Molecular signatures of atherosclerotic plaques: An up-dated panel of protein related markers.

Atherosclerosis remains the leading cause of ischemic syndromes such as myocardial infarction or brain stroke, mainly promoted by plaque rupture and subsequent arterial blockade. Identification of vulnerable or high-risk plaques constitutes a major challenge, being necessary to identify patients at risk of occlusive events in order to provide them with appropriate therapies. Clinical imaging tools have allowed the identification of certain structural indicators of prone-rupture plaques, including a necrotic lipidic core, intimal and adventitial inflammation, extracellular matrix dysregulation, and smooth muscle cell depletion and micro-calcification. Additionally, alternative approaches focused on identifying molecular biomarkers of atherosclerosis have also been applied. Among them, proteomics has provided numerous protein markers currently investigated in clinical practice. In this regard, it is quite uncertain that a single molecule can describe plaque rupture, due to the complexity of the process itself. Therefore, it should be more accurate to consider a set of markers to define plaques at risk. Herein, we propose a selection of 76 proteins, from classical inflammatory to recently related markers, all of them identified in at least two proteomic studies analyzing unstable atherosclerotic plaques. Such panel could be used as a prognostic signature of plaque instability.

Variation in allergen content in sublingual allergen immunotherapy with house dust mites.

BACKGROUND: Allergen immunotherapy is a treatment modality which can be applied using different vaccines. The aim of this study was to quantify and compare the allergen content of different house dust mites (HDM)' sublingual treatments and to review the evidence on their efficacy. METHODS: Five sublingual allergen immunotherapy (SLIT) products were ordered and purchased at an ordinary pharmacy and masked for blinding before the study was started. Detection of Dermatophagoides pteronyssinus and Dermatophagoides farinae allergens Der p 1, Der f 1, Der p 2 and Der f 2 was carried out by immunoblotting and fluorescent multiplex. A literature search for meta-analyses and systematic reviews that included SLIT-HDM products was performed. RESULTS: Der p 1 concentrations ranged from 0.6 to 14.5 µg/ml; similar figures were found for Der f 1 that ranged from 0.2 to 12.4 µg/ml. Der p 2+ Der f 2 ranged from 0.2 to 1.5 µg/ml. Data on efficacy are scarce for most of the five products. CONCLUSIONS: Substantial variations regarding allergen content were found among these five SLIT-HDM products. Therefore, it can be necessary to guarantee the quality of the SLIT-HDM products and to demonstrate their effectiveness before they are marketed. It seems necessary, for the moment, to take into account these characteristics of the products before prescribing.

Polyphosphate binds to human von Willebrand factor in vivo and modulates its interaction with glycoprotein Ib.

BACKGROUND: Polyphosphate, a phosphate polymer released by activated platelets, has recently been described as a potent modulator of blood coagulation and fibrinolysis. In blood plasma, polyphosphate binds to and alters the biological functions of factor XII, fibrin(ogen), thrombin and factor VII activating protease. OBJECTIVES: The aim of the present study is to investigate whether polyphosphate also binds to von Willebrand factor (VWF) and alters some of its activities. METHODS/RESULTS: When studying patients with type 1 von Willebrand disease (VWD) and their healthy relatives, we discovered a significant correlation between von Willebrand factor (VWF) and platelet polyphosphate levels. We have also found polyphosphate in preparations of VWF isolated from normal platelets and plasma. Surface plasmon resonance and electrophoretic mobility assays indicated that polyphosphate interacts with VWF in a dose- and time-dependent manner. Treatment of normal plasma with active exopolyphosphatase decreased the VWF ristocetin cofactor (VWF:RCo) activity, a functional measure of VWF binding to platelet glycoprotein receptor Ib. VWF collagen binding and multimerization were unaltered after polyphosphate depletion. Moreover, addition of polyphosphate increased the deficient VWF:RCo activity presented by plasma from patients with type 1 VWD. CONCLUSIONS: Our results reveal that a new role is played by polyphosphate in hemostasis by its interaction with VWF, and suggest that this polymer may be effective in the treatment of some types of VWD.

The effects of hepatitis C virus core protein on functional responses in the NK cell line YTS.

Hepatitis C virus infection affects more than 170 million people worldwide. More than 80% of the patients are not able to eliminate the virus and progress to a chronic infection that usually culminates in complications such as cirrhosis and/or hepatocellular carcinoma. Although the adaptive immune response has been widely shown to be essential for viral clearance, the role of natural killer (NK) cells is not clearly understood. In this study, the effect of HCV core protein is examined on NK cell function, i.e., cytotoxicity and cytokine secretion. The expression of core protein in the YTS NK cell line led to an increase in the percentage of apoptotic cells soon after transduction. The surviving cells exhibited decreased cytotoxicity associated with decreases in perforin and granzyme B expression. Furthermore, the HCV core protein-transduced YTS NK cells had reduced IFNγ production as well as an altered surface receptor expression pattern. These features may correspond to a state of functional anergy similar to that seen in T cells transduced with HCV core protein. Together, these data suggest that HCV core protein may alter NK cell function.

Regulation of interferon-gamma gene expression by nuclear factor of activated T cells.

Transcription factors of the nuclear factor of activated T cells (NFAT) family are thought to regulate the expression of a variety of inducible genes such as interleukin-2 (IL-2), IL-4, and tumor necrosis factor-alpha. However, it remains unresolved whether NFAT proteins play a role in regulating transcription of the interferon- gamma (IFN-gamma) gene. Here it is shown that the transcription factor NFAT1 (NFATc2) is a major regulator of IFN-gamma production in vivo. Compared with T cells expressing NFAT1, T cells lacking NFAT1 display a substantial IL-4-independent defect in expression of IFN-gamma mRNA and protein. Reduced IFN-gamma production by NFAT1(-/-)x IL-4(-/-) T cells is observed after primary in vitro stimulation of naive CD4+ T cells, is conserved through at least 2 rounds of T-helper cell differentiation, and occurs by a cell-intrinsic mechanism that does not depend on overexpression of the Th2-specific factors GATA-3 and c-Maf. Concomitantly, NFAT1(-/-)x IL-4(-/-) mice show increased susceptibility to infection with the intracellular parasite Leishmania major. Moreover, IFN-gamma production in a murine T-cell clone is sensitive to the selective peptide inhibitor of NFAT, VIVIT. These results suggest that IFN-gamma production by T cells is regulated by NFAT1, most likely at the level of gene transcription.

Genomic structure and chromosome location of the human gene encoding the zinc finger autoantigen ZNF330.

We have recently described a novel zinc finger cDNA, ZNF330, which was immunologically characterized as a new human autoantigen, highly conserved during evolution from nematodes to humans. The protein was found at the nucleolus and the cytoplasm in interphase and transiently associates with centromeres in mitosis as determined by immunofluorescence analysis. We now describe that the association of ZNF330 with the nucleolus but not with the cytoplasm is RNA-dependent as shown by RNAse treatment of fixed culture cells, since ZNF330 localization was unaffected by DNAse treatment. We also report the cloning, structural organization and chromosome location of the human ZNF330 gene. The gene is comprised of 10 exons and spans approximately 16 kb of genomic DNA. The conserved residues forming nine CXXC motifs are contained in exons 3 to 9. Several major transcription initiation sites were located 126, 124 and 121 bp upstream of the translation initiation codon ATG, as determined by primer extension analysis. The human ZNF330 gene was mapped by FISH to chromosome 4q31.1-->q31.2, the site of the FRA4C locus previously described as a common fragile site for acquired chromosome instability in humans.

Molecular analysis of the 5' region of human ribosomal transcription factor UBF.

Upstream binding factor, UBF, is a nucleolar autoantigen involved in the transcription of ribosomal DNA genes. Previously, human genomic clones served to demonstrate that an alternative pre-mRNA splicing of a single gene is used to form UBF1 and UBF2. Here, to complete characterizing the 5'end genomic organization of this nucleolar transcription factor, lambda clones containing the human UBF gene were isolated from a human placenta genomic library using a hamster UBF cDNA as a probe. An additional PCR product was isolated from HeLa genomic DNA to cover the first translated 60 nt of the gene containing the ATG initiation codon. We have also determined the transcription start site of the gene by primer extension analysis at nt 188 upstream from the start ATG codon. It served first, to identify an untranslated initial exon on the UBF gene covering the first 121 nt of human UBF cDNA, and also to establish the sequence of the proximal promoter. The human UBF promoter lacks a TATA and CAAT boxes but contains multiple binding sites for SP1, AP1, AP2, TFIID, NF-1 and a single site for NFAT-1. Consequently we have defined the first five exons of the human UBF gene covering 7.5kb. The complete gene now consists of 20 exons with intervening sequences and spans approximately 15kb of DNA.

Two-site interaction of nuclear factor of activated T cells with activated calcineurin.

Transcription factors belonging to the nuclear factor of activated T cells (NFAT) family regulate the expression of cytokine genes and other inducible genes during the immune response. The functions of NFAT proteins are directly controlled by the calcium- and calmodulin-dependent phosphatase calcineurin. Here we show that the binding of calcineurin to NFAT is substantially increased when calcineurin is activated with calmodulin and calcium. FK506.FKBP12 drug-immunophilin complexes inhibited the interaction of NFAT with activated calcineurin much more effectively than they inhibited the interaction with inactive calcineurin, suggesting that part of the interaction with activated calcineurin involved the enzyme active site. We have previously shown that NFAT is targeted to inactive calcineurin at a region distinct from the calcineurin active site (Aramburu, J., Garcia-Cozar, F. J., Raghavan, A., Okamura, H., Rao, A., and Hogan, P. G. (1998) Mol. Cell 1, 627-637); this region is also involved in NFAT binding to activated calcineurin, since binding is inhibited by an NFAT peptide spanning the calcineurin docking site on NFAT. The interacting surfaces are located on the catalytic domain of the calcineurin A chain and on an 86-amino acid fragment of the NFAT regulatory domain. NFAT binding to the calcineurin catalytic domain was inhibited by the calcineurin autoinhibitory domain and the RII substrate peptide, which bind in the calcineurin active site, as well as by the NFAT docking site peptide, which binds to a region of calcineurin distinct from the active site. We propose that, in resting cells, NFAT is targeted to a region of the calcineurin catalytic domain that does not overlap the calcineurin active site. Upon cell activation, displacement of the autoinhibitory domain by calmodulin binding allows NFAT to bind additionally to the calcineurin active site, thus positioning NFAT for immediate dephosphorylation at functional phosphoserine residues.

Defective B7 expression on antigen-presenting cells underlying T cell activation abnormalities in systemic lupus erythematosus (SLE) patients.

Defective T cell functions, including IL-2 production and proliferation, have been shown in SLE patients. After T cell stimulation (first signal), a costimulatory signal (second signal) is required to achieve complete T cell activation. Main costimulatory signals are provided to T cells by B7 antigens (CD80 and CD86, expressed on antigen-presenting cells (APC)) upon interaction with its receptor, the CD28 molecule expressed on T cells. The aim of this study was to investigate the role of CD28/B7 interactions in the impaired T cell responses of SLE patients. We show that stimulation of T cells with phytohaemagglutinin (PHA) in the presence, but not in the absence, of anti-CD28 MoAb or B7+ cells results in tyrosine phosphorylation of specific substrates, transcription of mRNA and production of IL-2 that is indistinguishable in SLE patients and healthy controls. Moreover, proliferation of costimulated T cells from SLE and controls was specifically abrogated by blocking the CD28/B7 interactions by means of addition to the culture of the CTLA4-Ig fusion protein. However, in most patients activated APC failed to up-regulate B7 molecules, giving rise to ineffective costimulatory signalling to T cells. These results indicate that the CD28/B7 costimulatory pathway is defective in SLE patients.