Improving medication adherence in rheumatoid arthritis (RA): a pilot study.

The aim of this exploratory pilot study was to adapt a psychological intervention to improve adherence to medication for patients with rheumatoid arthritis (RA). The approach draws on cognitive behavioural therapy (CBT) techniques, including motivational interviewing . The current study aimed to (i) adapt the intervention for patients with RA, (ii) assess its effectiveness in improving adherence to medication and (iii) evaluate patients' experience of the intervention. Participants were randomly allocated to either the 'intervention group' (N = 10), receiving up to six weekly sessions of 'Compliance Therapy', or to the 'wait-list control' group (N = 8), who received standard care. Data was collected pre intervention (baseline), post intervention and at six weeks post intervention (follow-up). Eighteen female participants with a mean age of 48.78 years (SD 15.12) took part in the study. Comparisons across the two time points for each group found that only those in the 'intervention' group demonstrated significant improvement in mean scores on adherence measures. Between-group comparisons were not significant. The pilot study suggests that an intervention based on CBT may improve adherence in patients with RA, but further research is required.

Joint counts in inflammatory arthritis.

OBJECTIVES: Counting the number of tender and swollen joints is an important aspect of assessing patients with an inflammatory arthritis. We provide a comprehensive overview of joint counts in inflammatory arthritis. This spans how they are undertaken, their use in clinical and research settings, their limitations and standardisation and who can perform them. METHODS: We reviewed the literature surrounding joint counts in inflammatory arthropathies, with a specific focus on rheumatoid arthritis (RA). RESULTS: The current widely used joint count assesses 28 peripheral joints. In RA these are usually incorporated in a composite score of disease activity, termed the disease activity score on a 28-joint count (DAS28). Assessing 28 joints has a strong 'floor-effect' with most patients in routine practice having low swollen and tender joint counts. Marked between-observer variation exists in joint count scores; although the variation in tender joint counts can be reduced by standardised training its impact on swollen joint counts is uncertain. Fibromyalgia can have a marked impact on tender joint count scores, resulting in a disproportionately high tender joint count to swollen joint count ratio. Although there is evidence that patient-assessed tender joint counts correlate well with those undertaken by physicians, patients are limited assessors of synovitis. CONCLUSIONS: Although joint counts provide an important objective measure of disease activity in clinical practice, they have a number of limitations. Future research may provide a more robust clinical assessment for disease activity in inflammatory arthropathies, which overcomes these issues.

Enhanced and persistent levels of interleukin (IL)-17⁺ CD4⁺ T cells and serum IL-17 in patients with early inflammatory arthritis.

Prognosis of patients with early inflammatory arthritis (EIA) is highly variable. The aim of this study was to compare, longitudinally and cross-sectionally, the levels of cytokine-expressing cells in peripheral blood (PB) from patients with EIA to those in established rheumatoid arthritis (RA) and healthy controls (HC). PB mononuclear cells from HC (n = 30), patients with EIA (n = 20) or RA (n = 38) were stimulated with phorbol myristate acetate (PMA)/ionomycin for 3 h, and stained for cell markers and cytokines. Serum cytokines and chemokines were measured by Luminex. Patients with EIA were reassessed at 6 and 12 months. The percentage of interleukin (IL)-17⁺ interferon (IFN)-γ⁻ CD4⁺ T cells [T helper type 17 (Th17)] was increased in RA and EIA versus HC. Serum IL-1ß, IL-2, IL-4 IL-17 and macrophage inflammatory protein (MIP)-1α were increased in RA and EIA versus HC. IL-1Ra, IL-15 and IFN-α were increased in EIA versus HC. IL-6 and tumour necrosis factor (TNF)-α was increased in RA but not EIA versus HC. Disease activity scores in EIA patients improved over 12 months' treatment. Th17 percentage at baseline was correlated with both rheumatoid factor (RF) titre and functional deficit at 12 months. Baseline levels of serum granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6 and IL-8 were correlated with Larsen score at 12 months. There were no significant changes in cytokine-expressing CD4⁺ T cells over time, although the percentage of IL-6⁺monocytes increased. IL-17⁺ CD4⁺ T cells and serum IL-17 levels are increased in EIA. IL-6-expressing monocytes increase during the first year of disease, irrespective of disease-modifying anti-rheumatic drug (DMARD) therapy. We observed incomplete clinical responses, suggesting EIA patients need more intensive early therapy.

Classifying idiopathic inflammatory myopathies: comparing the performance of six existing criteria.

OBJECTIVES: Various criteria have been proposed to classify the inflammatory myositides (IIMs) polymyositis (PM) and dermatomyositis (DM). However, none have received universal acceptance. Our aim was to assess the performance of the main criteria used to classify IIM. Specialist consultant diagnosis was considered the gold standard. METHODS: Patients attending King's College Hospital (KCH) or Reggio Emilia Hospital (REH) since 1990 with a diagnosis of IIM or non-inflammatory myopathy were identified, and their records and laboratory investigations retrospectively reviewed. Where the complete data required for the classification criteria or a final physician diagnosis was unavailable, patients were excluded. 52 patients with a specialist diagnosis of PM, DM, inclusion body myositis (IBM) or non-inflammatory myopathy were included. Agreement between specialist consultant diagnosis and classification criteria was measured using Cohen's kappa (κ) statistics. Sensitivity and specificity were also calculated. RESULTS: The Dalakas (2003) criteria demonstrated substantial agreement with specialist diagnosis: κ=0.69, sensitivity 77%, specificity 99%. The European Neuromuscular Centre criteria (ENMC) demonstrated fair agreement: κ=0.49, sensitivity 71%, specificity 82%. Other criteria performed less well. In particular, the Bohan and Peter criteria demonstrated a specificity of only 29%. CONCLUSIONS: The criteria of Dalakas (2003) agreed best with specialist consultant diagnosis. The criteria of Bohan and Peter demonstrated very poor specificity. Prospective studies are required to develop improved classification criteria.

Single, intra-articular treatment with 6 ml hylan G-F 20 in patients with symptomatic primary osteoarthritis of the knee: a randomised, multicentre, double-blind, placebo controlled trial.

OBJECTIVES: The primary objective was to compare a single, 6 ml, intra-articular injection of hylan G-F 20 with placebo in patients with symptomatic knee osteoarthritis. The safety of a repeat injection of hylan G-F 20 was also assessed. METHODS: Patients with primary osteoarthritis knee pain were randomly assigned to arthrocentesis plus a 6 ml intra-articular injection of either hylan G-F 20 or placebo in a prospective, double-blind (one injector/one blinded observer) study. RESULTS: were evaluated at 4, 8, 12, 18 and 26 weeks post-injection. The primary outcome criterion was change from baseline over 26 weeks in Western Ontario and McMaster Universities (WOMAC) Osteoarthritis Index A pain. Secondary outcome measures included WOMAC A1 and C, patient global assessment (PGA) and clinical observer global assessment (COGA) and Outcome Measures in Rheumatology, Osteoarthritis Research Society International responder rates. A 4-week, open, repeat treatment phase evaluated safety only. Results: A total of 253 patients (Kellgren-Lawrence grade II or III) was randomly assigned. Patients receiving hylan G-F 20 experienced statistically significantly greater improvements in WOMAC A pain scores (-0.15, SE 0.076, p = 0.047), and several of the secondary outcome measures (WOMAC A1, PGA and COGA), than patients receiving placebo. There was no difference between the safety results of the two groups. No increased risk of local adverse events was observed in the open, repeat treatment phase. CONCLUSIONS: This placebo-controlled study demonstrated that, in patients with knee osteoarthritis, a single 6 ml intra-articular injection of hylan G-F 20 is safe and effective in providing statistically significant, clinically relevant pain relief over 26 weeks, with a modest difference versus placebo.

Secular changes in functional disability, pain, fatigue and mental well-being in early rheumatoid arthritis. A longitudinal meta-analysis.

OBJECTIVES: To conduct a systematic review and longitudinal meta-analysis of early rheumatoid arthritis (RA) cohorts with long-term data on pain, fatigue or mental well-being. METHODS: Searches using PUBMED, EMBASE and PyscInfo were performed to identify all early RA cohorts with longitudinal measures of pain, fatigue or mental well-being, along with clinical measures. Using longitudinal meta-analyses, the progression of each outcome over the first 60-months was estimated. Cohorts were stratified based on the median recruitment year to investigate secular trends in disease progression. RESULTS: Of 7,319 papers identified, 75 met the inclusion criteria and 46 cohorts from 41 publications provided sufficient data on 18,046 patients for meta-analysis. The Disease Activity Scores (DAS28) and the Short-Form 36 (SF-36) Physical Component Score (PCS) indicated that post-2002 cohorts had statistically significant improvements over the first 60-months compared to pre-2002 cohorts, with standardised mean differences (SMD) of 0.86 (95% Confidence Intervals 0.34 to 1.37) and 0.76 (95% CI 0.25 to 1.27) respectively at month-60. However, post-2002 cohorts indicated statistically non-significant improvements in pain, fatigue, functional disability and SF-36 Mental Component Score (MCS) compared to pre-2002 cohorts, with SMD of 0.24 (95% CI -0.25 to 0.74), 0.38 (95% CI -0.11 to 0.88), 0.34 (95% CI -0.15-0.84) and -0.08 (95% CI -0.41 to 0.58) at month-60 respectively. CONCLUSIONS: Recent cohorts indicate improved levels of disease activity and physical quality of life, however this has not translated into similar improvements in levels of pain, fatigue and functional disability by 60-months.

A multi-biomarker disease activity score can predict sustained remission in rheumatoid arthritis.

BACKGROUND: Reliable assessment of remission is important for the optimal management of rheumatoid arthritis (RA) patients. In this study, we used the multi-biomarker disease activity (MBDA) test to explore the role of biomarkers in predicting point remission and sustained remission. METHODS: RA patients on > 6 months stable therapy in stable low disease activity (DAS28-ESR ≤ 3.2) were assessed every 3 months for 1 year. Baseline, intermittent (IR) and sustained (SR) remission were defined by DAS28-ESR, DAS28-CRP, simple disease activity index (SDAI), clinical disease activity index (CDAI) and ACR/EULAR Boolean criteria. Patients not fulfilling any remission criteria at baseline were classified as 'low disease activity state' (LDAS). Patients not fulfilling any remission criteria over 1 year were classified as 'persistent disease activity' (PDA). MBDA score was measured at baseline/3/6 months. The baseline MBDA score, the 6-month time-integrated MBDA score and MBDA biomarkers were used for analyses. The area under the receiver operating characteristic curve (AUROC) assessed the ability of the MBDA score to discriminate between remission and non-remission. Biomarkers were analysed at baseline using the Mann-Whitney test and over time using the Jonckheere-Terpstra trend test. RESULTS: Of 148 patients, 27% were in the LDAS, 65% DAS28-ESR remission, 51% DAS28-CRP remission, 40% SDAI remission, 43% CDAI remission and 25% ACR/EULAR Boolean remission at baseline. Over 1 year, 9% of patients were classified as PDA. IR and SR were achieved in 42%/47% by DAS28-ESR, 46%/29% by DAS28-CRP, 45%/20% by SDAI, 44%/21% by CDAI and 35%/9% by ACR/EULAR Boolean criteria, respectively. By all remission criteria, baseline MBDA score discriminated baseline remission (AUROCs 0.68-0.75) and IR/SR (AUROCs 0.65-0.74). The 6-month time-integrated MBDA score discriminated IR/SR (AUROCs 0.65-0.79). Baseline MBDA score and concentrations of IL-6, leptin, SAA and CRP were significantly lower in all baseline remission criteria groups vs LDAS. They and the 6-month time-integrated values were lower among patients who achieved IR/SR vs PDA over 1 year. CONCLUSIONS: This study demonstrated that the MBDA score and its biomarkers IL-6, leptin, SAA and CRP differentiated between small differences in disease activity (i.e. between low disease activity and remission states). They were also predictors of remission over 1 year.

Long-term management of the liver transplant patient: recommendations for the primary care doctor.

No official document has been published for primary care physicians regarding the management of liver transplant patients. With no official source of reference, primary care physicians often question their care of these patients. The following guidelines have been approved by the American Society of Transplantation and represent the position of the association. The data presented are based on formal review and analysis of published literature in the field and the clinical experience of the authors. These guidelines address drug interactions and side effects of immunosuppressive agents, allograft dysfunction, renal dysfunction, metabolic disorders, preventive medicine, malignancies, disability and productivity in the workforce, issues specific to pregnancy and sexual function, and pediatric patient concerns. These guidelines are intended to provide a bridge between transplant centers and primary care physicians in the long-term management of the liver transplant patient.

Crystal structure of cobra-venom phospholipase A2 in a complex with a transition-state analogue.

The crystal structure of a complex between a phosphonate transition-state analogue and the phospholipase A2 (PLA2) from Naja naja atra venom has been solved and refined to a resolution of 2.0 angstroms. The identical stereochemistry of the two complexes that comprise the crystal's asymmetric unit indicates both the manner in which the transition state is stabilized and how the hydrophobic fatty acyl chains of the substrate are accommodated by the enzyme during interfacial catalysis. The critical features that suggest the chemistry of binding and catalysis are the same as those seen in the crystal structure of a similar complex formed with the evolutionarily distant bee-venom PLA2.

Crystal structure of bee-venom phospholipase A2 in a complex with a transition-state analogue.

The 2.0 angstroms crystal structure of a complex containing bee-venom phospholipase A2 (PLA2) and a phosphonate transition-state analogue was solved by multiple isomorphous replacement. The electron-density map is sufficiently detailed to visualize the proximal sugars of the enzyme's N-linked carbohydrate and a single molecule of the transition-state analogue bound ot its active center. Although bee-venom PLA2 does not belong to the large homologous Class I/II family that encompasses most other well-studied PLA2s, there is segmental sequence similarity and conservation of many functional substructures. Comparison of the bee-venom enzyme with other phospholipase structures provides compelling evidence for a common catalytic mechanism.

Interfacial catalysis: the mechanism of phospholipase A2.

A chemical description of the action of phospholipase A2 (PLA2) can now be inferred with confidence from three high-resolution x-ray crystal structures. The first is the structure of the PLA2 from the venom of the Chinese cobra (Naja naja atra) in a complex with a phosphonate transition-state analogue. This enzyme is typical of a large, well-studied homologous family of PLA2S. The second is a similar complex with the evolutionarily distant bee-venom PLA2. The third structure is the uninhibited PLA2 from Chinese cobra venom. Despite the different molecular architectures of the cobra and bee-venom PLA2s, the transition-state analogue interacts in a nearly identical way with the catalytic machinery of both enzymes. The disposition of the fatty-acid side chains suggests a common access route of the substrate from its position in the lipid aggregate to its productive interaction with the active site. Comparison of the cobra-venom complex with the uninhibited enzyme indicates that optimal binding and catalysis at the lipid-water interface is due to facilitated substrate diffusion from the interfacial binding surface to the catalytic site rather than an allosteric change in the enzyme's structure. However, a second bound calcium ion changes its position upon the binding of the transition-state analogue, suggesting a mechanism for augmenting the critical electrophile.

Structures of free and inhibited human secretory phospholipase A2 from inflammatory exudate.

Phospholipase A2 (PLA2) participates in a wide range of cellular processes including inflammation and transmembrane signaling. A human nonpancreatic secretory PLA2 (hnps-PLA2) has been identified that is found in high concentrations in the synovial fluid of patients with rheumatoid arthritis and in the plasma of patients with septic shock. This enzyme is secreted from certain cell types in response to the proinflammatory cytokines, tumor necrosis factor or interleukin-1. The crystal structures of the calcium-bound form of this enzyme have been determined at physiological pH both in the presence [2.1 angstrom (A) resolution] and absence (2.2 A resolution) of a transition-state analogue. Although the critical features that suggest the chemistry of catalysis are identical to those inferred from the crystal structures of other extracellular PLA2s, the shape of the hydrophobic channel of hnps-PLA2 is uniquely modulated by substrate binding.

Crystal structure of the extracellular segment of integrin alpha Vbeta3.

Integrins are alphabeta heterodimeric receptors that mediate divalent cation-dependent cell-cell and cell-matrix adhesion through tightly regulated interactions with ligands. We have solved the crystal structure of the extracellular portion of integrin alphaVbeta3 at 3.1 A resolution. Its 12 domains assemble into an ovoid "head" and two "tails." In the crystal, alphaVbeta3 is severely bent at a defined region in its tails, reflecting an unusual flexibility that may be linked to integrin regulation. The main inter-subunit interface lies within the head, between a seven-bladed beta-propeller from alphaV and an A domain from beta3, and bears a striking resemblance to the Galpha/Gbeta interface in G proteins. A metal ion-dependent adhesion site (MIDAS) in the betaA domain is positioned to participate in a ligand-binding interface formed of loops from the propeller and betaA domains. MIDAS lies adjacent to a calcium-binding site with a potential regulatory function.

The frequency of remission and low disease activity in patients with rheumatoid arthritis, and their ability to identify people with low disability and normal quality of life.

OBJECTIVE: Treat-to-target in rheumatoid arthritis (RA) recommends targeting remission, with low disease activity (LDA) being an alternative goal. When deciding to target remission or LDA, important considerations are the likelihood of attaining them, and their impacts on function and health-related quality of life (HRQoL). We have addressed this by studying: (a) the frequency of remission and LDA/remission; (b) DAS28-ESR trends after remission; (c) ability of remission vs. LDA to identify patients with normal function (HAQ ≤ 0.5) and HRQoL (EQ-5D ≥ the normal population). METHODS: We studied 571 patients in two clinical trials, and 1693 patients in a 10-year routine care cohort. We assessed the frequency and sustainability of remission and LDA/remission, variability in DAS28-ESR after remission, and sensitivity/specificity of remission and LDA/remission at identifying patients with low disability levels and normal HRQoL using Receiver Operator Characteristic (ROC) curves. RESULTS: Point remission and remission/LDA were common (achieved by 35-58% and 49-74% of patients, respectively), but were rarely sustained (sustained remission and remission/LDA achieved by 5-9% and 9-16% of patients, respectively). Following attaining remission, DAS28-ESR levels varied substantially. Despite this, of those patients attaining point remission, the majority (53-61%) were in remission at study end-points. Whilst remission was highly specific at identifying patients with low disability (85-91%) it lacked sensitivity (51-57%); similar findings were seen for normal HRQoL (specificity 78-86%; sensitivity 52-59%). The optimal DAS28-cut-off to identify individuals with low disability and normal HRQoL was around the LDA threshold. CONCLUSIONS: Our findings support both the treat-to-target goals. Attaining remission is highly specific for attaining low disability and normal HRQoL, although many patients with more active disease also have good function and HRQoL. Attaining a DAS28-ESR ≤ 3.2 has a better balance of specificity and sensitivity for attaining these outcomes, with the benefit of being more readily achievable. Although sustaining these targets over time is rare, even attaining them on a one-off basis leads to better function and HRQoL outcomes for patients.

A systematic review and meta-analysis of efficacy and toxicity of disease modifying anti-rheumatic drugs and biological agents for psoriatic arthritis.

OBJECTIVE: Treatments for psoriatic arthritis (PsA) range from high-cost agents such as tumour necrosis factor (TNF) inhibitors evaluated in large randomised control trials (RCTs) and low-cost disease-modifying anti-rheumatic drugs (DMARDs) studied in less detail. We compared their efficacy and toxicity in a systematic review. METHODS: We searched Medline, PubMed and EmBase (1966-2006) for RCTs in PsA. We included RCTs that were randomised, placebo-controlled, in English, involved current treatments and only enrolled PsA patients. Efficacy was assessed by the numbers of patients withdrawn for lack of effect; toxicity by withdrawals for adverse events. RCTs were compared using risk ratios (RR) with 95% confidence intervals (CI). RESULTS: We identified 32 potentially relevant RCTs; 14 were excluded because they involved unused agents, were unblinded, were not placebo-controlled and enrolled patients with other diseases. 18 studies were included in the meta-analysis assessing DMARD monotherapy (11), DMARD combinations (one), TNF inhibitors (five) and alefacept (one). Treatment was more effective than placebo (RR = 0.35; 95% CI 0.25, 0.49) but caused more toxicity (RR = 2.33; 95% CI 1.61, 3.37). There was evidence that gold, sulfasalazine, leflunomide and TNF inhibitors were effective; gold and TNF inhibitors showed the largest effect sizes; TNF inhibitors had the best efficacy/toxicity ratio (number needed to harm/number needed to treat = 0.25); tolerability was least with gold and leflunomide. CONCLUSIONS: Efficacy/toxicity ratios were highest with TNF inhibitors followed by leflunomide, gold and sulfasalazine. Gold, though effective, has excessive toxicity and sulfasalazine, though of low toxicity, was also relatively ineffective.

Factorial randomised controlled trial of glucocorticoids and combination disease modifying drugs in early rheumatoid arthritis.

OBJECTIVE: Treating early active rheumatoid arthritis (RA) with disease modifying antirheumatic drug (DMARD) monotherapy achieves incomplete outcomes and intensive treatment seems preferable. As the relative benefits of combining two DMARDs, one DMARD with glucocorticoids and two DMARDs with glucocorticoids are uncertain we defined them in a factorial trial. METHODS: A 2-year randomised double-blind factorial trial in patients with RA within 2 years of diagnosis treated with methotrexate studied the benefits of added ciclosporin, 9 months intensive prednisolone or both (triple therapy). The primary outcome was the number of patients with new erosions. Secondary outcomes included Larsen's x-ray scores, disability, quality of life and adverse events. FINDINGS: 1391 patients were screened and 467 randomised. Over 2 years 132 (28%) changed therapy and 88 (19%) were lost to follow-up. The number of patients with new erosions was reduced by nearly half by adding ciclosporin or prednisolone (p = 0.01 and 0.03); both treatments reduced increases in Larsen's x-ray scores by over 2 units (p = 0.008 and 0.003). A further reduction in erosive damage was seen with combined use of both treatments. Their effects on erosive damage appeared independent. Triple therapy reduced disability and improved quality of life compared with methotrexate; ciclosporin and prednisolone acted synergistically. More patients withdrew because of adverse events with triple therapy, without an increase in serious adverse effects. CONCLUSIONS: This study confirms the existence of a "window of opportunity" in early RA, when intensive combination therapy produces sustained benefits on damage and disability. Although methotrexate-prednisolone combinations reduce erosive damage, the synergistic effect of two DMARDs is needed to improve quality of life.

Impact of intensive treatment and remission on health-related quality of life in early and established rheumatoid arthritis.

OBJECTIVES: To establish if using intensive treatment to reduce synovitis and attain remission in active rheumatoid arthritis (RA) improves all aspects of health-related quality of life (HRQoL). METHODS: A secondary analysis of two randomised clinical trials (CARDERA and TACIT) was undertaken. CARDERA randomised 467 patients with early active RA to different disease-modifying antirheumatic drug (DMARD) regimens, including high-dose tapering corticosteroids. TACIT randomised 205 established patients with active RA to combination DMARDs (cDMARDs) or tumour necrosis factor-α inhibitors (TNFis). Short-Form 36 (SF-36) measured HRQoL across eight domains, generating physical (PCS) and mental (MCS) component summary scores. Linear regression evaluated 6-month intensive treatment impacts. Mean SF-36 scores, stratified by end point disease activity category, were compared with age/gender-matched population scores. RESULTS: In CARDERA, intensive corticosteroid treatment gave significantly greater improvements in PCS but not MCS scores relative to placebo. In TACIT, all eight SF-36 domains had improvements from baseline exceeding minimal clinically important differences with cDMARDs and TNFis. Significantly greater improvements with TNFi relative to cDMARDs were reported in PCS only (p=0.034), after adjusting for covariates. Remission provided the best SF-36 profiles, but scores in physical functioning, role physical and general health in both trials remained below normative values. Patient global assessment of disease activity had a greater association with HRQoL than other disease activity score (DAS28) components. CONCLUSIONS: Intensive corticosteroid treatment in early RA improves physical but not mental health, relative to placebo. In established RA, cDMARDs and TNFi provide similar improvements in HRQoL. As remission optimises but fails to normalise HRQoL, a focus on treatment strategies targeting HRQoL is required. TRIAL REGISTRATION NUMBERS: CARDERA was registered as ISRCTN 32484878. TACIT was registered as ISRCTN 37438295; pre-results.

The influence of dietary selenium and vitamin E on glutathione peroxidase and glutathione in the rat.

The effect of dietary selenium (Se) and vitamin E supplementation on tissue reduced glutathione (GSH) and glutathione peroxidase activity has been studied in the rat. Increasing Se intake by 0.4 ppm gave significantly higher enzyme levels in all tissues studied, an effect not influenced by vitamin E intake. Further increasing Se to 4 ppm gave higher enzyme levels in red blood cells only, while in liver there was a significant decrease in enzyme activity probably reflecting Se hepatotoxicity. In the absence of Se supplements increasing dietary vitamin E to 100 mg/kg diet significantly increased enzyme activity but this effect was modified by simultaneous Se supplementation. Se intake had no effect on GSH levels. Rats on a high vitamin E intake 500 mg/kg had a significantly higher tissue GSH level. Dietary Se had a sparing effect on vitamin E, rats supplemented with Se having significantly raised plasma vitamin E levels. These results confirm the role of selenium in glutathione peroxidase and also show that vitamin E influences the activity of the enzyme.

The 2.4 A crystal structure of cholera toxin B subunit pentamer: choleragenoid.

Cholera toxin, a heterohexameric AB5 enterotoxin released by Vibrio cholera, induces a profuse secretory diarrhea in susceptible hosts. Choleragenoid, the B subunit pentamer of cholera toxin, directs the enzymatic A subunit to its target by binding the GM1 gangliosides exposed on the luminal surface of intestinal epithelial cells. The crystal structure of choleragenoid has been independently solved and refined at 2.4 A resolution by combining single isomorphous replacement with non-crystallographic symmetry averaging. The structure of the B subunits, and their pentameric arrangement, closely resembles that reported for the intact holotoxin, choleragen, the heat-labile enterotoxin from Escherichia coli, and for a choleragenoid-GM1 pentasaccharide complex. In the absence of the A subunit the central cavity of the B pentamer is a highly solvated channel. The binding of choleragenoid to the A subunit or to its receptor pentasaccharide modestly affects the local stereochemistry without perceptibly altering the subunit interface.

The three-dimensional crystal structure of cholera toxin.

The clinical manifestations of cholera are largely attributable to the actions of a secreted hexameric AB5 enterotoxin (choleragen). We have independently solved and refined the three-dimensional structure of choleragen at 2.5 A resolution. The structure of the crystalline toxin closely resembles that described for the heat-labile enterotoxin from Escherichia coli (LT) with which it shares 80% sequence homology. In both cases, the wedge-shaped A subunit is loosely held high above the plane of the pentameric B subunits by the tethering A2 chain. The most striking difference between the two toxins occurs at the carboxyl terminus of the A2 chain. Whereas the last 14 residues of the A2 chain of LT threading through the central pore of the B5 assembly form an extended chain with a terminal loop, the A2 chain of choleragen remains a nearly continuous alpha-helix throughout its length. The four carboxyl-terminal residues of the A2 chain (KDEL sequence), disordered in the crystal structure of LT, are clearly visible in choleragen's electron-density map. In the accompanying article we describe the three-dimensional structure of the isolated B pentamer of cholera toxin (choleragenoid). Comparison of the crystalline coordinates of choleragen, choleragenoid, and LT provides a solid three-dimensional foundation for further experimental investigation. These structures, along with those of related toxins from Shigella dysenteria and Bordetella pertussis, offer a first step towards the rational design of new vaccines and anti-microbial agents.