During ageing thymic function declines and is unable to meet the demand for peripheral T helper (Th) cell replenishment. Therefore, population maintenance of naive Th cells must be at least partly peripherally based. Such peripheral postthymic expansion of recent thymic emigrants (RTEs) during ageing consequently should lead to loss or dilution of T cell receptor excision circles (TRECs) from a subset of naive T cells. We have identified two subsets of naive Th cells in human adult peripheral blood characterized by a striking unequal content of TRECs, indicating different peripheral proliferative histories. TRECs are highly enriched in peripheral naive CD45RA+ Th cells coexpressing CD31 compared with peripheral naive CD45RA+ Th cells lacking CD31 expression, in which TRECs can hardly be detected. Furthermore we show that CD31−CD45RA+ Th cells account for increasing percentages of the naive peripheral Th cell pool during ageing but retain phenotypic and functional features of naive Th cells. As CD31 is lost upon T cell receptor (TCR) engagement in vitro, we hypothesize that TCR triggering is a prerequisite for homeostatically driven peripheral postthymic expansion of human naive RTEs. We describe here the identification of peripherally expanded naive Th cells in human adult blood characterized by the loss of CD31 expression and a highly reduced TREC content.

Voltage-dependent anion-selective channel (VDAC) is a b-barrel protein in the outer mitochondrial membrane that is necessary for metabolite exchange with the cytosol and is proposed to be involved in certain forms of apoptosis. We studied the biogenesis of VDAC in human mitochondria by depleting the components of the mitochondrial import machinery by using RNA interference. Here, we show the importance of the translocase of the outer mitochondrial membrane (TOM) complex in the import of the VDAC precursor. The deletion of Sam50, the central component of the sorting and assembly machinery (SAM), led to both a strong defect in the assembly of VDAC and a reduction in the steadystate level of VDAC. Metaxin 2-depleted mitochondria had reduced levels of metaxin 1 and were deficient in import and assembly of VDAC and Tom40, but not of three matrix-targeted precursors. We also observed a reduction in the levels of metaxin 1 and metaxin 2 in Sam50-depleted mitochondria, implying a connection between these three proteins, although Sam50 and metaxins seemed to be in different complexes. We conclude that the pathway of VDAC biogenesis in human mitochondria involves the TOM complex, Sam50 and metaxins, and that it is evolutionarily conserved.

In spite of thymic involution early in life, the numbers of naive CD4 + T cells only slowly decline in ageing humans implying peripheral post-thymic naive CD4 + T cell expansion. This proliferation may compensate for continuous activation and death of naive CD4 + T cells but may also have negative consequences for protective immunity. Here we show that naive CD4 + T cells that have proliferated in the periphery are characterized by a highly restricted oligoclonal TCR repertoire. Additionally these cells, which constitute the majority of naive CD4 + T cells in the elderly, display signatures of recent TCR engagement. Our results demonstrate for the first time that peripheral post-thymic proliferation of naive CD4 + T cells in healthy human individuals causes a significant contraction of the peripheral TCR repertoire. This age-dependent deterioration of CD4 + T cell immunity could entail ageing-associated autoimmunity, increased susceptibility to infection or cancer and decreased efficiency of vaccination.

Mitochondrial outer membrane permeabilization (MOMP) and release of mitochondrial intermembrane proteins like cytochrome c are critical steps in the control of apoptosis. Previous work has shown that MOMP depends on the functionally redundant multidomain proapoptotic proteins, Bak and Bax. Here we demonstrate that Bak and Bax are functionally non‐redundant during Neisseria gonorrhoeae (Ngo)‐ and cisplatin‐induced apoptosis. While the activation of Bak is caspase independent Bax activation needs Bak and active caspases. Silencing of either Bak or Bax resists both Ngo‐ and cisplatin‐ but not TNFα‐induced apoptosis. Activation of Bak is required to release cytochrome c from the mitochondria; however, Bax is still required to activate effector caspases. Thus, both Bak and Bax are necessary to accomplish DNA damage and Ngo‐induced apoptosis.

Methods to quantify Th cell reconstitution after immunosuppressive therapies such as hematopoietic stem cell transplantation are becoming a key issue since persistent Th cell deficiencies may result in severe complications and adverse events. We employed here cytometric monitoring of CD31+ thymus-naive Th cells for the direct assessment of human thymic function in 10 patients undergoing autologous stem cell transplantation for severe autoimmune diseases. High frequencies of posttransplant recurring naive Th cells coexpressed CD31 and stable long-term reconstitution with elevated absolute counts of CD31+ thymus-naive Th cells that were enriched with T cell receptor excision circles was demonstrated. Cytometric monitoring of CD31+ thymus-naive Th cells enables to directly evaluate human thymic function ex vivo.

SummaryChlamydia trachomatis is an obligate intracellular pathogen responsible for a high burden of human disease. Here, a loss-of-function screen using a set of lentivirally transduced shRNAs identified 14 human host cell factors that modulate C. trachomatis infectivity. Notably, knockdown of dynamin, a host GTPase, decreased C. trachomatis infectivity. Dynamin functions in multiple cytoplasmic locations, including vesicle formation at the plasma membrane and the trans-Golgi network. However, its role in C. trachomatis infection remains unclear. Here we report that dynamin is essential for homotypic fusion of C. trachomatis inclusions but not for C. trachomatis internalization into the host cell. Further, dynamin activity is necessary for lipid transport into C. trachomatis inclusions and for normal re-differentiation from reticulate to elementary bodies. Fragmentation of the Golgi apparatus is proposed to be an important strategy used by C. trachomatis for efficient lipid acquisition and replication within the host. Here we show that a subset of C. trachomatis-infected cells displayed Golgi fragmentation, which was concurrent with increased mitotic accumulation. Golgi fragmentation was dispensable for dynamin-mediated lipid acquisition into C. trachomatis inclusions, irrespective of the cell cycle phase. Thus, our study reveals a critical role of dynamin in host-derived lipid acquisition for C. trachomatis development.

T helper lymphocytes (Th cells) play a central role in cellular defence against pathogens as well as in cellular self tolerance. The activation of Th cells is a crucial process determining the course of a protective immune response. Dysregulated activation processes can lead to pathologic immune reactions and may induce autoimmune diseases. Thus, for example, chronic activation of Th cells can trigger autoimmune diseases such as rheumatoid arthritis. One fundamental feature of antigen-specific T-cell activation is the synthesis of cytokines, e.g. Interleukin- (IL-)2. Here we present results of our working group indicating for the first time that, at the level of the individual cell, IL-2 is expressed in a binary fashion, i.e. according to an all-or-none principle. The identification and characterization of such intracellular switches may provide new options to manipulate the fate of T cells and develop novel therapeutic strategies.

Japan Arthritis Res Ther 2003, 5(Suppl 3):1 (DOI 10.1186/ar800) Apoptosis is a principal mechanism in metazoans by which superfluous or potentially harmful cells are eliminated. Deregulation of this process leads to a variety of diseases such as cancer and autoimmune diseases. Stimuli that can induce apoptosis are relatively diverse, and include the death factors (Fas ligand, tumor necrosis factor and TRAIL), DNA damage, and oxidative stress. Regardless of the origin of the apoptotic stimulus, commitment to apoptosis leads to activation of caspases, a family of cysteine proteases. Cleavage of a select group of cellular substrates by caspases is responsible for the morphological and biochemical changes that characterize apoptotic cell death. The degradation of nuclear DNA into nucleosomal units is one of the features of apoptotic cell death, and is mediated by a caspase-activated DNase (CAD). Cells deficient in CAD undergo cell death without the DNA fragmentation, but CAD-null mice did not show any adverse phenotypes. A close examination of the apoptotic cells in these mice indicated that apoptotic cells are always in macrophages. It seems that at an early stage of apoptosis, the dying cells present an 'eat me signal' on their surface. This signal is recognized by macrophages for engulfment, and DNase II in the lysosomes of macrophages degrades DNA of apoptotic cells. Mice deficient in both CAD and DNase II genes were established, and the development of various organs was found to be severely impaired in these mutant mice. The mice accumulated a large amount of undigested DNA in macrophages in various tissues during development. This accumulation of DNA in macrophages activated the innate immunity to induce the expression of the interferon β gene. The interferon thus produced seems to be responsible for the impaired tissue development. These results indicate that the degradation of DNA during apoptotic cell death is an essential step of apoptosis to maintain mammalian homeostasis. Osteoarthritis (OA) has been considered a biomechanically driven, degenerative disease of cartilage. However, the OA disease process affects not only the cartilage, but also the entire joint structure; and within the bone, cartilage and synovium of affected joints, profound metabolic changes transpire, which include the production of growth factors, nitric oxide (NO), prostaglandins (PGs), leukotrienes (LTs), IL-1β, tumor necrosis factor alpha, IL-6, and IL-8. The autocrine production of IL-1β by OA cartilage has been of particular interest, since both ex vivo human and in vivo animal studies indicate that IL-1 antagonists effectively attenuate cartilage degradation. Microarray technology has demonstrated differential expression in OA cartilage of a variety of IL-1-induced, NFβB-dependent genes. Among IL-β-induced products of OA cartilage are various eicosanoids, which include E 2 , PGD 2 , LTB 4 , PGF 1α , PGF 2α and thromboxane. Treatment of OA cartilage with cyclooxygenase (COX) inhibitors increases LTB 4 production threefold to five...