Brief report: effect of ambrisentan treatment on exercise-induced pulmonary hypertension in systemic sclerosis: a prospective single-center, open-label pilot study.

OBJECTIVE: Exercise-induced pulmonary hypertension (ePH) may represent an early, clinically relevant phase in the spectrum of pulmonary vascular disease. The purpose of this pilot study was to describe the changes in hemodynamics and exercise capacity in patients with systemic sclerosis (SSc) spectrum-associated ePH treated with open-label daily ambrisentan. METHODS: Patients were treated with ambrisentan, 5 mg or 10 mg once daily, for 24 weeks. At baseline and 24 weeks, patients with SSc spectrum disorders exercised in a supine position, on a lower extremity cycle ergometer. All patients had normal hemodynamics at rest. We defined baseline ePH as a mean pulmonary artery pressure of >30 mm Hg with maximum exercise and a transpulmonary gradient (TPG) of >15 mm Hg. The primary end point was change in pulmonary vascular resistance (PVR) with exercise. Secondary end points included an improvement from baseline in 6-minute walking distance, health-related quality of life assessments, and cardiopulmonary hemodynamics. RESULTS: Of the 12 enrolled patients, 11 completed the study. At 24 weeks there were improvements in mean exercise PVR (85.8 dynes × second/cm(5) ; P = 0.003) and mean distance covered during 6-minute walk (44.5 meters; P = 0.0007). Improvements were also observed in mean exercise cardiac output (1.4 liters/minute; P = 0.006), mean pulmonary artery pressure (-4.1 mm Hg; P = 0.02), and total pulmonary resistance (-93.0 dynes × seconds/cm(5) ; P = 0.0008). Three patients developed resting pulmonary arterial hypertension during the 24 weeks. CONCLUSION: Exercise hemodynamics and exercise capacity in patients with SSc spectrum-associated ePH improved over 24 weeks with exposure to ambrisentan. Placebo-controlled studies are needed to confirm whether this is a drug-related effect and to determine optimal therapeutic regimens for patients with ePH.

Patterns and predictors of change in outcome measures in clinical trials in scleroderma: an individual patient meta-analysis of 629 subjects with diffuse cutaneous systemic sclerosis.

OBJECTIVE: To examine the range and responsiveness to change of clinical outcome measures and study predictors of clinical response in patients with diffuse cutaneous systemic sclerosis (dcSSc) in the context of clinical trials. METHODS: Data were combined from 629 patients with dcSSc who participated in 7 multicenter clinical therapeutic trials. Trials used common outcome measures: modified Rodnan skin thickness score (MRSS), Health Assessment Questionnaire disability index (HAQ DI), patient's global assessment of disease activity, pulmonary function tests (forced vital capacity, diffusing capacity for carbon monoxide), hand span, and oral aperture. RESULTS: The combined database included 629 patients (82% women, mean ± SD age 46.5 ± 11.8 years, mean ± SD disease duration 19.4 ± 15.9 months). Outcomes tended to improve during trials for patients with more severe disease at study entry and to worsen for patients with less severe disease at entry. Disease duration was mildly negatively predictive of change in MRSS at 6 months (r = -0.27, P < 0.001), and substantial bidirectional variation in change in MRSS and HAQ DI score was seen across the spectrum of disease duration. Sixty-three percent of patients with "early" disease (disease duration <18 months) had a decline in MRSS, and 37% had an increase in MRSS. Eighty-one percent of patients with "late" disease (disease duration ≥ 18 months) had a decline in MRSS, and 19% had an increase in MRSS. Multivariate mixed models did not demonstrate that any baseline variables were strongly predictive of subsequent outcome. CONCLUSION: Among patients with dcSSc enrolled in clinical trials, standard outcome measures tend to improve in those with more severe disease at study entry and to worsen in those with less severe disease at entry. Overall, the MRSS improves during trials, while HAQ DI scores and lung function are mostly static. None of these variables, including disease duration, reliably identifies groups of subjects whose MRSS will predictably increase or decrease in the course of a clinical trial. These findings have important implications for clinical trial design in scleroderma.

Is fascia iliaca compartment block or intravenous opioid analgesia better when positioning patients with fractured neck of femur for spinal anaesthesia?

Patients with a fractured neck of femur require effective analgesia to improve positioning before the administration of spinal anaesthetic. This article discusses the evidence to show whether fascia iliaca compartment block or intravenous opioid analgesia is preferable in this situation.

Systemic sclerosis and bilateral lung transplantation: a single centre experience.

Lung involvement is the leading cause of death in systemic sclerosis (SSc), but lung transplantation (LT) for systemic disease remains controversial. Our objective was to comprehensively evaluate post-LT outcomes for SSc compared to idiopathic pulmonary fibrosis (IPF). We retrospectively evaluated bilateral LT recipients (LTRs) with SSc or IPF at our centre between January 1, 2003 and December 31, 2007. The primary end-point was all-cause mortality at 1 yr post-LT. Secondary end-points included assessments of acute rejection (AR), pulmonary function, infection and chronic rejection. 14 patients with SSc and 38 patients with IPF underwent LT. Apart from a younger SSc cohort (53.2 versus 58.8 yrs; p = 0.02), the two groups were well matched. 1-yr all-cause mortality was no different between SSc (6.6%) and IPF (13.1%) groups, after adjusting for age (p = 0.62). Rates of (AR) ≥2 were significantly increased for the SSc compared with the IPF group (hazard ratio (HR) 2.91; p = 0.007). Other end-points, including chronic rejection, infection and pulmonary function, showed no differences. SSc LTRs experience similar survival 1 yr post-LT when compared to IPF. AR rates may be significantly higher in the SSc group. Longer follow-up is necessary to determine the effects of gastrointestinal dysfunction and AR on late allograft function in SSc LTR.

Stimulation with type I collagen induces changes in gene expression in peripheral blood mononuclear cells from patients with diffuse cutaneous systemic sclerosis (scleroderma).

An autoantigenic role for collagen type I (CI) has been suggested previously in diffuse cutaneous systemic sclerosis (dcSSc). Whether CI is indeed capable of affecting the immune system in dcSSc is not known. Patients with early (3 years or less) or late (>3 years) dcSSc and healthy controls donated blood. Peripheral blood mononuclear cells (PBMC) were cultured with or without CI, and expression of genes known for their involvement in autoimmune and inflammatory processes was assessed using cDNA arrays; results were confirmed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay for selected genes. Patients with early and late dcSSc were similarly different from healthy controls in basal gene expression. When cultured with CI, PBMC from patients with early dcSSc differed from healthy controls in expression of 34 genes, whereas PBMC from patients with late dcSSc differed from healthy controls in expression of only 29 genes. Direct comparisons of matched PBMC samples cultured with and without CI revealed differences in expression of eight genes in healthy controls, of five genes in patients with early dcSSc, and no differences in patients with late dcSSc. Thus, PBMC from patients with dcSSc respond differently than do PBMC from healthy controls when cultured with CI. Exposure to CI in culture of PBMC from patients in the early stage of dcSSc in contrast to PBMC from patients with late-stage dcSSc evokes a greater degree of activation of immune-related genes, suggesting that CI is more dominant as an autoantigen in early versus late dcSSc.

Differences in presentation of younger and older systemic sclerosis patients in clinical trials.

OBJECTIVES: To compare the characteristics of younger and older subjects with diffuse cutaneous systemic sclerosis (SSc) entering clinical trials. METHODS: Subjects were participants in three randomised interventional trials that shared relative uniformity of demographics and disease characteristics. Only subjects with diffuse cutaneous systemic sclerosis were evaluated. To maximise possible differences, the lowest (age<38 years) and highest quartiles (age>53 years) were used, and a total of 264 diffuse cutaneous SSc (dcSSc) subjects were identified. For the comparison between the two age groups, generalised linear mixed or linear models with adjustment for population norms, demographics and medications were employed to assess differences attributable to subject age. RESULTS: After adjustment for population norms and study effects, differences in diastolic blood pressure, alkaline phosphatase, AST, and creatinine phosphokinase (CK) were found between the two age groups. After further adjustment for demographics, disease duration and medications, older SSc patients still had significantly higher alkaline phosphatase (11 U/L higher), and lower CK (76 U/L lower) than younger patients (p<0.003 for all). All other variables were not significantly different in the two age groups. CONCLUSIONS: Clinical baseline differences exist between younger and older patients with SSc. However, after adjustment for population norms and potential confounders, including medications, only differences in alkaline phosphatise (only 11U/L) and CK (76 U/L) remain. Overall, older patients with SSc in clinical trials seem to be more similar to younger patients than was previously thought.

A computer-aided diagnosis system for quantitative scoring of extent of lung fibrosis in scleroderma patients.

OBJECTIVES: To evaluate an improved quantitative lung fibrosis score based on a computer-aided diagnosis (CAD) system that classifies CT pixels with the visual semi-quantitative pulmonary fibrosis score in patients with scleroderma-related interstitial lung disease (SSc-ILD). METHODS: High-resolution, thin-section CT images were obtained and analysed on 129 subjects with SSc-ILD (36 men, 93 women; mean age 48.8±12.1 years) who underwent baseline CT in the prone position at full inspiration. The CAD system segmented each lung of each patient into 3 zones. A quantitative lung fibrosis (QLF) score was established via 5 steps: 1) images were denoised; 2) images were grid sampled; 3) the characteristics of grid intensities were converted into texture features; 4) texture features classified pixels as fibrotic or non-fibrotic, with fibrosis defined by a reticular pattern with architectural distortion; and 5) fibrotic pixels were reported as percentages. Quantitative scores were obtained from 709 zones with complete data and then compared with ordinal scores from two independent expert radiologists. ROC curve analyses were used to measure performance. RESULTS: When the two radiologists agreed that fibrosis affected more than 1% or 25% of a zone or zones, the areas under the ROC curves for QLF score were 0.86 and 0.96, respectively. CONCLUSIONS: Our technique exhibited good accuracy for detecting fibrosis at a threshold of both 1% (i.e. presence or absence of pulmonary fibrosis) and a clinically meaningful threshold of 25% extent of fibrosis in patients with SSc-ILD.

EULAR recommendations for the treatment of systemic sclerosis: a report from the EULAR Scleroderma Trials and Research group (EUSTAR).

PURPOSE: The optimal treatment of systemic sclerosis (SSc) is a challenge because the pathogenesis of SSc is unclear and it is an uncommon and clinically heterogeneous disease affecting multiple organ systems. The aim of the European League Against Rheumatism (EULAR) Scleroderma Trials and Research group (EUSTAR) was to develop evidence-based, consensus-derived recommendations for the treatment of SSc. METHODS: To obtain and maintain a high level of intrinsic quality and comparability of this approach, EULAR standard operating procedures were followed. The task force comprised 18 SSc experts from Europe, the USA and Japan, two SSc patients and three fellows for literature research. The preliminary set of research questions concerning SSc treatment was provided by 74 EUSTAR centres. RESULTS: Based on discussion of the clinical research evidence from published literature, and combining this with current expert opinion and clinical experience, 14 recommendations for the treatment of SSc were formulated. The final set includes the following recommendations: three on SSc-related digital vasculopathy (Raynaud's phenomenon and ulcers); four on SSc-related pulmonary arterial hypertension; three on SSc-related gastrointestinal involvement; two on scleroderma renal crisis; one on SSc-related interstitial lung disease and one on skin involvement. Experts also formulated several questions for a future research agenda. CONCLUSIONS: Evidence-based, consensus-derived recommendations are useful for rheumatologists to help guide treatment for patients with SSc. These recommendations may also help to define directions for future clinical research in SSc.

European League Against Rheumatism (EULAR) Scleroderma Trial and Research group (EUSTAR) recommendations for the treatment of systemic sclerosis: methods of elaboration and results of systematic literature research.

OBJECTIVE: To describe methods and procedures used for the development of the European League Against Rheumatism (EULAR)/EULAR Scleroderma Trial and Research group (EUSTAR) recommendations for the treatment of systemic sclerosis. In particular, the results of a web-based Delphi exercise aimed at selection of research questions and evidence from systematic literature research, as parts of the development of these recommendations, are presented in detail. METHODS: In agreement with the EULAR standard operating procedures a Task Force was created that consisted of the EUSTAR board members, 10 systemic sclerosis (SSc) experts invited from outside the EUSTAR board and representing Europe, the USA and Japan, a clinical epidemiologist, 2 patients with SSc and 3 fellows for literature research. All EUSTAR centres were invited to contribute to the development of recommendations through submission and preliminary selection of the research questions. The systematic literature research was performed using the Pubmed, Medline, EMBASE and Cochrane databases. Retrieved trials were evaluated according to the Jadad classification, and the level of evidence was graded from 1 to 4. Outcome data for efficacy and adverse events were abstracted and effect size, number needed to treat (NNT) and number needed to harm (NNH) were calculated when appropriate. RESULTS: In all, 65 EUSTAR Centres provided 304 research questions concerning SSc treatment. These questions were aggregated, subdivided into 19 treatment categories and then subjected to preliminary selection by a web-based Delphi technique. The final set of 26 research questions was created by the Expert Committee based on the results of the Delphi exercise and the expert's experience. CONCLUSIONS: This paper is a comprehensive summary of the methods we used to build recommendations for the drug treatment of systemic sclerosis, combining an evidence based approach and expert opinion.

Development of a provisional core set of response measures for clinical trials of systemic sclerosis.

OBJECTIVE: To develop a provisional core set of response measures for clinical trials of systemic sclerosis (SSc). METHODS: The Scleroderma Clinical Trials Consortium (SCTC) conducted a structured, 3-round Delphi exercise to reach consensus on a core set of measures for clinical trials of SSc. Round 1 asked the SCTC investigators to list items in 11 pre-defined domains (skin, musculoskeletal, cardiac, pulmonary, cardio-pulmonary, gastrointestinal, renal, Raynaud phenomenon and digital ulcers, health-related quality of life and function, global health, and biomarkers) for SSc clinical trials. Round 2 asked respondents to rate the importance of the chosen items and was followed by a meeting, during which the Steering Committee discussed the feasibility, reliability, redundancy and validity of the items. Round 3 sought to obtain broader consensus on the core set measures. Members also voted on items that had data on feasibility but lacked data on reliability and validity, but may still be useful research outcome measures for future trials. RESULTS: A total of 50 SCTC investigators participated in round 1, providing 212 unique items for the 11 domains. In all, 46 (92%) participants responded in round 2 and rated 177 items. The ratings of 177 items were reviewed by the Steering Committee and 31 items from the 11 domains were judged to be appropriate for inclusion in a 1-year multi-centre clinical trial. In total, 40 SCTC investigators completed round 3 and ranked 30 of 31 items as acceptable for inclusion in the core set. The Steering Committee also proposed 14 items for a research agenda. CONCLUSION: Using a Delphi exercise, we have developed a provisional core set of measures for assessment of disease activity and severity in clinical trials of SSc.

Glucocorticoids administered in vivo inhibit human suppressor T lymphocyte function and diminish B lymphocyte responsiveness in in vitro immunoglobulin synthesis.

The effects of corticosteroid given in vivo on human lymphocyte subpopulation function were investigated using an in vitro system of pokeweek mitogen-stimulated immunoglobulin production. Peripheral blood lymphocytes were obtained from normal volunteers before and 4 h after the intravenous administration of methylprednisolone. Unfractioned peripheral blood lymphocytes showed a consistent decrease (mean congruent with 50%) in immunoglobulin and total protein synthesis after steroid administration. Utilizing separated thymus-derived (T) and bone marrow-derived (B) lymphocyte fractions, the pathophysiology of this alteration in immunoglobulin production was elucidated. B lymphocytes obtained after steroid treatment showed a markedly diminished immunoglobulin response (20% of normal) to normal T lymphocytes and to normal T cells that had been irradiated to remove suppressor T lymphocyte function. All major classes of immunoglobulin (IgG, IgM, and IgA) were affected. T lymphocytes procured after steroid administration were capable of providing normal amounts of T cell help for B cells in immunoglobulin production. However, suppressor T lymphocyte activity, observed with normal T lymphocytes at high T to B cell ratios, was absent from the post-steroid T lymphocytes. This loss of suppressor T lymphocyte function was not due to the presence of excess help as irradiated pre- and poststeroid T cells provided equal amounts of helper activity. On recombining the poststeroid treatment B cells, which are hyporesponsive in immunoglobulin synthesis, with the posttreatment T lymphocytes, which lack suppressor activity, diminished amounts of immunoglobulin were produced which correlate well with the effects observed with unseparated cells. Thus, corticosteroids have differential effects on the lymphocyte populations involved in immunoglobulin biosynthesis. B cell responsiveness is diminished, suppressor T lymphocyte activity is removed, and helper T lymphocyte function is unaffected.

Human lymphocyte subpopulations. Effect of corticosteroids.

Normal subjects given 60 mg of prednisone orally at 8:00 a.m. developed a transient lymphopenia at 2:00 p.m. To define the populations of lymphocytes affected the number and type of lymphocytes in the peripheral blood were assayed. "Late" and "early" spontaneous sheep red blood cell rosettes were used as markers for thymus-derived (T) lymphocytes and one of its subpopulations, respectively. Receptors for aggregated gammaglobulin and complement identified bursal-equivalent or bone marrow-derived (B) lymphocytes and one of its subpopulations, respectively. 6 h after administration of 60 mg of prednisone, the blood samples showed a decrease in proportion of T cells from 69.2 +/- 2.1% to 55.9 +/- 2.8% (average +/- SE) and an increase in B-cell proportion from 21.3 +/- 2.0% to 44.8 +/- 4.1%. The changes of "early" rosettes and complement receptor lymphocytes also paralleled these. In all cases the absolute numbers of T cells and of B cells were decreased by prednisone. The density gradient distribution of the lymphocytes did not change after prednisone. These data indicate that both T and B lymphocytes are affected by the prednisone but that the T cell lymphopenia was more pronounced. The lymphopenia might reflect either sequestration in the marrow and/or transient arrest of recirculation.

Structure of a human lysosomal sulfatase.

BACKGROUND: . Sulfatases catalyze the hydrolysis of sulfuric acid esters from a wide variety of substrates including glycosaminoglycans, glycolipids and steroids. There is sufficient common sequence similarity within the class of sulfatase enzymes to indicate that they have a common structure. Deficiencies of specific lysosomal sulfatases that are involved in the degradation of glycosamino-glycans lead to rare inherited clinical disorders termed mucopolysaccharidoses. In sufferers of multiple sulfatase deficiency, all sulfatases are inactive because an essential post-translational modification of a specific active-site cysteine residue to oxo-alanine does not occur. Studies of this disorder have contributed to location and characterization of the sulfatase active site. To understand the catalytic mechanism of sulfatases, and ultimately the determinants of their substrate specificities, we have determined the structure of N-acetylgalactosamine-4-sulfatase. RESULTS: . The crystal structure of the enzyme has been solved and refined at 2.5 resolution using data recorded at both 123K and 273K. The structure has two domains, the larger of which belongs to the alpha/beta class of proteins and contains the active site. The enzyme active site in the crystals contains several hitherto undescribed features. The active-site cysteine residue, Cys91, is found as the sulfate derivative of the aldehyde species, oxo-alanine. The sulfate is bound to a previously undetected metal ion, which we have identified as calcium. The structure of a vanadate-inhibited form of the enzyme has also been solved, and this structure shows that vanadate has replaced sulfate in the active site and that the vanadate is covalently linked to the protein. Preliminary data is presented for crystals soaked in the monosaccharide N-acetylgalactosamine, the structure of which forms a product complex of the enzyme. CONCLUSIONS: . The structure of N-acetylgalactosamine-4-sulfatase reveals that residues conserved amongst the sulfatase family are involved in stabilizing the calcium ion and the sulfate ester in the active site. This suggests an archetypal fold for the family of sulfatases. A catalytic role is proposed for the post-translationally modified highly conserved cysteine residue. Despite a lack of any previously detectable sequence similarity to any protein of known structure, the large sulfatase domain that contains the active site closely resembles that of alkaline phosphatase: the calcium ion in sulfatase superposes on one of the zinc ions in alkaline phosphatase and the sulfate ester of Cys91 superposes on the phosphate ion found in the active site of alkaline phosphatase.

A mitotic spindle requirement for DNA damage-induced apoptosis in Chinese hamster ovary cells.

Promiscuously reactive electrophilic agents induce DNA and other cellular damage. DNA repair-defective cells, when compared with genetically matched, repair-proficient parental cells, provide a means to distinguish cellular responses triggered by individual genetic lesions from other macromolecular damage. The Chinese hamster ovary (CHO) cell line EM9 is hypersensitive to the alkylating agent ethyl methanesulfonate (EMS) and is unable efficiently to repair DNA single strand breaks in contrast to parental AA8 cells. EM9 was used to examine how CHO cells couple unrepaired DNA strand breaks to loss of viability. Flow cytometry revealed that EMS-treated EM9 cells underwent prolonged cell cycle arrest in G2, followed by entry into mitosis, micronucleation, and apoptosis. EM9 cells synchronized in G1 prior to EMS treatment entered mitosis 24-36 h after release from synchrony, approximately 12 h after untreated control cells. Mitoses in EMS-treated cells were abnormal, involving multipolar mitotic spindles and elongated and/or incompletely condensed chromosomes. The mitotic spindle poison nocodazole reduced DNA damage-induced apoptosis by >60%, whereas the frequency of micronucleation was similar in the presence or absence of nocodazole. Flow cytometry revealed that nocodazole-treated cells sustained a second round of DNA replication without intervening mitosis. These results demonstrate that nuclear fragmentation and inappropriate DNA replication are insufficient to trigger apoptosis following DNA strand breakage and demonstrate a requirement for mitotic spindle assembly for this process in CHO cells.

A rapid four column purification of 2-deoxy-D-glucoside-2-sulphamate sulphohydrolase from human liver.

2-Deoxy-D-glucoside-2-sulphamate sulphohydrolase was extracted from human liver and purified 40 000-fold by a simple four column procedure. The purification was followed using a specific substrate isolated from an acid hydrolysate of heparin, O-(alpha-2-sulphamino-2-deoxy-D-glucopyranosyl)-(1 leads to 3)-L-[6,3H]idonic acid. Only one form of the enzyme was seen on either ion exchange chromatography or isoelectric focussing, with a pI of 6.8. The apparent Mr of the holoenzyme as determined by gel filtration was 190 000 +/- 20 000. Two other larger Mr protein peaks observed on gel filtration appear to be an inactive dimer of the 190 000 dalton peak and a larger aggregate near the exclusion limit of the column. On polyacrylamide disc gel electrophoresis in sodium dodecyl sulphate, with or without prior reduction, each protein peak from the gel filtration column electrophoresed as a single major band with an apparent Mr corresponding to 55 000 +/- 6000.

Genotype-phenotype correlations in mucopolysaccharidosis type I using enzyme kinetics, immunoquantification and in vitro turnover studies.

Fibroblasts from 16 patients with known alpha-L-iduronidase gene mutations and different clinical phenotypes of mucopolysaccharidosis type I (MPS I) were investigated in order to establish genotype/phenotype correlations. Enzyme kinetic studies were performed using the specific alpha-L-iduronidase substrate iduronosyl anhydro[1-3H]mannitol-6-sulfate. Specific residual enzyme activities were estimated using the kinetic parameters and an immunoquantification assay which determines levels of alpha-L-iduronidase protein. Cells were cultured in the presence of [35S]sulfate and the in vivo degradation of accumulated labelled glycosaminoglycans measured after different chase times. Residual enzyme activity and different amounts of residual enzyme protein were present in extracts from 9 of 16 cell lines covering a wide spectrum of clinical severity. Catalytic capacity, calculated as the product of kcat/Km and ng iduronidase protein per mg cell protein, was shown in most cases to be directly related to the severity of clinical phenotype, with up to 7% of normal values for patients with the attenuated form of MPS I (Scheie) and less than 0.13% for severely affected patients (Hurler) In vitro turnover studies allowed further refinement of correlations between genotype and phenotype. Scheie disease compared to Hurler disease patients were shown to accumulate smaller amounts of glycosaminoglycans that were also turned over faster. A combination of turnover and residual enzyme data established a correlation between the genotype, the biochemical phenotype and the clinical course of this lysosomal storage disorder.

Physiological, pharmacological and toxicological considerations of drug-induced structural cardiac injury.

The incidence of drug-induced structural cardiotoxicity, which may lead to heart failure, has been recognized in association with the use of anthracycline anti-cancer drugs for many years, but has also been shown to occur following treatment with the new generation of targeted anti-cancer agents that inhibit one or more receptor or non-receptor tyrosine kinases, serine/threonine kinases as well as several classes of non-oncology agents. A workshop organized by the Medical Research Council Centre for Drug Safety Science (University of Liverpool) on 5 September 2013 and attended by industry, academia and regulatory representatives, was designed to gain a better understanding of the gaps in the field of structural cardiotoxicity that can be addressed through collaborative efforts. Specific recommendations from the workshop for future collaborative activities included: greater efforts to identify predictive (i) preclinical; and (ii) clinical biomarkers of early cardiovascular injury; (iii) improved understanding of comparative physiology/pathophysiology and the clinical predictivity of current preclinical in vivo models; (iv) the identification and use of a set of cardiotoxic reference compounds for comparative profiling in improved animal and human cellular models; (v) more sharing of data (through publication/consortia arrangements) on target-related toxicities; (vi) strategies to develop cardio-protective agents; and (vii) closer interactions between preclinical scientists and clinicians to help ensure best translational efforts.

Identification and molecular characterization of alpha-L-iduronidase mutations present in mucopolysaccharidosis type I patients undergoing enzyme replacement therapy.

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder caused by a deficiency of alpha-L-iduronidase (IDUA). Mutations in the gene are responsible for the enzyme deficiency, which leads to the intralysosomal storage of the partially degraded glycosaminoglycans dermatan sulfate and heparan sulfate. Molecular characterization of MPS I patients has resulted in the identification of over 70 distinct mutations in the IDUA gene. The high degree of molecular heterogeneity reflects the wide clinical variability observed in MPS I patients. Six novel mutations, c.1087C>T (p.R363C), c.1804T>A (p.F602I), c.793G>C, c.712T>A (p.L238Q), c.1727+2T>A, and c.1269C>G (p.S423R), in a total of 14 different mutations, and 13 different polymorphic changes, including the novel c.246C>G (p.H82Q), were identified in a cohort of 10 MPS I patients enrolled in a clinical trial of enzyme-replacement therapy. Five novel amino acid substitutions and c.236C>T (p.A79V) were engineered into the wild-type IDUA cDNA and expressed. A p.G265R read-through mutation, arising from the c.793G>C splice mutation, was also expressed. Each mutation reduced IDUA protein and activity levels to varying degrees with the processing of many of the mutant forms also affected by IDUA. The varied properties of the expressed mutant forms of IDUA reflect the broad range of biochemical and clinical phenotypes of the 10 patients in this study. IDUA kinetic data derived from each patient's cultured fibroblasts, in combination with genotype data, was used to predict disease severity. Finally, residual IDUA protein concentration in cultured fibroblasts showed a weak correlation to the degree of immune response to enzyme-replacement therapy in each patient.

Mutational analysis of mucopolysaccharidosis type VI patients undergoing a trial of enzyme replacement therapy.

Mucopolysaccharidosis type VI (MPS VI), or Maroteaux-Lamy syndrome, is a lysosomal storage disorder caused by a deficiency of N-acetylgalactosamine-4-sulfatase (ARSB). Seven MPS VI patients were chosen for the initial clinical trial of enzyme replacement therapy. Direct sequencing of genomic DNA from these patients was used to identify ARSB mutations. Each individual exon of the ARSB gene was amplified by PCR and subsequently sequenced. Nine substitutions (c.289C>T [p.Q97X], c.629A>G [p.Y210C], c.707T>C [p.L236P], c.936G>T [p.W312C], c.944G>A [p.R315Q], c.962T>C [p.L321P], c.979C>T [p.R327X], c.1151G>A [p.S384N], and c.1450A>G [p.R484G]), two deletions (c.356_358delTAC [p.Y86del] and c.427delG), and one intronic mutation (c.1336+2T>G) were identified. A total of 7 out of the 12 mutations identified were novel (p.Y86del, p.Q97X, p.W312C, p.R327X, c.427delG, p.R484G, and c.1336+2T>G). Two of these novel mutations (p.Y86del and p.W312C) were expressed in Chinese hamster ovary cells and analyzed for residual ARSB activity and mutant ARSB protein. The two common polymorphisms c.1072G>A [p.V358M] and c.1126G>A [p.V376M] were identified among the patients, along with the silent mutation c.1191A>G. Cultured fibroblast ARSB mutant protein and residual activity were determined for each patient, and, together with genotype information, were used to predict the expected clinical severity of each MPS VI patient.

Small airways in progressive systemic sclerosis (PSS).

Obstructive disease involving peripheral airways has been noted in diffuse interstitial pulmonary disease, including sarcoidosis and cryptogenic fibrosing alveolitis. The possibility of obstruction of small airways in progressive systemic sclerosis (PSS) has been suggested by widespread bronchiolectasis and peribronchial fibrosis noted at necropsy. We performed pulmonary function studies in 39 subjects (22 nonsmokers and 17 smokers) with PSS, most of whom had functional evidence of interstitial pulmonary involvement (increased static recoil pressure and reduced diffusing capacity). The 1 second forced expiratory volume to forced vital capacity ratio (FEV1:FVC) was normal in all subjects. Although the severity of the restrictive process was greater in nonsmokers compared with that in smokers, the maximal mid-expiratory flow rate, closing volume, closing capacity, volume of isoflow, change in maximal expiratory flow at 50 per cent of vital capacity during 80 per cent helium--20 per cent oxygen breathing compared with air breathing (delta Vmax50), ratio of dynamic to static lung compliance at different breathing frequencies and upstream airway conductance at static recoil pressures of 5 and 10 cm H2O were nearly always normal in the nonsmokers but were frequently abnormal in the smokers with PSS. These findings suggest that diffuse interstitial pulmonary disease due to PSS generally does not lead to functional evidence of obstruction in peripheral airways and that when the latter is found it can likely be attributed to the effects of concomitant cigarette smoking.