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)     

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.     

Incorporating trnH-psbA to the core DNA barcodes improves significantly species discrimination within?southern African Combretaceae

Recent studies indicate that the discriminatory power of the core DNA barcodes (rbcLa + matK) for land plants may have been overestimated since their performance have been tested only on few closely related species. In this study we focused mainly on how the addition of complementary barcodes (nrITS and trnH-psbA) to the core barcodes will affect the performance of the core barcodes in discriminating closely related species from family to section levels. In general, we found that the core barcodes performed poorly compared to the various combinations tested. Using multiple criteria, we finally advocated for the use of the core + trnH-psbA as potential DNA barcode for the family Combretaceae at least in southern Africa. Our results also indicate that the success of DNA barcoding in discriminating closely related species may be related to evolutionary and possibly the biogeographic histories of the taxonomic group tested.     

A Global Trend towards the Loss of Evolutionarily Unique Species in Mangrove Ecosystems

The mangrove biome stands out as a distinct forest type at the interface between terrestrial, estuarine, and near-shore marine ecosystems. However, mangrove species are increasingly threatened and experiencing range contraction across the globe that requires urgent conservation action. Here, we assess the spatial distribution of mangrove species richness and evolutionary diversity, and evaluate potential predictors of global declines and risk of extinction. We found that human pressure, measured as the number of different uses associated with mangroves, correlated strongly, but negatively, with extinction probability, whereas species ages were the best predictor of global decline, explaining 15% of variation in extinction risk. Although the majority of mangrove species are categorised by the IUCN as Least Concern, our finding that the more threatened species also tend to be those that are more evolutionarily unique is of concern because their extinction would result in a greater loss of phylogenetic diversity. Finally, we identified biogeographic regions that are relatively species-poor but rich in evolutionary history, and suggest these regions deserve greater conservation priority. Our study provides phylogenetic information that is important for developing a unified management plan for mangrove ecosystems worldwide.     

Picking at the Paint: Viewing Contemporary Bushman Art as Art

In most writing on contemporary Bushman art the art is discussed as a tool of development, a tourism endeavor and an income generator. While these are aims both legitimate and beneficial to the communities involved, they also work (implicitly yet effectively) to separate the art, artist and subsequently the community out of which the art is produced from “the rest of us,” defining these communities as “other.” This article attempts to engage with contemporary Bushman art as art objects. Using criteria of judgment based on a functional semiotics of art, two works will be analyzed to prove that the works may be discussed as Art. The premise is that once people recognize the aesthetic intelligence imbued in these artworks they will be able to engage with the work in a way that is similarly intelligent and contemporary. The art and its people will then stand a better chance of being accepted into the realm of the everyday as opposed to being relegated to the sarcophagi of history. Once taken from this starting position, goals of community development and tourism endeavors linked to indigenous art can become that much more beneficial and life-changing for the communities concerned.     

Understanding the Potential Impact of a Combination HIV Prevention Intervention in a Hyper-Endemic Community

Objectives

Despite demonstrating only partial efficacy in preventing new infections, available HIV prevention interventions could offer a powerful strategy when combined. In anticipation of combination HIV prevention programs and research studies we estimated the population-level impact of combining effective scalable interventions at high population coverage, determined the factors that influence this impact, and estimated the synergy between the components.

Methods

We used a mathematical model to investigate the effect on HIV incidence of a combination HIV prevention intervention comprised of high coverage of HIV testing and counselling, risk reduction following HIV diagnosis, male circumcision for HIV-uninfected men, and antiretroviral therapy (ART) for HIV-infected persons. The model was calibrated to data for KwaZulu-Natal, South Africa, where adult HIV prevalence is approximately 23%.

Results

Compared to current levels of HIV testing, circumcision, and ART, the combined intervention with ART initiation according to current guidelines could reduce HIV incidence by 47%, from 2.3 new infections per 100 person-years (pyar) to 1.2 per 100 pyar within 4 years and by almost 60%, to 1 per 100 pyar, after 25 years. Short-term impact is driven primarily by uptake of testing and reductions in risk behaviour following testing while long-term effects are driven by periodic HIV testing and retention in ART programs. If the combination prevention program incorporated HIV treatment upon diagnosis, incidence could be reduced by 63% after 4 years and by 76% (to about 0.5 per 100 pyar) after 15 years. The full impact of the combination interventions accrues over 10–15 years. Synergy is demonstrated between the intervention components.

Conclusion

High coverage combination of evidence-based strategies could generate substantial reductions in population HIV incidence in an African generalized HIV epidemic setting. The full impact could be underestimated by the short assessment duration of typical evaluations.     

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.     

Targeted deletion of CD44v7 exon leads to decreased endothelial cell injury but not tumor cell killing mediated by interleukin-2-activated cytolytic lymphocytes

In the current study, we investigated the nature and role of CD44 variant isoforms involved in endothelial cell (EC) injury and tumor cell cytotoxicity mediated by IL-2-activated killer (LAK) cells. Treatment of CD44 wild-type lymphocytes with IL-2 led to increased gene expression of CD44 v6 and v7 variant isoforms and to significant induction of vascular leak syndrome (VLS). CD44v6-v7 knockout (KO) and CD44v7 KO mice showed markedly reduced levels of IL-2-induced VLS. The decreased VLS in CD44v6-v7 KO and CD44v7 KO mice did not result from differential activation and expansion of CD8+ T cells, NK, and NK-T cells or from altered degree of perivascular lymphocytic infiltration in the lungs. LAK cells from CD44v7 KO mice showed a significant decrease in their ability to adhere to and mediate lysis of EC but not lysis of P815 tumor cells in vitro. CD44v7-mediated lysis of EC by LAK cells was dependent on the activity of phosphatidylinositol 3-kinase and tyrosine kinases. Interestingly, IL-2-activated LAK cells expressing CD44hi but not CD44lo were responsible for EC lysis. Furthermore, lysis of EC targets could be blocked by addition of soluble or enzymatic cleavage of CD44v6-v7-binding glycosaminoglycans. Finally, anti-CD44v7 mAbs caused a significant reduction in the adherence to and killing of EC and led to suppression of IL-2-induced VLS. Together, this study suggests that the expression of CD44v7 on LAK cells plays a specific role in EC injury and that it may be possible to reduce EC injury but not tumor cell killing by specifically targeting CD44v7.     

Follicular dendritic cells and the persistence of HIV infectivity: the role of antibodies and Fcgamma receptors

Large quantities of HIV are found trapped on the surface of follicular dendritic cells (FDCs), and virus persists on these cells until they ultimately die. We recently found that FDCs maintain HIV infectivity for long periods in vivo and in vitro. Because FDCs trap Ags (and virus) in the form of immune complexes and are rich in FcgammaRs, we reasoned that Ab and FcgammaRs may be required for FDC-mediated maintenance of HIV infectivity. To investigate this hypothesis, HIV immune complexes were formed in vitro and incubated for increasing times with or without FDCs, after which the remaining infectious virus was determined by HIV-p24 production in rescue cultures. FDCs maintained HIV infectivity in vitro in a dose-dependent manner but required the presence of specific Ab for this activity regardless of whether laboratory-adapted or primary X4 and R5 isolates were tested. In addition, Abs against either virally or host-encoded proteins on the virion permitted FDC-mediated maintenance of HIV infectivity. We found that the addition of FDCs to HIV immune complexes at the onset of culture gave optimal maintenance of infectivity. Moreover, blocking FDC-FcgammaRs or killing the FDCs dramatically reduced their ability to preserve virus infectivity. Finally, FDCs appeared to decrease the spontaneous release of HIV-1 gp120, suggesting that FDC-virus interactions stabilize the virus particle, thus contributing to the maintenance of infectivity. Therefore, optimal maintenance of HIV infectivity requires both Ab against particle-associated determinants and FDC-FcgammaRs.