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BACKGROUND & AIMS: Biologic therapies may effectively treat Crohn's disease (CD), and pediatric patients who discontinue multiple biologics risk exhausting treatment options. The frequency and context of biologic discontinuation have not been well-characterized. We aimed to determine patterns of biologic use, discontinuation, and evaluation in pediatric patients with CD. METHODS: Pediatric patients with CD at 7 U.S. centers (2010-2020) were identified. Prospective ImproveCareNow registry data were supplemented with medical record abstraction. Biologics included monoclonal antibody and small molecule medications. Therapeutic drug monitoring (TDM) was considered induction if <14 weeks after biologic start, proactive if later during quiescent disease, and reactive during active disease. RESULTS: Of 823 patients included (median age, 13.0 years; 40% female), 86% started biologics (78% infliximab, 21% adalimumab, <1% others). Twenty-six percent used concomitant immunomodulators for ≥12 months. Most (85%) measured TDM including 47% induction, 69% proactive, and 24% reactive. Twenty-nine percent discontinued their first biologic after median 793 days because of inefficacy (34%), anti-drug antibodies (8%), adverse events (8%), or non-adherence (12%). If inefficacy, 86% underwent pre-discontinuation evaluation. If infliximab or adalimumab inefficacy and TDM was done, 62% had levels <10 µg/mL. Proactive TDM and concomitant immunomodulators were associated with 60% and 32% reduced biologic discontinuation. CONCLUSIONS: Most children with CD are treated with biologics; 25%-37% discontinue biologics, resulting in 1 in 12 using >2 biologics during pediatric care. Half of patients discontinued biologics without trial of high-dose therapy and 14% without any evaluation. Concomitant immunomodulator use and proactive TDM decreased risk of biologic discontinuation. Strategies are needed to preserve biologic efficacy and prevent biologic discontinuation.
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BACKGROUND AND AIMS: COVID-19 vaccination prevents severe disease in most patients with inflammatory bowel disease (IBD), but immunosuppressive medications can blunt serologic response. We followed adults with IBD for >1 year post-COVID-19 vaccination to describe factors associated with SARS-CoV-2 infection after vaccination, evaluate for a protective SARS-CoV-2 antibody level, characterize SARS-CoV-2 antibody persistence, and identify factors associated with humoral immune response durability. METHODS: Using a prospective cohort of COVID-19 immunized adults with IBD, we analyzed factors associated with SARS-CoV-2 infection after vaccination. We evaluated for an association between SARS-CoV-2 antibody level 12 weeks postvaccination and subsequent SARS-CoV-2 infection and assessed for a threshold of protection using receiver-operating characteristic curve analysis. We then conducted a separate analysis evaluating factors associated with persistence of SARS-CoV-2 antibodies 52 weeks postimmunization. RESULTS: Almost half (43%) of 1869 participants developed COVID-19 after vaccination, but most infections were mild, and <1% required hospitalization. Older age and corticosteroid use were associated with a decreased risk of SARS-CoV-2 infection postvaccination (50-59 years of age vs 18-29 years of age: adjusted hazard ratio, 0.57; 95% confidence interval, 0.44-0.74; steroid users vs nonusers: adjusted hazard ratio, 0.58; 95% confidence interval, 0.39-0.87). Most (98%) participants had detectable antibody levels at 52 weeks postvaccination. Antibody levels at 12 weeks and number of vaccine doses were positively associated with higher antibody levels at 52 weeks, while anti-tumor necrosis factor α therapy was negatively associated. CONCLUSIONS: COVID-19 vaccination generates an effective and durable protective response for the vast majority of adults with IBD, including vulnerable populations such as corticosteroid users and older individuals. Patients with IBD benefit from COVID-19 booster vaccination.
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Anticuerpos Antivirales , Vacunas contra la COVID-19 , COVID-19 , Enfermedades Inflamatorias del Intestino , SARS-CoV-2 , Humanos , COVID-19/prevención & control , COVID-19/inmunología , Masculino , Femenino , Persona de Mediana Edad , Enfermedades Inflamatorias del Intestino/inmunología , Adulto , Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/inmunología , Estudios Prospectivos , Anticuerpos Antivirales/sangre , SARS-CoV-2/inmunología , Vacunación , Anciano , Adulto JovenRESUMEN
INTRODUCTION: Obesity is common among patients with pediatric Crohn's disease (PCD). Some adult studies suggest obese patients respond less well to anti-tumor necrosis factor (TNF) treatment. This study sought compares anti-TNF response and anti-TNF levels between pediatric patients with normal and high body mass index (BMI). METHODS: The COMBINE trial compared anti-TNF monotherapy with combination therapy with methotrexate in patients with PCD. In this secondary analysis, a comparison of time-to-treatment failure among patients with normal BMI vs BMI Z -score >1, adjusting for prescribed anti-TNF (infliximab [IFX] or adalimumab [ADA]), trial treatment assignment (combination vs monotherapy), and relevant covariates. Median anti-TNF levels across BMI category was also examined. RESULTS: Of 224 participants (162 IFX initiators and 62 ADA initiators), 111 (81%) had a normal BMI and 43 (19%) had a high BMI. High BMI was associated with treatment failure among ADA initiators (7/10 [70%] vs 12/52 [23%], hazard ratio 0.29, P = 0.007) but not IFX initiators. In addition, ADA-treated patients with a high BMI had lower ADA levels compared with those with normal BMI (median 5.8 vs 12.8 µg/mL, P = 0.02). IFX trough levels did not differ between BMI groups. DISCUSSION: Overweight and obese patients with PCD are more likely to experience ADA treatment failure than those with normal BMI. Higher BMI was associated with lower drug trough levels. Standard ADA dosing may be insufficient for overweight children with PCD. Among IFX initiators, there was no observed difference in clinical outcomes or drug levels, perhaps due to weight-based dosing and/or greater use of proactive drug monitoring.
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Adalimumab , Índice de Masa Corporal , Enfermedad de Crohn , Quimioterapia Combinada , Infliximab , Metotrexato , Factor de Necrosis Tumoral alfa , Humanos , Enfermedad de Crohn/tratamiento farmacológico , Masculino , Femenino , Infliximab/uso terapéutico , Adalimumab/uso terapéutico , Niño , Adolescente , Metotrexato/uso terapéutico , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Insuficiencia del Tratamiento , Fármacos Gastrointestinales/uso terapéutico , Obesidad Infantil/complicaciones , Obesidad Infantil/tratamiento farmacológicoRESUMEN
Children with very early onset inflammatory bowel disease (VEO-IBD) may respond differently to coronavirus disease 2019 (COVID-19) immunization compared to healthy children or other patients with IBD. We recruited children with VEO-IBD <6 years of age and younger following receipt of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine. Demographics, IBD characteristics, medication use, adverse events (AEs) and IBD exacerbations were collected. Blood draws (optional) were obtained for measurement of antireceptor binding domain (RBD) IgG antibodies following vaccination. Of 41 participants, none required emergency department visit or hospitalization due to AE, and only one experienced IBD exacerbation. Detectable antibody was present in 19/19 participants who provided blood sample; 6/7 participants (86%) had durable humoral response 12 months postvaccination. Children with VEO-IBD experience robust humoral immune response to COVID-19 immunization. Severe AEs were rare. These findings provide reassurance that children with VEO-IBD respond well and safely to SARS-CoV-2 vaccination.
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COVID-19 , Enfermedades Inflamatorias del Intestino , Niño , Humanos , Inmunidad Humoral , Vacunas contra la COVID-19/efectos adversos , SARS-CoV-2 , COVID-19/prevención & control , Vacunación/efectos adversos , Inmunoglobulina G , Anticuerpos AntiviralesRESUMEN
BACKGROUND: Ephrin (EPH) receptors have been implicated in tumorigenesis and metastasis, but the functional understanding of mutations observed in human cancers is limited. We previously demonstrated reduced cell compartmentalisation for somatic EPHB1 mutations found in metastatic colorectal cancer cases. We therefore integrated pan-cancer and pan-EPH mutational data to prioritise recurrent EPHB1 mutations for functional studies to understand their contribution to cancer development and metastasis. METHODS: Here, 79,151 somatic mutations in 9,898 samples of 33 different tumour types were analysed with a bioinformatic pipeline to find 3D-mutated cluster pairs and hotspot mutations in EPH receptors. From these, 15 recurring EPHB1 mutations were stably expressed in colorectal cancer followed by confocal microscopy based in vitro compartmentalisation assays and phospho-proteome analysis. RESULTS: The 3D-protein structure-based bioinformatics analysis resulted in 63% EPHB1 mutants with compartmentalisation phenotypes vs 43% for hotspot mutations. Whereas the ligand-binding domain mutations C61Y, R90C, and R170W, the fibronectin domain mutation R351L, and the kinase domain mutation D762N displayed reduced to strongly compromised cell compartmentalisation, the kinase domain mutations R743W and G821R enhanced this phenotype. While mutants with reduced compartmentalisation also had reduced ligand induced receptor phosphorylation, the enhanced compartmentalisation was not linked to receptor phosphorylation level. Phosphoproteome mapping pinpointed the PI3K pathway and PIK3C2B phosphorylation in cells harbouring mutants with reduced compartmentalisation. CONCLUSIONS: This is the first integrative study of pan-cancer EPH receptor mutations followed by in vitro validation, a robust way to identify cancer-causing mutations, uncovering EPHB1 mutation phenotypes and demonstrating the utility of protein structure-based mutation analysis in characterization of novel cancer genes. Video Abstract.
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Neoplasias Colorrectales , Fosfatidilinositol 3-Quinasas , Humanos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Ligandos , Mutación , Recurrencia Local de Neoplasia , Proteínas Tirosina Quinasas Receptoras/metabolismoRESUMEN
Painful diabetic peripheral neuropathy (DPN) is a highly prevalent and disabling complication of diabetes that is often misdiagnosed and undertreated. The management of painful DPN involves treating its underlying cause via lifestyle modifications and intensive glucose control, targeting its pathogenesis, and providing symptomatic pain relief, thereby improving patient function and health-related quality of life. Four pharmacologic options are currently approved by the US Food and Drug Administration (FDA) to treat painful DPN. These include three oral medications (duloxetine, pregabalin, and tapentadol extended release) and one topical agent (capsaicin 8% topical system). More recently, the FDA approved several spinal cord stimulation (SCS) devices to treat refractory painful DPN. Although not FDA-approved specifically to treat painful DPN, tricyclic antidepressants, serotonin/norepinephrine reuptake inhibitors, gabapentinoids, and sodium channel blockers are common first-line oral options in clinical practice. Other strategies may be used as part of individualized comprehensive pain management plans. This article provides an overview of the most recent US guidelines for managing painful DPN, with a focus on the two most recently approved treatment options (SCS and capsaicin 8% topical system), as well as evidence for using FDA-approved and guideline-supported drugs and devices. Also discussed are unmet needs for this patient population, and evidence for potential future treatments for painful DPN, including drugs with novel mechanisms of action, electrical stimulation devices, and nutraceuticals.
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According to single-molecule localisation microscopy almost all plasma membrane proteins are clustered. We demonstrate that clusters can arise from variations in membrane topography where the local density of a randomly distributed membrane molecule to a degree matches the variations in the local amount of membrane. Further, we demonstrate that this false clustering can be differentiated from genuine clustering by using a membrane marker to report on local variations in the amount of membrane. In dual colour live cell single molecule localisation microscopy using the membrane probe DiI alongside either the transferrin receptor or the GPI-anchored protein CD59, we found that pair correlation analysis reported both proteins and DiI as being clustered, as did its derivative pair correlation-photoactivation localisation microscopy and nearest neighbour analyses. After converting the localisations into images and using the DiI image to factor out topography variations, no CD59 clusters were visible, suggesting that the clustering reported by the other methods is an artefact. However, the TfR clusters persisted after topography variations were factored out. We demonstrate that membrane topography variations can make membrane molecules appear clustered and present a straightforward remedy suitable as the first step in the cluster analysis pipeline.
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Antígenos CD59 , Membrana Celular , Receptores de Transferrina , Imagen Individual de Molécula , Imagen Individual de Molécula/métodos , Membrana Celular/metabolismo , Antígenos CD59/metabolismo , Receptores de Transferrina/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Análisis por Conglomerados , Microscopía Fluorescente/métodosRESUMEN
Perianal complications are common and morbid in children with Crohn's disease. In this review, we describe the epidemiology, the presentation and diagnosis, evaluation and management. We focus on updates such as the increasing frequency of biologic medications and MRI for evaluation. We also highlight controversies on the timing and approaches to surgical techniques. Finally, perianal disease requires the coordination of multidisciplinary care with nursing, radiology, gastroenterology, and surgery to optimize outcomes - both medical and patient-centered.
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Enfermedad de Crohn , Humanos , Enfermedad de Crohn/terapia , Enfermedad de Crohn/diagnóstico , Enfermedad de Crohn/epidemiología , Niño , Enfermedades del Ano/terapia , Enfermedades del Ano/diagnóstico , Enfermedades del Ano/epidemiología , Enfermedades del Ano/etiologíaRESUMEN
Due to the prevalence of chronic pain and high-impact chronic pain in the US, a significant percentage of the population is prescribed opioids for pain management. However, opioid use disorder is associated with reduced quality of life, along with fatal opioid overdoses, and is a significant burden on the US economy. Considering the clinical needs of patients with intractable chronic pain and the potential harms associated with prescribed and illicit opioids in our communities, having a deep understanding of current treatment options, supporting evidence, and clinical practice guidelines is essential for optimizing treatment selections. Buprenorphine is a Schedule III opioid with a unique mechanism of action, allowing effective and long-lasting analgesia at microgram doses with fewer negative side effects and adverse events, including respiratory depression, when compared with other immediate-release, long-acting, and extended-release prescription opioids. Due to its relatively lower risk for overdose and misuse, buprenorphine was recently added to the Clinical Practice Guideline for the Use of Opioids in the Management of Chronic Pain as a first-line treatment for chronic pain managed by opioids by the US Departments of Defense and Veterans Affairs, and the Department of Health and Human Services recommends that buprenorphine be made available for the treatment of chronic pain. In this narrative review, we discuss the different buprenorphine formulations, clinical efficacy, advantages for older adults and other special populations, clinical practice guideline recommendations, and payer considerations of buprenorphine and suggest that buprenorphine products approved for chronic pain should be considered as a first-line treatment for this indication.
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AIMS: Secondary mucosal colonisation by a carcinoma originating from a distant site is a pattern of metastasis to the intestines and hepatobiliary tract and a mimic of primary neoplasia. Although endometriosis is considered benign, its ability to spread widely underscores its quasi-neoplastic nature. After noting that endometriotic glands can colonise the colonic mucosa along the basement membrane, mimicking metastatic disease, we conducted an intradepartmental review of intestinal specimens showing endometriosis obtained from 2016 to 2023 to characterise and quantify the incidence of this phenomenon. METHODS: Material from 38 lower gastrointestinal specimens with a primary or ancillary diagnosis of endometriosis was identified from our surgical pathology database. Slides were reviewed, documenting the extent and micro-anatomic location affected by endometriosis, with a focus on identifying examples showing mucosal colonisation. RESULTS: The most common site of involvement was the distal colon (23 cases; 11 of rectum, 9 of sigmoid colon and 3 of rectosigmoid) followed by the appendix (N=10), cecum (N=2), small intestine (N=2) and 'colon not otherwise specified' (N=1). Mucosal involvement was identified in eight cases (21%), half of which demonstrated seamless colonisation of the epithelium by endometriotic glands. In two of these, the procedure was prompted by the presence of a rectal mass or stricture with concern for malignancy. CONCLUSION: Endometriosis occasionally (4/38; 10.5%) colonises colonic epithelium, potentially mimicking a metastasis or intraepithelial neoplasia/dysplasia. Although unusual, this phenomenon was observed in half of specimens from patients with mucosal involvement in whom a mass or stricture suggested malignancy, a potentially misleading pattern of endometriosis.
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Carcinoma , Endometriosis , Femenino , Humanos , Endometriosis/diagnóstico , Endometriosis/patología , Constricción Patológica/complicaciones , Constricción Patológica/patología , Colon/patología , Recto/patología , Carcinoma/patologíaRESUMEN
Pragmatic clinical research is 1 of the 5 focus areas of the Challenges in IBD Research 2024, a multidisciplinary effort by scientists, clinicians, patients, and funders to identify priorities for patient-centric research. This summary provides a comprehensive overview of current gaps in inflammatory bowel disease (IBD) clinical research and actionable approaches to address them. This review is focused on identifying research that is needed to achieve the best outcomes for patients in clinical practice. Research gaps include understanding the needs of understudied patient groups and addressing barriers to care so all patients receive optimal care, validating and using biomarkers to enable early diagnosis and result in better outcomes for adults and children with IBD, and determining the optimal sequencing of treatments (medical, surgical, adjunct) in children and adults. Inclusive pragmatic research is needed to address these gaps and lead to improvements in patient care and outcomes for all populations of patients with IBD.
Pragmatic clinical research focuses on improving evidence for how to best treat patients to improve quality of life and disease outcomes in real-world practice. This includes evaluating and improving healthcare delivery and decreasing barriers for all patients.
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Enfermedades Inflamatorias del Intestino , Humanos , Enfermedades Inflamatorias del Intestino/terapia , Enfermedades Inflamatorias del Intestino/diagnóstico , Investigación Biomédica , Biomarcadores/análisisRESUMEN
BACKGROUND: Despite advances in medical therapy, many children and adults with ileal Crohn's disease (CD) progress to fibrostenosis requiring surgery. We aimed to identify MRI and circulating biomarkers associated with the need for surgical management. METHODS: This prospective, multicenter study included pediatric and adult CD cases undergoing ileal resection and CD controls receiving medical therapy. Noncontrast research MRI examinations measured bowel wall 3-dimensional magnetization transfer ratio normalized to skeletal muscle (normalized 3D MTR), modified Look-Locker inversion recovery (MOLLI) T1 relaxation, intravoxel incoherent motion (IVIM) diffusion-weighted imaging metrics, and the simplified magnetic resonance index of activity (sMaRIA). Circulating biomarkers were measured on the same day as the research MRI and included CD64, extracellular matrix protein 1 (ECM1), and granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibodies (Ab). Associations between MRI and circulating biomarkers and need for ileal resection were tested using univariate and multivariable LASSO regression. RESULTS: Our study sample included 50 patients with CD undergoing ileal resection and 83 patients with CD receiving medical therapy; mean participant age was 23.9â ±â 13.1 years. Disease duration and treatment exposures did not vary between the groups. Univariate biomarker associations with ileal resection included log GM-CSF Ab (odds ratio [OR],â 2.87; Pâ =â .0009), normalized 3D MTR (OR, 1.05; Pâ =â .002), log MOLLI T1 (OR, 0.01; Pâ =â .02), log IVIM perfusion fraction (f; OR, 0.38; Pâ =â .04), and IVIM apparent diffusion coefficient (ADC; OR, 0.3; Pâ =â .001). The multivariable model for surgery based upon corrected Akaike information criterion included age (OR, 1.03; Pâ =â .29), BMI (OR, 0.91; Pâ =â .09), log GM-CSF Ab (OR, 3.37; Pâ =â .01), normalized 3D MTR (OR, 1.07; Pâ =â .007), sMaRIA (OR, 1.14; Pâ =â .61), luminal narrowing (OR, 10.19; Pâ =â .003), log C-reactive protein (normalized; OR, 2.75; Pâ =â .10), and hematocrit (OR, 0.90; Pâ =â .13). CONCLUSION: After accounting for clinical and MRI measures of severity, normalized 3D MTR and GM-CSF Ab are associated with the need for surgery in ileal CD.
Despite advances in medical therapy, many patients with ileal Crohn's disease progress to fibrostenosis requiring surgery. Our study has shown that GM-CSF autoantibodies and MRI biomarker sequences are associated with the need for ileal resection and may help guide management decisions.
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Biomarkers are used frequently for evaluation and monitoring of patients with Crohn's disease (CD). This American Gastroenterological Association (AGA) guideline is intended to support practitioners in decisions about the use of biomarkers for the management of CD. A multidisciplinary panel of content experts and guideline methodologists used the Grading of Recommendations Assessment, Development and Evaluation framework to formulate patient-centered clinical questions and review evidence on the performance of fecal calprotectin, serum C-reactive protein (CRP), and Endoscopic Healing Index in patients with established CD who were asymptomatic, had symptoms of varying severity, or were in surgically induced remission. Biomarker performance was assessed against the gold standard of endoscopic activity, defined as a Simple Endoscopic Score for Crohn's Disease ≥3. The panel used the Grading of Recommendations Assessment, Development and Evaluation Evidence-to-Decision framework to develop recommendations for use of biomarkers in various settings. Implementation considerations were formulated for each recommendation to inform clinical practice. The guideline panel made 11 conditional recommendations. In patients with CD in symptomatic remission, the panel suggests use of a biomarker- and symptom-based monitoring strategy over symptoms alone. In patients in symptomatic remission, a fecal calprotectin <150 µg/g and normal CRP rules out active inflammation, avoiding endoscopic evaluation for assessment of disease activity. However, elevated biomarkers in this setting merit confirmation with endoscopy before treatment adjustment. In patients with CD with mild symptoms, neither normal nor elevated biomarkers alone are sufficiently accurate to determine endoscopic activity. In patients with CD with moderate to severe symptoms, elevated fecal calprotectin or serum CRP suggests endoscopic activity, precluding routine endoscopic assessment for disease activity. In patients with CD in surgically induced remission in low-risk patients on pharmacologic prophylaxis, a normal fecal calprotectin reliably rules out endoscopic recurrence. In other postoperative settings, the panel suggests endoscopic assessment for establishing postoperative recurrence. In patients with CD, fecal calprotectin and serum CRP can inform disease management in both asymptomatic and symptomatic disease. Discordance between symptom assessment and biomarker value may merit endoscopic evaluation for confirmation of status of disease activity.
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Humanos , Inducción de Remisión , Enfermedades Inflamatorias del Intestino/diagnóstico , Proteína C-Reactiva/análisis , Enfermedades Inflamatorias del Intestino/diagnóstico por imagen , Endoscopía Gastrointestinal , Complejo de Antígeno L1 de Leucocito/análisisRESUMEN
Biomarkers are used frequently for noninvasive monitoring and treatment decision making in the management of patients with ulcerative colitis (UC). This American Gastroenterological Association (AGA) guideline is intended to support practitioners in decisions about the use of biomarkers for the management of UC. A multidisciplinary panel of content experts and guideline methodologists used the Grading of Recommendations Assessment, Development and Evaluation framework to prioritize clinical questions, identify patient-centered outcomes, and conduct an evidence synthesis on the clinical performance of serum C-reactive protein (CRP), fecal calprotectin, and fecal lactoferrin as biomarkers of disease activity in patients with established UC in symptomatic remission or with active symptoms. The guideline panel used the Evidence-to-Decision framework to develop recommendations for the use of biomarkers for monitoring and management of UC and provided implementation considerations for clinical practice. The guideline panel made 7 conditional recommendations. In patients with UC in symptomatic remission, the panel suggests the use of a biomarker- and symptom-based monitoring strategy over a symptom-based monitoring strategy. For patients in symptomatic remission, the panel suggests using fecal calprotectin <150 µg/g, normal fecal lactoferrin, and/or normal CRP to rule out active inflammation and avoid routine endoscopic assessment of disease. In patients with UC with moderate to severe symptoms, the panel suggests using fecal calprotectin >150 µg/g, elevated fecal lactoferrin, or elevated CRP to inform treatment decisions and avoid routine endoscopic assessment of disease. However, in patients in symptomatic remission but elevated biomarkers, and in patients with moderate to severe symptoms with normal biomarkers, the panel suggests endoscopic assessment of disease to inform treatment decisions. In patients with UC with mild symptoms, the panel suggests endoscopic assessment of disease activity to inform treatment decisions. The panel identified the use of a biomarker-based monitoring strategy over an endoscopy-based monitoring strategy as a knowledge gap. The panel also proposed key implementation considerations for optimal use of biomarkers, and identified areas for future research. In patients with UC, noninvasive biomarkers, including fecal calprotectin, fecal lactoferrin, and serum CRP can inform disease monitoring and management.
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Humanos , Biomarcadores , Colitis Ulcerosa/prevención & control , Lactoferrina/análisis , Endoscopía Gastrointestinal , Complejo de Antígeno L1 de Leucocito/análisisRESUMEN
The Src family tyrosine kinase Lck is essential for T cell development and T cell receptor (TCR) signaling. Lck is post-translationally fatty acylated at its N-terminus conferring membrane targeting and concentration in plasma membrane lipid rafts, which are lipid-based organisational platforms. Confocal fluorescence microscopy shows that Lck colocalizes in rafts with GPI-linked proteins, the adaptor protein LAT and Ras, but not with non-raft membrane proteins including the protein tyrosine phosphatase CD45. The TCR also associates with lipid rafts and its cross-linking causes coaggregation of raft-associated proteins including Lck, but not of CD45. Cross-linking of either the TCR or rafts strongly induces specific tyrosine phosphorylation of the TCR in the rafts. Remarkably, raft patching alone induces signalling events analogous to TCR stimulation, with the same dependence on expression of key TCR signalling molecules. Our results indicate a mechanism whereby TCR engagement promotes aggregation of lipid rafts, which facilitates colocalization of signaling proteins including Lck, LAT, and the TCR, while excluding CD45, thereby potentiating protein tyrosine phosphorylation and downstream signaling. We are currently testing this hypothesis as well as using imaging techniques such as fluorescence resonance energy transfer (FRET) microscopy to study the dynamics of proteins and lipids in lipid rafts in living cells undergoing signaling events. Recent data show that the key phosphoinositide PI(4,5)P2 is concentrated in T cell lipid rafts and that on stimulation of the cells it is rapidly converted to PI(3,4,5)P3 and diacylglycerol within rafts. Thus rafts are hotspots for both protein and lipid signalling pathways (AU)#S