three-Dimensional Trabecular Alignment Model

The Shear-slip Mesh Update Method (SSMUM) is being used in flow simulations involving large but regular displacements of one or more boundaries of the computational domain. We follow up the earlier discussion of the method with notes on practical implementation aspects. In order to establish a benchmark problem for this class of flow problems, we define and report results from a two-dimensional viscous flow around a rotating stirrer in a square chamber. The application potential of the method is demonstrated in the context of biomedical design problem, as we perform an analysis of blood flow in a centrifugal left ventricular assist device, or blood pump, which involves a rotating impeller in a non-axisymmetric housing.     

CURRICULUM GUIDE FOR RESEARCH ETHICS WORKSHOPS FOR COUNTRIES IN THE MIDDLE EAST

To help ensure the ethical conduct of research, many have recommended educational efforts in research ethics to investigators and members of research ethics committees (RECs). One type of education activity involves multi‐day workshops in research ethics. To be effective, such workshops should contain the appropriate content and teaching techniques geared towards the learning styles of the targeted audiences. To ensure consistency in content and quality, we describe the development of a curriculum guide, core competencies and associated learning objectives and activities to help educators organize research ethics workshops in their respective institutions. The curriculum guide is divided into modular units to enable planners to develop workshops of different lengths and choose content materials that match the needs, abilities, and prior experiences of the target audiences. The content material in the curriculum guide is relevant for audiences in the Middle East, because individuals from the Middle East who participated in a Certificate Program in research ethics selected and developed the training materials (e.g., articles, powerpoint slides, case studies, protocols). Also, many of the activities incorporate active‐learning methods, consisting of group work activities analyzing case studies and reviewing protocols. The development of such a workshop training curriculum guide represents a sustainable educational resource to enhance research ethics capacity in the Middle East.     

Ontogeny of the follicular dendritic cell phenotype and function in the postnatal murine spleen

Follicular dendritic cells (FDCs) represent a unique cell population of antigen trapping cells restricted to follicles within the secondary lymphoid tissues. FDCs appear to be involved in the formation of primary follicles during the ontogeny of lymphoid tissue. We sought to determine the kinetics and tissue distribution of cells in the spleen of newborn mice expressing various differentiation antigens restricted to FDCs using immunohistochemistry with monoclonal antibodies (mAb) against FDCs and in vivo immune complex binding and retention. The earliest FDC-specific marker displayed was the antigenic determinant recognized by the FDC-M1 mAb, which was detectable by Day 3 prior to follicle formation on cells located around the peripheral part of the developing white pulp. The appearance of CD21/35 (complement receptor Type 2 and 1, CR1.2) was observed at the end of the first week, revealing a focal pattern in B-cell-rich areas. In addition, at that time there were some FDC-M1-positive cells in the nonfollicular part of the periarteriolar region. The administration of anti-horseradish peroxidase antibody followed by soluble antigen HRP into 7-day-old newborn mice resulted in the trapping and retention of immune complexes onto FDCs even in the absence of Fcgamma receptors. The appearance of another FDC-specific marker, FDC-M2, was observed during the second week after birth and was restricted on the cells located in the same area as CR1.2 cells. The Fcgamma receptor Type II appeared on FDCs after the second postnatal week. The above sequence of phenotypic maturation could also be observed in newborns after lethal irradiation at Day 3. This indicates that not only mature FDCs but also their precursors are highly radioresistant, and their phenotypic maturation follows a programmed path that requires only a small number of mature B cells.