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.

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.

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.

Next-generation sequencing identifies major DNA methylation changes during progression of Ph+ chronic myeloid leukemia.

Little is known about the impact of DNA methylation on the evolution/progression of Ph+ chronic myeloid leukemia (CML). We investigated the methylome of CML patients in chronic phase (CP-CML), accelerated phase (AP-CML) and blast crisis (BC-CML) as well as in controls by reduced representation bisulfite sequencing. Although only ~600 differentially methylated CpG sites were identified in samples obtained from CP-CML patients compared with controls, ~6500 differentially methylated CpG sites were found in samples from BC-CML patients. In the majority of affected CpG sites, methylation was increased. In CP-CML patients who progressed to AP-CML/BC-CML, we identified up to 897 genes that were methylated at the time of progression but not at the time of diagnosis. Using RNA-sequencing, we observed downregulated expression of many of these genes in BC-CML compared with CP-CML samples. Several of them are well-known tumor-suppressor genes or regulators of cell proliferation, and gene re-expression was observed by the use of epigenetic active drugs. Together, our results demonstrate that CpG site methylation clearly increases during CML progression and that it may provide a useful basis for revealing new targets of therapy in advanced CML.

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.

Factor V Leiden mutation increases the risk for venous thromboembolism in cancer patients - results from the Vienna Cancer And Thrombosis Study (CATS).

BACKGROUND: Patients with cancer are at an increased risk for venous thromboembolism (VTE). The risk varies markedly in different patient populations. Factor V (FV) Leiden is the most common genetic risk factor for VTE, and the impact of FV Leiden on cancer-associated thrombosis is not yet fully elucidated. OBJECTIVE: To study the impact of FV Leiden on the risk of thrombosis in cancer patients. METHODS: In the prospective observational Vienna Cancer And Thrombosis Study (CATS), 982 patients were included and were followed until occurrence of VTE or death, for a maximum period of 2 years. FV Leiden was determined by genotyping at inclusion. Main outcome measures were symptomatic or lethal objectively confirmed VTE. RESULTS: Of the 982 patients, FV Leiden was diagnosed in 72 (7.3%, 70 were heterozygous and 2 were homozygous). Ten of 72 (13.9%) patients with FV Leiden developed VTE, whereas this was the case in 69 of 910 (7.6%) patients without FV Leiden. In multivariate analysis that included age, sex, different tumor types, tumor stage, newly diagnosed vs. recurrence of disease, and the treatment modalities, the hazard ratio was 2.0 (95% confidence interval 1.0-4.0). In Kaplan-Meier analysis, the probability for development of VTE was 13% in those with and 5.7% in those without FV Leiden after 6 months; after 1 year, the corresponding risks were 15% and 7.3%. CONCLUSIONS: FV Leiden is a genetically determined and thus disease-independent parameter, which is associated with VTE in cancer patients and could therefore be used for individual risk assignment.

No impact of endogenous prothrombotic conditions on the risk of central venous line-related thrombotic events in children: results of the KIDCAT study (KIDs with Catheter Associated Thrombosis).

BACKGROUND AND OBJECTIVE: Central venous lines (CVLs) are the major exogenous risk factor for deep venous thrombosis (DVT) in children. The study objective was to assess whether endogenous prothrombotic conditions contribute to the risk of CVL-related DVT in children. METHODS: This was a cohort study of consecutive children with heart disease requiring CVLs for perioperative care. CVLs were inserted percutaneously in the upper venous system and patients received prophylaxis with continuous unfractionated heparin (50 u kg(-1)  d(-1) ). Blood samples to test for prothrombotic conditions were collected prospectively and assayed in a blinded fashion. Outcome assessment was by screening for DVT by venography, venous ultrasound and echocardiography. RESULTS: The study population consisted of 90 children, median age 2.7 years (0 months-18 years). Prevalence rates of antithrombin deficiency, protein C deficiency, protein S deficiency, heterozygous factor V Leiden, prothrombin G20210A mutation, methylentetrahydrofolate C677TT genotype, hyperhomocysteinemia, lupus anticoagulant, anticardiolipin antibodies and increased levels of lipoprotein (a) were within the range reported for the general population. At least one prothrombotic condition was present in 38% of children and combined abnormalities in 8%. The incidence of DVT was 28% (25/90), and most DVTs were asymptomatic. None of the prothrombotic conditions showed a significant association with DVT. The population attributable risk (i.e. the risk of DVT in the overall population attributable to a specific condition) did not exceed 2.2%. CONCLUSION: Prothrombotic conditions did not have an important impact on the risk of DVT in children with short-term CVLs. The results of the study suggest that screening for prothrombotic conditions is not justified in this setting.

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+.

Biomarkers for arterial and venous thrombotic disorders.

The haemostatic system maintains the blood in a fluid state, but allows rapid clot formation at sites of vascular injury to prevent excessive bleeding. Unbalances within the haemostatic system can lead to thrombosis. Inspite of successful research our understanding of the disease pathogenesis is still incomplete. There is great hope that genetic, genomic, and epigenetic discoveries will enhance the diagnostic capability, and improve the treatment options. During the preceding 20 years, the identification of polymorphisms and the elucidation of their role in arterial and venous thromboses became an important area of research. Today, a large body of data is available regarding associations of single nucleotide polymorphisms (SNPs) in candidate genes with plasma concentrations and e. g. the risk of ischaemic stroke or myocardial infarction. However, the results for individual polymorphisms and genes are often controversial. It is now well established that besides acquired also hereditary risk factors influence the occurrence of thrombotic events, and environmental factors may add to this risk. Currently available statistical methods are only able to identify combined risk genotypes if very large patient collectives (>10,000 cases) are tested, and appropriate algorithms to evaluate the data have yet to be developed. Further research is needed to understand the functional effects of genetic variants in genes of blood coagulation proteins that are critical to the pathogenesis of arterial and venous thrombotic disorders. In this review genetic variants in selected genes of the haemostatic system and their relevance for arterial and venous thrombosis will be discussed.

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.

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.

Quantitative monitoring of BCR/ABL1 mutants for surveillance of subclone-evolution, -expansion, and -depletion in chronic myeloid leukaemia.

BACKGROUND: In chronic myeloid leukaemia (CML), clonal evolution with resistance to tyrosine kinase inhibitors (TKIs) is often triggered by BCR/ABL1 mutations. However, in the context of the complex pro-oncogenic signalling networks which ultimately lead to clonal expansion and disease progression, the exact contribution of BCR/ABL1 mutants remains uncertain. Recent data indicate that detection of BCR/ABL1 mutant subclones does not permit prediction of their expansion dynamics and their potential to become drivers of resistant disease. METHODS: To determine the patterns of clonal evolution and the distinct proliferation kinetics of individual BCR/ABL1 mutants during treatment, we employed ligase-dependent polymerase chain reaction (LD-PCR) analysis for quantitative surveillance of CML subclones with various tyrosine kinase domain (TKD) mutations including M244V, L248V, G250E, E255K, T315I, F317L-A/G, M351T and F359V. FINDINGS: Inadequate treatment responses were observed in 27 of 100 patients investigated and 16 were found to bear one or more BCR/ABL1 TKD mutations in separate subclones. Rapid subclone expansion upon onset or switch of TKI treatment was common and sometimes preceded corresponding changes in BCR/ABL1 transcript levels. Mutant subclones were found to respond differentially and sometimes unexpectedly to various treatment modalities. Decline and persistent depletion of specific mutation-bearing subclones in response to treatment could be documented by LD-PCR surveillance. INTERPRETATION: The observations show that quantitative monitoring of mutant BCR/ABL1 subclones by LD-PCR is a powerful tool for detection of clonal evolution, subclone-expansion and subclone-depletion and can contribute to optimised management of patients with CML.

Defective diacylglycerol-induced Ca2+ entry but normal agonist-induced activation responses in TRPC6-deficient mouse platelets.

BACKGROUND: Platelet adhesion, activation and aggregation at sites of vascular injury are essential processes for primary hemostasis. Elevation of the intracellular Ca(2+) concentration is a central event in platelet activation but the underlying mechanisms are not fully understood. Store-operated calcium entry (SOCE) through Orai1 was shown to be the main Ca(2+) influx pathway in murine platelets, but there are additional non-store-operated Ca(2+) (non-SOC) and receptor operated Ca(2+) (ROC) channels expressed in the platelet plasma membrane. OBJECTIVE: Canonical transient receptor potential (TRPC) channel 6 is found both in human and murine platelets and has been proposed to mediate diacylglycerol (DAG) activated ROCE but also a role in the regulation of SOCE has been suggested. METHODS: To investigate the function of TRPC6 in platelet Ca(2+) signaling and activation, we analyzed platelets from mice deficient in TRPC6 using a wide range of in vitro and in vivo assays. RESULTS: In the mutant platelets, DAG activated Ca(2+) influx was found to be abolished. However, this did not significantly affect SOCE or agonist induced Ca(2+) responses. Platelet function in vitro and in vivo was also unaltered in the absence of TRPC6. CONCLUSION: Our results indicate that DAG activated ROCE is mediated exclusively by TRPC6 in murine platelets, but this Ca(2+) influx has no major functional relevance for hemostasis and thrombosis. Further, in contrast to previous suggestions, based on studies with human platelets, TRPC6 appears to play an insignificant role in the regulation of SOCE in murine platelets.

The role of fibrinogen plasma levels, the -455G>A fibrinogen and the factor XIII A subunit (FXIII-A) Val34Leu polymorphism in cancer-associated venous thrombosis.

Venous thromboembolism (VTE) is a life-threatening complication in cancer patients. Identification of risk factors has been in focus in the past years. Functional single nucleotide polymorphisms (SNP) of coagulation factors known to influence the concentration or function may be considered to influence the risk of VTE in cancer patients. We evaluated the influence of fibrinogen plasma levels, the -455G>A SNP in the fibrinogen beta gene and the Val34Leu (163G>T) SNP in the factor XIII A-subunit (FXIII-A) gene on the risk of VTE. In 1,079 tumour patients recruited for the prospective Vienna Cancer and Thrombosis Study (CATS) fibrinogen levels were determined by the Clauss method. The FXIII-A Val34Leu and the fibrinogen -455G>A SNPs were tested by allele-specific PCR. The median follow-up time was 604 days, 83 thrombotic events occurred. The median fibrinogen level was 381 mg/dl (25th-75th percentile: 312 to 467). In a multivariable Cox model adjusted to chemotherapy, surgery, radiotherapy, age and sex, neither the fibrinogen concentration (hazard ratio [HR] =1.05, confidence interval [CI] 0.839-1.310 p=0.68), nor the -455G>A SNP (HR=0.77, 95%CI 0.491-1.197; p=0.24), nor the Val34Leu SNP (HR=0.99, 95%CI 0.646-1.542 p=0.99) were associated with occurrence of VTE. The fibrinogen concentration was not significantly different among the fibrinogen -455G or A genotype carriers (p = 0.33). Disseminated intravascular coagulation was observed in only five patients, none of these developed VTE. In conclusion, fibrinogen plasma levels, the fibrinogen -455G>A and the FXIII-A Val34Leu polymorphisms were not associated with VTE in our study.

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.

Maternal interleukin-6 (-174) C/C polymorphism is associated with chorioamnionitis and cystic periventricular leucomalacia of the preterm infant.

OBJECTIVE: To evaluate the association between maternal interleukin (IL)-6 G(-174)C polymorphism and cystic periventricular leukomalacia (cPVL) of the preterm newborn. STUDY DESIGN: After searching a local database, we recruited 132 preterm infants with diagnosis of cPVL, 44 Caucasian mothers were also recruited to participate in this candidate gene-association study at a single teritary care center. Data related to maternal IL-6 G(-174)C polymorphisms were compared with 41 controls, and furthermore compared with data from umbilical cord blood samples from a consecutive birth cohort of 395 healthy newborns, and published data from Caucasian populations including 1104 adults, respectively. In addition, subgroup analysis was performed in cases with either history of preterm premature rupture of the membranes (PPROM) or clinical chorioamnionitis (CCA). IL-6 genotyping was performed using an allele-specific polymerase chain reaction technique. RESULT: Frequencies of the IL-6 G(-174)C polymorphisms did not differ between cases (GG, 29.5%; GC, 54.5% and CC, 15.9%) and controls (GG, 34.2; GC, 51.2 and CC, 14.6%). Subgroup analysis of 31 cases with history of PPROM (GG, 25.8; GC, 54.8 and CC 19.4%) and controls did not reveal significant differences, but a significantly higher frequency of the CC genotype was found in 23 cases with a history of CCA (34.8%) compared with controls by either univariate (P=0.032; odds ratio 3.11, 95% confidence interval (CI) 1.11 to 8.68) or multivariate analysis (P=0.049, odds ratio 2.54, 95% CI 1.01 to 6.45). These data were confirmed by a comparing the CC genotype frequency to 395 term controls (CC 14.7%, P=0.005) and to the mean CC genotype frequency of 1104 Caucasian adults (CC 15.6%, P<0.0001). CONCLUSION: Frequencies of the IL-6 G(-174)C polymorphisms did not differ between groups. Subgroup analysis revealed an association of the CC genotype with CCA and cPVL in the preterm newborn.

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.