TLR signalling and adapter utilization in primary human in vitro differentiated adipocytes.

Toll-like receptors (TLRs) are central to innate immunity and yet their expression is widespread and not restricted to professional inflammatory cells. TLRs have been reported on adipocytes and have been implicated in obesity-associated pathologies such as diabetes. Why TLRs are found on adipocytes is not clear although one hypothesis is that they may coordinate energy utilization for the energy intensive process of an immune response. We have explored TLR signalling in primary human in vitro differentiated adipocytes and investigated the specific adapter molecules that are involved. Only lipopolysaccharide (LPS), poly(I:C), Pam3CSK4 and MALP-2 could induce the production of IL-6, IL-8 and MCP-1 by adipocytes. Poly(I:C) alone caused a strong induction of type I interferons, as assessed by IP-10 production. Using siRNA, it was confirmed that LPS-dependent signalling in adipocytes occurs via TLR4 utilizing the adapter molecules MyD88, Mal and TRIF and caused rapid degradation of IκBα. Pam3CSK4 signalling utilized TLR2, MyD88 and Mal (but not TRIF). However, the response to poly(I:C) observed in these cells appeared not to require TRIF, but MyD88 was required for induction of NFκB-dependent cytokines by Poly(I:C). Despite this, IκBα degradation could not be detected in poly(I:C) stimulated adipocytes at any time-point up to 4 h. Indeed, IL-6 transcription was not induced until 8-16 h after exposure. These data suggest that Pam3CSK4 and LPS signal via the expected routes in human adipocytes, whereas poly(I:C)/TLR3 signalling may act via a TRIF-independent, MyD88-dependent route.

Signalling, inflammation and arthritis: NF-kappaB and its relevance to arthritis and inflammation.

In the synovial cells of patients with RA, activation of the nuclear factor-kappaB (NF-kappaB) pathway results in the transactivation of a multitude of responsive genes that contribute to the inflammatory phenotype, including TNF-alpha from macrophages, matrix metalloproteinases from synovial fibroblasts and chemokines that recruit immune cells to the inflamed pannus. This is largely a consequence of activation of the 'canonical' NF-kappaB pathway that involves heterodimers of p50/p65. Whilst much information on the role of NF-kappaB in inflammation has been gleaned from genetic deficiency of the respective genes in mice, important differences exist in the signalling networks between human and murine immune cells and immortalized cell lines. Despite these differences at the molecular level, the importance of NF-kappaB in inflammation is undisputed and inhibition of the pathway is widely believed to have great potential as a therapeutic target in RA. Commercial effort has gone into developing inhibitors of NF-kappaB activation. However, inhibition of the NF-kappaB activation can result in an exacerbation of inflammation if TNF-alpha production by macrophages is not controlled. It will be important that such inhibitors are carefully monitored before their long-term use in chronic inflammatory conditions such as RA.

Haemostatic genetic risk factors in arterial thrombosis.

Haemostasis plays an integral role in arterial thrombotic disease. However, establishing which of the factors are risk factors has proven surprisingly difficult. Because of its technical simplicity and digital nature, the study of haemostatic polymorphisms as risk factors has grown in popularity. Once established as a risk factor, a genetic polymorphism has the potential to aid selective prophylaxis and therapy of disease. Numerous reports have now been published on polymorphisms of coagulation and fibrinolytic factors, of coagulation and fibrinolytic inhibitory proteins, and of platelet membrane glycoprotein receptors. This article describes the polymorphisms and evaluates the results of these studies using the premises of consistency of within-report genotype/phenotype/disease relationships and consistency of outcome between studies. Many studies have been only of association between polymorphisms and disease, a type of study that is prone to error. Furthermore, the collective outcome of these studies has primarily been inconsistent. It is concluded that despite the early promise of polymorphisms as risk factors, fresh approaches differing in scale and design are now required to clarify their possible importance.

Structural and functional implications of the intron/exon organization of the human endothelial cell protein C/activated protein C receptor (EPCR) gene: comparison with the structure of CD1/major histocompatibility complex alpha1 and alpha2 domains.

The endothelial cell protein C/activated protein C receptor (EPCR) is located primarily on the surface of the large vessels of the vasculature. In vitro studies suggest that it is involved in the protein C anticoagulant pathway. We report the organization and nucleotide sequence of the human EPCR gene. It spans approximately 6 kbp of genomic DNA, with a transcription initiation point 79 bp upstream of the translation initiation (Met) codon in close proximity to a TATA box and other promoter element consensus sequences. The human EPCR gene has been localized to 20q11.2 and consists of four exons interrupted by three introns, all of which obey the GT-AG rule. Exon I encodes the 5' untranslated region and the signal peptide, and exon IV encodes the transmembrane domain, the cytoplasmic tail, and the 3' untranslated region. Exons II and III encode most of the extracellular region of the EPCR. These exons have been found to correspond to those encoding the alpha1 and alpha2 domains of the CD1/major histocompatibility complex (MHC) class I superfamily. Flanking and intervening introns are of the same phase (phase I) and the position of the intervening intron is identically located. Secondary structure prediction for the amino acid sequence of exons II and III corresponds well with the actual secondary structure elements determined for the alpha1 and alpha2 domains of HLA-A2 and murine CD1.1 from crystal structures. These findings suggest that the EPCR folds with a beta-sheet platform supporting two alpha-helical regions collectively forming a potential binding pocket for protein C/activated protein C.

Identification of 19 protein S gene mutations in patients with phenotypic protein S deficiency and thrombosis. Protein S Study Group.

Protein S is a protein C-dependent and independent inhibitor of the coagulation cascade. Deficiency of protein S is an established risk factor for venous thromboembolism. We have used a strategy of specific amplification of the coding regions and intron/exon boundaries of the active protein S gene (PROS1) and direct single-strand solid phase sequencing, to seek mutations in 35 individuals with phenotypic protein S deficiency. Nineteen point mutations (16 novel) in 19 probands (or relatives of probands) with venous thromboembolism are reported here. Fifteen of the 19 mutations were expected to be causal and included 10 missense mutations (Lys9Glu, Glu26Ala, Gly54Glu, Cys145Tyr, Cys200Ser, Ser283Pro, Gly340Asp, Cys408Ser, Ser460Pro, and Cys625Arg). Three of the 15 mutations resulted in premature stop codons (delete T 635 producing a stop codon at position 126, Lys368stop and Tyr595stop) and two were at intron/exon boundaries (+1 G to A in intron d and +3 A to C in intron j). Of the remaining four mutations, three were within intronic sequence and one was a silent mutation within the coding region and did not alter amino acid composition. In two of the 10 missense mutations, reduced plasma protein S activity compared with antigen level suggested the presence of variant (type II) protein S.

Genetic and phenotypic analysis of a large (122-member) protein S-deficient kindred provides an explanation for the familial coexistence of type I and type III plasma phenotypes.

Protein S deficiency is a known risk factor for thrombosis. The coexistence of phenotypic type I (reduction in total and free antigen) and type III (reduction in free antigen only) protein S deficiencies in 14 of 18 families was recently reported. We investigated the cause of this phenotypic variation in the largest of these families (122 family members, including 44 affected individuals) using both molecular genetic and phenotypic analysis. We have identified a sole causative mutation (Gly295Val) in three family members representative of the variable phenotype. Complete cosegregation of the mutation with reduced free protein S antigen levels was found, regardless of the total antigen level. Analysis of phenotypic data showed high correlations between total protein S antigen and age in both normal and protein S-deficient family members, irrespective of gender. Free protein S antigen levels were not influenced by age, a finding explained by an association between beta-chain containing C4b-binding protein (C4bBP-beta+) antigen levels and age. We propose that the identified Gly295Val mutation causes quantitative, or type I, protein S deficiency, and that as age increases the total protein S antigen level normalizes with respect to the reference plasma pool, giving rise to a type III protein S-deficient phenotype.

Clarification of the risk for venous thrombosis associated with hereditary protein S deficiency by investigation of a large kindred with a characterized gene defect.

BACKGROUND: Protein S is an important regulatory protein of the coagulation cascade. The risk for venous thrombosis associated with protein S deficiency has been uncertain because all previous risk estimates used phenotypic evaluation alone, which can be ambiguous. OBJECTIVE: To quantitate the risk for thrombosis associated with a characterized protein S gene mutation that causes a Gly295-->Val substitution and protein S deficiency. DESIGN: Retrospective study of a single extended family. SETTING: University hospital referral center. PARTICIPANTS: A 122-member protein S-deficient family, in which 44 members had a recently characterized gene defect. MEASUREMENTS: Comprehensive history of thrombosis, history of exposure to acquired risk factors for thrombosis, levels of total and free protein S antigen, and genotype for the mutation causing the Gly295-->Val substitution. RESULTS: Kaplan-Meier analysis of thrombosis-free survival showed that the probability of remaining free of thrombosis at 30 years of age is 0.5 (95% CI, 0.33 to 0.66) for carriers of the Gly295-->Val mutation compared with 0.97 (CI, 0.93 to 1.0) for normal family members (P < 0.001). In a multivariate Cox regression model that included smoking and obesity, the mutation was a strong independent risk factor for thrombosis (hazard ratio, 11.5 [CI, 4.33 to 30.6]; P < 0.001). For free (but not total) protein S antigen levels, the distributions of persons with and persons without the mutation did not overlap. CONCLUSIONS: Protein S deficiency, as defined by the presence of a causative gene mutation or a reduced level of free protein S antigen, is a strong independent risk factor for venous thrombosis in a clinical affected family.