Human interleukin-27: wide individual variation in plasma levels and complex inter-relationships with interleukin-17A.

Although it is widely believed that interleukin (IL)-27 is anti-inflammatory, its role in controlling human immune responses is not fully established. In particular, its interactions with T helper type 17 (Th)17 cytokines are unclear. Our aims were to establish the relationships between IL-27 and proinflammatory cytokines, including IL-17A, in human sera and cultures of peripheral blood mononuclear cells. Plasma IL-27 levels in 879 healthy humans from 163 families varied widely, but with relatively low heritability (19%). Despite IL-27 including a subunit encoded by Epstein-Barr virus-induced gene 3 (EBI3), there was no correlation of levels with serological evidence of infection with the virus. Although IL-27 has been reported to inhibit IL-17A production, we demonstrated a strong positive correlation in sera, but lower correlations of IL-27 with other proinflammatory cytokines. We verified that IL-27 inhibited IL-17A production by human peripheral blood T cells in vitro, but not that it stimulated IL-10 secretion. Importantly, addition of IL-17A decreased IL-27 production by stimulated T cells but had the opposite effect on resting T cells. Together, these data suggest a model whereby IL-27 and IL-17A exerts complex reciprocal effects to boost inflammatory responses, but restrain resting cells to prevent inappropriate activation.

Novel genetic variants linked to coronary artery disease by genome-wide association are not associated with carotid artery intima-media thickness or intermediate risk phenotypes.

BACKGROUND: It is uncertain whether the novel single nucleotide polymorphisms (SNPs) that have recently been associated with coronary artery disease (CAD) in genome-wide studies also influence carotid atheroma and stroke risk. The mechanisms of their association with CAD are unknown; relationships to other cardiovascular phenotypes may give mechanistic clues. Carotid artery intima-media thickness (CIMT) is a subclinical marker of atherosclerosis associated with stroke. We investigated association of reported CAD risk variants with CIMT, and with other intermediate phenotypes that may implicate causative pathways. METHODS: We studied 1425 members of 248 British Caucasian families ascertained through a hypertensive proband. We genotyped CAD risk SNPs on chromosomes 9 (rs1333049, rs7044859, rs496892, rs7865618), 6 (rs6922269) and 2 (rs2943634) using TaqMan. Merlin software was used for family-based association testing. RESULTS: No significant association was found between genotype at any SNP and CIMT in 846 individuals with acceptable measurements. Nor were SNPs significantly associated with blood pressure, obesity, cholesterol, CRP, interleukin-6, TNF-alpha, or leptin. CONCLUSIONS: These novel CAD variants are not associated with CIMT and do not appear to mediate the risk of atherothrombosis through known risk factors.

ABO(H) blood groups and vascular disease: a systematic review and meta-analysis.

BACKGROUND: Associations between vascular disease and ABO(H) blood groups have a long history, but no consensus exists regarding its magnitude and significance, or whether it relates to all disorders equally. An accurate calculation of risk would allow direct assessment of whether the effects of non-O status on thrombosis risk are of the magnitude predicted by its effect on von Willebrand factor/FVIII levels. METHODS AND RESULTS: We conducted a systematic review and meta-analysis of studies reporting associations with non-O blood groups. This gave pooled odds ratios of 1.25 [95% confidence interval (CI) 1.14-1.36] for myocardial infarction (MI), 1.03 (95% CI 0.89-1.19) for angina, 1.45 (95% CI 1.35-1.56) for peripheral vascular disease, 1.14 (95% CI 1.01-1.27) for cerebral ischemia of arterial origin, and 1.79 (95% CI 1.56 to 2.05) for venous thromboembolism (VTE). However, restriction to prospective MI studies only did not confirm the association (OR 1.01; 95% CI 0.84-1.23), although these studies may have failed to capture early-onset disease. For VTE, using a combined group of OO/A(2)A(2)/A(2)O as index, the combination of A(1)A(1)/A(1)B/BB gave an OR of 2.44 (95% CI 1.79-3.33) and A(1)O/ BO/A(2)B an OR of 2.11 (95% CI 1.66-2.68). CONCLUSIONS: This study confirms the historical impression of linkage between some vascular disorders and non-O blood group status. Although the odds ratios are similar to those predicted by the effect of ABO(H) on von Willebrand factor levels, further work is required to assess risk prospectively and to refine the effect of reducing O(H) antigen expression on thrombosis. However, as non-O and particularly A(1)A(1), A(1)B, BB constitute a significant proportion of the population attributable fraction of VTE, there may be a role for more widespread adoption of ABO(H) typing in testing strategies.

Immunomodulation against leukemias and lymphomas: a realistic future treatment?

Immunotherapy offers the potential for cure of malignancy without the side effects too commonly seen with conventional chemotherapy. The efficacy of allogenic transplantation and monoclonal antibodies in hematological malignancies illustrate this principle and are now part of routine care. Newer cell based and molecular approaches aimed at stimulating cytotoxic activity against host derived tumor associated antigens are able to 'boost' anti-tumor immunity as judged by immunological assays in vitro. Although clinically meaningful responses were originally less evident, more promising results are now being reported. Our growing understanding of tumor immunology provide rationales for further improvements in the field.

Platelet and coagulation activation markers in myeloproliferative diseases: relationships with JAK2 V6I7 F status, clonality, and antiphospholipid antibodies.

BACKGROUND AND OBJECTIVES: Patients with myeloproliferative disease (MPD) have an increased risk of thrombosis. We studied markers of platelet and coagulation activation in a large cohort of patients with MPD (n = 118) and related this to Janus Kinase 2 (JAK2) V617 F mutation status, a marker of clonality, and the presence of antiphospholipid antibodies (APA), all of which have been associated with thrombosis in MPD. METHODS: D-dimer, thrombin-antithrombin complexes (TAT), prothrombin fragments 1 + 2 (F(1+2)), soluble E-selectin (sE-selectin), and soluble P-selectin (sP-selectin) levels were compared between patients and hypertensive controls (n = 127). Assays for lupus anticoagulant (LA), anticardiolipin antibodies (ACA), antibeta2 glycoprotein 1 antibodies (anti-beta(2)GP1), and antiprothrombin antibodies (alpha-Pro) were also performed. The JAK2 V617F mutation status was determined in the cohort using amplification refractory mutation system (ARMS) polymerase chain reaction. Disease clonality was determined in 54 patients using the HUMARA assay. RESULTS: sP-selectin was significantly increased in patients with MPD (P

Pyoderma gangrenosum as a cause of splenomegaly and association with a T-cell clone.

The spectrum of clinical presentation of haematological disease is wide. We highlight two features of this principle: a rare cause of a 'haematological' presentation and a possible haematological cause of a disease not normally considered as such. A case of systemic pyoderma gangrenosum presented with splenomegaly in the absence of a rash. A clonal gamma- and beta-T-cell receptor rearrangement was demonstrated. Such clones may be a general phenomenon involved in the pathogenesis of this condition.

The procoagulant potential of environmental particles (PM10).

BACKGROUND AND AIMS: Epidemiology studies have shown that cardiovascular (CV) disease is primarily responsible for the mortality associated with increased pulmonary environmental particle (PM10) exposure. The mechanisms involved in PM10 mediated CV effects are unknown although changes in plasma viscosity and in the homoeostasis of blood coagulation have been implicated. It was hypothesised that PM10 exposure would result in an inflammatory response and enhance the activation of the extrinsic coagulation mechanisms in pulmonary and vascular cells in culture. METHODS: Primary human monocyte derived macrophages and human umbilical cord vein endothelial, human alveolar type II epithelial (A549), and human bronchial epithelial (16HBE) cells were tested for their inflammatory and procoagulant response to PM10 exposure. IL-8, tissue factor (TF), and tissue plasminogen activator (tPA) gene expression and protein release, and coagulation enhancing ability of culture media were determined 6 and 24 hours following exposure. RESULTS: The culture media from macrophages and 16HBE bronchial epithelial cells, but not A549 cells, exposed to PM10 had an enhanced ability to cause clotting. Furthermore, H2O2 also increased the clotting activity. Apoptosis was significantly increased in macrophages exposed to PM10 and LPS as shown by annexin V binding. TF gene expression was enhanced in macrophages exposed to PM10, and HUVEC tissue factor and tPA gene and protein expression were inhibited. CONCLUSIONS: These data indicate that PM10 has the ability to alter macrophage, epithelial, and endothelial cell function to favour blood coagulation via activation of the extrinsic pathway and inhibition of fibrinolysis pathways.

Polymorphisms in the interleukin-10 and interferon gamma genes in Hodgkin lymphoma.

Genetic factors are known to be important in the development of Hodgkin lymphoma (HL). Interleukin-10 (IL-10) secretion by both malignant and reactive cells is thought to be important in the pathogenesis of HL especially Epstein-Barr virus (EBV) positive cases. Polymorphisms of the IL-10 gene have been reported to be associated with susceptibility to EBV infection. The cytotoxic response to EBV is determined by a Th1 biased immune response which is characterised by interferon gamma (IFNgamma) secretion. We therefore investigated polymorphisms in the IL-10 (-1082 G/A and -592 C/A) and IFNgamma (intron 1 CA repeat) genes as predisposing factors in the development 147 cases of HL. A difference of borderline statistical significance was demonstrated for the IFNgamma gene polymorphism but significance was lost when analysis was restricted to the common genotypes. No significant differences in the distributions of genotypes were found for the IL-10 gene polymorphisms. IL-10 and IFNgamma levels were also measured on 26 patients with HL. No statistically significant differences were detected when the results were analysed by genotype. We found little evidence IL-10 and IFNgamma genotypes predispose to the development of HL or influence the inflammatory host response.

Phosphodiesterase type 5 inhibitors for the treatment of erectile dysfunction in patients with diabetes mellitus.

Sildenafil, a phosphodiesterase 5 (PDE5) inhibitor, has become a first-line therapy for diabetic patients with erectile dysfunction (ED). The efficacy in this subgroup, based on the Global Efficacy Question, is 56% vs 84% in a selected group of non-diabetic men with ED. Two novel PDE5 inhibitors, tadalafil (Lilly ICOS) and vardenafil (Bayer), have recently completed efficacy and safety clinical trials in 'general' and diabetic study populations and are now candidates for US FDA approval. A summary analysis of the phase three clinical trials of sildenafil, tadalafil and vardenafil in both study populations is presented to provide a foundation on which the evaluation of the role of the individual PDE5 inhibitors for the treatment of patients with ED and DM can be built.

Estimation of mutation rate at human glycophorin A locus in hematopoietic stem cell progenitors.

Surveys of human mutant cells exhibit a few individuals with relatively high "outlying" values, which might be explained by rare mutations occurring during development. To estimate how commonly this occurs, mutant red cell frequencies at the glycophorin A locus in 135 neonates and 109 children and adolescents from three research centers are compared with simulations in which mutations arise from successive cycles of binary fission. The simulations predict the data most accurately when the mutation rate in stem cell precursors is about 2-4 x 10(-7) per division cycle, which is similar to previous estimates from adult stem cell divisions. If these mutation rates are accurate, and the number of stem cell divisions during adult life is as low as previously estimated, it is predicted that up to one-sixth of mutant stem cells over a lifetime arose in early life. However, these mutant stem cells would be difficult to detect in surveys because their distribution within the general population is so skewed.

Prothrombotic genotypes are not associated with pre-eclampsia and gestational hypertension: results from a large population-based study and systematic review.

DNA samples collected as part of a large population-based case-control study were genotyped to examine the associations of five prothrombotic gene polymorphisms with pre-eclampsia (PE) and gestational hypertension (GH). The polymorphisms studied were: G1691A in Factor V (Factor V Leiden; FVL), prothrombin G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T, plasminogen activator inhibitor-1 4G/5G and the platelet collagen receptor alpha2beta1 C807T. A group of 404 women who developed PE were retrospectively compared with 303 women with GH and 164 control women. The frequency of genotypes did not differ significantly between cases of PE or GH and controls for any of the five polymorphisms studied. We conclude that these prothrombotic genotypes are not associated with the development of PE or GH in our population. The systematic review supports our conclusion, for all but cases of severe disease. which appear to be associated with FVL and, to a lesser extent, MTHFR C677T. There is little value in antenatal screening for prothrombotic polymorphisms to predict the development of pre-eclampsia or gestational hypertension.

Monosomy for the most telomeric, gene-rich region of the short arm of human chromosome 16 causes minimal phenotypic effects.

We have examined the phenotypic effects of 21 independent deletions from the fully sequenced and annotated 356 kb telomeric region of the short arm of chromosome 16 (16p13.3). Fifteen genes contained within this region have been highly conserved throughout evolution and encode proteins involved in important housekeeping functions, synthesis of haemoglobin, signalling pathways and critical developmental pathways. Although a priori many of these genes would be considered candidates for critical haploinsufficient genes, none of the deletions within the 356 kb interval cause any discernible phenotype other than alpha thalassaemia whether inherited via the maternal or paternal line. These findings contrast with previous observations on patients with larger (> 1 Mb) deletions from the 16p telomere and therefore address the mechanisms by which monosomy gives rise to human genetic disease.

In vivo platelet activation in atherothrombotic stroke is not determined by polymorphisms of human platelet glycoprotein IIIa or Ib.

Platelet membrane glycoprotein polymorphisms are candidate risk factors for thrombosis, but epidemiological data are conflicting. Thus, demonstration of a genotype-dependent alteration in function is desirable to resolve these inconsistencies. We investigated in vivo platelet activation in acute thrombosis and related this to platelet genotype. Frequencies of the 1b and 2b alleles of the HPA 1a/1b and HPA 2a/2b platelet glycoprotein polymorphisms were determined in 150 (52 men/98 women, mean age 58.3 years) patients with atherothrombotic stroke, and the influence of genotype on markers of platelet activation was assessed. Platelet P-selectin (CD62P) expression and fibrinogen binding was measured using whole blood flow cytometry within 24 h of stroke and 3 months later in 77 patients who provided a repeat blood sample. Results were compared with matched controls. Neither the 1b allele [allele frequency 0.11 vs. 0.13, odds ratio (OR) confidence interval (CI) 0.8 (0.5-1.3)] nor the 2b allele [0.09 vs. 0.07, OR (CI) 1.4 (0.8-2.4)] was significantly over-represented in patients. Increased numbers of activated platelets were found following stroke (acute mean P-selectin expression 0.64% vs. control 0.35%, P < 0.001; acute mean fibrinogen binding 1.6% vs. control 0.9%, P < 0.001). Activation persisted in the convalescent phase (P < 0.001 and P = 0.005 vs. controls for P-selectin and fibrinogen respectively). Expression of P-selectin and fibrinogen was not influenced by either the HPA 1a/1b genotype (P > 0.95 for each marker, Scheffe's test) or the 2a/2b genotype (P > 0.95 for each). Although persisting platelet activation is seen in atherothrombotic stroke, it is independent of HPA 1a/1b and 2a/2b genotypes. These data suggest an underlying prothrombotic state, but do not support the polymorphisms studied as risk factors for thrombotic stroke in this population.

Assessment of mechanism of acquired skewed X inactivation by analysis of twins.

Skewed X-chromosome inactivation in peripheral blood granulocytes becomes more frequent with increasing age, affecting up to half of those over 75 years old. To investigate the mechanisms underlying this phenomenon, X-inactivation profiles in 33 monozygotic and 22 dizygotic elderly twin pairs were studied. Differential methylation-sensitive restriction enzyme cutting at a hypervariable locus in the human androgen receptor gene (HUMARA) was studied on purified granulocytes using T cells as controls. A large genetic effect on skewed granulocytic X inactivation was shown (P <.05); heritability was estimated to be 0.68. A minor part (SD.0151 relative allele frequency [ie, larger/smaller] units) of the observed variance is due to experimental error. A further contributor to acquired skewing is stochastic asymmetric stem cell division, which was modeled and shown as unlikely to account for a substantial part of variance. Two monozygotic twin pairs had X-inactivation ratios skewed markedly in opposite directions, evidence for a further stochastic mechanism, suggestive of a single overrepresented clone. In conclusion, all 3 suggested mechanisms contribute to acquired X inactivation but the dominant mechanism is genetic selection. The observed proportion of putatively clonal hematopoiesis is similar to the lifetime incidence of hematopoietic stem cell malignancy consistent with the concept that clonal hematopoiesis precedes stem cell malignancy.

Plasma homocysteine concentrations in the acute and convalescent periods of atherothrombotic stroke.

BACKGROUND AND PURPOSE: Homocysteine is a proposed causal risk factor for atherosclerosis, but this remains controversial. We measured fasting plasma homocysteine concentrations immediately after atherothrombotic stroke and in the convalescent period to investigate this controversy. METHODS: One hundred six patients (59 men and 47 women, mean age 57.2 [25 to 70] and 56.5 [26 to 69] years, respectively) were recruited within 24 hours of admission, and 82 patients were resampled at least 3 months later. Fasting total plasma homocysteine (tHcy) concentrations were measured by high-performance liquid chromatography. RESULTS: Median tHcy in the acute phase of stroke was not significantly higher than in matched control subjects (men 9.2 [range 4.4 to 22.8] versus 8.7 [4.9 to 20] micromol/L, P:=0.09, Mann-Whitney U: test; women 8.1 [4.8 to 32.3] versus 7.6 [3.3 to 14.4] micromol/L, P:=0.58). Median plasma concentrations increased significantly in the convalescent period (from 8.5 [4.8 to 19.2] to 10.1 [4.3 to 31.5] micromol/L, P:<0.001, Wilcoxon signed rank test) and were then significantly higher than in control subjects in both men and women (P:=0.03 and 0.05, respectively, Mann-Whitney U: test). This did not appear to be explained by alteration in the known covariates red-cell folate, serum B(12), or creatinine concentrations. CONCLUSIONS: Homocysteine concentrations are not elevated after recent atherothrombotic stroke but rise in the convalescent period. These data do not support the hypothesis that raised plasma homocysteine concentrations predate atherothrombotic stroke. Instead, they offer an explanation for the discrepancies between prospective and retrospective studies and suggest that elevated tHcy levels may be caused by the disease process itself.