High proportion of patients with bleeding of unknown cause in persons with a mild-to-moderate bleeding tendency: Results from the Vienna Bleeding Biobank (VIBB).

INTRODUCTION: Data on clinical characteristics and the prevalence of underlying coagulopathies in patients with mild-to-moderate bleeding disorders (MBDs) are scarce. AIM: We established the Vienna Bleeding Biobank (VIBB) to characterize and thoroughly investigate Austrian patients with MBDs. RESULTS: Four hundred eighteen patients (female = 345, 82.5%) were included. A platelet function defect (PFD) was diagnosed in 26 (6.2%) and a possible PFD in 30 (7.2%) patients. Eight patients (1.9%) were diagnosed with von Willebrand disease (VWD) (type 1 n = 6; type 2 n = 2), and 29 patients had low VWF (30-50 IU/dL). Deficiencies in factor VIII, IX, XI or XIII were found in 11 (2.6%), 3 (0.7%), 3 (0.7%) and 1 patient(s), 2 patients had dysfibrinogenaemia, and further 2 had possible PFD and FXI deficiency. Probable causal mutations were detected in 8 of 11 patients with FVIII deficiency, 2 of 3 patients with FIX deficiency and 2 of 8 patients with VWD. Three hundred three patients (72.5%) had normal results in the coagulation assays and were categorized as patients with bleeding of unknown cause (BUC). The bleeding score did not differ between patients with and without established diagnosis. A diagnosis of a bleeding disorder was more frequently made in men than in women (49.3% vs 22.9%). Male sex (OR 3.55, 95% CI: 2.02-6.22; P < .001) and blood group 0 (OR 1.86, 95% CI: 1.17-2.94; P = .008) were independently associated with diagnosis of a bleeding disorder. CONCLUSION: The high rate of patients with BUC despite in-depth haemostatic assessment underlines the incompleteness of available routine laboratory tests. Males with MBDs were more likely to be diagnosed with an established bleeding disorder than females.

Characterization of patients at high risk of melanoma in Austria.

BACKGROUND: Risk of melanoma is determined by genetic and exogenous factors. Only a few studies have included both characteristics in a comprehensive multivariable analysis. OBJECTIVES: To find determinants of patients at high risk of melanoma in Austria, including phenotype, genotype and lifestyle characteristics in comprehensive analyses. METHODS: In total, 1668 patients with melanoma from the M3 case-control study were studied. Overall, 567 participants were sequenced for CDKN2A, 232 for CDK4, 123 for MITF encoding the variant E318K and 964 for MC1R. RESULTS: Patients with melanoma with a positive family history (n = 190, 11·6%), multiple primary melanomas (n = 261, 15·7%) and younger age (< 50 years, n = 675, 40·5%) were defined as being at high risk. All other patients with melanoma were defined as the reference group. We found significant differences between those two groups and between the high-risk subgroups (positive family history, multiple primary melanomas and younger age). Pigmentation phenotype was associated with the high-risk group in general (childhood freckling, odds ratio 1·46, P = 0·007; blond/reddish hair colour, odds ratio 1·43, P = 0·011). Patients with a positive family history and patients with early-onset disease were similar regarding both their phenotypic characteristics and external factors. Established high-risk mutations in CDKN2A were found in cases with a positive family history (n = 12) or multiple melanomas (n = 2). Moreover, we found three patients carrying the MITF p.E318K variant, two with a CDK4 variant and seven with nonsynonymous MC1R variants with undescribed biological significance, of which four were predicted as damaging. CONCLUSIONS: Austrian patients could represent a reservoir for novel genetic variants. Further investigation of populations in Central and Eastern Europe might reveal more novel and disease-relevant variants.

Evaluation of the PC-1 K121Q and G2906C variants as independent risk factors for ischaemic stroke.

UNLABELLED: Overexpression of plasma cell membrane glycoprotein-1 (PC-1) inhibits insulin receptor tyrosine kinase activity and thus favours insulin resistance and atherosclerotic vascular disease. Recent findings indicate that the minor variant K121Q in the PC-1 gene confers an increased risk for early myocardial infarction independent of other established risk factors. We hypothesized that genetic variants in PC-1 may also influence the risk for cerebrovascular disease. AIM: Therefore, we assessed the association of the PC-1 K121Q variant in the coding region and a polymorphism (G2906C) in the 3' untranslated region of the PC-1 gene with the risk of stroke. PATIENTS: We analyzed 1014 patients with a history of ischaemic stroke from the Vienna stroke registry and 1001 control individuals without vascular disease. RESULTS, CONCLUSION: Genotype frequencies of both genetic variants were similar in patients and controls in the total study population. By multivariate analysis, no interactions were observed between the PC-1 genotype and established vascular risk factors. However, the PC-1 2906C allele was significantly more frequent in patients who suffered from stroke before the age of 40 years. In these patients the risk for ischaemic stroke was increased four-fold.

Interaction of CD31 with a heterophilic counterreceptor involved in downregulation of human T cell responses.

CD31 is a 130-kD glycoprotein of the immunoglobulin (Ig) superfamily expressed on the surface of endothelial cells, platelets, and several leukocyte subsets. Previous reports indicated that CD31 can mediate intercellular adhesion via both homophilic and heterophilic interaction mechanisms. Using a soluble recombinant CD31-Ig fusion protein (CD31 receptor globulin [Rg]), we demonstrate here that human CD31- T lymphocytes and CD4+CD31- T cell clones express a heterophilic CD31 ligand that is upregulated 18 h after activation. Interaction of CD31Rg with CD31- T helper cell (Th) clones was divalent cation independent but could be blocked by heparin, thus indicating that the CD31 counterreceptor on T cells can be distinguished from the ligands identified on other cell types. Moreover, a single chain protein of 120 kD was precipitated by CD31Rg from the lysates of CD31- Th clones. CD31Rg completely downregulated the proliferative response and cytokine production (interleukin-4, interferon-gamma, and tumor necrosis factor-alpha) of CD31- Th clones when the cells were maximally stimulated via immobilized CD3 monoclonal antibody. These results suggest that interaction of CD31 with a heterophilic counterreceptor on T lymphocytes can interfere with a positive regulatory pathway of T cell activation, or directly signal T cells to downregulate immune function.

Ligand-induced conformational change within the CD2 ectodomain accompanies receptor clustering: implication for molecular lattice formation.

CD2 mediates interaction between T cells and their cognate partners through its CD58-binding membrane-distal adhesion domain (D1) facilitating T cell receptor (TCR) triggering. A neoepitope defined by anti-CD2R monoclonal antibodies (mAbs) has suggested structural alteration within the CD2 ectodomain during T cell activation. Here, we map CD2R to the flexible CD2 linker region between D1 and the membrane-proximal extracellular domain (D2) and show that exposure of this conformational site is independent of temperature and metabolic energy. Co-ligation of CD2 and CD58 molecules on opposing cells within a conjugate pair induces CD2R and redistributes CD2 to the region of cell-cell contact. These CD2R+ molecules, in contrast to the CD2R-molecules, are tightly clustered on the T cell surface. Hence, a ligand-mediated increase in the D1-D2 interdomain angle apparently exposes CD2R, facilitates packing of CD2 molecules in a clustered array and is linked to CD2-mediated adhesion and activation events. Conformational alteration of this type may be generally important in ordered lattice formation involving surface receptors.

[Regulation of cytokine production by human T-lymphocytes in allergic immune response]. / Regulation der Zytokinproduktion humaner T-Lymphozyten in der allergischen Immunantwort.

A network of cytokines regulates the growth and function of the cells of the immune system. T cells possess a dominant role in this network since they are the main source of many cytokines. The production of different cytokines is specifically regulated by means of cell interactions and cytokines and depends largely on the state of differentiation of the T cell. The production of certain cytokines is, therefore, restricted to defined subpopulations of T cells. IL2, IL4, IL5, IL10, IL12 and Interferon gamma (IFN gamma) play a central role in allergic immune response. The production of these cytokines can be restricted to specialized T cells. Two different types of differentiated T cells can be characterised according to the pattern of cytokine production of T cells: IL2 and IFN gamma are typically produced by T helper 1 (Th 1) cells, whereas predominantly T helper 2 (Th 2) cells produce IL4, IL5 and IL10. The selective activation of either Th 1 or Th 2 type cells depends on the antigen and is influenced by cytokines produced partly by antigen-presenting cells and partly by T helper cells. Typically, allergens induce an immune response which is dominated by Th 2 cells. These cells then stimulate the production of IgE by B cells.

A p56lck-independent pathway of CD2 signaling involves Jun kinase.

The p56 Src family non-receptor tyrosine kinase has been shown to be critical for T lymphocyte differentiation and activation. Hence in the absence of p56, T cell receptor triggered activation does not occur. We now provide evidence for a CD2-based signaling pathway which, in contrast to that of the T cell receptor, is independent of p56. CD2-mediated interleukin-2 production occurs via activation of Jun kinase in cell lines lacking p56. Jun kinase then facilitates the binding of c-Jun/c-Fos heterodimers to the AP-1 consensus site and the subsequent transcriptional activity of the interleukin-2 promoter. These data elucidate differences between TCR and CD2 signaling pathways in the same T cells.

In vitro and in vivo activated T cells display increased sensitivity to PGE2.

In this study we report that in vitro activation of T cells increased the cyclic AMP response to subsequent prostaglandin E2 (PGE2) stimulation severalfold per cell. This sensitization of T cells to PGE2-induced cyclic AMP generation was observed when the T cells had been stimulated in vitro for 5 days with either the CD3 monoclonal antibody OKT3, phytohemagglutinin, or the combination phytohemagglutinin plus the phorbol ester PMA. Enhanced cyclic AMP generation following mitogenic activation was seen in response to both PGE2 and forskolin, direct activator of the adenylate cyclase, indicating that the amount of adenylate cyclase had increased during the in vitro activation course. In order to investigate whether various T cell subsets in general and in vivo activated T cells in particular would differ in their susceptibility to PGE2, we isolated CD4+, CD8+, CD4-CD8-, CD4+CD45RO+ ("memory"), and CD4+CD45RA+ ("virgin") T cells and studied PGE2-mediated inhibition of CD3-induced proliferation, as well as cyclic AMP generation in response to PGE2, respectively. We found that CD8+ T cells are more susceptible to PGE2 inhibition and produce more cyclic AMP than CD4+ T cells. Double-negative T cells (enriched for gamma delta T cell receptor positive cells) were found to be sensitive to PGE2 as well. Within the CD4+ T cell population, CD45RO+ ("memory") T cells were significantly more sensitive to PGE2-mediated suppression than CD45RA+ ("virgin") T cells. CD45RO+ cells required a 10-fold lower dose of PGE2 for half-maximum suppression of proliferation. However, no difference in cyclic AMP production could be demonstrated between these two subsets. We propose that substantial heterogeneity exists among peripheral blood T lymphocyte subsets regarding their sensitivity to the immunosuppressive action of PGE2 and that the sensitivity of individual cells changes in the course of an immune response.

High IL-4 production is a stable phenotype of CD8negCD45RAnegCD27neg T cells.

CD27neg T cells are found only among CD4pos-CD45ROpos T cells and represent a T cell subset functionally distinct from CD27pos T cells. We examined CD4posCD45ROpos T cells that were sorted into CD27neg and CD27pos populations for their cytokine production in response to different activation pathways. We found that CD27neg T cells are characterized by high IL-4 and low IL-2 production, regardless of whether the cells were activated through CD3 plus CD28, CD2 plus CD28, or PHA plus PMA. However, subpopulation-specific patterns of cytokines were the clearest demonstrable following CD2 plus CD28 stimulation. We conclude from these data that high IL-4 production is a stable phenotype of CD27neg T cells.

A critical role for p59(fyn) in CD2-based signal transduction.

TCR- but not CD2-triggered IL-2 production is p56(lck) dependent. To test the hypothesis that p59(fyn), a second src-family protein tyrosine kinase (PTK) expressed in T lymphocytes, might be an essential upstream component of the CD2 signaling pathway, we generated human (h) CD2 transgenic (tg) fyn(+/+) and fyn(-/-) mice. Clustering of hCD2 molecules on resting peripheral T lymphocytes results in Ca(2+) mobilization, activation of MAPK and cellular proliferation. In contrast, in the absence of p59(fyn), these CD2-initiated activities are markedly reduced, while TCR-triggered proliferation is unaffected. Several CD2 pathway components regulated by p59(fyn) have been identified including phospholipase C-gamma1 (PLC-gamma1), Vav, protein kinase C-theta isoform (PKC-theta), docking protein (Dok), focal adhesion kinase (FAK) and Pyk2. Decreased inducible PKC-theta catalytic activity and Vav phosphorylation likely account for diminished p38 and JNK activation in hCD2tg fyn(-/-) mice. Moreover, deficiency in fyn-dependent PLC-gamma1 catalytic activity may contribute to reduced PKC-alpha-dependent ERK activation. Of note, CD2-dependent Dok but not linker from activated T cells (LAT) tyrosine phosphorylation requires p59(fyn). Furthermore, that FAK and Pyk2 are target substrates implies that p59(fyn) may be an important regulator of T cell adhesion as well. Collectively, these data identify p59(fyn) as a key PTK in CD2-mediated activation of mature T lymphocytes.

Functional analysis of immunoreceptor tyrosine-based activation motif (ITAM)-mediated signal transduction: the two YxxL segments within a single CD3zeta-ITAM are functionally distinct.

Functional analysis of the immunoreceptor tyrosine-based activation motif (ITAM) derived from the membrane-proximal ITAM of CD3zeta demonstrates that mutations at either the tyrosine or leucine residues in the N-terminal YxxL segment of the ITAM abolish all signal transduction functions of this ITAM. In contrast, mutations at the tyrosine or leucine residues in the C-terminal YxxL segment abrogate signals for interleukin (IL)-2 production but do not prevent tyrosine phosphorylation of the N-terminal tyrosine of the ITAM, lck association with the ITAM, activation of phospholipase C-gamma1 or calcium mobilization. Cross-linking of chimeric receptors containing a C-terminal YxxL leucine mutation induces tyrosine phosphorylation of ZAP70 but without stable binding to the phosphorylated ITAM. These results indicate that the two YxxL segments in an ITAM are functionally distinct and that both are essential for ZAP70 binding and IL-2 production. Furthermore, tyrosine phosphorylation of ZAP70 per se is not sufficient to trigger the downstream events leading to IL-2 production. Substitution of an alanine for the bulky side chain at the Y+1 position of the N-terminal YxxL segment reduces the receptor cross-linking requirement necessary to achieve cellular activation and the absolute dependence on lck in this process. Our results reveal that both the number of ITAM as well as the specific amino acid residues within a single ITAM determine the extent of chimeric receptor cross-linking required to trigger tyrosine phosphorylation-dependent signaling events.

Crosslinking of CD27 in the presence of CD28 costimulation results in T cell proliferation and cytokine production.

Monoclonal antibodies recognizing CD27, a member of the nerve growth factor receptor family, have been observed to enhance or inhibit PHA- or CD3 mAb-stimulated T cell proliferation. In this study, we investigate the effects of CD27 stimulation on the proliferation and cytokine production of T cells stimulated simultaneously through CD3, CD2, or CD28. Here we report that simultaneous crosslinking of CD27 plus CD28 results in T cell proliferation and in the production of IL-2, IL-4, IL-10, and IFN-gamma. CD27 plus CD28 stimulation thus introduces a novel Ag-independent pathway of T cell activation. We further show that all major T cell subsets (CD4+, CD8+, CD4+CD45RA+, or CD4+CD45RO+ T cells) respond to CD27 plus CD28-mediated costimulation. Synergistic effects on T cell stimulation are also found when CD27 is crosslinked on cells stimulated simultaneously through CD3 or CD2. In further experiments we show that crosslinking of CD27 elevates cytoplasmic free Ca2+ levels. Finally, both the CD3 plus CD27 and the CD28 plus CD27-stimulated T cell proliferation is sensitive to inhibition by CsA. Taken together, these data emphasize the importance of CD27 stimulation in the context of simultaneously received signals through CD3, CD2 or, most importantly, through CD28. Furthermore, signaling through CD27 appears to involve Ca(2+)-dependent mechanisms sensitive to CsA.

CD2 signaling in T cells involves tyrosine phosphorylation and activation of the Tec family kinase, EMT/ITK/TSK.

Ligation of the CD2 cell surface glycoprotein expressed on T lymphocytes and NK cells induces protein tyrosine phosphorylation and activation of the Src kinases, LCK and FYN. We show here that in Jurkat T leukemia cells and in peripheral blood T cells, CD2 stimulation also leads to tyrosine phosphorylation and activation of the Tec family kinase, EMT/ITK/TSK. Activation of EMT by CD2 was induced by mitogenic pairs of CD2 mAb, certain single CD2 mAb followed by secondary antibody cross-linking, and CD58-bearing sheep red blood cells. With the use of different Jurkat cell mutants it was demonstrated that CD2-mediated activation of EMT required expression of LCK, but not require surface expression of the CD3 zeta chain. Receptor-mediated activation of LCK does not in itself lead to activation of this Tec kinase since induction of LCK by ligation of CD4 or CD5 did not result in activation of EMT. The activation of EMT during CD2 signaling suggests an important role for this kinase in CD2 co-stimulation of T cell responses.

Single human T cells stimulated in the absence of feeder cells transcribe interleukin-2 and undergo long-term clonal growth in response to defined monoclonal antibodies and cytokine stimulation.

The two-signal model of T-cell activation postulates that T lymphocytes require at least two distinct signals for activation. This model has been established with bulk cultures of T cells in which T-cell-T-cell interaction can occur, possibly delivering further unrecognized costimulatory signals. The signal requirements of single T cells for the induction of clonal cell growth or the transcription of cytokines would best be studied in a cell cloning system in the absence of feeder cells; however, such an experimental system has not been reported so far. In this study, we report the long-term cloning of human resting peripheral blood CD4+CD45RO- T cells under feeder cell-free conditions in response to CD3 and CD28 stimulation in the presence of exogenous interleukin-2 (IL-2). Cloning efficiency ranged from 40% to 60% depending on the presence of additional cytokines IL-1 and IL-6. Single-call polymerase chain reaction showed that transcription of IL-2 occurred in cells stimulated through CD3 plus CD28 alone. T cells grown in response to CD3 plus CD28 plus IL-2 stimulation produced both IL-4 and interferon-gamma (IFN-gamma) on restimulation (Th0 cells) and could be functionally differentiated into Th1- or Th2-type cells by the addition of IFN-gamma or IL-4, respectively, during cell cloning. These data show on the single-cell level a two-signal model of T- cell activation for the transcription of IL-2. In addition, these experiments show that IFN-gamma and IL-4 exert their T-cell-differentiating effects directly on the T cell without any further need for antigen-presenting cells. Together, our experiments show the feasability of a defined long-term clonal cell culture system to study the growth and differentiation of human T lymphocytes.

Anti-interleukin-1 alpha autoantibodies in hemodialysis patients.

IL-1 activity is increased in hemodialysis patients and interest has recently been focused on IL-1 antagonism in various clinical settings. We studied the presence of anti-IL-1 alpha autoantibodies in sera from 49 hemodialysis patients, 159 kidney graft recipients and 89 chronic renal failure patients without renal replacement therapy. Within the three month study period 32.6% of the hemodialysis patients were found to present with anti-IL-1 alpha autoantibodies, in contrast to 5.6% of kidney graft recipients, 8.9% of chronic renal failure patients, and only 1.4% of healthy subjects. The presence of these autoantibodies was neither associated with primary kidney disease nor with the type of dialysis membrane we used. In addition, in antibody positive patients a pronounced increase of IL-1 alpha serum levels within a dialysis session from 14.8 +/- 4.7 pg/ml to 26.4 +/- 11.2 pg/ml (P less than 0.0005) was observed, contrasting to the more even increase from 14.1 +/- 3.1 pg/ml to 19.3 +/- 12.7 pg/ml (P less than 0.05) in the antibody negative group. Neither clinical symptoms due to adverse effects of IL-1 alpha nor some influence on erythropoiesis mediated by IL-1 alpha could be envisaged. Thus, we believe, that anti-IL-1 alpha autoantibodies, present in high frequency in hemodialysis patients, have a neutralizing effect on IL-1 alpha in these patients.

Association of an ABCB1 gene haplotype with pharmacoresistance in temporal lobe epilepsy.

The multidrug transporter P-glycoprotein is suspected of contributing to pharmacoresistance in temporal lobe epilepsy (TLE). To assess the role of functional variations in its coding gene (ABCB1) the authors genotyped 210 patients with TLE who were stratified according to their degree of drug resistance. They identified a common haplotype that when present in the homozygous state significantly increased the risk for pharmacoresistance.

CD99 engagement on human peripheral blood T cells results in TCR/CD3-dependent cellular activation and allows for Th1-restricted cytokine production.

We have assessed the functional effect of CD99 engagement on resting human peripheral blood (PB) T cells. CD99, as detected by the mAb 3B2/TA8, is constitutively expressed on all PB T cells and becomes further up-regulated upon cellular activation. In this study we demonstrate that cross-linking of the CD99 molecule with the agonistic mAb 3B2/TA8 cooperates with suboptimal TCR/CD3 signals, but not with phorbol ester, ionomycin, or CD28 mAb stimulation, to induce proliferation of resting PB T cells. Comparable stimulatory effects were observed with the CD99 mAb 12E7. Characterization of the signaling pathways involved revealed that CD99 engagement leads to the elevation of intracellular Ca2+, which is dependent on the cell surface expression of the TCR/CD3 complex. No CD99 mAb-induced calcium mobilization was observed on TCR/CD3-modulated or TCR/CD3-negative T cells. To examine the impact of CD99 stimulation on subsequent cytokine production by T cells, we cross-linked CD99 molecules in the presence of a suboptimal TCR/CD3 trigger followed by determination of intracellular cytokine levels. Significantly, T cell lines as well as Th1 and Th0 clones synthesized TNF-alpha and IFN-gamma after this treatment. In contrast, Th2 clones were unable to produce IL-4 or IFN-gamma when stimulated in a similar fashion. We conclude that CD99 is a receptor that mediates TCR/CD3-dependent activation of resting PB T cells and specifically induces Th1-type cytokine production in polyclonally activated T cell lines, Th1 and Th0 clones.

A cdc15-like adaptor protein (CD2BP1) interacts with the CD2 cytoplasmic domain and regulates CD2-triggered adhesion.

A human CD2 cytoplasmic tail-binding protein, termed CD2BP1, was identified by an interaction trap cloning method. Expression of CD2BP1 is restricted to hematopoietic tissue, being prominent in T and natural killer (NK) cells, with long (CD2BP1L) and short (CD2BP1S) variants arising by alternative RNA splicing. Both CD2BP1 molecules are homologous to Schizosaccharomyces pombe cdc15, and include a helical domain, variable length intervening PEST sequence and C-terminal SH3 domain. Although the CD2BP1 SH3 domain binds directly to the CD2 sequence, KGPPLPRPRV (amino acids 300-309), its association is augmented markedly by the CD2BP1 N-terminal segment. Upon ligand-induced clustering of surface CD2 molecules, CD2BP1 redistributes from a cytosolic to a surface membrane compartment, co-localizing with CD2. In turn, CD2-stimulated adhesion is downregulated by CD2BP1, apparently through coupling of the protein tyrosine phosphatase (PTP)-PEST to CD2. These findings offer the first molecular view into the control processes for T cell adhesion.