The identification of CCL18 as biomarker of disease activity in localized scleroderma.

BACKGROUND: Localized Scleroderma (LoS) encompasses a group of idiopathic skin conditions characterized by (sub)cutaneous inflammation and subsequent development of fibrosis. Currently, lack of accurate tools enabling disease activity assessment leads to suboptimal treatment approaches. OBJECTIVE: To investigate serum concentrations of cytokines and chemokines implicated in inflammation and angiogenesis in LoS and explore their potential to be utilized as biomarker of disease activity. Additionally, to investigate the implication of potential biomarkers in disease pathogenesis. METHODS: A 39-plex Luminex immuno-assay was performed in serum samples of 74 LoS and 22 Healthy Controls. The relation between a validated clinical measure of disease activity (mLoSSI) and serum analytes was investigated. Additionally, gene and protein expression were investigated in circulating cells and skin biopsies. RESULTS: From the total of 39, 10 analytes (CCL18, CXCL9, CXCL10, CXCL13, TNFRII, Galectin-9, TIE-1, sVCAM, IL-18, CCL19) were elevated in LoS serum. Cluster analysis of serum samples revealed CCL18 as most important analyte to discriminate between active and inactive disease. At individual patient level, CCL18 serum levels correlated strongest with mLoSSI-scores (rs = 0.4604, P < 0.0001) and in longitudinal measures CCL18 concentrations normalised with declining disease activity upon treatment initiation. Additionally, CCL18 was elevated in LoS serum, and not in (juvenile) dermatomyositis or spinal muscular atrophy. Importantly, CCL18 gene and protein expression was increased at the inflammatory border of cutaneous LoS lesions, with normal expression in unaffected skin and circulating immune cells. CONCLUSION: CCL18 is specific for disease activity in LoS thereby providing relevance as a biomarker for this debilitating disease.

Expression of insulin/IGF-I hybrid receptors is increased in skeletal muscle of patients with chronic primary hyperinsulinemia.

The insulin receptor (IR) shares structural and functional homology with the IGF-I receptor (IGF-IR). Hybrid receptors composed of an IR alphabeta-heterodimer and an IGF-IR alphabeta-heterodimer are formed in tissues expressing both molecules. Hybrids behave as IGF-IR rather than IR with respect to ligand binding affinity, receptor autophosphorylation, and hormone internalization and degradation. Factors regulating hybrid formation in vivo are unknown. We recently reported that in skeletal muscle of NIDDM patients, expression of hybrids is increased and correlated with a decrease in IR number and an increase in fasting insulin levels. However, it is not clear whether increased expression of hybrid receptors is a primary defect specifically associated with NIDDM or a secondary event caused by hyperinsulinemia. To address this issue, we used a quantitative microwell-based immunoassay to measure hybrid receptor abundance in skeletal muscle of 11 normal subjects and 12 patients with insulinoma, a state of primary nongenetically determined hyperinsulinemia. Total insulin binding was lower in insulinoma patients than in normal subjects (0.70 +/- 0.18 vs. 4.59 +/- 0.77; P < 0.0001). Total IGF-I binding did not differ between the two groups (0.81 +/- 0.27 and 0.85 +/- 0.10, respectively). The amount of hybrids, expressed as bound/total (B/T), was higher in patients with insulinoma than in normal subjects (0.57 +/- 0.19 vs. 0.36 +/- 0.03; P < 0.0006) and was inversely correlated with total insulin binding (r = -0.64, P < 0.0004). Increased abundance of hybrid receptors was positively correlated with insulin levels (r = -0.82, P < 0.0009) and inversely correlated with insulin-mediated glucose uptake (r = -0.80, P < 0.01). No correlations were observed between insulin-mediated glucose uptake and maximal specific insulin binding (r = 0.19, P = 0.64). These results indicate that insulin-induced IR downregulation may lead to the formation of a higher proportion of hybrid receptors, whose abundance is negatively correlated with in vivo insulin sensitivity. These results, therefore, support a role for insulin in the regulation of hybrid receptors formation and suggest that increased expression of hybrids in NIDDM may be a secondary event caused by hyperinsulinemia rather than a primary defect.

Evidence for glucose/hexosamine in vivo regulation of insulin/IGF-I hybrid receptor assembly.

Hybrid receptors composed of an insulin alphabeta-hemireceptor and a type 1 IGF alphabeta-hemireceptor are formed in tissues expressing both molecules. We recently reported an increased hybrid receptor expression in skeletal muscle of type 2 diabetic patients that is inversely correlated with in vivo insulin sensitivity. It is unclear whether these changes were due to primary abnormalities or to secondary derangements acting in vivo, such as hyperglycemia. To address this, we determined abundance of hybrids in skeletal muscle from three groups of rats: controls, diabetic (90% pancreatectomy), and diabetic treated with phlorizin to normalize plasma glucose levels. We found that the abundance of hybrid receptors was higher in diabetic rats compared with control and phlorizin-treated diabetic rats (percentage of 125I-insulin bound versus total added radioactivity [B/T] = 1.8+/-0.11, 0.4+/-0.01, and 0.32+/-0.04, respectively; P < 0.0001). Fasting plasma glucose levels were positively correlated with hybrids abundance (r = 0.77, P < 0.002). Hybrid receptor protein content, assessed by immunoblotting, was 2.4-fold higher in diabetic rats as compared with control and phlorizin-treated diabetic rats. Because it has been shown that some of the regulatory effects of glucose may be mediated by the glucosamine pathway, we subsequently determined the effect of an in vivo glucosamine infusion on hybrid receptor formation. We found that abundance of hybrids was significantly higher in muscle from glucosamine-treated rats compared with control rats (B/T = 0.17+/-0.02 and 0.11+/-0.01, respectively; P < 0.009). Quantitation of hybrid content by immunoblotting revealed that their abundance was 1.9-fold higher in glucosamine-treated rats. The results demonstrate that 1) elevated glucose levels in diabetic rats are associated with increased expression of hybrid receptors in muscle, 2) correction of hyperglycemia with phlorizin completely reverses increased expression of hybrids, and 3) glucosamine infused into control rats mimics the effects of hyperglycemia on hybrid receptor formation. Thus, the results support the hypothesis that glucose acting, at least in part, through the glucosamine pathway may play an important role in regulating hybrid receptor assembly in vivo.

Increased abundance of insulin/insulin-like growth factor-I hybrid receptors in skeletal muscle of obese subjects is correlated with in vivo insulin sensitivity.

We reported that in noninsulin-dependent diabetes melitus (NIDDM) patients expression of insulin/insulin-like growth factor I (IGF-I) hybrid receptors is increased in insulin target tissues. Whether this is a defect associated with NIDDM or represents a generalized abnormality associated with insulin resistant states is still unsettled. To address this, we applied a microwell-based immunoassay to measure abundance of insulin receptors, type 1 IGF receptors, and hybrid receptors in muscle of eight normal and eight obese subjects. Maximal insulin binding to insulin receptors was lower in obese than in control subjects (B/T = 1.8 +/- 0.20 and 2.6 +/- 0.30; P < 0.03, respectively) and was negatively correlated with insulinemia (r = -0.60; P < 0.01). Maximal IGF-I binding to type 1 IGF receptors was higher in obese than in controls (B/T = 1.9 +/- 0.20 and 0.86 +/- 0.10; P < 0.0001, respectively) and was negatively correlated with plasma IGF-I levels (r = -0.69; P < 0.003). Hybrid receptor abundance was higher in obese than in normal subjects (B/T = 1.21 +/- 0.14 and 0.44 +/- 0.06; P < 0.0003, respectively) and was negatively correlated with insulin binding (r = -0.60; P < 0.01) and positively correlated with IGF-I binding (r = 0.92; P < 0.0001). Increased abundance of hybrids was correlated with insulinemia (r = 0.70; P < 0.002) and body mass index (r = 0.71; P < 0.0019), whereas it was negatively correlated with in vivo insulin sensitivity measured by ITT (r = -0.67; P < 0.016). These results indicate that downregulation of insulin receptors or upregulation of type 1 IGF receptors because of changes in plasma insulin and IGF-I levels may result in modifications in hybrid receptor abundance.

Increased abundance of insulin/IGF-I hybrid receptors in adipose tissue from NIDDM patients.

Insulin/IGF-I hybrid receptors composed of an insulin receptor (IR) alphabeta-hemireceptor and a type 1 IGF receptor (IGF-IR) alphabeta-hemireceptor are formed in tissues expressing both molecules. To date there is a limited information about the proportion of hybrids in tissues of normal or diabetic subjects. In this study, we determined the abundance of hybrids in fat from control and NIDDM subjects by using a microwell-based immunoassay. Microwells coated with MA-20 anti-IR or alpha-IGF-IR-PA anti-IGF-IR antibody were incubated with tissue extracts. Immunoadsorbed receptors were incubated with 125I-insulin or 125I-IGF-I in the presence or absence of unlabeled ligands, and hybrids were quantitated as the fraction of 125I-IGF-I binding immunoadsorbed with MA-20. Abundance of hybrids was increased in NIDDM patients as compared with controls (B/T = 1.29 +/- 0.18 and 0.52 +/- 0.06%; P < 0.008, respectively), and it was inversely correlated with both IR number (r = -0.65; P < 0.002), and in vivo insulin sensitivity measured by insulin tolerance test (r = -0.75; P < 0.005), whereas it was positively correlated with insulinemia (r = 0.63; P < 0.003). Insulin binding affinity was lower in NIDDM subjects than in controls (ED50 = 1.87 +/- 0.32 and 0.54 +/- 0.20 nmol/l; P < 0.009, respectively), and was correlated with the percentage of hybrids. Maximal IGF-I binding was significantly greater in NIDDM patients than controls and was positively correlated with the percentage of hybrids whereas IGF-I binding affinity did not differ between the two groups. Results show that expression of hybrids is increased in fat of NIDDM patients compared to control subjects and is correlated with in vivo insulin sensitivity thus raising the possibility that alterations in expression of hybrids which bind IGF-I with higher affinity than insulin may contribute, at least in part, to insulin resistance.

Specificity of the G1 block induced by ethionine in human lymphocytes in vitro: a flow cytometric analysis.

Ethionine is the ethyl analogue of the amino acid methionine. The agent is well known to have a weak demethylating activity. In addition, its capacity to reversibly block the cell cycle progression in G1 human lymphocytes (HL) without interfering with blastic transformation has been reported. In order to better understand the mechanism by which the agent is able to induce cell cycle block, experiments have been performed by using flow cytometry, in HL. In particular the hypothesis of the involvement of a specific target at the G0/G1 boundary was tested by treating HL at different times after blastic transformation. Starting from the 40th hour after PHA stimulation, ethionine loses its blocking capacity in such a way that cells challenged by the agent do not differ from controls in any one of the tested cell cycle-related parameters. We suggest the agent exerts its blocking activity at a specific stage of the transformation pathway.

Towards effective and safe immunotherapy after allogeneic stem cell transplantation: identification of hematopoietic-specific minor histocompatibility antigen UTA2-1.

Donor T cells directed at hematopoietic system-specific minor histocompatibility antigens (mHags) are considered important cellular tools to induce therapeutic graft-versus-tumor (GvT) effects with low risk of graft-versus-host disease after allogeneic stem cell transplantation. To enable the clinical evaluation of the concept of mHag-based immunotherapy and subsequent broad implementation, the identification of more hematopoietic mHags with broad applicability is imperative. Here we describe novel mHag UTA2-1 with ideal characteristics for this purpose. We identified this antigen using genome-wide zygosity-genotype correlation analysis of a mHag-specific CD8(+) cytotoxic T lymphocyte (CTL) clone derived from a multiple myeloma patient who achieved a long-lasting complete remission after donor lymphocyte infusion from an human leukocyte antigen (HLA)-matched sibling. UTA2-1 is a polymorphic peptide presented by the common HLA molecule HLA-A*02:01, which is encoded by the bi-allelic hematopoietic-specific gene C12orf35. Tetramer analyses demonstrated an expansion of UTA2-1-directed T cells in patient blood samples after several donor T-cell infusions that mediated clinical GvT responses. More importantly, UTA2-1-specific CTL effectively lysed mHag(+) hematopoietic cells, including patient myeloma cells, without affecting non-hematopoietic cells. Thus, with the capacity to induce relevant immunotherapeutic CTLs, it's HLA-A*02 restriction and equally balanced phenotype frequency, UTA2-1 is a highly valuable mHag to facilitate clinical application of mHag-based immunotherapy.

Activation of TrkA tyrosine kinase in embryonal carcinoma cells promotes cell compaction, independently of tyrosine phosphorylation of catenins.

Cadherins are transmembrane receptors whose extracellular domain mediates homophilic cell-cell interactions, while their cytoplasmic domain associates with a family of proteins known as catenins. Although the mechanisms that regulate the assembly and functional state of cadherin-catenin complexes are poorly understood, current evidence supports a role for protein tyrosine kinase activity in regulating cell adhesion and migration. Tyrosine phosphorylation of catenins is thought to mediate loss of intercellular adhesion promoted by activation of receptor tyrosine kinases in epithelial cells. Here, we show that activation of ectopically expressed TrkA, the tyrosine kinase receptor for nerve growth factor (NGF), stimulates embryonal carcinoma P19 cells to develop extensive intercellular contacts and to assemble into closely packed clusters. Thus, activation of receptor tyrosine kinases can differentially regulate adhesiveness by cell-type-specific mechanisms. Furthermore, activation of TrkA in P19 and epithelial MDCK cells induces tyrosine phosphorylation of p120(ctn) and of beta-catenin, irrespective of the elicited cellular response. The selective Src tyrosine kinase inhibitor PP2, however, suppresses NGF- or HGF-induced tyrosine phosphorylation of catenins in both P19 and MDCK cells without interfering with the acquisition of a compacted or scattered phenotype. These findings provide a cogent argument for considering that tyrosine phosphorylation of catenins is dispensable for their interaction with cadherins and, ultimately, for the modulation of cadherin-based cell adhesion by receptor tyrosine kinases.

Population variation analysis at nine loci containing expressed trinucleotide repeats.

The polymorphisms of nine loci containing reiterated CAG repeats were examined in four populations from three continents. Their normal variation was analysed across populations or in subsets of loci grouped according to either the presence/absence of disease-associated expansions or CAG interruptions. A unifying feature of the allele distributions of all loci in all populations was the marked non-normality. Significantly larger numbers of alleles, average lengths, length ranges and variances in repeat number were observed in loci with vs. without known expansions. Significantly longer alleles were found at loci with vs. without interruption of the (CAG)n motif. The nine loci detected levels of inter-population variability comparable to other loci. Altogether the data are at odds with a model assuming that autosomal expressed trinucleotides accumulate variation exclusively by insertion/deletion of a single unit.

Analysis of (CAG)n size heterogeneity in somatic and sperm cell DNA from intermediate and expanded Huntington disease gene carriers.

The length of the CAG repeat responsible for Huntington disease has been analysed by two PCR methods in blood and sperm DNA of 13 expansion carriers, two carriers of intermediate alleles, and four normal subjects. The two methods consistently confirmed size heterogeneity, more pronounced in sperm and confined to the CAG stretch. Based on densitometric scanning of films, four indexes addressed to different features of the PCR pattern were used to quantitate mosaicism. These revealed strong correlations with CAG size and intergenerational instability. However, mosaicism did not show a greater similarity in sibs who shared the same HD chromosome, nor was correlated with instability in the proband's pedigree. Our data do not support the hypothesis that cis-acting factors play a major role in the instability and leave the CAG size per se as the major determinant of sperm cell CAG instability.

Insulin receptor substrate (IRS) transduction system: distinct and overlapping signaling potential.

Insulin receptor substrate (IRS) proteins play a central role in maintaining basic cellular functions such as growth and metabolism. They act as an interface between multiple growth factor receptors possessing tyrosine kinase activity, such as the insulin receptor, and a complex network of intracellular signalling molecules containing Src homology 2 (SH2) domains. Four members (IRS-1, IRS-2, IRS-3, IRS-4) of this family have been identified which differ in their subcellular distribution and interaction with SH2 domain proteins. In addition, differential IRS tissue- and developmental-specific expression patterns may contribute to specificity in their signaling potential.

The sulfonylurea glimepiride regulates intracellular routing of the insulin-receptor complexes through their interaction with specific protein kinase C isoforms.

Sulfonylureas may stimulate glucose metabolism by protein kinase C (PKC) activation. Because interaction of insulin receptors with PKC plays an important role in controlling the intracellular sorting of the insulin-receptor complex, we investigated the possibility that the sulfonylurea glimepiride may influence intracellular routing of insulin and its receptor through a mechanism involving PKC, and that changes in these processes may be associated with improved insulin action. Using human hepatoma Hep-G2 cells, we found that glimepiride did not affect insulin binding, insulin receptor isoform expression, and insulin-induced receptor internalization. By contrast, glimepiride significantly increased intracellular dissociation of the insulin-receptor complex, degradation of insulin, recycling of internalized insulin receptors, release of internalized radioactivity, and prevented insulin-induced receptor down-regulation. Association of PKC-betaII and -epsilon with insulin receptors was increased in glimepiride-treated cells. Selective depletion of cellular PKC-betaII and -epsilon by exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) or treatment of cells with PKC-betaII inhibitor G06976 reversed the effect of glimepiride on intracellular insulin-receptor processing. Glimepiride increased the effects of insulin on glucose incorporation into glycogen by enhancing both sensitivity and maximal efficacy of insulin. Exposing cells to TPA or G06976 inhibitor reversed these effects. Results indicate that glimepiride increases intracellular sorting of the insulin-receptor complex toward the degradative route, which is associated with both an increased association of the insulin receptor with PKCs and improved insulin action. These data suggest a novel mechanism of action of sulfonylurea, which may have a therapeutic impact on the treatment of type 2 diabetes.