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Trial Registration: These studies were conducted before clinical trial registration was required; therefore, clinical trial registration numbers are not available.

Background: Understanding why patients with severe asthma do not follow healthcare provider (HCP) advice to adjust treatment is critical to achieving personalised disease management., Methods: We reviewed patient choice to follow HCP advice to adjust asthma treatment in a UK-based randomised, controlled, single-blind (study participant), multicentre, parallel group 48-week clinical study comparing biomarker-directed treatment adjustment with standard care in severe asthma., Results: Of 1572 treatment advisories (291 participants), instructions were followed in 1377 cases (87.6%). Patients were more likely to follow advice to remain on treatment (96.7%) than to either reduce (70.3%) or increase (67.1%) their treatment, with 64% of patients following all treatment advice. Multivariate analysis associated belonging to an ethnic minority group (OR 3.10, 95% CI 1.68-5.73) and prior study medication changes (two or more changes: OR 2.77, 95% CI 1.51-5.10) with failure to follow treatment advice. In contrast, emergency room attendance in the prior year (OR 0.54, 95% CI 0.32-0.92) was associated with following treatment advice. The largest effect was seen with transition onto or off oral corticosteroids (OR 29.28, 95% CI 16.07-53.36) when compared with those requested to maintain treatment. Centre was also an important determinant regarding the likelihood of patients to follow treatment advice., Conclusions: Belonging to an ethnic minority group and multiple prior treatment adjustments were associated with not following HCP treatment advice. Patients also responded differently to HCP advice across UK specialist centres. These findings have implications for the generalisability of models of care in severe asthma and require further focused studies., Competing Interests: Conflict of interest: J. Busby has nothing to disclose. Conflict of interest: J.G. Matthews has nothing to disclose. Conflict of interest: R. Chaudhuri reports grants, personal fees for advisory board work and nonfinancial support for meeting attendance from AstraZeneca, personal fees for advisory board work from GlaxoSmithKline and Novartis, personal fees for advisory board work and nonfinancial support for meeting attendance from Chiesi, nonfinancial support for meeting attendance from Napp Pharmaceuticals, outside the submitted work. Conflict of interest: I.D. Pavord reports personal fees for lectures, advisory board honoraria, sponsorship to attend scientific meetings, and payments for organising educational events from AstraZeneca, personal fees for lectures, advisory board honoraria, and sponsorship to attend scientific meetings from Boehringer Ingelheim and GlaxoSmithKline, personal fees for lectures from Aerocrine and Chiesi, personal fees for lectures and advisory board honoraria from Almirall and Novartis, advisory board honoraria from Genentech, Regeneron, Sanofi, Circassia and Knopp, personal fees for lectures, payments for organising educational events, and sponsorship to attend scientific meetings from Teva, grants from the National Institute for Health Research, outside the submitted work. Conflict of interest: T.C. Hardman has nothing to disclose. Conflict of interest: J.R. Arron has patents “Diagnosis and treatments relating to Th2 inhibition” (US 9,684,000 B2) and “Diagnosis and treatments relating to Th2 inhibition” (US 9,995,755 B2) issued with rights assigned to Genentech, a member of the Roche group, and is an employee of Genentech, Inc. and owns stocks/options in the Roche group. Conflict of interest: P. Bradding reports grants from Genentech, other (consultancy work on behalf of the University of Leicester) from Roche and Boehringer Ingelheim, outside the submitted work. Conflict of interest: C.E. Brightling reports grants and personal fees from GlaxoSmithKline, AstraZeneca, Sanofi, Novartis, Chiesi, Genentech, Gossamer, Mologic and 4D Pharma, all paid to the institution and outside the submitted work. Conflict of interest: D.F. Choy is an employee of Genentech, Inc. and owns stocks/options in the Roche group. Conflict of interest: D.C. Cowan has nothing to disclose. Conflict of interest: R. Djukanovic reports receiving fees for lectures at symposia organised by Novartis, AstraZeneca and Teva, consultation for Teva and Novartis as member of advisory boards, and participation in a scientific discussion about asthma organised by GlaxoSmithKline; he is a co-founder and current consultant, and has shares in Synairgen, a University of Southampton spin-out company. Conflict of interest: C.E. Hanratty has nothing to disclose. Conflict of interest: T.W. Harrison reports grants, personal fees and nonfinancial support from AstraZeneca, grants and personal fees from GlaxoSmithKline, personal fees from Vectura, Synairgen and Chiesi, outside the submitted work. Conflict of interest: C.T. Holweg is an employee and stock owner of Genentech Inc., a member of the Roche Group, outside the submitted work. Conflict of interest: P.H. Howarth reports employment by GlaxoSmithKline. Conflict of interest: S.J. Fowler reports personal fees for lectures and support to attend an international conference from AstraZeneca, grants and personal fees for lectures from Boehringer Ingelheim, personal fees for lectures, participation in an advisory board, and support to attend an national conference from Chiesi, personal fees for lectures from GlaxoSmithKline, Novartis and Teva, outside the submitted work. Conflict of interest: J.L. Lordan has nothing to disclose. Conflict of interest: A.H. Mansur reports personal and institution payment for talks, advisory board meetings, sponsorship to attend conferences and education grants for service developments from GlaxoSmithKline, AstraZeneca, Novartis, Sanofi, Teva and others, outside the submitted work. Conflict of interest: A. Menzies-Gow reports grants and personal fees from AstraZeneca, personal fees from Novartis, GlaxoSmithKline, Sanofi and Vectura, personal fees and nonfinancial support from Teva and Boehringer Ingelheim, outside the submitted work. Conflict of interest: R.M. Niven has received lecture fees, advisory board activity and been supported to attend international meetings/conferences in the last 5 years by the following companies: AstraZeneca, Boehringer, Boston Scientific, Chiesi, Napp, Novartis and Teva. None of these have any relevance to the manuscript; R.M. Niven owns no shares or stocks and is not personally or in any family way connected to any pharmaceutical companies. Conflict of interest: D.S. Robinson has nothing to disclose. Conflict of interest: S.M. Walker has nothing to disclose. Conflict of interest: A. Woodcock reports personal fees for lectures from GlaxoSmithKline, personal fees for lectures and consultancy from Novartis, personal fees for consultancy from Chiesi, other (chairing research projects) from Reacta Biotech, Axalbion and Medicines Evaluation Unit, outside the submitted work. Conflict of interest: L.G. Heaney reports other (sponsorship for meeting attendance) from AstraZeneca, Boehringer Ingelheim, Chiesi, GlaxoSmithKline and Napp Pharmaceutical, personal fees for lectures and advisory board work from Novartis, Hoffman la Roche/Genentech Inc., Sanofi, GlaxoSmithKline, AstraZeneca, Teva, Theravance and Circassia, grants from Medimmune, Novartis UK, Roche/Genentech Inc., and GlaxoSmithKline, and is academic lead for the Medical Research Council Stratified Medicine UK Consortium in severe asthma, which involves industrial partnerships with Amgen, Genentech/Hoffman la Roche, AstraZeneca, Medimmune, GlaxoSmithKline, Aerocrine and Vitalograph, outside the submitted work., (Copyright ©The authors 2022.)

Background: Patients with difficult-to-control asthma consume 50-60% of healthcare costs attributed to asthma and cost approximately five-times more than patients with mild stable disease. Recent evidence demonstrates that not all patients with asthma have a typical type 2 (T2)-driven eosinophilic inflammation. These asthmatics have been called 'T2-low asthma' and have a minimal response to corticosteroid therapy. Adjustment of corticosteroid treatment using sputum eosinophil counts from induced sputum has demonstrated reduced severe exacerbation rates and optimized corticosteroid dose. However, it has been challenging to move induced sputum into the clinical setting. There is therefore a need to examine novel algorithms to target appropriate levels of corticosteroid treatment in difficult asthma, particularly in T2-low asthmatics. This study examines whether a composite non-invasive biomarker algorithm predicts exacerbation risk in patients with asthma on high-dose inhaled corticosteroids (ICS) (± long-acting beta agonist) treatment, and evaluates the utility of this composite score to facilitate personalized biomarker-specific titration of corticosteroid therapy., Methods/design: Patients recruited to this pragmatic, multi-centre, single-blinded randomised controlled trial are randomly allocated into either a biomarker controlled treatment advisory algorithm or usual care group in a ratio of 4:1. The primary outcome measure is the proportion of patients with any reduction in ICS or oral corticosteroid dose from baseline to week 48. Secondary outcomes include the rate of protocol-defined severe exacerbations per patient per year, time to first severe exacerbation from randomisation, dose of inhaled steroid at the end of the study, cumulative dose of inhaled corticosteroid during the study, proportion of patients on oral corticosteroids at the end of the study, proportion of patients who decline to progress to oral corticosteroids despite composite biomarker score of 2, frequency of hospital admission for asthma, change in the 7-item Asthma Control Questionnaire (ACQ-7), Asthma Quality of Life Questionnaire (AQLQ), forced expiratory volume in 1 s (FEV1), exhaled nitric oxide, blood eosinophil count, and periostin levels from baseline to week 48. Blood will also be taken for whole blood gene expression; serum, plasma, and urine will be stored for validation of additional biomarkers., Discussion: Multi-centre trials present numerous logistical issues that have been addressed to ensure minimal bias and robustness of study conduct., Trial Registration: ClinicalTrials.gov, NCT02717689 . Registered on 16 March 2016.

Background: In phase 2 trials, lebrikizumab, an anti-interleukin-13 monoclonal antibody, reduced exacerbation rates and improved FEV 1 in patients with uncontrolled asthma, particularly in those with high concentrations of type 2 biomarkers (eg, periostin or blood eosinophils). We undertook replicate phase 3 studies to assess the efficacy and safety of lebrikizumab in patients with uncontrolled asthma despite inhaled corticosteroids and at least one second controller medication., Methods: Adult patients with uncontrolled asthma, pre-bronchodilator FEV 1 40-80% predicted, and stable background therapy were randomly assigned (1:1:1) with an interactive voice-web-based response system to receive lebrikizumab 37·5 mg or 125 mg, or placebo subcutaneously, once every 4 weeks. Randomisation was stratified by screening serum periostin concentration, history of asthma exacerbations within the last 12 months, baseline asthma medications, and country. The primary efficacy endpoint was the rate of asthma exacerbations over 52 weeks in biomarker-high patients (periostin ≥50 ng/mL or blood eosinophils ≥300 cells per μL), analysed with a Poisson regression model corrected for overdispersion with Pearson χ 2 that included terms for treatment group, number of asthma exacerbations within the 12 months before study entry, baseline asthma medications, geographic region, screening periostin concentration, and blood eosinophil counts as covariates. Both trials are registered at ClinicalTrials.gov, LAVOLTA I, number NCT01867125, and LAVOLTA II, number NCT01868061., Findings: 1081 patients were treated in LAVOLTA I and 1067 patients in LAVOLTA II. Over 52 weeks, lebrikizumab reduced exacerbation rates in biomarker-high patients in the 37·5 mg dose group (rate ratio [RR] 0·49 [95% CI 0·34-0·69], p<0·0001) and in the 125 mg dose group (RR 0·70 [0·51-0·95], p=0·0232) versus placebo in LAVOLTA I. Exacerbation rates were also reduced in biomarker-high patients in both dose groups versus placebo in LAVOLTA II (37·5 mg: RR 0·74 [95% CI 0·54-1·01], p=0·0609; 125 mg: RR 0·74 [0·54-1·02], p=0·0626). Pooling both studies, the proportion of patients who experienced treatment-emergent adverse events (79% [1125 of 1432 patients] for both lebrikizumab doses vs 80% [576 of 716 patients] for placebo), serious adverse events (8% [115 patients] for both lebrikizumab doses vs 9% [65 patients] for placebo), and adverse events leading to study drug discontinuation (3% [49 patients] for both lebrikizumab doses vs 4% [31 patients] for placebo) were similar between lebrikizumab and placebo. The following serious adverse events were reported in the placebo-controlled period: one event of aplastic anaemia and five serious adverse events related to raised concentrations of eosinophils in patients treated with lebrikizumab and one event of eosinophilic pneumonia in the placebo group., Interpretation: Lebrikizumab did not consistently show significant reduction in asthma exacerbations in biomarker-high patients. However, it blocked interleukin-13 as evidenced by the effect on interleukin-13-related pharmacodynamic biomarkers, and clinically relevant changes could not be ruled out., Funding: F Hoffmann-La Roche., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Periostin is a matricellular protein that has been implicated in many disease states. It interacts with multiple signaling cascades to modulate the expression of downstream genes that regulate cellular interactions within the extracellular matrix. This review focuses on the role of periostin in respiratory diseases, including asthma and idiopathic pulmonary fibrosis, and its potential to help guide treatment or assess prognosis. Epithelial injury is a common feature of many respiratory diseases, resulting in the secretion, among others, of periostin, which is subsequently involved in airway remodeling and other aspects of pulmonary pathophysiology. In asthma, periostin is recognized as a biomarker of type 2 inflammation; POSTN gene expression is up-regulated in bronchial epithelial cells by IL-13 and IL-4. Serum periostin has been evaluated for the identification of patients with increased clinical benefit from treatment with anti-IL-13 (lebrikizumab, tralokinumab) and anti-IgE (omalizumab) therapy and may be prognostic for increased risk of asthma exacerbations and progressive lung function decline. Furthermore, in asthma, periostin may regulate subepithelial fibrosis and mucus production and may serve as a systemic biomarker of eosinophilic airway inflammation. Periostin is also highly expressed in the lungs of patients with idiopathic pulmonary fibrosis, and its serum levels may predict clinical progression. Overall, periostin contributes to multiple pathogenic processes across respiratory diseases, and peripheral blood levels of periostin may have utility as a biomarker of treatment response and disease progression.

Background: Periostin levels are associated with airway eosinophilia and are suppressed by corticosteroid treatment in asthma. This study sought to determine the relationship between serum and sputum periostin, airway inflammatory phenotype and asthma control., Methods: Adults with poorly-controlled asthma (n = 83) underwent a clinical assessment, sputum induction and blood sampling. Dispersed sputum was used for a differential cell count and periostin assessment (ELISA). Serum periostin was determined by the Elecsys® immunoassay., Results: Periostin levels were significantly higher in serum (median (IQR) of 51.6 (41.8, 62.6) ng/mL) than in sputum (1.1 (0.5, 2.0) ng/mL) (p < 0.001). Serum and sputum periostin were significantly higher in patients with eosinophilic asthma (n = 37) compared with non-eosinophilic asthma. Both serum and sputum periostin levels were significantly associated with proportion of sputum eosinophils (r = 0.422, p < 0.001 and r = 0.364, p = 0.005 respectively) but were not associated with asthma control. In receiver operator characteristic curve analysis, the area under the curve (AUC) for serum periostin (n = 83) was 0.679, p = 0.007. Peripheral blood eosinophils assessed in 67 matched samples, had a numerically greater AUC of 0.820 compared with serum periostin, p = 0.086 for the detection of eosinophilic asthma., Conclusion: In poorly-controlled asthma, sputum and serum periostin levels are significantly related to sputum eosinophil proportions while their ability to predict the presence of eosinophilic asthma is modest.

Introduction: In a subset of patients with asthma, standard-of-care treatment does not achieve disease control, highlighting the need for novel therapeutic approaches. Lebrikizumab is a humanised, monoclonal antibody that binds to and blocks interleukin-13 activity., Methods: LUTE and VERSE were replicate, randomised, double-blind, placebo-controlled studies, evaluating multiple doses of lebrikizumab in patients with uncontrolled asthma despite the use of medium-to-high-dose inhaled corticosteroid and a second controller. Patients received lebrikizumab 37.5, 125, 250 mg or placebo subcutaneously every four weeks. The primary endpoint was the rate of asthma exacerbations during the placebo-controlled period. Analyses were performed on prespecified subgroups based on baseline serum periostin levels. Following the discovery of a host-cell impurity in the study drug material, protocols were amended to convert from phase III to phase IIb. Subsequently, dosing of study medication was discontinued early as a precautionary measure. The data collected for analysis were from a placebo-controlled period of variable duration and pooled across both studies., Results: The median duration of treatment was approximately 24 weeks. Treatment with lebrikizumab reduced the rate of asthma exacerbations, which was more pronounced in the periostin-high patients (all doses: 60% reduction) than in the periostin-low patients (all doses: 5% reduction); no dose-response was evident. Lung function also improved following lebrikizumab treatment, with greatest increase in FEV1 in periostin-high patients (all doses: 9.1% placebo-adjusted improvement) compared with periostin-low patients (all doses: 2.6% placebo-adjusted improvement). Lebrikizumab was well tolerated and no clinically important safety signals were observed., Conclusions: These data are consistent with, and extend, previously published results demonstrating the efficacy of lebrikizumab in improving rate of asthma exacerbations and lung function in patients with moderate-to-severe asthma who remain uncontrolled despite current standard-of-care treatment., Trial Registration Numbers: The LUTE study was registered under NCT01545440 and the VERSE study under NCT01545453 at http://www.clinicaltrials.gov., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Periostin, also termed osteoblast-specific factor 2, is a matricellular protein with known functions in osteology, tissue repair, oncology, cardiovascular and respiratory systems, and in various inflammatory settings. However, most of the research to date has been conducted in divergent and circumscribed areas meaning that the overall understanding of this intriguing molecule remains fragmented. Here, we integrate the available evidence on periostin expression, its normal role in development, and whether it plays a similar function during pathologic repair, regeneration, and disease in order to bring together the different research fields in which periostin investigations are ongoing. In spite of the seemingly disparate roles of periostin in health and disease, tissue remodeling as a response to insult/injury is emerging as a common functional denominator of this matricellular molecule. Periostin is transiently upregulated during cell fate changes, either physiologic or pathologic. Combining observations from various conditions, a common pattern of events can be suggested, including periostin localization during development, insult and injury, epithelial-mesenchymal transition, extracellular matrix restructuring, and remodeling. We propose mesenchymal remodeling as an overarching role for the matricellular protein periostin, across physiology and disease. Periostin may be seen as an important structural mediator, balancing appropriate versus inappropriate tissue adaption in response to insult/injury.

Introduction: Rheumatoid arthritis (RA) is a complex and clinically heterogeneous autoimmune disease. Currently, the relationship between pathogenic molecular drivers of disease in RA and therapeutic response is poorly understood., Methods: We analyzed synovial tissue samples from two RA cohorts of 49 and 20 patients using a combination of global gene expression, histologic and cellular analyses, and analysis of gene expression data from two further publicly available RA cohorts. To identify candidate serum biomarkers that correspond to differential synovial biology and clinical response to targeted therapies, we performed pre-treatment biomarker analysis compared with therapeutic outcome at week 24 in serum samples from 198 patients from the ADACTA (ADalimumab ACTemrA) phase 4 trial of tocilizumab (anti-IL-6R) monotherapy versus adalimumab (anti-TNFα) monotherapy., Results: We documented evidence for four major phenotypes of RA synovium - lymphoid, myeloid, low inflammatory, and fibroid - each with distinct underlying gene expression signatures. We observed that baseline synovial myeloid, but not lymphoid, gene signature expression was higher in patients with good compared with poor European league against rheumatism (EULAR) clinical response to anti-TNFα therapy at week 16 (P =0.011). We observed that high baseline serum soluble intercellular adhesion molecule 1 (sICAM1), associated with the myeloid phenotype, and high serum C-X-C motif chemokine 13 (CXCL13), associated with the lymphoid phenotype, had differential relationships with clinical response to anti-TNFα compared with anti-IL6R treatment. sICAM1-high/CXCL13-low patients showed the highest week 24 American College of Rheumatology (ACR) 50 response rate to anti-TNFα treatment as compared with sICAM1-low/CXCL13-high patients (42% versus 13%, respectively, P =0.05) while anti-IL-6R patients showed the opposite relationship with these biomarker subgroups (ACR50 20% versus 69%, P =0.004)., Conclusions: These data demonstrate that underlying molecular and cellular heterogeneity in RA impacts clinical outcome to therapies targeting different biological pathways, with patients with the myeloid phenotype exhibiting the most robust response to anti-TNFα. These data suggest a path to identify and validate serum biomarkers that predict response to targeted therapies in rheumatoid arthritis and possibly other autoimmune diseases., Trial Registration: ClinicalTrials.gov NCT01119859

Objective: Personalised healthcare is contingent on the identification of biomarkers that represent disease relevant pathways and predict drug response. The authors aimed to develop a gene expression signature in synovial tissue that could enrich clinical response of rheumatoid arthritis (RA) patients to rituximab., Methods: The authors studied synovial gene expression using high-throughput quantitative real-time-PCR in 20 RA patients who underwent arthroscopy before and after treatment with rituximab. Several objective approaches were used to explore patterns in the data and to find genes associated with changes in disease activity due to treatment., Results: This analysis revealed two patient populations associated with distinct clinical, laboratory and histological features and, importantly, showed enrichment for response (60% non-responders vs 90% responders). A composite baseline gene score (GS) correlated with change in disease activity score (ΔDAS) between baseline and month 3 (r=0.74, p=0.0002), but also with ΔDAS at later time-points (month 9, r=0.54, p=0.016; month 15, r=0.45, p=0.06; month 21, r=0.72, p=0.003). Notably, the GS significantly correlated with baseline erythrocyte sedimentation rate (r=0.69, p=0.0008), but not with other DAS components. The GS genes represented T cell, macrophage, remodelling and interferon-α biology. Responders demonstrated higher expression of macrophage and T cell genes, while non-responders showed higher expression of interferon-α and remodelling genes., Conclusions: This study reveals a baseline synovial GS that correlates with early and late clinical responses to rituximab. The GS biology suggests that T cells and macrophages are important for response to B cell depleting therapy, while expression of remodelling and interferon-α genes correlates with poor response.

Objective: Rituximab significantly improves the signs and symptoms of rheumatoid arthritis (RA) and slows the progression of joint damage. The aim of this study was to identify clinical characteristics and biomarkers that identify patients with RA in whom the clinical benefit of rituximab may be enhanced., Methods: The study group comprised 1,008 RA patients from 2 independent randomized placebo-controlled phase III clinical trials (REFLEX [Randomized Evaluation of Long-Term Efficacy of Rituximab in Rheumatoid Arthritis] and SERENE [Study Evaluating Rituximab's Efficacy in Methotrexate Inadequate Responders]). A novel threshold selection method was used to identify baseline candidate biomarkers present in at least 20% of patients that enriched for placebo-corrected American College of Rheumatology 50% improvement (ACR50 response; a high clinical efficacy bar) at week 24 after the first course of rituximab., Results: The presence of IgM rheumatoid factor (IgM-RF), IgG-RF, IgA-RF, and IgG anti-cyclic citrullinated peptide (anti-CCP) antibodies together with an elevated C-reactive protein (CRP) level were associated with enhanced placebo-corrected ACR50 response rates in the REFLEX patients with RA who had an inadequate response to anti-tumor necrosis factor therapies. These findings were independently replicated using samples from patients in SERENE who had an inadequate response to disease-modifying antirheumatic drug treatment. The combination of an elevated baseline CRP level together with an elevated level of any RF isotype and/or IgG anti-CCP antibodies was further associated with an enhanced benefit to rituximab., Conclusion: The presence of any RF isotype and/or IgG anti-CCP autoantibodies together with an elevated CRP level identifies a subgroup of patients with RA in whom the benefit of rituximab treatment may be enhanced. Although the clinical benefit of rituximab was greater in the biomarker-positive population compared with the biomarker-negative population, the clinical benefit of rituximab compared with placebo was also clinically meaningful in the biomarker-negative population., (Copyright © 2011 by the American College of Rheumatology.)

An important goal for personalized health care is the identification of biomarkers that predict the likelihood of treatment responses. Here, we tested the hypothesis that quantitative mRNA assays for B lineage cells in blood could serve as baseline predictors of therapeutic response to B cell depletion therapy in subjects with rheumatoid arthritis (RA). In samples from the REFLEX trial of rituximab in inadequate responders to antibodies to tumor necrosis factor-α, a 25% subgroup of treated subjects with elevated baseline mRNA levels of IgJ, a marker for antibody-secreting plasmablasts, showed reduced clinical response rates. There were no significant efficacy differences in the placebo arm subjects stratified by this marker. Prospective testing of the IgJ biomarker in the DANCER and SERENE rituximab clinical trial cohorts and the SCRIPT ocrelizumab cohort confirmed the utility of this marker to predict nonresponse to anti-CD20 therapy. A combination mRNA biomarker, IgJhiFCRL5lo, showed improved test performance over IgJhi alone. This study demonstrates that baseline blood levels of molecular markers for late-stage B lineage plasmablasts identify a ~20% subgroup of active RA subjects who are unlikely to gain substantial clinical benefit from anti-CD20 B cell depletion therapy.

Background: Treatment of acute rejection (AR) in heart transplantation relies on histopathological grading of endomyocardial biopsies according to International Society for Heart and Lung Transplantation guidelines. Intragraft gene expression profiling may be a way to complement histological evaluation., Methods and Results: Transcriptional profiling was performed on 26 endomyocardial biopsies, and expression patterns were compared with the 1990 International Society for Heart and Lung Transplantation AR grades. Importantly, transcriptional profiles from settings with an equivalent AR grade appeared the same. In addition, grade 0 profiles could not be distinguished from 1A profiles, and grade 3A profiles could not be distinguished from 3B profiles. Comparing the AR groupings (0+1A, 1B, and 3A+3B), 0+1A showed more striking differences from 1B than from 3A+3B. When these findings were extrapolated to the 2005 revised guidelines, the combination of 1A and 1B into a single category (1R) appears to have brought together endomyocardial biopsies with different underlying processes that are not evident from histological evaluation. Grade 1B was associated with upregulated immune response genes, as 1 categorical distinction from grade 1A. Although grade 1B was distinct from the clinically relevant AR grades 3A and 3B, all of these grades shared a small number of overlapping pathways consistent with common physiological underpinnings., Conclusion: The gene expression similarities and differences identified here in different AR settings have the potential to revise the clinical perspective on acute graft rejection, pending the results of larger studies.

Despite antiviral prophylaxis, a high percentage (over 90%) of heart transplant patients experience active cytomegalovirus (CMV) infection, diagnosed by detection of viral DNA in peripheral blood polymorphonuclear leukocytes within the first few months posttransplantation. Viral DNA was detected in mononuclear cells prior to detection in granulocytes from CMV-seropositive recipients (R+) receiving a heart from a CMV-seropositive donor (D+). Based on assessment of systemic infection in leukocyte populations, both R+ subgroups (R+/D- and R+/D+) experienced a greater infection burden than the R-/D+ subgroup, which was aggressively treated because of a higher risk of acute CMV disease. Despite widespread systemic infection in all at-risk patient subgroups, CMV DNA was rarely (< 3% of patients) detected in transplanted heart biopsy specimens. The R+ patients more frequently exceeded the 75th percentile of the CMV DNA copy number distribution in leukocytes (110 copies/10(5) polymorphonuclear leukocytes) than the R-/D+ subgroup. Therefore, active systemic CMV infection involving leukocytes is common in heart transplant recipients receiving prophylaxis to reduce acute disease. Infection of the transplanted organ is rare, suggesting that chronic vascular disease attributed to CMV may be driven by the consequences of systemic infection.

Background: Nephrotoxicity is a major side effect of calcineurin inhibitors (CNI). Earlier we reported 8% of our heart transplant recipients reaching end-stage renal failure (ESRF). Now, with an extended follow up of 20 years, we re-evaluated the development of ESRF and studied its influence on survival and the impact of polymorphisms in codon 10 and 25 of the promoter region of transforming growth factor (TGF)-beta on the risk of ESRF., Methods: In all, 465 patients were transplanted between June 1984 and June 2005. All were on maintenance CNI treatment. Development of ESRF was studied in 402/465 (86.5%) patients surviving at least one year. Their median follow up was eight years, total observation time of 3,414 years. TGF-beta polymorphisms in codon 10 (Leu to Pro) and codon 25 (Arg to Pro) were analyzed with real-time polymerase chain reaction in a cohort of 237 patients, with an observation time of 2,329 years., Results: Ten-year survival of patients surviving at least one year was 58.5%. Seventy-three patients (18.2%) developed ESRF. Dialysis-free survival was 60% at 15 years. The relative risk for ESRF in Pro carriers was 2.9 (CI 1.5-5.8) compared to patients with the Leu/Leu genotype (P = 0.002), while Pro carriers had a RR of 2.6 (CI 1.4-4.8) compared to the Arg/Arg25 genotype (P = 0.002). Survival of patients with ESRF was 1.5 years (median)., Conclusion: We found a highly significant association between TGF-beta polymorphisms and CNI induced ESRF after heart transplantation (HTx). Pro carriers of either codon 10 or 25 had a 2.6 to 2.9 times increased risk of developing ESRF. As ESRF after HTx results in high mortality rates these patients should no longer receive CNI-based immunosuppression.

Background: Anticytomegalovirus (CMV) prophylaxis prevents the acute disease but its impact on subclinical infection and allograft outcome is unknown. We sought to determine whether CMV prophylaxis administered for three months after heart transplant would improve patient outcomes., Methods: This prospective cohort study of 66 heart transplant recipients compared aggressive CMV prophylaxis (n = 21, CMV hyperimmune globulin [CMVIG] plus four weeks of intravenous ganciclovir followed by two months of valganciclovir); with standard prophylaxis (n = 45, intravenous ganciclovir for four weeks). Prophylaxis was based on pretransplant donor (D) and recipient (R) CMV serology: R-/D+ received aggressive prophylaxis; R+ received standard prophylaxis. Outcome measures were: CMV infection assessed by DNA-polymerase chain reaction on peripheral blood polymorphonuclear leukocytes, acute rejection, and cardiac allograft vascular disease (CAV) assessed by intravascular ultrasound. All patients completed one year of follow-up. RESULTS.: CMV infection was subclinical in all but four patients (two in each group). Aggressively treated patients had a lower incidence of CMV infection (73 +/- 10% vs. 94 +/- 4%; P = 0.038), and an independent reduced relative risk for acute rejection graded > or =3A (relative risk [95% CI] = 0.55 [0.26-0.96]; P = 0.03), as compared with the standard prophylaxis group. Aggressively prophylaxed patients also showed a slower progression of CAV, in terms of coronary artery lumen loss (lumen volume change=-21 +/- 13% vs. -10+/-14%; P = 0.05); and vessel shrinkage (vessel volume change = -15 +/- 11% vs. -3 +/- 18%; P = 0.03)., Conclusions: Prolonged (val)ganciclovir plus CMVIG reduces viral levels, acute rejection, and allograft vascular disease, suggesting a role for chronic subclinical infection in the pathophysiology of the most common diseases affecting heart transplant recipients.

We investigated the functional promoter repeat polymorphism (GT)(n) in the oxidative stress-induced enzyme heme oxygenase-1 in relation to heart failure and complications after transplantation. No influence of the polymorphism on the development of heart failure, graft survival, acute rejection, or transplant atherosclerosis was found.

Peri-operative tissue injury triggers the development of Transplant Coronary Artery Disease (TCAD). Animal studies have shown that induction of heme oxygenase (HO)-1 protects the donor organ from the development of TCAD. To investigate the role of HO-1 in TCAD after clinical heart transplantation, we measured intragraft mRNA expression of HO-1, HIF-1alpha, TGF-beta, FLIP, and the Bcl-2/Bax balance. Immunohistochemical staining of HO-1 was performed to determine its origin. Myocardial biopsies taken at the end of the transplantation procedure (time 0), at 1 week and at 10 months after transplantation were studied from recipients with or without angiographic signs of accelerated TCAD, diagnosed after 1 year. At time 0, no differences in mRNA expression for any of the measured parameters were found between TCAD positive and negative patients. At 1 week, mRNA expression of HO-1 and TGF-beta was higher in grafts that developed accelerated TCAD (p=0.001 and p=0.0002). These higher mRNA levels were accompanied by a pro-apoptotic shift in Bcl-2/Bax (p=0.02), suggesting proneness for apoptosis via the mitochondrial pathway. Immunohistochemical staining showed that HO-1 was mainly produced by infiltrating macrophages. At 10 months, again HO-1 and TGF-beta levels were high in TCAD positive patients (p=0.02 and p=0.05), but the expression of apoptotic markers was comparable at this time point. Our results suggest that a higher HO-1 by macrophages in our patient population might be an adaptive response to tissue injury and inflammation, reflecting damage due to the transplantation procedure that finally results in TCAD.

IL-2 and IFN-gamma are associated with acute rejection (AR) and graft vascular disease (GVD) after clinical heart transplantation. Polymorphisms in the genes of IL-2 (T-330G in the promoter) and IFN-gamma (CA repeat in the first intron) influence the production levels of these cytokines. Therefore, these polymorphisms might have an effect on the outcome after transplantation. To investigate possible effects of genetic variations in IL-2 and IFN-gamma genes on AR and GVD, we analyzed the IL-2 T-330G and the IFN-gamma CA repeat polymorphism in DNA of 301 heart transplant recipients. No associations were found for allele or genotype distributions between patients with or without AR (IL-2 allele frequency: P=0.44, genotype distribution: P=0.46; IFN-gamma allele frequency P=0.10, genotype distribution 12 repeats allele: P=0.21). Also, no associations were found analyzing the number (0 vs. 1 vs. >or=1) of AR (IL-2 allele frequency: P=0.59; genotype distribution: P=0.37; IFN-gamma allele frequency: P=0.27, genotype distribution 12 repeats allele: P=0.41) or analyzing the polymorphisms in patients with AR within the first month or thereafter (IL-2 allele frequency: P=0.45, genotype distribution: P=0.38; IFN-gamma allele frequency: P=0.21, genotype distribution 12 repeats allele: P=0.41). Analyzing both polymorphisms in relation to GVD, resulted in comparable allele and genotype distributions (IL-2 allele frequency: P=0.75; genotype distribution: P=0.77; IFN-gamma allele frequency: P=0.70, genotype distribution 12 repeats allele: P=0.63). In conclusion, we did not detect an association between the IL-2 T-330G promoter polymorphism and CA repeat polymorphism in the first intron of the IFN-gamma gene and AR or GVD after heart transplantation.

Background: To examine whether genetic factors are involved in the development of acute rejection (AR), we investigated a (CA)m(CT)n repeat in the 3'-flanking region of the interleukin (IL)-2 gene., Method: We genotyped 290 heart transplant recipients with and without AR (International Society for Heart and Lung Transplantation criteria > or =3A) and 101 controls., Results: The frequency of allele 135 of the repeat and its genotype distribution (carriers/noncarriers) were significantly associated with freedom from AR (P=0.03 and P=0.02, respectively). We also found interaction between allele 135 and HLA-DR matching. More carriers of allele 135 with no or one mismatch remained free from AR compared to patients without the allele (P=0.01). This was not found in the HLA-DR group with two mismatches., Conclusion: HLA-DR matching might only be effective in reducing AR after heart transplantation in recipients who carry allele 135 of the (CA)m(CT)n repeat in the 3'-flanking region of the IL-2 gene.

Background: The regulatory cytokine transforming growth factor (TGF)-beta1 is thought to play a role in atherosclerotic heart disease as well as in idiopathic cardiomyopathy. The production of TGF-beta1 is genetically controlled as polymorphisms in the signaling sequence of the TGF-beta1 gene leucine(10)-->proline and arginine(25)-->proline are involved in the regulation of the protein production level. We investigated whether these polymorphisms are associated with end-stage heart failure caused by dilated cardiomyopathy (CMP) or ischemic heart disease (IHD)., Methods: We determined polymorphisms using sequence specific oligonucleotide probing (SSOP) in genomic DNA samples from heart transplant recipients (n = 253) and controls (n = 94). Indications for transplantation were dilated CMP (n = 109) and IHD (n = 144)., Results: We found a difference in TGF-beta1 codon 10 genotype distribution among patients with IHD, dilated CMP, and controls (p = 0.034; chi(2) test). Patients with dilated CMP differed from patients with IHD (p = 0.044) and healthy controls (0.017). The genotype distribution between patients with IHD and controls was comparable. For codon 25, we found no difference in genotype distribution., Conclusions: The Leu(10)-->Pro (codon 10) polymorphism in the TGF-beta1 gene is associated with end-stage heart failure caused by dilated CMP and not with IHD. This observation suggests that TGF-beta1 is involved in the pathogenesis of CMP.

Background: The multifunctional cytokine transforming growth factor- (TGF) beta1 is thought to play a role in the pathogenesis of graft vascular disease (GVD). Polymorphisms at codon 10, (Leu10-->Pro) and codon 25 (Arg25-->Pro) in the signal sequence of the TGF-beta1 gene regulate the production and secretion of the protein. We investigated whether these polymorphisms are risk factors for the development of GVD after clinical heart transplantation., Method: TGF-beta1 polymorphisms, Leu10-->Pro and Arg25-->Pro, were determined in DNA from heart transplant recipients (n=252) and their donors (n=213), using sequence-specific oligonucleotide probing. GVD was angiographically diagnosed 1 year after transplantation. In addition other potential risk factors including underlying disease, recipient and donor age, recipient and donor gender, number of acute rejections in the first year, cold ischemia time, and HLA mismatches were analyzed by univariate and multivariate logistic regression analysis., Results: Univariate analysis showed that the recipient TGF-beta1 polymorphism Leu10-->Pro, (P=0.056, chi2 test), underlying disease (P=0.01, chi2 test), number of acute rejections in the first-year (P=0.03, analysis of variance), and donor age (P<0.001, analysis of variance) were risk factors for the development of GVD. The TGF-beta1 Arg25-->Pro polymorphism was not a risk factor. Also in the multivariate analysis, the recipient TGF-beta1 codon 10 polymorphism was associated with GVD, with patients homozygous for Pro at greatest risk (odds ratio 7.7, P=0.03). Apart for the recipient TGF-beta1 Leu10-->Pro polymorphism, donor age appeared to be an independent risk factor for the development of GVD at 1 year. Patients with older donor hearts were at greater risk than patients receiving grafts from younger donors (odds ratio 1.1/year, P<0.001)., Conclusion: Recipient TGF-beta1 Leu10-->Pro polymorphism and higher donor age are independent risk factors for the development of GVD after clinical heart transplantation.

Background: To determine whether genetic factors are involved in the development of renal dysfunction due to cyclosporine nephrotoxicity, we analyzed 2 polymorphisms in the signal sequence of the transforming growth factor (TGF)-beta 1 gene; codon 10 (Leu(10) --> Pro) and codon 25 (Arg(25) --> Pro)., Method: Using sequence specific oligonucleotide probing, we analyzed both TGF-beta1 gene polymorphisms in cardiac allograft recipients (n = 168) who survived at least 1 year with minimal follow-up of 7 years. Patients received cyclosporine and steroids as maintenance immunosuppressive therapy. Renal dysfunction was defined as a serum creatinine > or = 250 micromol/liter., Results: Renal dysfunction was observed in 2% (3/168) of the patients at 1 year, in 7% (11/160) at 3 years, in 12% (18/152) at 5 years, and in 20% (26/131) at 7 years post-transplantation. The genotypic distributions for TGF-beta1 codon 10 were 7% Pro/Pro, 61% Pro/Leu, and 32% Leu/Leu, and for codon 25 these percentages were 1% Pro/Pro, 12% Pro/Arg, and 87% Arg/Arg. We found an association between the TGF-beta 1 genotype encoding proline at codon 10 and renal dysfunction. At 7 years post-transplantation, 26% (23/89) of the patients with the heterozygous Pro/Leu or homozygous Pro/Pro genotype had renal dysfunction vs only 7% (3/42) of the patients with the homozygous Leu/Leu genotype (p = 0.017). For the TGF-beta1 codon 25 genotypes, we found no association between TGF-beta 1 genotypes and renal dysfunction., Conclusion: Our data support the hypothesis that TGF-beta 1 is involved in the process leading to renal insufficiency in cyclosporine-treated cardiac allograft recipients. In these patients the presence of TGF-beta 1 Pro(10) might be a risk factor.

Background: Despite anti-CD25 (interleukin [IL]-2 receptor alpha chain) monoclonal antibody (mAb) therapy, rejection can still occur. T-cell activation through the IL-2 receptor beta and gamma chains by IL-2 or other growth factors may contribute to this rejection. Recently, we have demonstrated that the T-cell growth factor IL-15 was abundantly present in rejecting cardiac grafts during anti-CD25 mAb treatment., Methods: To test whether IL-2- and IL-15-responsive T cells play an active role in rejection during anti-CD25 mAb therapy, we measured the frequency of IL-2- and IL-15-proliferative T cells in peripheral blood from treated patients during rejection (n=12). Measurements were made by limiting dilution analysis in the absence and presence of extra in vitro-added mouse anti-human CD25 mAb., Results: In the absence of anti-CD25 mAb, the frequencies of peripheral T cells responding to recombinant human (rh)IL-2 and rhIL-15 from patients were lower than those measured in samples of healthy controls (n=7): median of IL-2-responding T cells 78 per 10(6) (range 31-210 per 10(6)) vs. 154 per 10(6) (122-484 per 10(6), P=0.008) and median of IL-15-responding T cells 62 per 10(6) (range 19-207 per 10(6)) vs. 129 per 10(6) (range 79-192 per 10(6), P=0.02), respectively. In the presence of extra in vitro-added anti-CD25 mAb, frequencies of IL-2-responding T cells from patients significantly decreased, although a considerable number of T cells still proliferated on rhIL-2 (median 85%, range 46-100%). In contrast, the frequencies of IL-15 T cells still responding remained stable (median 2%, range 0-50%, P<0.001)., Conclusions: Treatment with anti-CD25 mAbs cannot provide complete suppression of T-cell function because significant numbers of IL-2- and IL-15-responsive T cells remain present in the peripheral blood of allograft recipients during anti-CD25 mAb treatment.

Background: Despite blockade of the interleukin-2/interleukin 2 receptor (IL-2/IL-2R) pathway by the murine anti-CD25 (i.e., IL-2R alpha chain) monoclonal antibody BT563, cardiac rejection can still occur. In these cases, growth factors other than IL-2 may contribute to allograft rejection. We studied the expression of IL-15, a macrophage-derived cytokine associated with T-cell activation, which interacts with the beta and gamma chains of the IL-2R during rejection episodes under anti-CD25 therapy., Methods: We measured intragraft IL-15 mRNA expression and the number of IL-15- and CD68-positive cells in posttransplantation endomyocardial biopsies (EMBs; n=45) and in nontransplanted, donor-heart specimens (n=11) by competitive template reverse transcription-polymerase chain reaction and immunohistochemistry, respectively., Results: IL-15 mRNA expression was present in the majority of posttransplantation EMB specimens (91%, 41/45) and in nontransplanted donor-heart specimens (91%, 10/11). Relative IL-15 mRNA levels were neither associated with transplantation nor with rejection status. After transplantation, the number of IL-15- and CD68-positive cells significantly increased (P<0.001), but IL-15-positive cell counts did not reflect the histological rejection grade. Anti-CD25 treatment, in contrast to its effects on the IL-2/IL-2R complex, had no influence on intragraft IL-15 mRNA and protein production. In rejection EMB specimens, during (n=5) and after (n=8) anti-CD25 therapy, no differences in relative IL-15 mRNA levels, or in IL-15- and CD68-positive cell counts, were measured., Conclusions: After heart transplantation, high numbers of IL-15- and CD68-positive cells infiltrate the graft. This phenomenon is independent of the rejection status. IL-15 remains present during blockade of the IL-2/IL-2R pathway by anti-CD25 monoclonal antibodies, and it may participate in T cell-dependent donor-directed immune responses, thereby explaining the occurrence of rejection in the absence of IL-2.

Background: To determine mechanisms that trigger graft vascular disease (GVD) after heart transplantation, we studied parameters that reflect both early and late intragraft allogeneic reactions., Method: With reverse transcriptase-polymerase chain reaction analysis, mRNA expression of interleukin-2 (IL-2), interleukin-4, interleukin-6, interleukin-10, interferon-gamma, platelet-derived growth factor-alpha, and transforming growth factor-beta was measured in endomyocardial biopsy (EMB) specimens obtained from 34 recipients during the first acute rejection episode (n = 29) or at a comparable time after transplantation for patients without rejection (n = 5) and at time of assessment of GVD by coronary angiography at 1 year (n = 34)., Results: At the time of assessment of GVD, mRNA expression of IL-2, interleukin-4, and interleukin-6 were barely detectable, whereas messenger coding for interferon-gamma, interleukin-10, transforming growth factor-beta, and platelet-derived growth factor-alpha genes were constitutively expressed. Moreover, intragraft mRNA patterns of cytokines and growth factors between patients with GVD (n = 17) or without GVD (n = 17) were comparable. In contrast, during the first acute rejection episode a completely different pattern was found. Development of GVD was associated with IL-2 mRNA expression and not with the other cytokines analyzed. IL-2 mRNA was present in 77% of rejection EMB specimens obtained from patients with GVD versus 33% of the EMB specimens obtained from patients without GVD (p = 0.03) and not detectable in EMB specimens obtained from patients with no rejection. Also nonimmunologic risk factors such as longer ischemia time (median 193 vs 141 minutes; p = 0.002) and higher donor age (median 32 vs 23 years; p = 0.02) were associated with GVD. But no relation was found between these nonimmunologic risk factors and IL-2-positive acute rejections., Conclusions: Nonspecific risk factors and IL-2-positive rejections may independently trigger GVD after clinical heart transplantation.

Background: Anti-interleukin (IL)-2 receptor (IL-2R) antibodies have been used as rejection prophylaxis after organ transplantation. Despite this induction treatment, acute rejections may occur. We wondered whether these rejections developed via the IL-2/IL-2R pathway., Methods: In a prospective trial using BT563, a murine IgG1 anti-IL-2R antibody, for rejection prophylaxis after heart transplantation, 20 patients were treated in combination with cyclosporine from the day of transplantation (group A). As a control group, 31 patients were also treated with BT563, but in these patients, cyclosporine treatment was initiated on day 3 (group B)., Results: Three patients from group A and two patients from group B died in the first postoperative month (of causes not related to acute rejection) and were left out of the analysis of rejection incidence. Freedom from acute rejection at 1 week after transplantation in group A (14/17; 82%) was lower than in group B (16/29; 55%), although the difference did not reach statistical significance. There was no difference in either the number of acute rejection episodes at 12 weeks or the required rejection treatments between groups A and B. Infectious complications were evenly distributed in both groups. Immunohistochemistry showed that during acute rejection, in the presence of circulating BT563, IL-2R-bearing cells were present in only one of five rejection biopsies (20%), whereas these cells were often present (6/8, or 75%) in rejections occurring in the absence of BT563. The presence of BT563 was associated with a similar difference in the mRNA expression of IL-2 (2/5 vs. 6/8)., Conclusions: Apparently, despite adequate blockade of the IL-2/IL-2R pathway, patients may develop acute rejection, reflecting the redundancy of the cytokine network. The ever-present IL-15 may well be a candidate for overtaking the role of IL-2.