Connective tissue activation. xxxv. detection of connective tissue activating peptide–iii isoforms in synovium from osteoarthritis and rheumatoid arthritis patients: patterns of interaction with other synovial cytokines in cell culture

Objective. To determine whether extracts of unincubated osteoarthritis (OA) and rheumatoid arthritis (RA) synovial tissue contain connective tissue activating peptide–III (CTAP-III) isoforms and prostaglandin E₂ (PGE₂), and whether such extracts have growth-promoting activity, and to determine whether binary combinations of CTAP-III with other cytokines reported to be present in synovial tissue lead to synergistic, additive, or inhibitory effects on growth. Methods. Acid–ethanol extracts of human synovium were examined for growth-promoting activity by measuring formation of ¹⁴C-glycosaminoglycan (¹⁴CGAG) and ³H-DNA in synovial cell cultures; PGE₂ was measured by enzyme immunoassay, and CTAP-III isoforms were identified by Western blotting of extracted proteins separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Growth-promoting activity of CTAP-III and other cytokines was tested in synovial cultures treated with the agonists singly and in binary combination, by measuring changes in synthesis of ¹⁴C-GAG and ³H-DNA. Results. Platelet-derived CTAP-III and a cleavage isoform with the electrophoretic mobility of CTAP-III–des 1–15/neutrophil-activating peptide–2 (NAP-2) and PGE₂ were found in biologically active extracts of synovial samples from patients with RA and OA. Five growth factors (recombinant epidermal growth factor [rEGF], recombinant interleukin-1β [rIL-1β], basic fibroblast growth factor [bFGF], PGE₁, and PGE₂) in binary combination with CTAP-III showed synergism in stimulating GAG synthesis; two (recombinant platelet-derived growth factor type BB [rPDGF-BB] and recombinant transforming growth factor β [rTGFβ]) had an additive effect. In combination with CTAP-III, rEGF and rPDGF-BB had a synergistic effect in promoting DNA synthesis, rTGFβ and rbFGF had an additive effect, and rIL-1β, PGE₁, and PGE₂ were antagonistic. Conclusions. The results suggest that, in addition to endogenous factors, CTAP-III and other plateletderived cytokines may play roles in regulating synovial cell metabolism in RA and OA, and that combinations of growth factors may be more significant than single agents in amplification or suppression of important cell functions.     

Connective tissue activation XXXVIII: heparin/heparanase activity of human platelets resides in a high molecular weight protein,not in connective tissue activating peptide III

OBJECTIVE: Connective tissue activating protein-III (CTAP-III), with molecular weight 9278 Da and isoforms including CTAP-III des 1-15 (neutrophil activating peptide-2, NAP-2) and other amino terminal deletion isoforms, has been isolated from human platelets and characterized. Platelets have also been shown to possess significant heparin/heparanase activity. We investigated whether human platelet heparin/heparanase activity derives from CTAP-III. METHODS: Radial immunodiffusion measurement showed substantial amounts of CTAP-III in plasma from outdated platelet packs. A convenient method for measurement of heparin/heparanase activity is described, and with this method platelet associated plasma was investigated for heparin/heparanase activity assayed against 3H-heparin and 35S-heparan sulfate. RESULTS: Removal of CTAP-III from platelet associated plasma with an immunospecific immunoaffinity column did not remove the heparin/heparanase activity from the plasma. Highly purified CTAP-III eluted from an immunospecific affinity column lacked heparin/heparanase activity. CONCLUSION: Human platelet heparin/heparanase activity resides not in CTAP-III but in a protein or proteins with molecular weight >/= 55 kDa.     

Connective tissue activation. XXXV. Detection of connective tissue activating peptide-III isoforms in synovium from osteoarthritis and rheumatoid arthritis patients: patterns of interaction with other synovial cytokines in cell culture.

OBJECTIVE. To determine whether extracts of unincubated osteoarthritis (OA) and rheumatoid arthritis (RA) synovial tissue contain connective tissue activating peptide-III (CTAP-III) isoforms and prostaglandin E2 (PGE2), and whether such extracts have growth-promoting activity, and to determine whether binary combinations of CTAP-III with other cytokines reported to be present in synovial tissue lead to synergistic, additive, or inhibitory effects on growth. METHODS. Acid-ethanol extracts of human synovium were examined for growth-promoting activity by measuring formation of 14C-glycosaminoglycan (14C-GAG) and 3H-DNA in synovial cell cultures; PGE2 was measured by enzyme immunoassay, and CTAP-III isoforms were identified by Western blotting of extracted proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Growth-promoting activity of CTAP-III and other cytokines was tested in synovial cultures treated with the agonists singly and in binary combination, by measuring changes in synthesis of 14C-GAG and 3H-DNA. RESULTS. Platelet-derived CTAP-III and a cleavage isoform with the electrophoretic mobility of CTAP-III-des 1-15/neutrophil-activating peptide-2 (NAP-2) and PGE2 were found in biologically active extracts of synovial samples from patients with RA and OA. Five growth factors (recombinant epidermal growth factor [rEGF], recombinant interleukin-1 beta [rIL-1 beta], basic fibroblast growth factor [bFGF], PGE1, and PGE2) in binary combination with CTAP-III showed synergism in stimulating GAG synthesis; two (recombinant platelet-derived growth factor type BB [rPDGE-BB] and recombinant transforming growth factor beta [rTGF beta]) had an additive effect. In combination with CTAP-III, rEGF and rPDGF-BB had a synergistic effect in promoting DNA synthesis, rTGF beta and rbFGF had an additive effect, and rIL-1 beta, PGE1, and PGE2 were antagonistic. CONCLUSIONS. The results suggest that, in addition to endogenous factors, CTAP-III and other platelet-derived cytokines may play roles in regulating synovial cell metabolism in RA and OA, and that combinations of growth factors may be more significant than single agents in amplification or suppression of important cell functions.     

Connective tissue activating peptide III induction of synthesis and secretion of plasminogen activator by synovial fibroblasts

Connective tissue activating peptide III (CTAP-III) is a platelet factor that induces, in cultured connective tissue cells, activities observed in chronic inflammation. In this study we measured plasminogen activator secretion by synovial fibroblasts after stimulation by CTAP-III. Increased plasminogen activator secretion was observed 24—48 hours after stimulation. Induction was prevented by dexamethasone (10^-9—10^-7M), cycloheximide (1 μg/ml) and, variably, by actinomycin D (0.3 μg/ml), but not by cytosine arabinoside (10^-4M). This is the first evidence that CTAP-III induces degradative as well as proliferative activity by connective tissue cells.     

Connective tissue activation

Connective tissue activating peptide-III (CTAP-III) isolated from human platelets is a potent mitogen for human connective tissue cells in culture in addition to stimulating glycosaminoglycan synthesis, glucose consumption, and lactate formation. The amino acid composition of apparently homogeneous CTAP-III was determined, confirming the presence of two disulfide links and providing a calculated molecular weight of 11,633 daltons. Comparison of the mitogenic activity of serum and plasma-serum suggests that CTAP-III is a major mitogenic component of human serum. Seventeen strains of human connective tissue cells (synovial, cartilage, dermal and thyroid) incorporated [³H]-thymidine at up to 30 times control at levels under the influence of microgram quantities of CTAP-III and caused detectable increases in thymidine incorporation at levels as low as 10–29 ng/ml. Prostaglandin E₁ (0.01 μg/ml) and dibutyryl cyclic AMP (25 μg/ml) potentiated the glycosaminoglycan stimulating effect of CTAP-III, but not its mitogenic effect. Cycloheximide and actinomycin D blocked the biologic actions of CTAP-III. Cortisol and penicillamine had little effect on the mitogenic activity of CTAP-III, whereas antirheumatic agents such as acetylsalicylic acid and phenylbutazone opposed the mitogenic activity when added to cultures at clinically relevant concentrations. A weak antiheparin factor secreted by platelets, low affinity platelet factor 4 (LA-PF₄), was shown to be similar to CTAP-III in biologic actions, electrophoretic mobility, amino acid composition, and antigenic determinants.     

Down-regulation of neutrophil functions by the ELR(+) CXC chemokine platelet basic protein

The platelet-derived neutrophil-activating peptide 2 (NAP-2, 70 amino acids) belongs to the ELR(+) CXC subfamily of chemokines. Similar to other members of this group, such as IL-8, NAP-2 activates chemotaxis and degranulation in neutrophils (polymorphonuclear [PMN]) through chemokine receptors CXCR-1 and CXCR-2. However, platelets do not secrete NAP-2 as an active chemokine but as the C-terminal part of several precursors that lack PMN-stimulating capacity. As we have previously shown, PMN themselves may liberate NAP-2 from the precursor connective tissue-activating peptide III (CTAP-III, 85 amino acids) by proteolysis. Instead of inducing cell activation, continuous accumulation of the chemokine in the surroundings of the processing cells results in the down-regulation of specific surface-expressed NAP-2 binding sites and in the desensitization of chemokine-induced PMN degranulation. Thus, NAP-2 precursors may be regarded as indirect mediators of functional desensitization in neutrophils. In the current study we investigated the biologic impact of another major NAP-2 precursor, the platelet basic protein (PBP, 94 amino acids). We show that PBP is considerably more potent than CTAP-III to desensitize degranulation and chemotaxis in neutrophils. We present data suggesting that the high desensitizing capacity of PBP is based on its enhanced proteolytic cleavage into NAP-2 by neutrophil-expressed cathepsin G and that it involves efficient down-regulation of surface-expressed CXCR-2 while CXCR-1 is hardly affected. Correspondingly, we found PBP and, less potently, CTAP-III to inhibit CXCR-2- but not CXCR-1- dependent chemotaxis of neutrophils toward NAP-2. Altogether our findings demonstrate that the anti-inflammatory capacity of NAP-2 is governed by the species of its precursors.     

Elevated plasma levels of beta-thromboglobulin and platelet factor 4 in patients with rheumatic disorders and cutaneous vasculitis

The efficacy of plasma levels of β-thromboglubulin (β-TG) and platelet factor 4 (PF4) as markers of the presence and activity of vasculiditic processes in rheumatic diseases were evaluated, first by serial measurement of their levels in a patient with rheumatoid arthritis and a chronic leg ulcer in the course of treatment, and second in 11 patients with rheumatoid arthritis without cutaneous vasculitis, and in nine patients with a variety of rheumatic diseases with cutaneous vasculitis. In the former, plasma levels of β-TG and PF4 were elevated and slowly reduced in parallel with healing, raised again after relapse, and normalized after disappearance of the leg ulcer. In the latter, both plasma levels were elevated in all of the nine patients with cutaneous vascular lesions and in one of the 11 patients rheumatoid arthritis without skin lesions. Levels of β-TG and PF4 may be useful to estimate the presence of vascular lesions in rheumatic disorders. Received: 5 November 2001 / Accepted: 18 June 2002 Correspondence and offprint requests to: Dr Toshihide Yamamoto, Kishiwada Tokushukai Hospital, 4-22-38 Isonokami-cho, Kishiwada, Osaka 596-0001, Japan. Tel: 81-724-38-8781; Fax: 81-724-37-7395; E-mail: ikyoku@kishiwada.tokushukai.or.jp     

Suppression of glucose-6-phosphate-isomerase induced arthritis by oral administration of transgenic rice seeds expressing altered peptide ligands of glucose-6-phosphate-isomerase

Objective: To investigate the effects of transgenic rice seeds expressing the altered peptide ligand (APL) of human glucose-6-phosphate-isomerase (hGPI_325–339) in mice model of GPI-induced arthritis (GIA).

Methods: We generated transgenic rice expressing T-cell epitope of hGPI_325–339 and APL12 contained in the seed endosperm. The transgenic rice seeds were orally administered prophylactically before the induction of GIA. The severity of arthritis and titers of serum anti-GPI antibodies were evaluated. We examined for IL-17 production in splenocytes and inguinal lymph node (iLN) cells, and analyzed the expression levels of functional molecules in splenocytes.

Results: Prophylactic treatment of GIA mice with APL12 transgenic (APL12-TG) rice seeds significantly reduced the severity of arthritis and titers of serum anti-GPI antibodies compared with non-transgenic (Non-TG) rice-treated mice. APL12-TG and hGPI_325–339 transgenic (hGPI_325–339-TG) rice seeds improved the histopathological arthritis scores and decreased IL-17 production compared with non-TG rice-treated mice. APL12-TG rice-treated GIA mice showed upregulation of Foxp3 and GITR protein in CD4?⁺?CD25?⁺?Foxp3^+?cells in the spleen compared with non-TG rice- and hGPI_325–339-TG rice-treated mice.

Conclusion: APL12-TG rice seeds improved the severity of GIA through a decrease in production of IL-17 and anti-GPI antibodies via upregulation of Foxp3 and GITR expression on Treg cells in spleen.     

Mast cells and neutrophils proteolytically activate chemokine precursor CTAP-III and are subject to counterregulation by PF-4 through inhibition of chymase and cathepsin G

The CXC chemokines platelet factor 4 (PF-4/CXCL4) and connective tissue-activating peptide III (CTAP-III) are released by activated human platelets in micromolar concentrations. So far, neutrophils have been recognized to cleave the precursor CTAP-III to form the active chemokine neutrophil-activating peptide 2 (NAP-2/CXCL7) through limited proteolysis by membrane-associated cathepsin G. Here we show for the first time that activated human skin mast cells (MCs) convert CTAP-III into biologically active NAP-2 through proteolytic cleavage by released chymase. A direct comparison on a cell number basis revealed that unstimulated MCs exceed the CTAP-III-processing potency of neutrophils about 30-fold, whereas MCs activated by IgE cross-linking exhibit even 1000-fold higher CTAP-III-processing capacity than fMLP-stimulated neutrophils. Intriguingly, PF-4 counteracted MC- as well as neutrophil-mediated NAP-2 generation at physiologically relevant concentrations. Addressing the underlying mechanism, we obtained evidence that PF-4 acts as an inhibitor of the CTAP-III-processing enzymes cathepsin G and chymase without becoming cleaved itself as a competitive substrate. Because cleavage of the CTAP-III-unrelated substrate substance P was also affected by PF-4, our results suggest a regulatory role for PF-4 not only in NAP-2 generation but also in neutrophil- and MC-mediated processing of other physiologically relevant inflammatory mediators.     

Circulating high molecular weight adiponectin isoform is heritable and shares a common genetic background with insulin resistance in nondiabetic White Caucasians from Italy: evidence from a family‐based study

Abstract. Menzaghi C, Salvemini L, Paroni G, De Bonis C, Mangiacotti D, Fini G, Doria A, Di Paola R, Trischitta V (IRCCS “Casa Sollievo della Sofferenza”, San Giovanni Rotondo (FG), Italy, Joslin Diabetes Center and Harvard Medical School, Boston, MA, USA, “Sapienza” University; IRCCS Casa Sollievo della Sofferenza‐Mendel Institute, Rome, Italy). Circulating HMW adiponectin isoform is heritable and shares a common genetic background with insulin resistance in nondiabetic White Caucasians from Italy: evidence from a family‐based study. J Intern Med 2010; 267 : 287–294. Objective. Reduced circulating adiponectin levels contribute to the aetiology of insulin resistance. Adiponectin circulates in three different isoforms: high molecular weight (HMW), medium molecular weight (MMW) and low molecular weight (LMW) isoforms. The genetics of adiponectin isoforms is mostly unknown. Our aim was to investigate whether and to which extent circulating adiponectin isoforms are heritable and whether they share common genetic backgrounds with insulin resistance‐related traits. Methods. In a family‐based sample of 640 nondiabetic White Caucasians from Italy, serum adiponectin isoforms concentrations were measured by ELISA. Three single nucleotide polymorphisms (SNPs) in the ADIPOQ gene previously reported to affect total adiponectin levels (rs17300539, rs1501299 and rs677395) were genotyped. The heritability of adiponectin isoform levels was assessed by variance component analysis. A linear mixed effects model was used to test the association between SNPs and adiponectin isoforms. Bivariate analyses were conducted to study genetic correlations between adiponectin isoforms levels and other insulin resistance‐related traits. Results. All isoforms were highly heritable (h² = 0.60–0.80, P = 1.0 × 10^?13–1.0 × 10^?23). SNPs rs17300539, rs1501299 and rs6773957 explained a significant proportion of HMW variance (2–9%, P = 1.0 × 10^?3–1.0 × 10^?5). In a multiple‐SNP model, only rs17300539 and rs1501299 remained associated with HMW adiponectin (P = 3.0 × 10^?4 and 2.0 × 10^?2). Significant genetic correlations (P = 1.0 × 10^?2–1.0 × 10^?5) were observed between HMW adiponectin and fasting insulin, homeostasis model assessment of insulin resistance, HDL cholesterol and the metabolic syndrome score. Only rs1501299 partly accounted for these genetic correlations. Conclusion. Circulating levels of adiponectin isoforms are highly heritable. The genetic control of HMW adiponectin is shared in part with insulin resistance‐related traits and involves, but is not limited to, the ADIPOQ locus.     

Anti–interleukin-6 autoantibodies in rheumatic diseases

Objective. To investigate the presence and the roles of anti–interleukin-6 (anti–IL-6) autoantibodies in rheumatic diseases, and to further elucidate clinical and pathophysiologic significance of anticytokine autoantibodies. Methods. Anti–IL-6 IgG autoantibodies were measured by the ¹²⁵I–IL-6 binding activity of IgG, which was isolated from serum by protein A–Sepharose. Results. Nine of 52 sera (17.3%) from patients with systemic sclerosis (SSc) contained anti–IL-6 antibodies, whereas only 1.9% of sera from normal subjects and 0–5% of sera from patients with other rheumatic diseases were positive for the antibodies. Moreover, anti–IL-6 autoantibodies were found predominantly among patients with the limited form of SSc (42.9%), compared with those with the diffuse form (7.9%). Conclusion. Anti–IL-6 IgG autoantibodies were detected in patients with SSc, particularly those with the limited form of the disease, at a significantly increased frequency compared with normal subjects and patients with other rheumatic diseases. These results suggest that the development of anti–IL-6 autoantibodies and IL-6 may have a role in the pathophysiology of SSc.     

Significant correlation between thrombospondin 1 and serine proteinase expression in rheumatoid synovium

Objective. Thrombospondin 1 (TSP1) is a potent active site inhibitor of leukocyte elastase and cathepsin G. This effect is markedly dependent on the disulfidebond conformation of TSPI, with one isoform, TSPI^0.1, being the most potent. The aims of this study were to examine the expression of different disulfide-bonded isoforms of TSP1 in inflammatory environments in which elastase and cathepsin G are present in variable amounts, and to determine the relationship between these proteinases and their potential inhibitor. Methods. Immunohistochemical staining and histomorphometric analysis were used to examine adjacent sections of synovial tissue from patients with rheumatoid arthritis (RA), osteoarthritis (OA), and meniscal trauma (MT), for expression of TSPl and the TSPl^0.1 isoform, elastase, cathepsin G, and chymase. Results. TSPl localized to vessels and cells within the synovium. TSPl expression was highly up-regulated in RA (mean density 98 cells and vessels / mm², compared with 13 / mm² in OA and 17 / mm² in MT). The TSPl^0.1 isoform was found virtually exclusively in RA, with 44% of vascular TSPl staining being due to the TSPl^0.1 isoform in RA, as compared with 7% in OA (P = 0.0047). Elastase- and cathepsin G-positive cells were abundant in RA, with mean densities of 106 cells / mm² and 103 cells / mm², respectively, compared with 2 cells / mm² and 11 cells / mm² in OA. There was a wide range of both TSPl and proteinase expression within the RA group, but samples containing large numbers of elastase- and cathepsin G-positive cells also showed high expression of TSPl, especially TSPl^0.1,. A strong correlation was found between elastase or cathepsin G densities and TSPl^0.1 expression in blood vessels (r = 0.86 and r = 0.76 respectively, P < 0.01). Conclusion. TSPl^0.1, with the most potent inhibitory activity in vitro, is specifically up-regulated in RA, and this up-regulation is in proportion to the numbers of surrounding leukocytes containing elastase and cathepsin G. One role of TSPl may be to act as a matrix-based regulator of leukocyte-derived serine proteinases in vivo.     

Immunolocalization of the multi-sarco/endoplasmic reticulum Ca2+ATPase system in human platelets

We recently identified a multi-SERCA (sarco/endoplasmic reticulum Ca²⁺ ATPase) system in haemopoietic cells comprising the SERCA 2b, SERCA 3 and a new monoclonal anti-Ca²⁺ ATPase antibody (PL/IM 430) recognizable SERCA isoforms. We have now investigated the subcellular localization of these enzymes in human platelets by Western blotting of subcellular membrane fractions and by immunoelectron microscopy. We precisely defined the recognition specificity of the polyclonal anti-SERCA 2b, anti-SERCA 3, anti-SERCA 1 antibodies as well as of the monoclonal antibody PL/IM 430 by testing their recognition of the tryptic fragments of the SERCA isoforms. The analysis of fragmented membranes enriched in plasma membrane and intracellular membrane components by Western blotting showed that the SERCA 2b and the SERCA 3 isoforms were found in both the plasma membrane and the intracellular membrane fractions, whereas the PL/IM 430 recognizable SERCA isoform was restricted to membranes associated with the plasma membrane fraction. The immunoelectron microscopical study of the SERCA isoforms in resting platelets showed that: (i) the SERCA 2b isoform was expressed in membranes associated with the plasma membrane and open canalicular system, some α-granules and in unidentified membranes; (ii) the SERCA 3 isoform was found associated with plasma and intracellular membranes; and (iii) the PL/IM 430 recognizable SERCA isoform was observed only in structures associated with the cytoplasmic face of the plasma membranes, as confirmed by flow cytometry. Finally, since the PL/IM 430 antibody was raised against intracellular membranes, we looked for a potential membrane redistribution during the isolation procedure used for the preparation of the immunizing membranes. Neuraminidase treatment indeed induced a translocation of the PL/IM 430 recognizable SERCA isoform from plasma to intracellular membranes. Thus, the multi-SERCA system in platelets: (i) is distributed over different platelet membranes, (ii) presents a sub-compartmental organization with some overlapping, and (iii) is partly associated with motile membranes, reflecting an unrecognized level of complexity of Ca²⁺ stores in these cells.     

Autoantibodies to glycyl–transfer RNA synthetase in myositis. Association with dermatomyositis and immunologic heterogeneity

Objective. To elucidate the clinical significance and immunologic heterogeneity of anti–glycyl–transfer RNA (tRNA) synthetase antibodies in polymyositis/dermatomyositis (PM/DM). Methods. Sera from 345 patients with rheumatic diseases, including 91 with myositis, were examined using immunoprecipitation assays. Autoantibodies to aminoacyl-tRNA synthetases were further analyzed with 2-dimensional RNA fractionation and via inhibition of in vitro aminoacylation. Results. Serum from 1 patient with DM and interstitial lung disease immunoprecipitated glycyl-tRNA synthetase along with only 1 of 4 associated tRNAs, in comparison with control anti–glycyl-tRNA synthetase antibodies, which bound the enzyme along with all 4 associated tRNAs. Immunoblotting findings and a lack of in vitro inhibition aminoacylation of tRNA^gly by serum from this patient also suggested differences between the epitope specificity of this serum and that of other sera with anti–glycyl-tRNA synthetase antibodies. Conclusion. This identification of antibodies to glycyl-tRNA synthetase from a patient with DM underscores the association of this specificity with the disease. The finding that these antibodies bound an epitope outside the active site of the synthetase enzyme, in contrast to most anti–aminoacyl-tRNA synthetases, emphasizes the immunologic heterogeneity of these autoantibodies.     

Increased levels of neutrophil-activating peptide-2 in acute coronary syndromes: possible role of platelet-mediated vascular inflammation.

OBJECTIVES: We sought to investigate the role of the CXC chemokine neutrophil-activating peptide-2 (NAP-2) in atherogenesis and plaque destabilization. BACKGROUND: Chemokines are involved in atherogenesis, but the role of NAP-2 in atherosclerotic disorders is unclear. Based on its potential pro-atherogenic properties, we hypothesized a pathogenic role for NAP-2 in coronary artery disease. METHODS: We tested this hypothesis by differential experimental approaches including studies in patients with stable (n = 40) and unstable angina (n = 40) and healthy control subjects (n = 20). RESULTS: The following results were discovered: 1) patients with stable, and particularly those with unstable, angina had markedly raised plasma levels of NAP-2 compared with control subjects, accompanied by increased expression of CXC receptor 2 in monocytes; 2) platelets, but also peripheral blood mononuclear cells (PBMCs), released large amounts of NAP-2 upon stimulation, with a particularly prominent PBMC response in unstable angina; 3) NAP-2 protein was detected in macrophages and smooth muscle cells of atherosclerotic plaques and in monocytes and platelets of coronary thrombi; 4) in vitro, recombinant and platelet-derived NAP-2 increased the expression of adhesion molecules and chemokines in endothelial cells; and 5) whereas aspirin reduced plasma levels of NAP-2, statin therapy increased NAP-2 with stimulating effects both on platelets and leukocytes. CONCLUSIONS: Our findings suggest that NAP-2 has the potential to induce inflammatory responses within the atherosclerotic plaque. By its ability to promote leukocyte and endothelial cell activation, such a NAP-2-driven inflammation could promote plaque rupture and acute coronary syndromes.     

Plasminogen Activator Inhibitor (PAI-1) Activity is Elevated in Asian and Caucasian Subjects with Non-insulin-dependent (Type 2) Diabetes but not in Those with Impaired Glucose Tolerance (IGT) or Non-diabetic Asians

In order to study the plasminogen activator inhibitor activity (PAI-1) in subjects at different risk of non-insulin-dependent diabetes and ischaemic heart disease we examined 89 subjects with diet controlled NIDDM (49 Caucasian, 40 Asian), 29 with impaired glucose tolerance (IGT) (13 Caucasian, 16 Asian), and 149 with normal glucose tolerance (67 Caucasian, 82 Asian). Diabetes was diagnosed by WHO criteria and highly specific, monoclonal antibody-based assays were used to measure insulin, intact proinsulin, and des 31,32 proinsulin. Subjects with NIDDM were significantly more obese, had more central distribution of obesity, higher fasting plasma specific insulin concentrations (NIDDM median 74 pmol l⁻¹ vs IGT 41 pmol l⁻¹, p < 0.01 and vs normals 34 pmol l⁻¹, p < 0.001) and higher PAI-1 activity than normals and those with IGT (NIDDM 23.0 ± 6.9 vs IGT 16.8 ± 5.0, p < 0.001 and vs normals 17.1 ± 6.9 AU ml⁻¹, p < 0.001). However, PAI-1 activity was not significantly different between Asian and Caucasian normals (17.5 ± 7.3 vs 16.5 ± 6.4 AU ml⁻¹, p = ns) and diabetic (22.8 ± 7.3 vs 23.1 ± 6.6 AU ml⁻¹, p = ns) subjects. In addition to relationships with obesity and plasma triglyceride, PAI-1 activity, after controlling for age, sex, body mass index, and waist–hip ratio, was related to fasting insulin (partial r = 0.22, p < 0.001), intact proinsulin (partial r = 0.36, p < 0.001), and des 31,32 proinsulin concentrations (partial r = 0.33, p < 0.001) as measured by highly specific assays. The association of PAI-1 with diabetes was weakened but remained statistically significant (p = 0.042) after controlling for age, sex, ethnicity, obesity, plasma triglyceride, and all insulin-like molecules. We conclude that, although PAI-1 activity is raised in subjects with diet-treated NIDDM, it is normal in subjects with IGT and non-diabetic Asians, populations at high risk of NIDDM and ischaemic heart disease. Raised PAI-1 activity may play an important role in the pathogenesis of macrovascular disease in subjects with NIDDM, but is unlikely to explain excess risk of ischaemic heart disease in Asians and those with impaired glucose tolerance.     

Fewer bone disease events, improvement in bone remodeling, and evidence of bone healing with bortezomib plus melphalan-prednisone vs. melphalan-prednisone in the phase III VISTA trial in multiple myeloma

Objectives: Bone disease is a key presenting feature of myeloma. This post hoc analysis of the phase III VISTA trial of bortezomib plus melphalan–prednisone (VMP) vs. MP in previously untreated myeloma patients assessed clinical bone disease events and changes in alkaline phosphatase (ALP), a marker for osteoblast activation, and serum Dickkopf‐1 (DKK‐1), an inhibitor of osteoblast differentiation, during treatment. Methods: Patients received nine 6‐wk cycles of VMP (bortezomib 1.3 mg/m², days 1, 4, 8, 11, 22, 25, 29, 32, cycles 1–4, days 1, 8, 22, 29, cycles 5–9, plus melphalan 9 mg/m² and prednisone 60 mg/m², days 1–4, cycles 1–9; N = 344) or MP alone (N = 338). Results: Rates of bisphosphonates use during treatment (73% vs. 82%), progression because of worsening bone disease (3% vs. 11%), and requirement for subsequent radiotherapy (3% vs. 8%) were lower with VMP vs. MP. Median maximum ALP increase was significantly higher with VMP vs. MP overall (49.7% vs. 30.3%, P = 0.029), and higher by response group (complete response [CR]: 68.7% vs. 43.9%; partial response [PR]: 41.5% vs. 31.2%). Greater maximum ALP increase was strongly associated with achievement of CR (P ≤ 0.0001) and CR/PR (P ≤ 0.01). Median DKK‐1 decreased with VMP by 694.4 pg/mL and increased with MP by 1273.3 pg/mL from baseline to day 4 (P = 0.0069). Available radiologic data revealed evidence of bone healing in 6/11 VMP‐treated patients, who achieved best responses of three CR, one PR, and two stable disease. Conclusions: These results suggest a positive effect of bortezomib on bone metabolism and potentially bone healing in myeloma.     

Connective tissue activation

Human synovial fibroblasts in culture have been stimulated to augment hyaluronate synthesis and glucose utilization by connective tissue activating peptides (CTAP) extracted from human spleen, lymphocytes, platelets, granulocytes, and tumor cells. The platelet-derived mediator CTAP-III also stimulated DNA synthesis in synovial fibroblasts, but CTAP-I from lymphocytes and spleen did not. The present study demonstrates the mitogenic potential of a granulocyte mediator (CTAP-PMN). Normal granuiocytes were prepared with Ficoll-diatrizoate gradients, platelet contamination being estimated by phase microscopy and by radioimmunoassay for the platelet-specific protein, β thromboglobulin. CTAP-PMN preparations derived from 4 × 10⁷ cells/ml stimulated culture ³H-thymidine incoporation to 3.56 ± 1.32 (SD) times controls levels. Although exposure of praparations to thiols reduced their mitogenicity, CTAP-PMN was relatively heat-stable. SDS gel eletrophoresis of active fractions suggested a molecular weight between 12,700 and 12, 700 daltons. In doulbe immunodiffusion, antisera to CTAP-III showed no reactivity with CTAP-PMN. CTAP-PMN or other granulocyte factors capable of stimulating fibroblast DNA synthesis may play a role in chronic proliferative synovitis or in other settings where exudative inflammation is accompanied by connective tissue growth.