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.

Parameters influencing FVIII pharmacokinetics in patients with severe and moderate haemophilia A.

In haemophilia A patients factor VIII (FVIII) recovery and half-life can vary substantially. There are parameters known to modulate FVIII pharmacokinetics (PK), but they explain only about 34% of the variability. The aim of this study was to identify new parameters that influence FVIII PK and thus to expand the current knowledge. FVIII PK were determined in 42 haemophilia A patients (37 severe, 5 moderate) without inhibitor. Patients' characteristics and laboratory parameters were evaluated for an association with FVIII PK. We analysed plasma levels of low-density lipoprotein receptor-related protein 1 (LRP1) and protein C (PC) activity, which had been hypothesized to influence FVIII activity. Furthermore, four variations in intron 6 of the LRP1 gene, which had been shown to influence LRP1, were investigated. FVIII half-life differed widely from 6.2 to 20.7 h, with a median of 10.0 h. Patients with blood group O had shorter FVIII half-life compared to patients with non-O blood group (median FVIII half-life 9.0 h vs. 10.4 h, P = 0.018). Age was significantly associated with FVIII half-life (r = 0.32, P = 0.035). Besides age, also VWF antigen (r = 0.52, P < 0.001) and blood group (r = -0.37, P = 0.015) was associated with FVIII half-life. No correlation was found with FVIII- or LRP1-genotype, LRP1 or PC concentrations. Our data showed large differences in FVIII PK between individual patients and revealed age, blood group and VWF levels as important determining factors for FVIII half-life. FVIII genotype or levels of LRP1 or PC had no influence on FVIII PK.

The KIT D816V allele burden predicts survival in patients with mastocytosis and correlates with the WHO type of the disease.

KIT D816V is present in a majority of patients with systemic mastocytosis (SM). We determined the KIT D816V allele burden by quantitative real-time PCR in bone marrow and peripheral blood of 105 patients with mastocytosis. KIT D816V was detected in 92/105 patients (88%). Significant differences in the median allele burden were observed between disease subgroups: cutaneous mastocytosis (0.042%), indolent SM (0.285%), smoldering SM (5.991%), aggressive SM (9.346%), and SM with associated hematologic non-mast cell lineage disease (3.761%) (P < 0.001). The KIT D816V burden also correlated with serum tryptase (R = 0.5, P < 0.005) but not with mast cell infiltration in bone marrow or mediator symptoms. Moreover, the allele burden was of prognostic significance regarding survival (P < 0.01). Patients responding to cytoreductive therapy showed a significant decrease in KIT D816V (P < 0.05). To conclude, the KIT D816V burden correlates with the variant of mastocytosis, predicts survival, and is a valuable follow-up parameter in SM.

The association of the Thr715Pro P-selectin genotype with levels of P-selectin in platelet concentrates.

BACKGROUND AND OBJECTIVES: P-selectin is stored in the alpha granules of platelets and in the Weibel Palade bodies of endothelial cells; upon activation, it is translocated to the cell surface and released into the plasma in soluble form. One variant of the P-selectin gene, the Thr715Pro polymorphism, is strongly associated with the plasma levels of soluble P-selectin. In platelet concentrates soluble P-selectin can be regarded mainly platelet derived. MATERIALS AND METHODS: The relation of the genotype with soluble P-selectin, platelet expressed P-selectin and the sum of all forms of P-selectin - comprising soluble P-selectin, platelet surface P-selectin and P-selectin from the alpha granules - was assessed in fresh whole blood and in apheresis platelets suspended in 35% plasma/65% SSP+ obtained from 89 platelet donors. RESULTS: Levels of total P-selectin were genotype associated (P = 0·025); likewise, in fresh whole blood there was an association of soluble P-selectin with genotype (P = 0·02). In platelets suspended in additive solution, however, levels of the storage-associated or TRAP-6 agonist induced increase of platelets' P-selectin were not associated with the genotype. A correlation between levels of soluble P-selectin and surface expression of P-selectin was observed on day 3 of storage in Thr715Thr individuals (P < 0·0001), but not in heterozygotes (Thr715Pro, P = 0·2). CONCLUSION: The donors' genotype has only little influence on levels of soluble P-selectin in apheresis platelets suspended in 35% plasma/65% SSP+.

Oncostatin M is a FIP1L1/PDGFRA-dependent mediator of cytokine production in chronic eosinophilic leukemia.

BACKGROUND: Chronic eosinophilic leukemia (CEL) is a myeloproliferative neoplasm characterized by expansion of neoplastic eosinophils, tissue infiltration, and organ damage. In a subset of these patients, the FIP1L1/PDGFRA (F/P) oncoprotein is detectable. F/P exhibits constitutive tyrosine kinase activity and activates a number of signaling pathways. So far, however, little is known about the role of F/P-dependent proteins in the pathogenesis of CEL. METHODS: A screen for F/P-dependent cytokines was performed in growth factor-dependent human cell lines lentivirally transduced with F/P. Signal transduction pathways were characterized in Ba/F3 cells with doxycycline-inducible expression of F/P and in EOL-1 cells. Cytokine expression was confirmed in patients' material by immunohistochemistry, immunofluorescence, and confocal microscopy. Gene expression analysis, proliferation assays, and chemotaxis assays were used to elucidate paracrine interactions between neoplastic eosinophils and stromal cells. RESULTS: We show that F/P upregulates expression of oncostatin M (OSM) in various cell line models in a STAT5-dependent manner. Correspondingly, neoplastic eosinophils in the bone marrow were found to overexpress OSM. OSM derived from F/P + cells stimulated proliferation of stromal cells. Moreover, OSM-containing supernatants from F/P + cells were found to upregulate production of stromal cell-derived factor-1 (SDF-1)/CXCL12 in human fibroblasts. SDF-1, in turn, induced migration of EOL-1 cells in a dose-dependent manner. CONCLUSIONS: We have identified a F/P-driven paracrine interaction between neoplastic eosinophils and stromal cells that may contribute to tissue fibrosis and accumulation of neoplastic eosinophils in CEL.

Monitoring neutrophils and platelets during casein-induced anaphylaxis in an experimental BALB/c mouse model.

BACKGROUND: With respect to the cellular players, mast cells and basophils have been well studied in experimental murine systemic anaphylaxis models, but the role of neutrophils and platelets is not fully understood today. OBJECTIVE: We tested the hypothesis that neutrophils and platelets might participate in an antigen-induced anaphylaxis model. METHODS: BALB/c mice were sensitized intraperitoneally with alum-adsorbed casein. A period of 2 weeks later, mice were challenged with 100 µg casein intravenously and immediate hypersensitivity reactions were assessed by rectal temperature measurements and monitoring the physical activity. Subsequently, leucocytes were counted in the peripheral blood as well as quantified in situ in typical shock organs like lung, liver and spleen, heart and kidney. RESULTS: Mice sensitized with casein showed casein-specific IgG1, IgE, and IgG2a. When sensitized mice were specifically challenged with casein they developed immediate hypersensitivity reactions including drop of temperature and reduced activity. Furthermore, pronounced peripheral neutropenia and reduced platelet counts correlated with the severity of the hypersensitivity reactions. In the histological analyses of collected tissues we observed lung interstitial neutrophilia using Gr-1 staining. These events occurred specifically in mice sensitized and challenged with casein, in contrast to control groups. CONCLUSIONS: On the basis of our data we suggest that in addition to mast cells and basophils, neutrophils and platelets participate in the anaphylactic response in this BALB/c mouse model. Platelet and neutrophils expressing relevant immunoglobulin receptors may therefore have a synergistic effect with allergen specific IgE as well as IgG antibodies in food-induced anaphylaxis. We suggest that management of these cells could be of clinical importance to handle anaphylaxis.

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.

Determinants of factor VIII plasma levels in carriers of haemophilia A and in control women.

Factor VIII (FVIII) levels show a considerable variability in female carriers of haemophilia A. Presently, the reasons for this are poorly understood. The aim of the study was to elucidate the influence of genetic and non-genetic parameters on FVIII plasma levels in carriers (n = 42). Results were compared with age-matched healthy women without carriership of haemophilia A (n = 42). Each carrier was tested for the family-specific mutation, ABO blood group, FVIII level, von Willebrand factor (VWF) antigen and activity and C-reactive protein (CRP). FVIII levels were lower in carriers compared to non-carriers [74% (51-103) vs. 142% (109-169), P < 0.001]. No statistically significant differences were observed between the two groups with respect to VWF activity, prothrombin-time, hs-CRP, fibrinogen, body mass index (BMI), age and smoking status as well as the distribution of ABO blood groups. In non-carriers, FVIII was statistically significantly correlated with BMI, activated partial thromboplastin time (APTT), VWF antigen, hs-CRP and fibrinogen. In carriers, significant correlations between FVIII and APTT, VWF antigen and activity were found, whereas BMI, hs-CRP or fibrinogen did not correlate with FVIII. In non-carriers, the association of FVIII with ABO blood groups was statistically significant (P = 0.006), but not in carriers of haemophilia A (P = 0.234). The type of FVIII gene mutation did not influence FVIII levels. Carrier status is the major determinant of a carrier;s FVIII plasma level. Factors known to influence FVIII levels in the general population do not significantly affect FVIII activity in carriers, neither does the type of mutation influence FVIII levels.

Relative seroprevalence of human herpes viruses in patients with chronic lymphocytic leukaemia.

BACKGROUND: Herpes virus infections may have a significant role in chronic lymphocytic leukaemia (CLL) due to their ability to modulate the host's immune system. MATERIALS AND METHODS: We examined the seroprevalence of four herpes viruses [Cytomegalovirus (CMV), Epstein-Barr Virus (EBV), human herpes virus (HHV)-6 and -7] in a cohort of European CLL patients (cohort 1, n = 100) in relation to the immunoglobulin variable heavy (IGHV) chain gene use and compared serological results with those obtained from age- and gender-matched healthy adults (n = 100). RESULTS: CMV-seroprevalence was significantly higher in CLL cohort 1 (79%) than in the control cohort (57%, P = 0.001); the seroprevalence of EBV (89% vs. 94%), HHV-6 (73% vs. 60%), or HHV-7 (35% vs. 35%) was not. In CLL cohort 1, use of IGHV3-30 was more prevalent among CMV-seropositive and of IGHV3-21 among HHV-7-seronegative cases. To investigate the generalizability of these findings, we investigated the herpes virus seroprevalence in a second cohort of age-matched CLL patients from a different geographical area (USA, n = 100, cohort 2). In cohort 2, CMV-seroprevalence was comparable with that of the control cohort (53%). Seroprevalence of EBV, HHV-6 and HHV-7 were 85%, 88% and 73% respectively. In CLL cohort 2, use of IGHV3-30 or IGHV3-21 was not associated with any of the herpes viruses investigated. CONCLUSIONS: CMV-seropositivity is associated with CLL in selected patient cohorts. However, the considerable variation in herpes virus-specific seropositivity between geographically distinct CLL cohorts indicates that seropositivity for any of the four human herpes viruses investigated is not generally associated with CLL.

Impact of Escherichia coli K12 and O18:K1 on human platelets: Differential effects on platelet activation, RNAs and proteins.

Blood platelets can interact with bacteria, possibly leading to platelet activation, cytokine and microparticle release and immune signalling. Besides, bacteria can also affect the platelet RNA content. We investigated the impact of non-pathogenic K12 and pathogenic O18:K1 Escherichia (E.) coli strains on platelet activation, RNA expression patterns, and selected proteins. Depending on bacteria concentration, contact of platelets with E. coli K12 lead to an increase of P-selectin (24-51.3%), CD63 (15.9-24.3%), PAC-1 (3.8-14.9%) and bound fibrinogen (22.4-39%) on the surface. E. coli O18:K1 did not affect these markers. Sequencing analysis of total RNA showed that E. coli K12 caused a significant concentration change of 103 spliced mRNAs, of which 74 decreased. For the RNAs of HMBS (logFC = +5.73), ATP2C1 (logFC = -3.13) and LRCH4 (logFC = -4.07) changes were detectable by thromboSeq and Tuxedo pipelines. By Western blot we observed the conversion of HMBS protein from a 47 kDA to 40 kDa product by E. coli K12, O18:K1 and by purified lipopolysaccharide. While ATP2C1 protein was released from platelets, E. coli either reduced the secretion or broke down the released protein making it undetectable by antibodies. Our results demonstrate that different E. coli strains influence activation, RNA and protein levels differently which may affect platelet-bacteria crosstalk.

Clinical and prognostic significance of histamine monitoring in patients with CML during treatment with imatinib (STI571).

BACKGROUND: Although imatinib is highly effective in chronic myeloid leukemia (CML), drug-resistance may occur. Therefore, monitoring of minimal residual disease (MRD) during treatment with imatinib is important. However, most MRD-parameters are expensive and require special technology. We determined the value of histamine as MRD-marker in CML. PATIENTS AND METHODS: Histamine levels were measured serially in whole blood samples before and during imatinib therapy in 80 CML patients by radioimmunoassay. RESULTS: Histamine levels were highly upregulated in CML at diagnosis compared to healthy controls, and correlated with the presence of basophils. During treatment with imatinib, histamine levels decreased and returned to normal levels in those achieving a complete cytogenetic response (CCR). Loss of CCR during therapy was invariably accompanied by an increase in histamine. Moreover, a histamine level of >100 ng/ml three or six months after start of imatinib was associated with a significantly reduced probability of survival (p<0.05). Whereas basophils were found to correlate well with histamine during imatinib, no correlations were found between histamine and Ph+ metaphases or histamine and BCR/ABL. CONCLUSION: Histamine-monitoring during treatment with imatinib is of prognostic significance.

Evidence of a U-shaped association between factor XII activity and overall survival.

INTRODUCTION: The clinical relevance of decreased coagulation factor XII (FXII) plasma activity as a risk factor for both venous and arterial thrombosis is still discussed controversially. The current study evaluated the predictive value of FXII levels for all-cause mortality in a large Viennese patient cohort. PATIENTS AND METHODS: Individuals, whose FXII activity levels were determined for suspected coagulation disorders or thrombophilia screening between 1991-2003 were included in this study (n = 8936, 51% male, 49% female, median age 43 years). Death/survival was determined by record linkage with the Austrian Death Registry. The median observation period was 5 years covering a total of 46 400 person years; the death rate was 17.1%. For Cox regression analysis, FXII plasma activity was divided into 11 categories of 10% steps with the category of > 100% FXII serving as a reference category. RESULTS: With decreasing FXII plasma activity, hazard ratios for all-cause mortality gradually increased linearly from 1.0 in the > 100% category to 1.5 (95% CI: 1.2-1.9) in the 80-90% category to 4.7 (95% CI: 3.4-6.5) in the 10-20% category. Similar results were obtained, when only vascular mortality or death as a result of ischemic heart disease was considered. No significant increase in all-cause mortality (HR: 1.4, 95%CI 0.7-2.8) was observed in the small group of FXII-deficient subjects [0-10% category (n = 58)]. CONCLUSIONS: This study first demonstrates a strong and almost linear association of FXII plasma activity between 90% and 10% with all-cause mortality in a large Viennese patient cohort. Interestingly, mortality rates are not increased when FXII activity is below 10%, resulting in a U-shaped survival curve.

4G4G genotype of the plasminogen activator inhibitor-1 promoter polymorphism associates with disseminated intravascular coagulation in children with systemic meningococcemia.

BACKGROUND: Meningococcal disease may present as sepsis, meningitis or a combination of both. Impaired fibrinolysis and massive elevation of the plasminogen activator inhibitor-1 (PAI-1) is a characteristic feature of meningococcal sepsis. We and others have reported an association between mortality and the functional 4G/5G promoter polymorphism of the PAI-1 gene in children with meningococcal sepsis. OBJECTIVE: Multicenter study to investigate the association of the 4G/5G PAI-1 polymorphism and disseminated intravascular coagulation (DIC) in children with meningococcal disease in a Central European population. PATIENTS/METHODS: Blood samples and clinical information of 326 previously healthy children with meningococcal infection were collected from 95 pediatric hospitals in Germany, Switzerland, Italy, and Austria from 2000 to 2002. RESULTS: DIC, defined as platelet counts below 100 G L(-1), increased D-dimer levels and prolonged prothrombin time, was significantly associated with the 4G4G genotype [31 of 63 (49%) vs. 55 of 175 (31%), P = 0.014], resulting in a hazard ratio (HR) of 1.5 (95% confidence interval 1.1-2.1) to develop DIC. Carriers of the 4G4G genotype showed significantly lower platelet counts (183 G L(-1) vs. 227 G L(-1), P = 0.009) on admission. Fibrinogen and C-reactive protein levels were not associated with the PAI-1 4G/5G polymorphism, nor were white blood cell counts. CONCLUSIONS: Our data show a correlation between the 4G4G genotype of the PAI-1 gene and development of DIC in meningococcal infection.

Low-density lipoprotein receptor-related protein 1 polymorphism 663 C > T affects clotting factor VIII activity and increases the risk of venous thromboembolism.

BACKGROUND: Clotting factor (F) VIII is an independent risk factor for primary and recurrent venous thromboembolism (VTE). The causes for high plasma FVIII levels are not fully understood, but an involvement of genetic factors has been demonstrated. A multifunctional endocytic receptor, low-density lipoprotein receptor-related protein 1 (LRP1), mediates cellular uptake and subsequent degradation of FVIII and may contribute to variations in FVIII levels. OBJECTIVE: We assessed the association of a genetic variation of LRP1 (663C > T) with basal FVIII levels and the risk of venous thrombosis in a group of high-risk patients and in healthy controls. PATIENTS AND METHODS: One hundred and fifty-two patients with a history of recurrent VTE (median age 56 years, 47% women) were compared with 198 age- and sex-matched controls (median age 53 years, 50% women). The LRP1 663C > T genotype was analyzed by mutagenic separated polymerase chain reaction assay and heterozygosity was confirmed by sequence analysis. RESULTS: LRP1 663C > T genotype distribution differed significantly between patients (663CC n = 138, 663CT n = 14) and controls (663CC n = 190, 663CT n = 8; P = 0.048). In multivariable linear regression analysis including LRP1 663C > T, ABO blood group, von Willebrand factor antigen, C-reactive protein and age, LRP1 663CT was independently associated with FVIII activity (P = 0.02). LRP1 663CT was also associated with increased odds for VTE following adjustment for blood group O, FV Leiden and the prothrombin variation 20210G > A in multivariate analysis (odds ratio 3.3, 95% CI 1.3-8.5). CONCLUSIONS: According to our data the LRP1 663C > T polymorphism influences plasma FVIII levels independently of blood group, C-reactive protein and von Willebrand factor and is significantly associated with the risk of VTE.

Polymorphisms in the coagulation factor VII gene and risk of primary intracerebral hemorrhage.

Data concerning genetic factors that may influence the risk of primary intracerebral hemorrhage (PICH) are scarce. One previous study, indicated that the carriers of the (-323)Ins allele of the coagulation factor VII (FVII) have an increased risk of PICH. Another recent study, tested the effect of apolipoprotein E. We analyzed, whether the (-401)G --> T polymorphism, which is in linkage disequilibrium (LD) with the 10-bp Ins/Del polymorphism at position (-323), or the (-402)G --> A polymorphisms of the FVII gene are associated with an increased risk for PICH. We performed a small case-control study in 85 patients with PICH and in 85 healthy control subjects. To each patient a control was individually matched for age, gender, and hypertension. We did not find any significant differences in allele frequencies for the A allele of the FVII (-402)G --> A polymorphism (0.25 vs. 0.25; P = 0.900, OR = 1, ns.) nor for the T Allele of the FVII (-401)G --> T polymorphism (0.09 vs. 0.12; P = 0.480, OR = 1.38, ns.). The analysis of haplotype distributions did not reveal significant differences. Our results do not support the hypothesis that the investigated polymorphisms in the FVII gene are significantly associated with the risk for PICH.

Association of low-grade inflammation with nephropathy in type 2 diabetic patients: role of elevated CRP-levels and 2 different gene-polymorphisms of proinflammatory cytokines.

BACKGROUND: Chronic inflammatory processes are thought to play a key role in the development of micro- and macrovascular complications in type 2 diabetes mellitus. An association between low -grade inflammation and type 2 diabetes has been described in some studies. We assayed the association of two frequent polymorphisms in proinflammatory cytokines: the interleukin 6 G(-174)C promoter polymorphism [IL-6G(-174)C], the exon 2 interleukin receptor antagonist insertion deletion polymorphism [IL1RA]) and serum CRP levels with the prevalence of diabetic nephropathy in patients suffering from type 2 diabetes mellitus. SUBJECTS AND METHODS: A total of 141 patients with type 2 diabetes mellitus, with and without diabetic nephropathy was genotyped for the above mentioned polymorphisms: 66 with normoalbuminuria, 31 with microalbuminuria and 44 with macroalbuminuria. CRP levels were analysed by a high sensitivity - immunnephelometric assay. RESULTS: While a significant association be-tween macroalbuminuria and CRP could be observed (p<0,015), no associations were found between IL-6G(-174)C or IL1RA genotype and any stage of nephropathy. CRP-levels were similar in the 3 different IL-6G(-174)C genotypes as well as in the 2 IL1RA genotypes. CONCLUSIONS: In type 2 diabetic subjects elevated CRP levels are associated with an increased prevalence of albuminuria. The two investigated proinflammatory polymorphisms do not seem to contribute to initiation of nephropathy in type 2 diabetic patients but we cannot exclude effects of these polymorphisms on course of nephropathy.

C-reactive protein 3' UTR +1444C>T polymorphism in patients with spontaneous venous thromboembolism.

OBJECTIVE: Data on C-reactive protein (CRP) as a risk indicator of venous thromboembolism are conflicting. A recent study reported higher CRP levels in homozygous carriers of a novel CRP gene polymorphism at the 3' UTR (CRP +1444C>T). We investigated, whether homozygosity for CRP +1444C>T is associated with an increased risk of spontaneous venous thromboembolism (VTE). METHODS AND RESULTS: CRP +1444C>T genotype and plasma levels were assessed in 128 patients with deep venous thrombosis (DVT, 70 females/58 males), 105 with pulmonary embolism (PE, 58 females/47 males) and 122 healthy individuals (60 females/62 males). CRP +1444TT was significantly associated with increased CRP plasma levels in healthy individuals. CRP +1444TT was more frequent (14%) among controls than DVT patients (9%, p=0.26) or PE patients (6%, p=0.05), respectively. No significant deviation from Hardy-Weinberg equilibrium was observed in patients (p=0.8) or controls (p=0.3), respectively. CRP +1444C>T was not significantly associated with CRP levels in patients with VTE. CONCLUSIONS: Homozygous carriers of the CRP 3' UTR +1444C>T polymorphism do not have a significantly increased risk of VTE. Our data support the assumption that a clinically relevant association between CRP and VTE is missing.

High expression of lipoprotein lipase in poor risk B-cell chronic lymphocytic leukemia.

We investigated the pattern of lipoprotein lipase (LPL) expression in B-cell chronic lymphocytic leukemia (B-CLL) and assessed its prognostic relevance. Expression of LPL mRNA as well as protein was highly restricted to leukemic B cells. The intensity of intracellular immunoreactivity of LPL was higher in samples of patients with unmutated immunoglobulin heavy-chain variable region genes (IGV(H)) compared to those with mutated IGV(H) genes. LPL mRNA levels in peripheral blood mononuclear cells (PBMNC) from 104 CLL patients differed by 1.5 orders of magnitude between cases with mutated (N=51) or unmutated (N=53) IGV(H) (median: 1.33 vs 45.22 compared to normal PBMNC). LPL expression correlated strongly with IGV(H) mutational status (R=0.614; P<0.0001). High LPL expression predicted unmutated IGV(H) status with an odds ratio of 25.90 (P<0.0001) and discriminated between mutated and unmutated cases in 87 of 104 patients (84%). LPL expression was higher in patients with poor risk cytogenetics. High LPL expression was associated with a shorter treatment-free survival (median 40 vs 96 months, P=0.001) and a trend for a shorter median overall survival (105 months vs not reached). Our data establish LPL as a prognostic marker and suggest functional consequences of LPL overexpression in patients with B-CLL.

Templated nucleotide addition and immunoglobulin JH-gene utilization in t(11;14) junctions: implications for the mechanism of translocation and the origin of mantle cell lymphoma.

The t(11;14)(q13;q32) between the BCL-1 and immunoglobulin heavy chain gene (IgH) loci in mantle cell lymphoma (MCL) are believed to be mediated by the mechanism of V(D)J recombination similar to the t(14; 18) in follicular lymphoma (FL). We have recently shown that the t(14;18) event creates staggered double-strand breaks in the BCL-2 locus, and that the t(14;18) junctions contain templated nucleotide insertions (T-nucleotides; U. Jäger et al., Blood, 95: 3520-3529, 2000). Reasoning that the earlier (pregerminal center) B-cell origin of MCL might be reflected in a different molecular structure of the chromosomal breakpoints, we PCR-amplified diagnostic samples from 93 patients. Thirty-six samples (39%) were positive for the direct (BCL-1/J(H)) and 23 for both direct and reciprocal (D(H)/BCL-1) junctions. The breaks on chromosome 14 exhibited features of V(D)J-mediated recombination as shown by D(H) and J(H) coding end processing. However, duplications of BCL-1 sequences in 39% of the 23 patients indicate staggered double-strand breaks in the major translocation cluster region (MTC). This is incompatible with V(D)J recombination and indicates a different mechanism of cleavage. The use of J(H)6 in the junctions (39%) was similar to that in the immunoglobulin genes of normal B cells and B-CLL, but considerably less than in FL. Only 2 of 36 samples contained a BCL-1/DJ(H) rearrangement, which was indicative of a previous DJ(H) rearrangement. Most importantly, 19% of the BCL-1/IgH junctions with inserts of > or =5 nucleotides contained error-prone copies (T-nucleotides) of 8-12 nucleotides originating from the surrounding BCL-1 or IgH regions, a lower rate than in FL. No correlation was found between the addition of T-nucleotides and the rate of somatic mutation in the immunoglobulin genes. We conclude that the t(11;14) and t(14;18) use the same basic mechanism of translocation including V(D)J-mediated recombination, double-strand staggered breaks, and template-dependent, error-prone DNA-synthesis. However, the distinct differences in the utilization of J(H) regions suggest that the t(11;14) occurs predominantly during an attempted primary D(H)-J(H) rearrangement in early B cells, whereas the t(14;18) mostly occurs during secondary rearrangement. This is in agreement with the pregerminal center B-cell origin of MCL.

Platelet-borne complement proteins and their role in platelet-bacteria interactions.

Essentials Platelets play an important role in pathogen recognition. Platelets contain several complement factors and can interact with E. coli. Platelet's complement protein C3 differs from plasmatic C3 in its electrophoretic mobility. Upon contact with bacteria, platelets are activated and can enhance complement activation. SUMMARY: Background The role of platelets in immune defense is increasingly being recognized. Platelets bind complement proteins from plasma, initiate complement activation, and interact with bacteria. However, the contribution of platelets to complement-mediated defense against bacterial infections is not known in detail. Objectives To assess platelet interactions with Escherichia coli strains, and evaluate the contributions of platelet complement proteins to host defense. Methods We studied the cell-cell interactions of a pathogenic and a non-pathogenic E. coli strain with platelet concentrates, washed platelets and manually isolated platelets by flow cytometry and ELISA. The presence of complement proteins and complement RNA in megakaryocytes and platelets was analyzed by PCR, RT-PCR, confocal microscopy, and western blotting. Results Incubation with E. coli leads to platelet activation, as indicated by the expression of CD62P and CD63 on the platelet surface. RNA and protein analyses show that megakaryocytes and platelets contain complement C3, and that platelet C3 migrates differently on polyacrylamide gels than plasmatic C3. Activation of platelets by bacteria leads to translocation of C3 to the cell surface. This translocation is not induced by thrombin receptor activating peptide or lipopolysaccharide. Interaction of platelets with E. coli occurs even in the absence of plasma proteins, and is independent of platelet toll-like receptor 4 and α2b ß3 (glycoprotein IIbIIIa). Conclusion Platelets contain a specific form of C3. Importantly, they can modulate immune defense against bacteria by enhancing plasmatic complement activation.