A -436C>A polymorphism in the human FAS gene promoter associated with severe childhood malaria.

Human genetics and immune responses are considered to critically influence the outcome of malaria infections including life-threatening syndromes caused by Plasmodium falciparum. An important role in immune regulation is assigned to the apoptosis-signaling cell surface receptor CD95 (Fas, APO-1), encoded by the gene FAS. Here, a candidate-gene association study including variant discovery at the FAS gene locus was carried out in a case-control group comprising 1,195 pediatric cases of severe falciparum malaria and 769 unaffected controls from a region highly endemic for malaria in Ghana, West Africa. We found the A allele of c.-436C>A (rs9658676) located in the promoter region of FAS to be significantly associated with protection from severe childhood malaria (odds ratio 0.71, 95% confidence interval 0.58-0.88, p(empirical) = 0.02) and confirmed this finding in a replication group of 1,412 additional severe malaria cases and 2,659 community controls from the same geographic area. The combined analysis resulted in an odds ratio of 0.71 (95% confidence interval 0.62-0.80, p = 1.8×10⁻7, n = 6035). The association applied to c.-436AA homozygotes (odds ratio 0.47, 95% confidence interval 0.36-0.60) and to a lesser extent to c.-436AC heterozygotes (odds ratio 0.73, 95% confidence interval 0.63-0.84), and also to all phenotypic subgroups studied, including severe malaria anemia, cerebral malaria, and other malaria complications. Quantitative FACS analyses assessing CD95 surface expression of peripheral blood mononuclear cells of naïve donors showed a significantly higher proportion of CD69+CD95+ cells among persons homozygous for the protective A allele compared to AC heterozygotes and CC homozygotes, indicating a functional role of the associated CD95 variant, possibly in supporting lymphocyte apoptosis.

Interleukin-2 inhibits FMS-like tyrosine kinase 3 receptor ligand (flt3L)-dependent development and function of conventional and plasmacytoid dendritic cells.

Steady-state development of plasmacytoid dendritic cells (pDCs) and conventional dendritic cells (cDCs) requires the ligand for FMS-like tyrosine kinase 3 receptor (flt3L), but little is known about how other cytokines may also control this process. In this study, we show that IL-2 inhibits the development of both pDCs and cDCs from bone marrow cells under flt3L stimulation, by acting on lineage(-) flt3(+) precursors. This inhibition of DC development by IL-2 requires IL-2Rα and IL2Rß. IL-2Rα is specifically expressed in one stage of the DC precursor: the monocyte and DC progenitors (MDPs). Furthermore, more MDPs are found in flt3L-stimulated bone marrow cultures when IL-2 is present, suggesting that IL-2 may be inhibiting DC development at the MDP stage. Consistent with our in vitro findings, we observe that nonobese diabetic (NOD) mice, which express less IL-2 compared with diabetes-resistant NOD.Idd3/5 mice, have more splenic pDCs. Additionally, DCs developed in vitro in the presence of flt3L and IL-2 display reduced ability to stimulate T-cell proliferation compared with DCs developed in the presence of flt3L alone. Although the addition of IL-2 does not increase the apoptosis of DCs during their development, DCs developed in the presence of IL-2 are more prone to apoptosis upon interaction with T cells. Together our data show that IL-2 can inhibit both the development and the function of DCs. This pathway may have implications for the loss of immune tolerance: Reduced IL-2 signaling may lead to increased DC number and T-cell stimulatory capacity.

Increased expression of guanylate binding protein-1 in lesional skin of patients with cutaneous lupus erythematosus.

The large GTPase human guanylate binding protein-1 (GBP-1) is a key mediator of angiostatic effects of inflammation and is induced by interferon (IFN)-α and IFN-γ in endothelial cells (ECs). The aim of this study was to investigate whether GBP-1 is a marker of skin lesions in patients with cutaneous lupus erythematosus (CLE). Western blotting revealed that GBP-1 was in vitro induced by IFN-α and -γ in primary keratinocytes obtained from healthy controls. Moreover, we found that this protein was expressed by keratinocytes and ECs in primary and ultraviolet (UV)-induced skin lesions from patients with various subtypes of CLE, when compared to non-lesional skin. No GBP-1 expression was noted in skin biopsy specimens 24 or 72 h after UV irradiation prior to lesion formation in patients with CLE or in healthy control specimens with or without UV irradiation. Initial findings suggest that GBP-1 is not expressed in other skin diseases with different inflammatory aetiology, such as atopic dermatitis. We conclude that GBP-1 expression is closely associated with skin lesions in patients with CLE, suggesting a contribution of GBP-1 in the pathogenesis of this disease.

Activation-induced internalization differs for the tetraspanins CD9 and Tspan8: Impact on tumor cell motility.

Exosomes are most important intercellular communicators and tetraspanins/tetraspanin-complexes have been suggested to play an important role in exosomal target cell selection. We have shown that only exosomes expressing a Tspan8-CD49d complex preferentially bind endothelial cells, which initiates angiogenesis. This finding was unexpected as in the exosome donor cell Tspan8 is associated with CD49c and the tetraspanins CD9 and CD151. In view of the discussed therapeutic power of exosomes as message/drug transporter, it became important to clarify the mechanisms accounting for the distinct Tspan8-web in the cell membrane versus exosomes. We therefore compared the route of Tspan8 and Tspan8-chimera internalization, where the N- and/or C-terminal regions were exchanged with the corresponding regions of CD9 or CD151. Activation-induced Tspan8-internalization proceeds more rapidly than CD9 internalization and is accompanied by disassembly of the Tspan8-CD9-CD151 membrane complex in resting cells. Tspan8-internalization relies on the association of the Tspan8 N-terminal region with intersectin-2, a multimodular complex involved in clathrin-coated pit internalization. Internalization and recovery of Tspan8 in early endosomes is further promoted by the recruitment of CD49d such that only in PMA-activated cells a Tspan8-INS2-CD49d-clathrin complex is recovered in cholesterol-depletion-resistant membrane microdomains. PMA-induced Tspan8-internalization promotes cell migration, but reduces matrix and cell adhesion. Thus, stimulation initiates tetraspanin-web rearrangements, which have strong functional consequences for the cell, exosome-delivery and exosome target selection. This knowledge will be essential for generating tailored therapeutic exosomes.

Anti-annexin 1 antibodies: a new diagnostic marker in the serum of patients with discoid lupus erythematosus.

Annexin 1 is an anti-inflammatory molecule and has also been described to be a common target of autoantibodies. In this study, we determined whether antibodies against annexin 1 can be detected in sera of patients with cutaneous lupus erythematosus (CLE). Levels of anti-annexin 1 antibodies were evaluated by a new established enzyme-linked immunosorbent assay and found to be significantly higher in sera of patients with CLE when compared to normal healthy donors (NHD). Moreover, the percentage of sera positively tested for anti-annexin 1 antibodies was elevated in patients with CLE when compared to NHD. In particular, the percentage of positive sera for anti-annexin 1 antibodies was significantly higher in patients with discoid lupus erythematosus (DLE); however, disease activity did not correlate with the antibody levels. The results of this study indicate that anti-annexin 1 antibodies in sera of patients with DLE might be a valuable aid in the diagnosis of this subtype.

CD127(low/-) and FoxP3(+) expression levels characterize different regulatory T-cell populations in human peripheral blood.

It has recently been suggested that a lack of CD127 expression can be used to identify human CD4(+) regulatory T cells (Tregs), especially when combined with CD25. Therefore, we analyzed CD4(+)CD25(+)CD127(low/-) cells and compared their frequency and expression pattern with those of FoxP3(+) Tregs using multiparameter flow cytometry analysis. We obtained human peripheral blood cells from 20 normal healthy donors and determined the number of CD25(+)CD127(low/-) cells as a percentage of CD4(+) T cells in the same panel used for CD4(+)CD25(+)FoxP3(+) cells. In contrast to CD4(+)CD25(+)FoxP3(+) cells, gating of a clear CD4(+)CD25(+)CD127(low/-) population was difficult. Moreover, we demonstrated that there was a high percentage (34.0+/-15.1%) of CD127(low/-) cells that did not express FoxP3 and, conversely, that there was a high percentage (30.3+/-7.4%) of CD127(+) cells that expressed FoxP3, suggesting that these markers did not represent the same population of Tregs. These data were also confirmed in blood samples from patients with systemic scleroderma. Thus, isolation of pure Treg populations for in vitro functional studies is a challenge, explaining the varying results by different groups in clinical studies. The data from this study have important implications for carrying out a quantitative analysis of Tregs in human samples.