Testing venous carbohydrate-deficient transferrin or capillary phosphatidylethanol with concurrent ethyl glucuronide and ethyl palmitate hair tests to assess historical and recent alcohol use.

Hair biomarkers, ethyl glucuronide (EtG) and ethyl palmitate (EtPa), together with blood biomarker tests, carbohydrate-deficient transferrin (CDT) or phosphatidylethanol (PEth), are commonly used in identifying patterns of alcohol consumption as they possess different windows of detection. The detection of EtG in hair samples is mainly used in combination with EtPa when hair cosmetic treatments such as hair colouring and bleaching affect EtG levels. The main purpose of our study was to investigate the differences in frequency distribution of positive CDT and PEth results indicating alcohol had been used, when EtG and EtPa were not detected, where evidence of abstinence is paramount. Of the total 602 cases, for 179 (29.7%), neither EtG nor EtPa markers were detected. Of these, 0.5% of the cases produced positive CDT. However, 18.6% produced positive PEth, a significantly higher proportion. A similar pattern emerges when results are evaluated according to whether hair had been either cosmetically treated or untreated. When hair was untreated, one case produced positive CDT, and 19.3% were positive for PEth (median of 51 ng/ml). No cases of positive CDT results, but 20.8% of PEth were positive (median of 106.5 ng/ml) when hair samples had been cosmetically treated. Whether EtG or EtPa markers were detected or not, significantly higher proportions of PEth than CDT were seen. The results of this study substantiate the case for using hair EtG in conjunction with a PEth test, rather than CDT test, for efficient monitoring of recent and historical alcohol consumption.

Cross-species/cross-modality physiologically based pharmacokinetics for biologics: 89Zr-labelled albumin-binding domain antibody GSK3128349 in humans.

Two-pore physiologically-based pharmacokinetics (PBPK) for biologics describes the tissue distribution and elimination kinetics of soluble proteins as a function of their hydrodynamic radius and the physiological properties of the organs. Whilst many studies have been performed in rodents to parameterize the PBPK framework in terms of organ-specific lymph flow rates, similar validation in humans has been limited. This is mainly due to the paucity of the tissue distribution time course data for biologics that is not distorted by target-related binding. Here, we demonstrate that a PBPK model based on rodent data provided good to satisfactory extrapolation to the tissue distribution time course of 89Zr-labeled albumin-binding domain antibody (AlbudAb™) GSK3128349 in healthy human volunteers, including correct prediction of albumin-like plasma half-life, volume of distribution, and extravasation half-life. The AlbudAb™ used only binds albumin, and hence it also provides information about the tissue distribution kinetics and turnover of that ubiquitous and multifunctional plasma protein.

The Landscape of Early Clinical Gene Therapies outside of Oncology.

The field of cell and gene therapy (GT) is expanding rapidly and there is undoubtedly a wave of enthusiasm and anticipation for what these treatments could achieve next. Here we assessed the worldwide landscape of GT assets currently in early clinical development (clinical trial phase 1/2 or about to enter clinical trial). We included all gene therapies, i.e., strategies that modify an individual's protein make-up by introducing exogenous nucleic acid or nucleic acid modifiers, regardless of delivery. Unmodified cell therapies, oncology therapies (reviewed elsewhere), and vaccine programs (distinct therapeutic strategy) were not included. Using a December 31, 2018 cutoff date, we identified 336 gene therapies being developed for 138 different indications covering 165 genetic targets. In all, we found that the early clinical GT landscape comprises a very disparate group of drug candidates in terms of indications, organizations, and delivery methods. We also highlight interesting trends, revealing the evolution of the field toward in vivo therapies and adeno-associated virus vector-based delivery systems. It will be interesting to witness what proportion of this current list effectively translates into new medicines.

The biodistribution and clearance of AlbudAb, a novel biopharmaceutical medicine platform, assessed via PET imaging in humans.

Conjugation or fusion to AlbudAbs™ (albumin-binding domain antibodies) is a novel approach to extend the half-life and alter the tissue distribution of biological and small molecule therapeutics. To understand extravasation kinetics and extravascular organ concentrations of AlbudAbs in humans, we studied tissue distribution and elimination of a non-conjugated 89Zr-labeled AlbudAb in healthy volunteers using positron emission tomography/computed tomography (PET/CT). METHODS: A non-conjugated AlbudAb (GSK3128349) was radiolabeled with 89Zr and a single 1 mg (~ 15 MBq) dose intravenously administered to eight healthy males. 89Zr-AlbudAb tissue distribution was followed for up to 7 days with four whole-body PET/CT scans. 89Zr-AlbudAb tissue concentrations were quantified in organs of therapeutic significance, measuring standardized uptake value and tissue/plasma ratios. Plasma pharmacokinetics were assessed by gamma counting and LC-MS/MS of blood samples. RESULTS: 89Zr-AlbudAb administration and PET/CT procedures were well tolerated, with no drug-related immunogenicity or adverse events. 89Zr-AlbudAb rapidly distributed throughout the vasculature, with tissue/plasma ratios in the liver, lungs, and heart relatively stable over 7 days post-dose, ranging between 0.1 and 0.5. The brain tissue/plasma ratio of 0.025 suggested minimal AlbudAb blood-brain barrier penetration. Slowly increasing ratios in muscle, testis, pancreas, and spleen reflected either slow AlbudAb penetration and/or 89Zr residualization in these organs. Across all tissues evaluated, the kidney tissue/plasma ratio was highest (0.5-1.5 range) with highest concentration in the renal cortex. The terminal half-life of the 89Zr-AlbudAb was 18 days. CONCLUSION: Evaluating the biodistribution of 89Zr-AlbudAb in healthy volunteers using a low radioactivity dose was successful (total subject exposure ~ 10 mSv). Results indicated rapid formation of reversible, but stable, complexes between AlbudAb and albumin upon dosing. 89Zr-AlbudAb demonstrated albumin-like pharmacokinetics, including limited renal elimination. This novel organ-specific distribution data for AlbudAbs in humans will facilitate a better selection of drug targets to prosecute using the AlbudAb platform and significantly contribute to modeling work optimizing dosing of therapeutic AlbudAbs in the clinic.

Student pharmacists' performance and perceptions on an evidence-based medicine objective structured clinical examination.

BACKGROUND AND PURPOSE: Studies have examined evidence-based medicine (EBM) focused objective structured clinical examinations (OSCEs) in medical training, but data are lacking in pharmacy trainees. This study sought to assess student pharmacists' performance on and perceptions of a novel EBM OSCE. EDUCATIONAL ACTIVITY AND SETTING: This EBM OSCE included answering a drug-information inquiry, researching background questions, calling a simulated provider to acquire specific patient information, developing a foreground clinical question, reviewing pre-appraised trial synopses, and applying evidence to write a recommendation. Pharmacy faculty served as simulated providers and assessed students on knowledge/analytical (AC) and global communication (GC) skills. Students completed a worksheet (WS) that included developing a patient, intervention, comparison, outcome (PICO) statement, trial selection, and clinical recommendation. After OSCE completion, students were surveyed regarding perceptions of their performance and OSCE applicability. Outcomes assessed were performance scores (AC, GC, WS) and student perceptions. FINDINGS: One-hundred twenty-nine students completed the survey and were included in analysis. AC, WS, and GC performance [median (IQR)] were 75.0 (37.8), 86.4 (36.9), and 88.9 (22.2), respectively, on a 100-point scale. On the WS, 89% of students developed a suitable searchable clinical question and 61% selected the correct trial synopsis to apply to the case. Students felt literature application and WS development were most challenging. A majority of students felt this OSCE increased comfort in engaging with providers (74%) and that these skills correlate with real clinical scenarios (77%). SUMMARY: OSCEs can be a valuable tool for simulating clinical scenarios and assessing student pharmacists' EBM skills.

Improving Pharmacy Calculations Using an Instructional Design Model.

Objective. To describe an evidence-based instructional design to improve performance and foster retention of pharmacy students' calculation skills longitudinally across the curriculum. Methods. Gagne's nine events of instructional design were employed in a longitudinal pharmacy calculations curriculum. Mean pharmacy calculation examination scores from four courses spanning the didactic curriculum for four different academic years (before and after the redesign) were compared. Results. Students demonstrated more stable outcomes with consistently higher means after the redesign, which may indicate improved retention. Additionally, the post-redesign classes have experienced fewer failures (score of <80%). Conclusion. Using an instructional design model to optimize immediate instructional outcomes is an effective method of enhancing retention of calculation skills over time.

Treatment of Adults with Idiopathic Recurrent Pericarditis: Novel Use of Immunotherapy.

Idiopathic recurrent pericarditis (IRP) can be challenging to treat. Even after guideline-directed first-line treatment consisting of aspirin (ASA) or a nonsteroidal antiinflammatory drug (NSAID) in combination with colchicine therapy, recurrences still occur in greater than 20% of patients. Many patients then require treatment with long-term corticosteroids, which is not a favorable option due to their short- and long-term adverse effects. Because it is theorized that the pathophysiology of IRP may possess autoimmune sequelae, the use of immunotherapy for the treatment of IRP has emerged. In this review, we describe the literature associated with immunotherapy used to treat IRP in an adult population as well as provide an overview of the safety and monitoring parameters for each agent. The most common immunotherapies used after patients have had multiple recurrences of IRP are anakinra, intravenous immunoglobulin (IVIG), and azathioprine. In most cases, these immunotherapies are adjunctive therapy, with the goal of tapering and discontinuing immunosuppressive corticosteroids. After reviewing the data, anakinra resulted in more patients discontinuing corticosteroids and prevented further recurrences of pericarditis. IVIG resulted in symptom resolution and no further recurrences in most of the patients. Azathioprine was associated with more than half of patients becoming recurrence free; however, many patients required a restart of corticosteroids due to recurrence. Clinicians should be aware of the adverse effects of immunotherapy, ranging from mild gastrointestinal events to risk of infection and serious blood dyscrasias that may require diligent monitoring. The use of immunotherapy for the treatment of adults with IRP should be restricted to patients who have multiple recurrences. Ideally, immunotherapy would be adjunctive to first-line combination therapy with ASA/NSAID plus colchicine, with the goal of tapering and discontinuing immunosuppressive corticosteroids. Furthermore, clinicians should consider cost, drug-drug and drug-disease interactions, and safety, as well as the quality of the retrospective evidence before considering any immunotherapy.

GLP1-RA Add-on Therapy in Patients with Type 2 Diabetes Currently on a Bolus Containing Insulin Regimen.

Adding glucagon-like peptide-1 receptor agonists (GLP-1 RAs) to basal insulin regimens has become a guideline-recommended treatment option for uncontrolled type 2 diabetes. However, limited data exist to support the use of GLP-1 RAs with insulin regimens, including bolus insulin in patients with type 2 diabetes. The primary objectives of this review were to identify if the combination of a GLP-1 RA and an insulin regimen containing bolus insulin resulted in improvements in HbA1c , weight loss, reduction in insulin doses, and to evaluate the side effect profile of this combination in terms of nausea and hypoglycemia risk. Eight studies using exenatide twice/day, liraglutide, and dulaglutide were reviewed ranging in average duration of follow-up from 3 to 15 months. Seven studies showed that addition of a GLP-1 RA was associated with significant HbA1c reductions ranging from 0.4% to 1.64% from baseline to follow-up. Patients in all eight studies had significant weight loss in the GLP-1 RA arm from baseline to follow-up ranging from 0.87 to 10.2 kg. In all the studies, total daily bolus insulin doses decreased 25-67% from baseline to follow-up. In some studies, a portion of patients were able to discontinue bolus insulin all together after initiation of a GLP-1 RA. In addition, in two randomized trials included in the review, the GLP-1 RA arm showed significant improvement in HbA1c and weight compared with the control group who received basal/bolus regimens. Nausea was identified in 7-42% of participants using GLP-1 RAs with insulin. Data support the use of GLP-1 RAs added to insulin regimens already containing bolus insulin for glycemic control, weight loss, and reduction or discontinuation of bolus insulin.

Changes in Student Performance and Confidence with a Standardized Patient and Standardized Colleague Interprofessional Activity.

OBJECTIVE: To assess the impact of a standardized patient and standardized colleague interprofessional activity on student performance and perceived confidence in communicating with patients and physicians. METHODS: Students in the third professional year were presented with a practice and final activity including a standardized patient interaction, SOAP note preparation, and standardized colleague interaction. Student performance was measured by assessment rubrics on practice and final activities. Students' perceived confidence was measured via presurvey and postsurvey. RESULTS: Students performed significantly better from the practice to the final activity with regard to communicating with patients, SOAP note, and the overall activity with a mean difference (95% CI) of 9.2 (6.9-11.5), 3.6 (1.3-5.8), and 3.9 (2.0-5.7), respectively. There was a positive significant change from presurvey to postsurvey in students' confidence talking to patients and physicians on majority of questions. CONCLUSION: This study demonstrates that active learning and integrated assessments improve overall student performance. Integration of interprofessional education also has positive effects on students' perceived confidence.

Pharmacokinetic and Pharmacodynamic Characterisation of an Anti-Mouse TNF Receptor 1 Domain Antibody Formatted for In Vivo Half-Life Extension.

Tumour Necrosis Factor-α (TNF-α) inhibition has been transformational in the treatment of patients with inflammatory disease, e.g. rheumatoid arthritis. Intriguingly, TNF-α signals through two receptors, TNFR1 and TNFR2, which have been associated with detrimental inflammatory and beneficial immune-regulatory processes, respectively. To investigate if selective TNFR1 inhibition might provide benefits over pan TNF-α inhibition, tools to investigate the potential impact of pharmacological intervention are needed. Receptor-deficient mice have been very insightful, but are not reversible and could distort receptor cross-talk, while inhibitory anti-TNFR1 monoclonal antibodies have a propensity to induce receptor agonism. Therefore, we set out to characterise a monovalent anti-TNFR1 domain antibody (dAb) formatted for in vivo use. The mouse TNFR1 antagonist (DMS5540) is a genetic fusion product of an anti-TNFR1 dAb with an albumin-binding dAb (AlbudAb). It bound mouse TNFR1, but not human TNFR1, and was an antagonist of TNF-α-mediated cytotoxicity in a L929 cell assay. Surprisingly, the dAb did not compete with TNF-α for TNFR1-binding. This was supported by additional data showing the anti-TNFR1 epitope mapped to a single residue in the first domain of TNFR1. Pharmacokinetic studies of DMS5540 in mice over three doses (0.1, 1.0 and 10 mg/kg) confirmed extended in vivo half-life, mediated by the AlbudAb, and demonstrated non-linear clearance of DMS5540. Target engagement was further confirmed by dose-dependent increases in total soluble TNFR1 levels. Functional in vivo activity was demonstrated in a mouse challenge study, where DMS5540 provided dose-dependent inhibition of serum IL-6 increases in response to bolus mouse TNF-α injections. Hence, DMS5540 is a potent mouse TNFR1 antagonist with in vivo pharmacokinetic and pharmacodynamic properties compatible with use in pre-clinical disease models and could provide a useful tool to dissect the individual contributions of TNFR1 and TNFR2 in homeostasis and disease.

Pharmacokinetic characteristics, pharmacodynamic effect and in vivo antiviral efficacy of liver-targeted interferon alpha.

Interferon alpha (IFNα) is used for the treatment of hepatitis B virus infection, and whilst efficacious, it is associated with multiple adverse events caused by systemic exposure to interferon. We therefore hypothesise that targeting IFN directly to the intended site of action in the liver would reduce exposure in blood and peripheral tissue and hence improve the safety and tolerability of IFNα therapy. Furthermore we investigated whether directing IFN to the reservoir of infection in the liver may improve antiviral efficacy by increasing local concentration in target organs and tissues. Our previous results show that the mIFNα2 fused to an ASGPR specific liver targeting antibody, DOM26h-196-61, results in a fusion protein which retains the activity of both fusion partners when measured in vitro. In vivo targeting of the liver by mIFNα2-DOM26h-196-61, hereafter referred to as targeted mIFNα2, was observed in microSPECT imaging studies in mice. In this study we show by pharmacokinetic analysis that antibody mediated liver-targeting results in increased uptake and exposure of targeted mIFNα2 in target tissues, and correspondingly reduced uptake and exposure in systemic circulation, clearance organs and non-target tissues. We also show that cytokine activity and antiviral activity of liver-targeted IFN is observed in vivo, but that, contrary to expectations, liver-targeting of mIFNα2 using ASGPR specific dAbs actually leads to a reduced pharmacodynamic effect in target organs and lower antiviral activity in vivo when compared to non-targeted mIFNα2-dAb fusions.

Predictors of glycaemic control among HIV-positive veterans with diabetes.

Antiretroviral therapy has decreased HIV-related mortality. However, the incidence of diabetes as a co-morbidity is increasing as HIV-positive patients age. The purpose of this study was to assess the correlation between markers of HIV-infection and diabetes and to determine the proportion of patients achieving an haemoglobin A1c (HbA1c) goal <7% according to specific antiretroviral therapy regimens and adherence. In this retrospective study, HIV-positive veterans with diabetes from 2007 to 2012 were identified. Patients were required to be on the same antiretroviral therapy and diabetes regimen for ≥3 months. In 56 patients, it was identified that for each unit increase in log10 viral load, HbA1c increased 0.67 units (p = 0.0085). Only 38% of patients prescribed a protease inhibitor-based regimen vs. 56% of patients not on a protease inhibitor-based regimen achieved an HbA1c goal (p = 0.1864). Additionally, patients on an insulin-based regimen and patients that were less adherent were less likely to be at HbA1c goal (p = 0.018 and p = 0.0378, respectively). Patients with higher viral loads and patients that were less adherent to antiretroviral therapy were more likely to have a higher HbA1c demonstrating that poor adherence to antiretroviral therapy leads to poor control of both disease states.

Selective tumor necrosis factor receptor I blockade is antiinflammatory and reveals immunoregulatory role of tumor necrosis factor receptor II in collagen-induced arthritis.

OBJECTIVE: Tumor necrosis factor (TNF) signals via 2 receptors, TNFR type I (TNFRI) and TNFRII, with distinct cellular distribution and signaling functions. In rheumatoid arthritis (RA), the net effect of TNFR signaling favors inflammatory responses while inhibiting the activity of regulatory T cells. TNFRII signaling has been shown to promote Treg cell function. To assess the relative contributions of TNFRI and TNFRII signaling to inflammatory and regulatory responses in vivo, we compared the effect of TNF blockade, hence TNFRI/II, versus TNFRI alone in collagen-induced arthritis (CIA) as a model of RA. METHODS: Mice with established arthritis were treated for 10 days with anti-mouse TNFRI domain antibody (dAb; DMS5540), an isotype control dAb (DMS5538), or murine TNFRII genetically fused with mouse IgG1 Fc domain (mTNFRII-Fc) beginning on the day of arthritis onset, and disease progression was monitored. Systemic cytokine concentrations and numbers of T cell subsets in lymph nodes and spleens were measured, and intrinsic Treg cell function was determined by ex vivo suppression assays. RESULTS: Progression of CIA was suppressed similarly by TNFRI (DMS5540) and TNFRI/II (mTNFRII-Fc) blockade. However, blockade of TNFRI/II led to increased effector T cell activity, which was not observed after selective TNFRI blockade, suggesting an immunoregulatory role of TNFRII. In support of this, TNFRI blockade, but not TNFRI/II blockade, expanded and activated Treg cells. Furthermore, a dramatic increase in expression of the Treg cell signature genes FoxP3 and TNFRII was observed in joints undergoing remission, which supports the notion that these molecules have a physiologic role in the resolution of inflammation. CONCLUSION: We propose that a therapeutic strategy that targets TNFRI while sparing TNFRII has the potential to both inhibit inflammation and promote Treg cell activity, which might be superior to TNF blockade.

Selective blockade of tumor necrosis factor receptor I inhibits proinflammatory cytokine and chemokine production in human rheumatoid arthritis synovial membrane cell cultures.

OBJECTIVE: To determine whether selective blockade of tumor necrosis factor receptor I (TNFRI) affects spontaneous proinflammatory cytokine and chemokine production in ex vivo-cultured human rheumatoid arthritis synovial membrane mononuclear cells (MNCs) and to compare this response to that of TNF ligand blockade using etanercept. METHODS: A bispecific, single variable-domain antibody (anti-TNFRI moiety plus an albumin binding moiety [TNFRI-AlbudAb]) was used to selectively block TNFRI. Inhibition of TNFα-mediated responses in cell lines expressing TNFRI/II confirmed TNFRI-AlbudAb potency, human rhabdomyosarcoma cell line KYM-1D4 cytotoxicity, and human umbilical vein endothelial cell (HUVEC) vascular cell adhesion molecule 1 (VCAM-1) upregulation. Eighteen RA synovial membrane MNC suspensions were cultured for 2 days or 5 days, either alone or in the presence of TNFRI-AlbudAb, control-AlbudAb, or etanercept. Proinflammatory cytokines and chemokines in culture supernatants were measured by enzyme-linked immunosorbent assays. A mixed-effects statistical analysis model was used to assess the extent of TNFRI selective blockade, where the results were expressed as the percentage change with 95% confidence intervals (95% CIs). RESULTS: TNFRI-AlbudAb inhibited TNFα-induced KYM-1D4 cell cytotoxicity (50% inhibition concentration [IC50 ] 4 nM) and HUVEC VCAM-1 up-regulation (IC50 12 nM) in a dose-dependent manner. In ex vivo-cultured RA synovial membrane MNCs, selective blockade of TNFRI inhibited the production of proinflammatory cytokines and chemokines to levels similar to those obtained with TNF ligand blockade, without inducing cellular toxicity. Changes in cytokine levels were as follows: -23.5% (95% CI -12.4, -33.2 [P = 0.004]) for granulocyte-macrophage colony-stimulating factor, -33.4% (95% CI -20.6, -44.2 [P ≤ 0.0001]) for interleukin-10 (IL-10), -17.6% (95% CI 3.2, -34.2 [P = 0.0880]) for IL-1ß, and -19.0% (95% CI -3.4, -32.1 [P = 0.0207]) for IL-6. Changes in chemokine levels were as follows: -34.2% (-14.4, -49.4 [P = 0.0030]) for IL-8, -56.6% (-30.7, -72.9 [P = 0.0011]) for RANTES, and -24.9% (2, -44.8 [P = 0.0656]) for monocyte chemotactic protein 1. CONCLUSION: In ex vivo-cultured RA synovial membrane MNCs, although a limited role of TNFRII cannot be ruled out, TNFRI signaling was found to be the dominant pathway leading to proinflammatory cytokine and chemokine production. Thus, selective blockade of TNFRI could potentially be therapeutically beneficial over TNF ligand blockade by retaining the beneficial TNFRII signaling.

Liver-targeting of interferon-alpha with tissue-specific domain antibodies.

Interferon alpha (IFNα) is used for the treatment of hepatitis C infection and whilst efficacious it is associated with multiple adverse events including reduced leukocyte, erythrocyte, and platelet counts, fatigue, and depression. These events are most likely caused by systemic exposure to interferon. We therefore hypothesise that targeting the therapeutic directly to the intended site of action in the liver would reduce exposure in blood and peripheral tissue and hence improve the safety and tolerability of IFNα therapy. We genetically fused IFN to a domain antibody (dAb) specific to a hepatocyte restricted antigen, asialoglycoprotein receptor (ASGPR). Our results show that the murine IFNα2 homolog (mIFNα2) fused to an ASGPR specific dAb, termed DOM26h-196-61, could be expressed in mammalian tissue culture systems and retains the desirable biophysical properties and activity of both fusion partners when measured in vitro. Furthermore a clear increase in in vivo targeting of the liver by mIFNα2-ASGPR dAb fusion protein, compared to that observed with either unfused mIFNα2 or mIFNα2 fused to an isotype control dAb VHD2 (which does not bind ASGPR) was demonstrated using microSPECT imaging. We suggest that these findings may be applicable in the development of a liver-targeted human IFN molecule with improved safety and patient compliance in comparison to the current standard of care, which could ultimately be used as a treatment for human hepatitis virus infections.

Anti-serum albumin domain antibodies in the development of highly potent, efficacious and long-acting interferon.

Serum albumin-binding domain antibodies (AlbudAbs) have previously been shown to greatly extend the serum half-life of the interleukin-1 receptor antagonist IL-1ra. We have subsequently extended this approach to look at the in vitro activity, in vivo efficacy and pharmacokinetics of an agonist molecule, interferon (IFN)-alpha2b, fused to an AlbudAb. Here we describe this molecule and show that in this format AlbudAb half-life extension technology displays significant advantages in comparison with other methods of half-life extension, in particular genetic fusion to serum albumin. When compared directly IFN-alpha2b fused to an Albudab shows higher potency, increased serum half-life and greater efficacy than human serum albumin fused to IFN-alpha2b. AlbudAbs are therefore an ideal platform technology for creation of therapeutics with agonist activity and long serum half-lives.

Architecture of secondary lymphoid tissue in sheep experimentally challenged with scrapie.

Scrapie is a transmissible spongiform encephalopathy in which there is an accumulation of the abnormal form of the prion protein, PrPsc, in the lymphoreticular system and nervous system. There is a particular accumulation of PrPsc on follicular dendritic cells within the germinal centre of B-cell follicles. Because accumulation of PrPsc in the nervous system leads to neuronal cell loss we have examined PrPsc accumulation in the prescapular and mesenteric lymph nodes in relation to lymph node architecture of scrapie-challenged sheep. We demonstrate that an accumulation of PrPsc in the lymph node fails to result in gross defects in the microanatomy and phenotype of T- and B-cell areas in the lymph nodes.

Candidate T cell epitopes of the human La/SSB autoantigen.

OBJECTIVE: To identify T cell epitopes of the human La autoantigen involved in the generation of anti-Ro/La autoantibodies. METHODS: Molecular techniques were used for HLA typing of 219 white patients with systemic lupus erythematosus and 125 white patients with primary Sjögren's syndrome. Anti-Ro/La antibody levels were measured by enzyme-linked immunosorbent assay. Peripheral blood mononuclear cell responses to an overlapping series of synthetic 15-mer peptides spanning the entire La sequence were examined in pools or individually in conventional 7-day proliferation assays. RESULTS: HLA typing confirmed that the HLA-DR3/DQ2 haplotype is closely associated with the occurrence of anti-Ro/La antibodies, and that the frequency of HLA-DR1 and DR4 haplotypes is reduced among antibody-positive patients. We identified 3 regions of the La sequence likely to contain T cell epitopes and 1 peptide, La 49-63, that generated a low-level but clear-cut T cell proliferative response. The HLA restrictions of these responses mirrored the HLA association data from the cohort study. Among individuals who were HLA-DR3 positive, there was no difference between patients and controls in the proliferative response to the La 49-63 peptide. CONCLUSION: Our data suggest that these are naive T cell responses, and that the identification of T cell epitopes involved in the generation of anti-Ro/La autoantibodies should focus on alternative candidate antigens.