Combined aerobic exercise and enzyme replacement therapy rejuvenates the mitochondrial-lysosomal axis and alleviates autophagic blockage in Pompe disease.

A unifying feature in the pathogenesis of aging, neurodegenerative disease, and lysosomal storage disorders is the progressive deposition of macromolecular debris impervious to enzyme catalysis by cellular waste disposal mechanisms (e.g., lipofuscin). Aerobic exercise training (AET) has pleiotropic effects and stimulates mitochondrial biogenesis, antioxidant defense systems, and autophagic flux in multiple organs and tissues. Our aim was to explore the therapeutic potential of AET as an ancillary therapy to mitigate autophagic buildup and oxidative damage and rejuvenate the mitochondrial-lysosomal axis in Pompe disease (GSD II/PD). Fourteen weeks of combined recombinant acid α-glucosidase (rhGAA) and AET polytherapy attenuated mitochondrial swelling, fortified antioxidant defense systems, reduced oxidative damage, and augmented glycogen clearance and removal of autophagic debris/lipofuscin in fast-twitch skeletal muscle of GAA-KO mice. Ancillary AET potently augmented the pool of PI4KA transcripts and exerted a mild restorative effect on Syt VII and VAMP-5/myobrevin, collectively suggesting improved endosomal transport and Ca(2+)- mediated lysosomal exocytosis. Compared with traditional rhGAA monotherapy, AET and rhGAA polytherapy effectively mitigated buildup of protein carbonyls, autophagic debris/lipofuscin, and P62/SQSTM1, while enhancing MnSOD expression, nuclear translocation of Nrf-2, muscle mass, and motor function in GAA-KO mice. Combined AET and rhGAA therapy reactivates cellular clearance pathways, mitigates mitochondrial senescence, and strengthens antioxidant defense systems in GSD II/PD. Aerobic exercise training (or pharmacologic targeting of contractile-activity-induced pathways) may have therapeutic potential for mitochondrial-lysosomal axis rejuvenation in lysosomal storage disorders and related conditions (e.g., aging and neurodegenerative disease).

Assessment of nociceptin/orphanin FQ and micro-opioid receptor mRNA in the human right atrium.

BACKGROUND: The expression of micro (mu: MOP) and nociceptin/orphanin FQ (NOP) receptors in the human myocardium is controversial. In this polymerase chain reaction (PCR)-based study using human right atrial biopsies, we have (i) probed for mRNA encoding NOP receptor and its endogenous peptide precursor, ppN/OFQ, and mRNA encoding MOP and (ii) attempted to correlate expression with cardiac function. METHODS: mRNA encoding MOP, NOP, and the precursor for NOP (ppN/OFQ) was assessed by quantitative real-time PCR (Q-PCR) using validated TaqMan primers and compared with a housekeeper (glyceraldehyde-3-phosphate dehydrogenase, GAPDH). Q-PCR data are expressed as the difference in cycle threshold (DeltaC(t)=C(tGene of interest)-C(tGAPDH): high value, low expression) and patients were grouped according to left ventricular ejection fraction (LVEF). RESULTS: Forty patients were recruited; NOP, MOP, and ppN/OFQ mRNA were measured in 38, 29, and 10 patients, respectively. DeltaC(t) (median and range) values for NOP and MOP were 10.9 (7.8-13.7) and 16.0 (12.3-18.9), respectively, representing low expression of MOP and approximately 34-fold more NOP. MOP mRNA was not detected in seven samples and with DeltaC(t) values of approximately 20, ppN/OFQ was considered absent. When patients were grouped into normal (>50%) and impaired (<50%) LVEF, there was no difference between the groups for either NOP or MOP. In some patients, intraoperative LVEF was estimated using transoesophageal echocardiography, and there was no correlation with either NOP or MOP. CONCLUSIONS: The human right atrium of patients with coronary artery disease and heart failure expresses mRNA encoding NOP and possibly low levels of MOP. This does not correlate with degree of cardiac dysfunction. In addition, the atrium does not express ppN/OFQ mRNA.

Urotensin II receptor expression in human right atrium and aorta: effects of ischaemic heart disease.

BACKGROUND: Urotensin II (UII) and its receptor UT are involved in control of the cardiovascular system and are implicated in heart failure. We measured UT expression by quantitative PCR (Q-PCR) in atrial and aortic tissue, and plasma UII while simultaneously assessing cardiac function in 40 patients undergoing coronary artery bypass surgery. METHODS: RNA extracted from atrial and aortic samples was probed with specific Q-PCR UT and housekeeper (glyceraldehyde-3-phosphate dehydrogenase, GAPDH) TaqMan primers. Plasma UII was measured using radioimmunoassay. Left ventricular ejection fraction (LVEF) was measured using preoperative trans-thoracic echocardiography and ventriculography, and intraoperatively using transoesophageal echocardiography. Q-PCR data are expressed as difference in cycle threshold (DeltaC(t)=C(tUT)-C(tGAPDH): high number indicates low expression). RESULTS: There was no difference in DeltaC(t) in either atrium or aorta between patients with normal (LVEF >50%) or those with impaired (LVEF <50%) preoperative systolic function. There was a weak negative correlation (r(2)=0.245, P=0.031) between intraoperative LVEF and DeltaC(t) in 19 patients possibly indicating down-regulation of UT with worsening LVEF. Atria expressed significantly more UT than aorta (P=0.011). In the absence of non-diseased controls, plasma UII was higher than a historical control group. CONCLUSIONS: This is the first study to simultaneously measure UT (mRNA), UII, and cardiovascular function. Collectively, these pilot data may suggest a down-regulation of UT within the right atrium of patients with heart failure.

Proposed high-risk screening protocol for Fabry disease in patients with renal and vascular disease.

Fabry disease is a complex, multisystemic and clinically heterogeneous disease with prominent urinary excretion of globotriaosylceramide (Gb(3)), the principal substrate of the deficient enzyme, alpha-galactosidase A. Some measure of specific treatment is possible with enzyme replacement therapy, which can be applied safely and effectively to Fabry patients. Incidence estimations of Fabry disease vary widely from 1:55 000 to 1:3000 male births. The true incidence is likely to be higher than originally thought, owing to the existence of milder variants of the disease. The main complications of Fabry disease are a 100-fold increased risk of ischaemic stroke, cardiac disease, a wide variety of arrhythmias, valvular dysfunction and cardiac vascular disease, as well as progressive renal failure usually associated with significant proteinuria. These clinical manifestations are non-specific and are often mistaken for symptoms of other disorders, thus complicating the confirmation of diagnosis. Other clinical features of the disease are often absent (angiokeratoma), subtle (corneal opacities and hypohidrosis), or unaccompanied by specific physical findings (acroparaesthesias) indicating the true nature of the underlying disease. We propose the hypothesis that alpha-galactosidase A deficiency is a modifiable cardiovascular risk factor in the general population. This hypothesis may be tested by a non-invasive high-risk screening protocol for Fabry patients with ischaemic strokes and a variety of cardiac, and renal complications. These patients would benefit from diagnosis, appropriate treatment, follow-up and surveillance. Early detection of Fabry patients would also benefit affected relatives, many of whom do not have a clear diagnosis of their clinical condition.

Intracellular calcium signalling patterns reflect the differentiation status of human T cells.

Stimulation of T lymphocytes results in the calcium-dependent activation and repression of a large number of genes. However, the functional response made by different T cell subsets is heterogeneous, as their differentiation results in alterations in their sensitivity to activation and in the secretion of cytokines. Here we have investigated the patterns of calcium responses in CD4 and CD8 T cell subsets to help explain their different responses to activation. CD4(+) CD45RA(+) T cells isolated freshly from human blood gave a sustained calcium signal after stimulation, but this was smaller than elicited in CD4(+) CD45RO(+) cells. On in vitro differentiation of CD4(+) CD45RA(+) cells to CD45RO(+), the level of the cytoplasmic calcium response rose initially, but then declined steadily during further rounds of differentiation. The proportion producing an oscillatory calcium response or not responding was increased and differentiation was accompanied by a shift in the calcium between intracellular pools. CD8(+) T cells gave a smaller calcium response than paired CD4(+) T cells and showed a difference in the numbers of cells giving a transient, rather than sustained, calcium signal. The increase in oscillating cells in the CD4(+) CD45RO(+) population may reflect the heterogeneity of this population, particularly in terms of cytokine production. The changing patterns of calcium responses in T cells as they differentiate may explain variation in the cellular response to activation at different stages in their lifespan and emphasize the importance of the both the quantity and the quality of the calcium signal in determining the outcome of T cell activation.

Nociceptin and urotensin-II concentrations in critically ill patients with sepsis.

BACKGROUND: The systemic inflammatory response to infection (sepsis) involves widespread organ dysfunction, including changes in immune modulation, cardiovascular derangements, and neural activation. Two neuropeptide/receptor systems, nociceptin/orphanin FQ (N/OFQ) which acts at the non-classical opioid receptor NOP and urotensin-II (U-II) which acts at the urotensin receptor (UT), have been implicated in neural, immune, and cardiovascular system function. In this study, we make measurements of these peptides in critically ill patients. METHODS: Plasma samples from 21 critically ill patients with sepsis were collected over four consecutive days. Plasma N/OFQ and U-II concentrations were determined by radioimmunoassay and compared with biochemical and clinical markers of illness severity, including serum creatinine, bilirubin, platelet and white cell counts, admission APACHE II and serial SOFA scores. RESULTS: Median (inter-quartile range) admission plasma N/OFQ concentrations in sepsis were higher in patients who died within 30 days (n=4) compared with survivors (n=17); 3.0 (2.5-5.0) vs 1.0 (1.0-2.5) pg ml(-1) (P=0.028). Plasma N/OFQ concentrations were increased in a subgroup of five patients who had undergone major gastrointestinal surgery. There were no significant changes in plasma U-II concentrations. There were no correlations between plasma U-II and N/OFQ concentrations and markers of illness severity and organ system dysfunction. CONCLUSIONS: Plasma N/OFQ concentrations were increased in critically ill patients with sepsis who had undergone major gastrointestinal surgery and in patients who subsequently died. Further work is required to clarify the significance of plasma N/OFQ concentrations in sepsis.

Hemolymph osmolality and cation concentrations in Litopenaeus vannamei during exposure to artificial sea salt or a mixed-ion solution: relationship to potassium flux.

Interest in culturing the Pacific white shrimp Litopenaeus vannamei in low-salinity and brackish-well waters has led to questions about the ability of this species to osmo- and ionoregulate in environments containing low concentrations of ions and in environments with ionic ratios that differ from those found in sea water. After seven days, hemolymph osmolality and potassium, sodium and calcium values were all significantly affected by salinity (as artificial sea salt) with values decreasing with decreasing salinity. These decreases were small, however, relative to decreases in salinity, indicating iono- and osmoregulation with adjustment for gradients. The hemolymph osmolality and sodium and calcium concentrations in shrimp exposed to either 2 g/L artificial sea salt or 2 g/L mixed-ion solution (a mixture of sodium, potassium, calcium, and magnesium chlorides that approximate the concentrations and ratios of these cations found in 2 g/L dilute seawater) did not differ significantly. However, hemolymph potassium levels were significantly lower in shrimp held in the mixed-ion environment. Potassium influx rates were similar in shrimp held in either artificial sea salt or mixed ions. The results of this study indicate that salinity affects hemolymph-cation concentrations and osmolality. Further, differential potassium-influx rates do not appear to be the basis for low hemolymph potassium levels observed in shrimp held in mixed-ion environments.

Development of an interactive learning tool for teaching rheumatology--a simulated clinical case studies program.

OBJECTIVES: To promote independent self-study involving problem solving and decision analysis in the undergraduate medical curriculum, we have developed a series of interactive web-based clinical case studies. METHODS: An initial needs assessment was performed to determine students' attitudes to e-learning. From these results we designed a series of 30 interactive case studies for delivery from a web-server. RESULTS: A survey of 59 undergraduate students believed that online teaching resources were a useful supplement to existing teaching and they could see a positive use for e-learning. The interactive case studies program was well received by a broad range of respondents (n = 84) of different abilities and backgrounds who felt that the program was realistic and clearly presented in an intuitive manner. CONCLUSIONS: The recent increases in numbers of medical undergraduates, the trend towards student-centred learning and the emphasis on patient-related teaching means a great pressure on teachers and resources in medical schools. The case studies program we have developed was effective and well received by both biomedical and medical students. This approach may provide a way to increase the exposure of students to clinical cases involving interactive diagnostic and treatment procedures, that mimic real-world scenarios, but with fewer resource implications.

Automated classification and analysis of the calcium response of single T lymphocytes using a neural network approach.

The gene activities in T lymphocytes that regulate immune responses are influenced by Ca2+ ([Ca2+]i). The intracellular calcium signals are highly heterogeneous and vitally important in determining the immune outcome. The signals in individual cells can be measured using fluorescence microscopy but to group the cells into classes with similar signal kinetics is currently laborious. Here, we demonstrate a method for the automated classification of the responses into four categories formerly identified by an expert's inspection. This method comprises characterising the response by a second-order model, performing frequency analysis, and using derived features as inputs to two multilayer perceptron neural networks (NNs). We compare the algorithm's performance on an example data set against the human classification: it was found to classify identically more than 70% of the data, despite small sample sizes in two categories and significant overlap between the other two classes. The group characterized by an oscillating signal showed the presence of a number of frequencies, which may be important in determining gene activation. A classification threshold enables the automatic identification of patterns with a low-classification certainty. Future refinement of the algorithm may allow the identification of more classes, which may be important in different immune responses associated with disease.

Nailfold capillary microscopy in healthy children and in childhood rheumatic diseases: a prospective single blind observational study.

OBJECTIVES: To develop an objective method of nailfold capillaroscopy (NFC), applicable to a wide age range of paediatric patients. To compare the morphological characteristics of the nailfold capillaries in different rheumatology patient groups and controls. METHODS: A colour digital video camera attached to a stereomicroscope was used to capture nailfold capillary images. Computerised image processing was used to analyse and store data. Subsequent quantitative and qualitative morphological analysis was performed in the following paediatric patient and control groups: 18 children with connective tissue diseases (CTD: juvenile dermatomyositis, systemic sclerosis, and undifferentiated connective tissue disease), eight with systemic lupus erythematosus, nine with primary Raynaud's disease, three with primary vasculitis, 15 with juvenile idiopathic arthritis, 17 healthy children and 20 healthy adults. Images were analysed by a single assessor who was unaware of the patient details. RESULTS: The NFC technique was simple to perform and gave reproducible results, although some intra- and intersubject variation was noted. Capillary density and width was age related, with younger children having fewer and wider capillaries than older children and adults. Linear capillary density was significantly higher in healthy adults (mean (SD) 8.6 (1.6) capillaries/mm) compared with healthy children (HC 6.9 (0.9) capillaries/mm). The group with CTD had the most abnormal findings, with lower linear density (4.9 (1.7) capillaries/mm) and increased capillary loop width (10.7 (7.3) mm) compared with HC (3.5 (1.7) mm). In addition, 11/18 (61%) patients in the CTD group had more than two definitely abnormal capillaries in at least two nailfolds, an abnormality not seen in other subjects. Two qualitative measures, the degree of avascularity and general disarrangement of capillary pattern, were more commonly observed in the CTD group than in HC. The proportion of tortuous capillaries did not differ significantly between study groups. CONCLUSIONS: This study is unique in measuring objective quantitative and qualitative parameters of the nailfold vasculature across a wide spectrum of age and disease. Differences in capillary morphology and frequency in children with CTD compared with other paediatric diseases and healthy controls were demonstrated. In the clinical situation, an assessment of the general degree of disarrangement may offer a fast tool for assessment of the nailfold vasculature which correlates well with NFC data.

Development and assessment of a World Wide Web site for systemic lupus erythematosus patient information.

Patient education is an important component of the management of chronic diseases such as SLE. We have investigated the value of the World Wide Web as a medium for delivery of SLE patient information. Volunteers recruited from the clinic and from the website completed interviews and questionnaires aimed at defining their information needs. A new website was then established and its impact on users tested using knowledge questionnaires. The new website was used extensively (20-30 users each day) over the 24 month period of study until April 2001. A total of 510 participants completed an online questionnaire that showed that for some users it was their first use of the internet to gather lupus information, but the majority (58.9%) accessed it at least monthly for this purpose. We also found that, while most users (56.9%) found current disease information was at an appropriate level, 37.5% thought it was too basic. Knowledge questionnaires from 42 participants before and after using the site showed a significant rise in users' knowledge of the areas covered by the site. As far as we are aware this study is the first to show that a patient-oriented website can have a positive effect on disease knowledge. The relative ease with which good quality information can be disseminated via the web suggests that this medium is likely to be less costly and perhaps more educationally effective than printed information, and so is likely to become a primary vehicle for patient education. The website tested can be found at: www.rheumatology.bham.ac.uk/lupus/intro.html.

Computerized information-gathering in specialist rheumatology clinics: an initial evaluation of an electronic version of the Short Form 36.

OBJECTIVES: Longitudinal outcome data are important for research and are becoming part of routine clinical practice. We assessed an initial version of an electronic Short Form 36 (SF-36), a well-established health assessment questionnaire, in comparison with standard paper forms, in two specialist rheumatology clinics. METHODS: Out-patients (20 with systemic lupus erythematosus and 31 with vasculitis) were randomly selected to complete either paper (n=29) or electronic and paper SF-36 versions (n=51) before and after consultation (paper vs paper comparison). Data were evaluated as the response correlation, internal consistency, missing data, patient satisfaction and preference. RESULTS: There were very good correlations in SF-36 responses (P<0.001) between the paper and electronic forms and the paper and paper forms. Internal reliability coefficients (Cronbach's alpha) showed good internal consistency for all reported responses in either computer or paper forms. There were no missing data in the computerized version but 24% of patients failed to answer all of the paper form questions. Ease of use of the computer version was rated highly by 71% of all the respondents, and 69% would prefer to use the computer version in future. DISCUSSION: Computerized data collection is acceptable to patients and feasible in clinical settings. It provides responses that are at least comparable to those to the paper form, improves data capture and is available immediately.

Immune responses to native beta(2)-glycoprotein I in patients with systemic lupus erythematosus and the antiphospholipid syndrome.

OBJECTIVE: To identify HLA class II associations with anti beta(2)-glycoprotein I (beta2GPI) antibodies in a cohort of Caucasian patients with systemic lupus erythematosus (SLE) and to determine whether these HLA genotypes act as restriction elements for lymphocyte proliferation to native human beta2GPI in vitro. METHODS: Anti-beta2GPI antibodies were detected in patient sera using enzyme-linked immunosorbent assays (ELISAs). HLA class II alleles (DRB1, DQB1) were determined by polymerase chain reaction-based DNA genotyping. In vitro peripheral blood mononuclear cell (PBMC) responses to native human beta2GPI were measured in a 7-day proliferation assay. RESULTS: We identified three groups of Caucasian SLE patients using these ELISAs. In group 1, 16 out of 18 SLE patients (89%) with anti-beta2GPI antibodies were positive for HLA-DRB1*0401/4/8, DR11 or DRB1*1302 (P=0.001 vs controls) compared with 23 out of 53 patients (43%) in group 2 with anti-cardiolipin antibodies only, 57 out of 151 patients (38%) in group 3 (SLE patients without anticardiolipin antibodies) and 109 out of 225 controls (48%). Fourteen patients with anti-beta2GPI antibodies had greater median stimulation indices to beta2GPI in vitro compared with the 15 controls studied (P=0.04). CONCLUSION: The HLA class II and PBMC proliferation data suggest that beta2GPI may be both a T- and B-cell autoantigen in SLE.

Accelerated atherogenesis in autoimmune rheumatic diseases.

The observation that systemic inflammatory rheumatic diseases such as rheumatoid arthritis (RA) are associated with a significantly increased rate of cardiovascular disease, which often occurs at a younger age than in the normal population, is particularly important given the increasing interest in the role of inflammation in atherogenesis in the general population. This review examines the accumulating evidence for accelerated atherogenesis of RA and updates the hypothesis that vasculitis plays a major role in this. Endothelial dysfunction (ECD), widely regarded as initial lesion in atherogenesis, has been shown to occur commonly in primary vasculitis. This ECD is a diffuse event, demonstrable in more than one vascular bed. It is not simply due to scarring in the vessel wall, related to the focal inflammation of the underlying vasculitis, since it may be reversed by suppression of the immune inflammation. However, the mechanisms for this ECD differ from that of the primary vasculitis. Preliminary evidence suggests that inflammatory mediators such as CRP, TNF, or sphingolipids may be involved. The diffuse ECD of vasculitis may have important consequences for both the progression of the primary disease and for cardiovascular events. A model for the role of vasculitis-induced ECD in the accelerated atherogenesis of rheumatic diseases is presented. These concepts are discussed together with the messages they suggest for 'idiopathic' atherosclerosis in the general population.

Effects of phytanic acid on the vitamin E status, lipid composition and physical properties of retinal cell membranes: implications for adult Refsum disease.

Adult Refsum disease is an inherited disorder in which phytanic acid accumulates in tissues and serum. Two hypotheses have been proposed to explain the pathogenesis of this condition. The molecular distortion hypothesis suggests that phytanic acid may alter membrane composition and structure, thereby affecting membrane function(s). The anti-metabolite hypothesis suggests that an accumulation of phytanic acid in membranes may interfere with vitamin E function. These two hypotheses were investigated by studying the effects of modulating phytanic acid and alpha-tocopherol concentrations on the fatty acid composition and certain physical parameters of cultured retinal cells. Results showed that (a) the phospholipid fraction of retinal cells readily incorporated phytanic acid, (b) the incorporation of phytanic acid increased membrane fluidity, (c) there was no competition for uptake between phytanic acid and alpha-tocopherol, and (d) the incorporation of phytanic acid did not increase the susceptibility of membranes to lipid peroxidation in vitro. These results obtained with cultured retinal cells suggest that the molecular distortion hypothesis, but not the anti-metabolite hypothesis, could explain the pathogenesis of adult Refsum disease. In vitro tissue culture models can, however, only approximate to the much more complex situation that occurs in vivo.

Role of oligosaccharide residues of IgG1-Fc in Fc gamma RIIb binding.

Engagement of Fc gamma receptors (Fc gamma Rs) with the Fc region of IgG elicits immune responses by leukocytes. The recent crystal structure of Fc gamma RIII in complex with IgG-Fc has provided details of molecular interactions between these components (Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000) Nature 406, 267-273). One of the most intriguing issues is that glycosylation of IgG-Fc is essential for the recognition by Fc gamma Rs although the carbohydrate moieties are on the periphery of the Fc gamma RIII-Fc interface. To better understand the role of Fc glycosylation in Fc gamma R binding we prepared homogeneous glycoforms of IgG-Fc (Cri) and investigated the interactions with a soluble form of Fc gamma RIIb (sFc gamma RIIb). A 1:1 complex stoichiometry was observed in solution at 30 degrees C (K(d), 0.94 microm; Delta G, -8.4 kcal mol(-1); Delta H, -6.5 kcal mol(-1); T Delta S, 1.9 kcal mol(-1); Delta C(p), -160 cal mol(-1) K(-1)). Removal of terminal galactose residues did not alter the thermodynamic parameters significantly. Outer-arm GlcNAc residues contributed significantly to thermal stability of the C(H)2 domains but only slightly to sFc gamma RIIb binding. Truncation of 1,3- and 1,6-arm mannose residues generates a linear trisaccharide core structure and resulted in a significantly decreased affinity, a less exothermic Delta H, and a more negative Delta C(p) for sFc gamma RIIb binding, which may result from a conformational change coupled to complex formation. Deglycosylation of the C(H)2 domains abrogated sFc gamma RIIb binding and resulted in the lowest thermal stability accompanied with noncooperative unfolding. These results suggest that truncation of the oligosaccharides of IgG-Fc causes disorder and a closed disposition of the two C(H)2 domains, impairing sFc gamma RIIb binding.

Provision of Internet-based rheumatology education (http://rheuma.bham.ac.uk).

OBJECTIVES: The Internet is becoming an important way of delivering medical information, and if used appropriately may assist in improving patients' self-management of their disease. We have established an arthritis education website ('Arthritis Help') and investigated its use over the last 2 yr. METHODS: Computer-generated log-file analysis and on-line questionnaires were used to create user profiles of our website. RESULTS: An average of 288 people visited our site each day, predominantly from America and the UK (49% of users). The typical questionnaire respondent (n = 770) was an American female with arthritis, aged 30+ yr, accessing the Internet from home. Typically, respondents had previously obtained information from medical staff or in written form, but were now more likely to use the Internet. One hundred and sixty-seven out of 585 respondents found our site to be useful, prompting them to seek more information (29%), change their behaviour or engage in more effective discussions with their physician (15%). CONCLUSIONS: These data indicate that it is possible to use the Internet to deliver medical information to its target audience, and that this process can have some impact on the way disease is self-managed. This information may aid more focused website design to maximize the use and potential benefits of such a resource.

Liquid chromatographic assay for a glucose tetrasaccharide, a putative biomarker for the diagnosis of Pompe disease.

A HPLC method associated with butyl-p-aminobenzoate derivatization has been developed for the analysis of a tetraglucose oligomer, Glcalpha1-6Glcalpha1-4Glcalpha1-4Glc, designated Glc(4), in biological fluids. This tetraglucose, normally excreted in the urine, has previously been shown to be elevated in a number of pathological conditions including Pompe disease (glycogen storage disease type II), which is caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase. Concentrations of Glc(4) in both urine and plasma were established for the age ranges of <1, 1-5, 6-10, 11-20, and >20 years, both in normal individuals and in a cohort of 21 patients with enzymatically confirmed Pompe disease. The Glc(4) concentration decreased with age in both groups, but all the patients had elevated Glc(4) levels compared with age-matched controls. Electrospray tandem mass spectrometry was employed to establish the homogeneity of the HPLC peak for Glc(4) and to investigate the identity of other unusual oligosaccharides excreted in patient urine. Our results demonstrate that this method is suitable for application in clinical laboratories to help establish the diagnosis of Pompe disease.

Dysregulated intracellular Ca2+ stores and Ca2+ signaling in synovial fluid T lymphocytes from patients with chronic inflammatory arthritis.

OBJECTIVE: Peripheral blood (PB) T cells from rheumatoid arthritis (RA) patients proliferate poorly to mitogen, a change that is related to decreased intracellular Ca2+ ([Ca2+]i) signaling after T cell receptor (TCR) stimulation. We hypothesized that this was, in part, due to the effect of mediators of inflammation and predicted that greater changes in [Ca2+]i signaling would be seen in synovial fluid (SF) T cells. We also examined the mechanisms underlying the altered [Ca2+]i signals. METHODS: Paired PB and SF T cells from patients with chronic inflammatory arthritis were stimulated with mitogen to assess the magnitude of the [Ca2+]i signal in cell populations by fluorometry, the pattern of the [Ca2+]i signal in individual cells in a single-cell ion-imaging system, and the spatial distribution of Ca2+ within intracellular organelles. RESULTS: There was a significantly smaller [Ca2+]i signal after phytohemagglutinin protein stimulation of SF T cells (peak rise in [Ca2+]i signal PB versus SF 200 nM versus 180 nM; P < 0.05). In single SF T cells, a change in the pattern of the [Ca2+]i signal and a reduction in the number of responding cells was seen. These changes were a magnification of those seen in RA PB compared with control PB T cells. The contribution of Ca2+ release from intracellular stores to the final [Ca2+]i signal in PB and SF T cells was equal, but there was a significant increase in the Ca2+ remaining in the endoplasmic reticulum (ER) in SF T cells after TCR activation (PB versus SF 6 nM versus 19 nM; P < 0.05). Non-ER Ca2+ stores were not similarly affected. CONCLUSION: We found abnormalities in the magnitude, pattern, and spatial distribution of [Ca2+]i signaling in T cells from SF of patients with chronic inflammatory arthritis. A reduction in the number of responding SF T cells may partly explain some of our observations. However, we propose that the observed redistribution of SF Ca2+ stores may underlie the altered [Ca2+]i signaling, thus making these cells hyporesponsive to mitogen. The inflammatory environment of the joint and the late stage of differentiation of SF T cells are both likely to contribute to these changes in [Ca2+]i signaling, resulting in aberrant T cell function and promotion of disease chronicity.