The protein tyrosine phosphatase N22 gene is associated with juvenile and adult idiopathic inflammatory myopathy independent of the HLA 8.1 haplotype in British Caucasian patients.

OBJECTIVE: To examine single-nucleotide polymorphisms (SNPs) of the protein tyrosine phosphatase N22 gene (PTPN22) and to study the relationship between PTPN22 and the HLA region in patients with idiopathic inflammatory myopathies (IIMs). METHODS: PTPN22 SNPs were assessed in a large, cross-sectional, case-control study from the UK involving patients with adult or juvenile IIM, comprising patients with polymyositis (PM) (n=114), dermatomyositis (DM) (n=102), myositis associated with another connective tissue disease (myositis-CTD overlap syndrome) (n=64), or juvenile DM (n=101), in comparison with 748 control subjects. Seventeen PTPN22 SNPs were genotyped using the Sequenom MassArray iPLEX platform. Serotyping for myositis-specific/myositis-associated autoantibodies (MSAs/MAAs) was performed by radioimmunoprecipitation. RESULTS: A significant association was noted between the R620W variant (rs2476601) and IIM (corrected P [Pcorr]=0.0009 versus controls), and specifically with the clinical subgroup of PM (Pcorr=0.003 versus controls). A weaker association was noted with juvenile DM (Pcorr=0.009 versus controls). No significant associations were noted after stratification by serologic subgroups. The association with the R620W variant was independent of alleles forming the HLA 8.1 haplotype. No other PTPN22 SNPs were associated with IIM. The PTPN22 haplotype containing the R620W T allele was the only haplotype significantly associated with IIM. CONCLUSION: The R620W variant is a significant risk factor for IIM, independent of the HLA 8.1 haplotype. Unlike that in the HLA region, risk is not increased in individuals possessing MSAs/MAAs. These results are further evidence that the PTPN22 gene confers autoimmune susceptibility.

Tumour necrosis factor-alpha single nucleotide polymorphisms are not independent of HLA class I in UK Caucasians with adult onset idiopathic inflammatory myopathies.

OBJECTIVE: To investigate haplotype tagging single nucleotide polymorphisms (SNPs) in the tumour necrosis factor alpha (TNF-alpha) gene, in UK Caucasian idiopathic inflammatory myopathy (IIM) patients. METHODS: A cross-sectional, case-control study of four TNF-alpha SNPs was undertaken, comparing cases of polymyositis (PM) (n = 121), dermatomyositis (DM) (n = 109) and myositis overlapping with other connective tissue diseases (CTD-overlap) (n = 73) with normal subjects (n = 177). Subgroup analyses were undertaken after stratifying for myositis specific/associated antibodies. RESULTS: The TNF-308A allele demonstrated a strong association with each myositis disease subgroup vs controls [PM, odds ratio (OR) 2.8, 95% confidence interval 1.9-4.3; DM, OR 2.5, 1.6-3.8; CTD-overlap, OR 3.3, 2.1-5.1]. The TNF-308GA/AA genotype frequency was significantly increased vs controls (PM, OR 3.7, 2.1-6.3; DM, OR 3.2, 1.8-5.5; CTD-overlap, OR 5.0, 2.6-9.6) suggesting a dominant model. The association was strongest in patients possessing anti-aminoacyl transfer RNA synthetase (anti-synthetase) (OR 5.1, 3.3-8.0) or -PM-Scl (OR 5.0, 2.7-8.9) antibodies. The -1031T allele was also a significant risk factor in DM (OR 2.2, 1.4-3.6), anti-synthetase (OR 2.9, 1.6-5.3) and -PM-Scl (OR 5.6, 1.9-6.4) antibody positive patients. The TNF-308A association was lost after adjusting for HLA-B*08, but remained independent of HLA-DQB1*02 (both are alleles forming part of the common ancestral haplotype). The HLA-B*08/TNF-308A/DRB1*03/DQA1*05/DQB1*02 haplotype was a risk factor in all myositis subgroups vs controls (OR 3.0, 1.8-5.3). CONCLUSIONS: TNF-308A and -1031T alleles are significant risk factors in the IIMs. In the IIMs, the TNF-308A allele is part of the common ancestral haplotype, but is not independent of HLA-B*08.

Monocyte chemotactic protein-1 single nucleotide polymorphisms do not confer susceptibility for the development of adult onset polymyositis/dermatomyositis in UK Caucasians.

OBJECTIVES: Polymyositis (PM) and dermatomyositis (DM) form part of the idiopathic inflammatory myopathies (IIMs). The chemokine monocyte chemotactic protein-1 (MCP-1) is expressed at sites of the T cell inflammatory response in the IIMs. We thus investigate whether genetic markers in the MCP-1 gene confer disease susceptibility for the development of PM and DM. METHODS: DNA samples were analysed from a group of 195 UK Caucasian IIM patients, comprising 103 PM and 92 DM. Their results were compared with those of 162 ethnically matched controls. The polymorphic positions of three single nucleotide polymorphisms (SNPs) and one insertion-deletion sequence within regions coding for MCP-1 were tested. The SNPs examined were located in intron 1 (rs2857657, C/G), exon 2 (rs4586, A/G) and the 3 ' untranslated region (rs13900, C/T). The insertion-deletion sequence was located in intron 1 (rs3917887, AGCTCCTCCTTCTC/-). Each SNP was tested for Hardy-Weinberg equilibrium and allelic/genotypic associations. Haplotype frequencies were estimated using the Expectation/Maximization algorithm. RESULTS: There was strong linkage disequilibrium present between three out of these four markers. The majority of controls were in Hardy Weinberg equilibrium. No allelic, genotypic or haplotypic associations were detected when comparing PM or DM cases to controls, or when PM and DM were compared with each other. CONCLUSIONS: Genetic markers in the MCP-1 gene do not demonstrate significant genetic associations with the IIMs, and do not discriminate PM from DM in a UK Caucasian population.

Cytotoxicity of colloidal CdSe and CdSe/ZnS nanoparticles.

Cytotoxicity of CdSe and CdSe/ZnS nanoparticles has been investigated for different surface modifications such as coating with mercaptopropionic acid, silanization, and polymer coating. For all cases, quantitative values for the onset of cytotoxic effects in serum-free culture media are given. These values are correlated with microscope images in which the uptake of the particles by the cells has been investigated. Our data suggest that in addition to the release of toxic Cd(2+) ions from the particles also their surface chemistry, in particular their stability toward aggregation, plays an important role for cytotoxic effects. Additional patch clamp experiments investigate effects of the particles on currents through ion channels.

Ion channel drug discovery and research: the automated Nano-Patch-Clamp technology.

Unlike the genomics revolution, which was largely enabled by a single technological advance (high throughput sequencing), rapid advancement in proteomics will require a broader effort to increase the throughput of a number of key tools for functional analysis of different types of proteins. In the case of ion channels -a class of (membrane) proteins of great physiological importance and potential as drug targets- the lack of adequate assay technologies is felt particularly strongly. The available, indirect, high throughput screening methods for ion channels clearly generate insufficient information. The best technology to study ion channel function and screen for compound interaction is the patch clamp technique, but patch clamping suffers from low throughput, which is not acceptable for drug screening. A first step towards a solution is presented here. The nano patch clamp technology, which is based on a planar, microstructured glass chip, enables automatic whole cell patch clamp measurements. The Port-a-Patch is an automated electrophysiology workstation, which uses planar patch clamp chips. This approach enables high quality and high content ion channel and compound evaluation on a one-cell-at-a-time basis. The presented automation of the patch process and its scalability to an array format are the prerequisites for any higher throughput electrophysiology instruments.

Simultaneous optical and electrical recording of single gramicidin channels.

We report here an approach for simultaneous fluorescence imaging and electrical recording of single ion channels in planar bilayer membranes. As a test case, fluorescently labeled (Cy3 and Cy5) gramicidin derivatives were imaged at the single-molecule level using far-field illumination and cooled CCD camera detection. Gramicidin monomers were observed to diffuse in the plane of the membrane with a diffusion coefficient of 3.3 x 10(-8) cm(2)s(-1). Simultaneous electrical recording detected gramicidin homodimer (Cy3/Cy3, Cy5/Cy5) and heterodimer (Cy3/Cy5) channels. Heterodimer formation was observed optically by the appearance of a fluorescence resonance energy transfer (FRET) signal (irradiation of Cy3, detection of Cy5). The number of FRET signals was significantly smaller than the number of Cy3 signals (Cy3 monomers plus Cy3 homodimers) as expected. The number of FRET signals increased with increasing channel activity. In numerous cases the appearance of a FRET signal was observed to correlate with a channel opening event detected electrically. The heterodimers also diffused in the plane of the membrane with a diffusion coefficient of 3.0 x 10(-8) cm(2)s(-1). These experiments demonstrate the feasibility of simultaneous optical and electrical detection of structural changes in single ion channels as well as suggesting strategies for improving the reliability of such measurements.

High quality ion channel analysis on a chip with the NPC technology.

In evaluating ion channel function, electrophysiology, e.g., patch clamping, provides the highest information content. For the analysis of ion channel-modulating compounds, one variant of the patch-clamp technique, the whole-cell configuration, is particularly useful. We present here patch-clamp recordings in the whole-cell configuration and single channel recordings performed with planar patch-clamp chips, which are microstructured from borosilicate glass substrate. The chips are used in the Port-a-Patch, an ion channel research/screening instrument that enables automated patch-clamp experiments on a single cell. A software runs the experiment by executing user-determined protocols for cell positioning, as well as for electrical stimulation and current readout. In various electrophysiological experiments, the high quality of recordings and the versatility of the perfusion of the recorded cells are demonstrated. Quantitative pharmacological experiments are performed on sodium channels expressed in HEK cells using solution volumes in the low microliter range. The exceptionally low volume consumption in the experiments make the system attractive for work on rare or expensive compounds. Due to the low volumes necessary, a rapid solution exchange is facilitated, which is shown on RBL cells. The patch-clamp chip enables a rapid and precise perfusion, allowing sophisticated investigations on ion channel function with the Port-a-Patch.

Microstructured glass chip for ion-channel electrophysiology.

We present a technique by which it is possible to produce a planar sensor for ion channel electrophysiology from glass substrates. Apertures with diameters in the low micrometer to submicrometer range are achieved by irradiation of a glass chip with a single heavy ion and subsequent wet track etching. The function of the device is demonstrated by recordings of single channel currents mediated by the model ion channel gramicidin A in lipid bilayers spanning the micromachined aperture.

Systemic sclerosis with renal crisis and pulmonary hypertension: a report of eleven cases.

OBJECTIVE: To describe a series of systemic sclerosis (SSc) patients with the unusual combination of scleroderma renal crisis (SRC) and pulmonary hypertension (PHT) without interstitial lung disease. METHODS: The medical records of 2,459 SSc patients in the University of Pittsburgh Scleroderma Databank first evaluated between 1972 and 1999 were reviewed. RESULTS: Eleven patients (0.45%) had both SRC and PHT. All had been evaluated since 1979, when angiotensin-converting enzyme (ACE) inhibitor therapy for SRC became available. Seven had SSc with limited cutaneous involvement, and 4 had SSc with diffuse cutaneous involvement. SRC occurred first in all patients except 1, in whom the onsets of SRC and PHT were simultaneous. SRC preceded PHT by a mean of 4.3 years. Four patients had anti-Th/To antibody, 5 had anti-RNA polymerase III antibody, 2 had anti-U3 RNP antibody, and none had anticentromere or antitopoisomerase I antibody. Ten of the 11 patients died, 8 from PHT. Ten patients were being treated with ACE inhibitor drugs when PHT developed. CONCLUSION: In SSc, SRC and PHT are not mutually exclusive complications. SSc patients surviving SRC who have serum antibodies to Th/To, RNA polymerase III, or U3 RNP are at increased risk to develop PHT. ACE inhibitor therapy did not prevent the development of PHT.

Isolated pulmonary hypertension in diffuse cutaneous systemic sclerosis successfully treated with long-term plasma exchange.

Isolated pulmonary hypertension (PHT) in patients with diffuse cutaneous systemic sclerosis (dSSc) is one of the most severe complication. Here we report the case of a 22 year-old white woman with anti-U3RNP antibody-positive dSSc complicated by severe, isolated PHT successfully treated with long-term plasma exchange. This beneficial effect persisted for two years, even after plasma exchange discontinuation. This is the first observation of isolated PHT in dSSc responsive to plasma exchange therapy.

Studies of HLA-DR and DQ alleles in systemic sclerosis patients with autoantibodies to RNA polymerases and U3-RNP (fibrillarin).

OBJECTIVE: To determine the clinical and immunogenetic features of systemic sclerosis (SSc) patients with anti-RNA polymerase (RNAP) or anti-fibrillarin antibodies. METHODS: DNA typing for HLA-DR and DQ alleles was performed in 292 patients with SSc, including 81 with anti-RNAP and 24 with anti-fibrillarin antibodies. The remaining patients had anti-topoisomerase I (anti-topo I; 71), anti-centromere (ACA; 56), anti-Th/To (28), or other antinuclear (32) antibodies. RESULTS: Significant associations were observed in the patients with anti-topo I, ACA, and anti-Th/To antibodies, similar to those previously reported. No significant HLA associations were detected in the 81 patients with anti-RNAP. although weak associations were noted when this group was subdivided on the basis of immunofluorescence staining pattern; i.e., HLA-DR4 was increased in patients with strong nucleolar staining and HLA-DR3 was more frequent in patients with nucleoplasm staining only. No HLA-DR or DQ associations were observed in 24 patients with anti-fibrillarin antibodies. CONCLUSION: The identification of HLA associations in SSc patients with anti-RNAP antibodies may only be possible when the individual antibody specificities recognized by these sera are identified. It may then be possible to classify these patients into distinct clinical and immunogenetic subgroups.

Systemic sclerosis sine scleroderma: demographic, clinical, and serologic features and survival in forty-eight patients.

OBJECTIVE: To describe the demographic, clinical, and laboratory features and natural history of patients with systemic sclerosis sine scleroderma (ssSSc), and to compare them with those of patients with SSc and limited cutaneous involvement (IcSSc). METHODS: The University of Pittsburgh Scleroderma Databank served as the data source. Patients were divided into those who had no skin thickening (ssSSc) and those who had skin thickening only distal to elbows or knees and/or of the face (IcSSc) during their disease course. These two groups were compared with regard to demographic characteristics, clinical, laboratory, and serologic features, and survival rates. Chi-square and Student's t-test analyses were performed, and Fisher's exact test was used as appropriate. RESULTS: Of 555 consecutive patients without diffuse cutaneous SSc, 48 (9%) had ssSSc and 507 (91%) had IcSSc. The ssSSc patients had a mean total disease duration of 18.6 years (15.1 years before study entry and 3.5 years of followup after study entry), and had not developed IcSSc or another connective tissue disease. Other than the absence of skin thickening, the ssSSc group had no significant differences in individual internal organ involvements, laboratory features, serum autoantibody type, or survival rate compared with patients with IcSSc. Within the category of lung involvement, patients with ssSSc had a significantly greater frequency of dyspnea with mild exertion or at rest, and a tendency toward reduced carbon monoxide diffusing capacity (<70% of predicted normal) and primary pulmonary arterial hypertension. Patients with IcSSc had significantly more frequent individual manifestations of digital pitting scars, digital-tip ulcers, telangiectasia, and calcinosis than those with ssSSc, in part related to increased time of observation. Puffy fingers and finger joint contractures were detected significantly more often in IcSSc patients. CONCLUSION: Systemic sclerosis sine scleroderma should be included in the spectrum of SSc with limited cutaneous involvement and should not be considered a distinct or separate disorder.

Anti-RNA polymerase III antibodies in the diagnosis of scleroderma renal crisis sine scleroderma.

We describe the use of antibodies to RNA polymerase III in the diagnosis of scleroderma in 2 patients who presented with renal crisis without other clinical features of the condition. Both presented with accelerated hypertension, rapidly progressive acute renal failure, microangiopathic hemolytic anemia, and thrombocytopenia. One patient developed digital infarcts in the course of his initial illness. Neither showed evidence of skin thickening at presentation. Nailfold capillaroscopy was normal in one patient and showed capillary dropout in the other. Renal biopsy showed findings consistent with thrombotic microangiopathy and both had anti-RNA polymerase III antibodies.

A novel cell-cycle-dependent 350-kDa nuclear protein: C-terminal domain sufficient for nuclear localization.

We have screened human scleroderma patients for immunoreactivity with the components of the nucleus and the mitotic apparatus. We announce the identification of a novel cell-cycle-dependent nuclear protein using serum from a CREST patient AH. AH protein first appears at the nucleus of G2-phase and associates with the centrosome throughout the cell cycle. As chromosomes condense during the prophase, AH protein becomes enriched at the kinetochores. During mitosis, AH protein progressively disperses from the kinetochore and becomes diffusely localized in the cytoplasm and in telophase; it appears to be enriched within the intracellular bridge. Molecular cloning and transfection studies reveal that the 350-kDa AH protein contains a coiled-coil and a globular domain at the C-terminus that is sufficient for nuclear localization.

Elevated serum soluble interleukin-2 receptor levels in subacute cutaneous lupus erythematosus.

Subacute cutaneous lupus erythematosus (SCLE) is a subset of lupus erythematosus which is characterized by unique cutaneous manifestations and immunological abnormalities. Soluble IL-2 receptor (sIL-2 R) is the shed product of membrane Il-2 R, a product of T cell activation. It is measured by enzyme-linked immunosorbent assay (ELISA) and has been found to correlate well with disease activity in systemic lupus erythematosus (SLE) patients. The objective of the present work was to determine whether there is a correlation between sIL-2 R levels and disease activity in SCLE. Serum samples were obtained from 25 SCLE patients and then measured for sIL-2 R levels of ELISA. Fifteen of 25 SCLE patients tested had normal levels of sIL-2 R, while 10 of 25 SCLE patients had elevated sIL-2 R levels. The serum sIL-2 R level in SCLE patients correlated well with disease activity and the number of American College of Rheumatology criteria for SLE. These findings indicate that sIL-2 R levels can be used as a valuable laboratory parameter in managing SCLE patients.