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INTRODUCTION: During the coronavirus disease 2019, pandemic clinical practice had to change, and this study trialled a diagnostic questionnaire to assess patients with dizziness over the telephone. METHODS: All 115 patients awaiting otorhinolaryngological assessment for balance were randomised to receive a dizziness questionnaire in the post prior to their telephone consultation or not. Consultation outcomes were recorded by the clinicians conducting the consultation. Follow-up data were collected in June 2022 for final outcomes. RESULTS: 82/115 patients had consultations with complete data collection: 35 in the questionnaire group (QG) and 47 in the no questionnaire group (NQG), with a 70% response rate in the QG. Clinicians made a diagnosis in 27/35 QG consultations versus 27/47 NQG consultations. Nine out of 35 QG patients required additional investigations compared to 34/47 in the NQG (p < 0.05). Only 6/35 QG patients needed additional telephone follow-up, compared to 20/47 NQG patients (p < 0.05). CONCLUSION: Using a diagnostic questionnaire increased clinicians' ability to come to a diagnosis in telephone consultations.
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COVID-19 , Consulta Remota , Humanos , Pandemias , Mareo/diagnóstico , Teléfono , Encuestas y CuestionariosRESUMEN
Dysfunction of interleukin-10 producing regulatory B cells has been associated with the pathogenesis of autoimmune diseases, but whether regulatory B cells can be therapeutically induced in humans is currently unknown. Here we demonstrate that a subset of activated B cells expresses CD25, and the addition of low-dose recombinant IL-2 to in vitro stimulated peripheral blood and splenic human B cells augments IL-10 secretion. Administration of low dose IL-2, aldesleukin, to patients increases IL-10-producing B cells. Single-cell RNA sequencing of circulating immune cells isolated from low dose IL2-treated patients reveals an increase in plasmablast and plasma cell populations that are enriched for a regulatory B cell gene signature. The transcriptional repressor BACH2 is significantly down-regulated in plasma cells from IL-2-treated patients, BACH2 binds to the IL-10 gene promoter, and Bach2 depletion or genetic deficiency increases B cell IL-10, implicating BACH2 suppression as an important mechanism by which IL-2 may promote an immunoregulatory phenotype in B cells.
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Interleucina-10 , Interleucina-2 , Humanos , Interleucina-2/efectos adversos , Interleucina-10/metabolismo , Linfocitos B , Células Plasmáticas , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismoRESUMEN
We report the case of a woman in her 30s who was referred to the ear, nose and throat department with sudden onset left-sided sensorineural hearing loss (SNHL), left anterior uveitis and erythematous lower limb lesions with bilateral pitting oedema. Based on her symptoms, an underlying inflammatory systemic disease was suspected. Autoantibodies were negative but an X-ray and high-resolution CT scan of the chest were suggestive of sarcoidosis, which was confirmed on endoscopic bronchial biopsy. Following treatment with a course of oral steroids, the patient's hearing has improved but she still suffers from episodes of uveitis. While immune-mediated inner ear disorders are a recognised cause of SNHL, sarcoidosis is a very rare cause. This case demonstrates the importance of screening for systemic autoimmune aetiology in SNHL and highlights the importance of an effective multidisciplinary team in the diagnosis and management of these patients.
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Pérdida Auditiva Sensorineural , Pérdida Auditiva Súbita , Sarcoidosis , Autoanticuerpos , Femenino , Pérdida Auditiva Sensorineural/diagnóstico , Pérdida Auditiva Sensorineural/etiología , Pérdida Auditiva Súbita/complicaciones , Humanos , Sarcoidosis/complicaciones , Sarcoidosis/diagnósticoRESUMEN
To investigate the role of lymphotoxin (LT) in Sjögren's syndrome (SS) and in mucosal associated lymphoid tissue (MALT)-lymphoma, we made transgenic mice (Amy1-LTαß) that targeted LTα and LTß to the salivary and lacrimal glands. Amy1-LTαß mice developed atrophic salivary and lacrimal glands that contained tertiary lymphoid organs (TLOs) and had reduced tear production. Amy1-LTαß mice developed cervical lymphadenopathy but not MALT-lymphoma. TLO formation in the salivary and lacrimal glands of Amy1-LTαß was not sufficient to induce autoimmunity as measured by autoantibody titres.
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Aparato Lagrimal/patología , Linfadenopatía/patología , Linfotoxina-alfa/metabolismo , Glándulas Salivales/patología , Estructuras Linfoides Terciarias/patología , Animales , Linfadenopatía/genética , Linfoma de Células B de la Zona Marginal/genética , Linfoma de Células B de la Zona Marginal/inmunología , Linfoma de Células B de la Zona Marginal/patología , Linfotoxina-alfa/genética , Ratones , Ratones Transgénicos , Síndrome de Sjögren/genética , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/patología , Lágrimas/metabolismo , Estructuras Linfoides Terciarias/genéticaRESUMEN
BACKGROUND: Type 1 diabetes (T1D) results from loss of immune regulation, leading to the development of autoimmunity to pancreatic ß cells, involving autoreactive T effector cells (Teffs). Tregs, which prevent autoimmunity, require IL-2 for maintenance of immunosuppressive functions. Using a response-adaptive design, we aimed to determine the optimal regimen of aldesleukin (recombinant human IL-2) to physiologically enhance Tregs while limiting expansion of Teffs. METHODS: DILfrequency is a nonrandomized, open-label, response-adaptive study of participants, aged 18-70 years, with T1D. The initial learning phase allocated 12 participants to 6 different predefined regimens. Then, 3 cohorts of 8 participants were sequentially allocated dose frequencies, based on repeated interim analyses of all accumulated trial data. The coprimary endpoints were percentage change in Tregs and Teffs and CD25 (α subunit of the IL-2 receptor) expression by Tregs, from baseline to steady state. RESULTS: Thirty-eight participants were enrolled, with thirty-six completing treatment. The optimal regimen to maintain a steady-state increase in Tregs of 30% and CD25 expression of 25% without Teff expansion is 0.26 × 106 IU/m2 (95% CI -0.007 to 0.485) every 3 days. Tregs and CD25 were dose-frequency responsive, Teffs were not. The commonest adverse event was injection site reaction (464 of 694 events). CONCLUSIONS: Using a response-adaptive design, aldesleukin treatment can be optimized. Our methodology can generally be employed to immediately access proof of mechanism, thereby leading to more efficient and safe drug development. TRIAL REGISTRATION: International Standard Randomised Controlled Trial Number Register, ISRCTN40319192; ClinicalTrials.gov, NCT02265809. FUNDING: Sir Jules Thorn Trust, the Swiss National Science Foundation, Wellcome, JDRF, and NIHR Cambridge Biomedical Research Centre.
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Diabetes Mellitus Tipo 1/tratamiento farmacológico , Interleucina-2/análogos & derivados , Linfocitos T Reguladores/efectos de los fármacos , Adolescente , Adulto , Anciano , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/inmunología , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Estudios de Factibilidad , Femenino , Humanos , Interleucina-2/administración & dosificación , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Activación de Linfocitos/efectos de los fármacos , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/administración & dosificación , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Resultado del Tratamiento , Adulto JovenRESUMEN
Genome-wide association studies have identified a genetic variant at 3p14.3 (SNP rs1354034) that strongly associates with platelet number and mean platelet volume in humans. While originally proposed to be intronic, analysis of mRNA expression in primary human hematopoietic subpopulations reveals that this SNP is located directly upstream of the predominantly expressed ARHGEF3 isoform in megakaryocytes (MK). We found that ARHGEF3, which encodes a Rho guanine exchange factor, is dramatically upregulated during both human and murine MK maturation. We show that the SNP (rs1354034) is located in a DNase I hypersensitive region in human MKs and is an expression quantitative locus (eQTL) associated with ARHGEF3 expression level in human platelets, suggesting that it may be the causal SNP that accounts for the variations observed in human platelet traits and ARHGEF3 expression. In vitro human platelet activation assays revealed that rs1354034 is highly correlated with human platelet activation by ADP. In order to test whether ARHGEF3 plays a role in MK development and/or platelet function, we developed an Arhgef3 KO/LacZ reporter mouse model. Reflecting changes in gene expression, LacZ expression increases during MK maturation in these mice. Although Arhgef3 KO mice have significantly larger platelets, loss of Arhgef3 does not affect baseline MK or platelets nor does it affect platelet function or platelet recovery in response to antibody-mediated platelet depletion compared to littermate controls. In summary, our data suggest that modulation of ARHGEF3 gene expression in humans with a promoter-localized SNP plays a role in human MKs and human platelet function-a finding resulting from the biological follow-up of human genetic studies. Arhgef3 KO mice partially recapitulate the human phenotype.
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Plaquetas/metabolismo , Polimorfismo de Nucleótido Simple , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Animales , Plaquetas/citología , Diferenciación Celular/fisiología , Tamaño de la Célula , Estudios de Cohortes , Femenino , Sangre Fetal , Regulación de la Expresión Génica , Humanos , Masculino , Volúmen Plaquetario Medio , Megacariocitos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Regiones Promotoras GenéticasRESUMEN
INTRODUCTION: Type 1 diabetes (T1D) is caused by autoimmune destruction of the insulin-producing ß cells in the pancreatic islets, leading to insulinopenia and hyperglycaemia. Genetic analyses indicate that alterations of the interleukin-2 (IL-2) pathway mediating immune activation and tolerance predispose to T1D, specifically the polymorphic expression of the IL-2 receptor-α chain (CD25) on T lymphocytes. Replacement of physiological doses of IL-2 could restore self-tolerance and prevent further autoimmunity by enhancing the function of CD4(+) T regulatory cells (Tregs) to limit the activation of auto reactive T effector cells (Teffs). In this experimental medicine study, we use an adaptive trial design to determine the optimal dosing regimen for IL-2 to improve Treg function while limiting activation of Teffs in participants with T1D. METHODS AND ANALYSIS: The Adaptive study of IL-2 dose frequency on Tregs in type 1 diabetes(DILfrequency) is a mechanistic, non-randomised, repeat dose open-label, response-adaptive study of 36 participants with T1D. The objective is to establish the optimal dose and frequency of ultra-low dose IL-2: to increase Treg frequency within the physiological range, to increase CD25 expression on Tregs, without increasing CD4(+) Teffs. DILfrequency has an initial learning phase where 12 participants are allocated to six different doses and frequencies followed by an interim statistical analysis. After analysis of the learning phase, the Dose and Frequency Committee will select the optimal targets for Treg frequency, Treg CD25 expression and Teff frequency. Three groups of eight participants will be treated consecutively in the confirming phase. Each dose and frequency selected will be based on statistical analysis of all data collected from the previous groups. ETHICS: Ethical approval for DILfrequency was granted on 12 August 2014. RESULTS: The results of this study will be reported, through peer-reviewed journals, conference presentations and an internal organisational report. TRIAL REGISTRATION NUMBERS: NCT02265809, ISRCTN40319192, CRN17571.
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Diabetes Mellitus Tipo 1/inmunología , Interleucina-2/administración & dosificación , Linfocitos T Reguladores/efectos de los fármacos , Adolescente , Adulto , Anciano , Femenino , Humanos , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Immune-deficient mice, reconstituted with human stem cells, have been used to analyze human immune responses in vivo. Although they have been used to study immune responses to xenografts, allografts, and pathogens, there have not been models of autoimmune disease in which the mechanisms of the pathologic process can be analyzed. We have found that reconstituted "humanized" mice treated with anti-CTLA-4 Ab (ipilimumab) develop autoimmune disease characterized by hepatitis, adrenalitis, sialitis, anti-nuclear Abs, and weight loss. Induction of autoimmunity involved activation of T cells and cytokine production, and increased infiltration of APCs. When anti-CTLA-4 mAb-treated mice were cotreated with anti-CD3 mAb (teplizumab), hepatitis and anti-nuclear Abs were no longer seen and weight loss did not occur. The anti-CD3 blocked proliferation and activation of T cells, release of IFN-γ and TNF, macrophage infiltration, and release of IP-10 that was induced with anti-CTLA-4 mAb. We also found increased levels of T regulatory cells (CD25(+)CD127(-)) in the spleen and mesenteric lymph nodes in the mice treated with both Abs and greater constitutive phosphorylation of STAT5 in T regulatory cells in spleen cells compared with mice treated with anti-CTLA-4 mAb alone. We describe a model of human autoimmune disease in vivo. Humanized mice may be useful for understanding the mechanisms of biologics that are used in patients. Hepatitis, lymphadenopathy, and other inflammatory sequelae are adverse effects of ipilimumab treatment in humans, and this study may provide insights into this pathogenesis and the effects of immunologics on autoimmunity.
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Anticuerpos Monoclonales Humanizados/farmacología , Enfermedades Autoinmunes/terapia , Modelos Animales de Enfermedad , Trasplante de Células Madre/métodos , Linfocitos T/inmunología , Glándulas Suprarrenales/efectos de los fármacos , Glándulas Suprarrenales/inmunología , Glándulas Suprarrenales/metabolismo , Animales , Anticuerpos Monoclonales/toxicidad , Enfermedades Autoinmunes/inducido químicamente , Enfermedades Autoinmunes/inmunología , Citocinas/sangre , Citocinas/inmunología , Citocinas/metabolismo , Citometría de Flujo , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Ipilimumab , Hígado/efectos de los fármacos , Hígado/inmunología , Hígado/metabolismo , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Fosforilación/efectos de los fármacos , Fosforilación/inmunología , Factor de Transcripción STAT5/inmunología , Factor de Transcripción STAT5/metabolismo , Análisis de Supervivencia , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Trasplante Heterólogo , Pérdida de Peso/efectos de los fármacos , Pérdida de Peso/inmunologíaRESUMEN
BACKGROUND: Serious infections of the head and neck cause lymphedema that can lead to airway compromise and oropharyngeal obstruction. Lymphangiogenesis occurs in the head and neck during infection and after immunization. The goal of this project was to develop tools to image lymphatic vessels in living animals and to be able to isolate individual lymphatic endothelial cells in order to quantify changes in single cells caused by inflammation. METHODS: The ProxTom transgenic red-fluorescent reporter mouse was developed specifically for the purpose of imaging lymphatic vessels in vivo. Prox1 is a transcription factor that is necessary for lymphangiogenesis in development and for the maintenance of lymphatics in adulthood. Mice were immunized and their lymphatic vessels in lymph nodes were imaged in vivo. Individual lymphatic endothelial cells were isolated by means of their fluorescence. RESULTS: The ProxTom transgene has the red-fluorescent reporter td-Tomato under the control of Prox1 regulatory elements. tdTomato was faithfully expressed in lymphatic vessels coincident with endogenous Prox1 expression. We show lymphangiogenesis in vivo after immunization and demonstrate a method for the isolation of lymphatic endothelial cells by their tdTomato red-fluorescence. CONCLUSIONS: The faithful expression of the red-fluorescent reporter in the lymphatic vessels of ProxTom means that these mice have proven utility for in vivo study of lymphatic vessels in the immune response. ProxTom has been made available for distribution from the Jackson Laboratory: http://jaxmice.jax.org/strain/018128.html .
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Células Endoteliales/metabolismo , Inflamación/metabolismo , Linfangiogénesis , Vasos Linfáticos/metabolismo , Animales , Células Endoteliales/inmunología , Células Endoteliales/patología , Citometría de Flujo , Cabeza , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Inmunización/métodos , Inflamación/genética , Inflamación/inmunología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Vasos Linfáticos/inmunología , Vasos Linfáticos/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente , Cuello , Oxazolona/inmunología , Regiones Promotoras Genéticas/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína Fluorescente RojaRESUMEN
Lymphatic vessels (LVs) are important structures for antigen presentation, for lipid metabolism, and as conduits for tumor metastases, but they have been difficult to visualize in vivo. Prox1 is a transcription factor that is necessary for lymphangiogenesis in ontogeny and the maintenance of LVs. To visualize LVs in the lymph node of a living mouse in real time, we made the ProxTom transgenic mouse in a C57BL/6 background using red fluorescent LVs that are suitable for in vivo imaging. The ProxTom transgene contained all Prox1 regulatory sequences and was faithfully expressed in LVs coincident with endogenous Prox1 expression. The progenies of a ProxTom × Hec6stGFP cross were imaged using two-photon laser scanning microscopy, allowing the simultaneous visualization of LVs and high endothelial venules in a lymph node of a living mouse for the first time. We confirmed the expression of Prox1 in the adult liver, lens, and dentate gyrus. These intensely fluorescent mice revealed the expression of Prox1 in three novel sites: the neuroendocrine cells of the adrenal medulla, megakaryocytes, and platelets. The novel sites identified herein suggest previously unknown roles for Prox1. The faithful expression of the fluorescent reporter in ProxTom LVs indicates that these mice have potential utility in the study of diseases as diverse as lymphedema, filariasis, transplant rejection, obesity, and tumor metastasis.
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Médula Suprarrenal/metabolismo , Plaquetas/metabolismo , Proteínas de Homeodominio/metabolismo , Vasos Linfáticos/metabolismo , Megacariocitos/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Células Cultivadas , Citoplasma/metabolismo , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/fisiología , Genotipo , Glicoproteínas/metabolismo , Proteínas de Homeodominio/genética , Proteínas Luminiscentes/metabolismo , Ganglios Linfáticos/metabolismo , Proteínas de Transporte de Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genética , Proteína Fluorescente RojaRESUMEN
Cells undergoing apoptosis are efficiently located and engulfed by phagocytes. The mechanisms by which macrophages, the professional scavenging phagocytes of apoptotic cells, are attracted to sites of apoptosis are poorly defined. Here we show that CX3CL1/fractalkine, a chemokine and intercellular adhesion molecule, is released rapidly from apoptotic lymphocytes, via caspase- and Bcl-2-regulated mechanisms, to attract macrophages. Effective chemotaxis of macrophages to apoptotic lymphocytes is dependent on macrophage fractalkine receptor, CX3CR1. CX3CR1 deficiency caused diminished recruitment of macrophages to germinal centers of lymphoid follicles, sites of high-rate B-cell apoptosis. These results provide the first demonstration of chemokine/chemokine-receptor activity in the navigation of macrophages toward apoptotic cells and identify a mechanism by which macrophage infiltration of tissues containing apoptotic lymphocytes is achieved.
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Apoptosis , Quimiocina CX3CL1/metabolismo , Quimiotaxis , Linfocitos/metabolismo , Macrófagos/fisiología , Animales , Linfoma de Burkitt , Caspasas , Línea Celular Tumoral , Células Cultivadas , Humanos , Ganglios Linfáticos , Linfocitos/citología , Ratones , Ratones Endogámicos BALB C , Proteínas Proto-Oncogénicas c-bcl-2RESUMEN
In Burkitt's lymphoma (BL), apoptosis occurs at high frequency alongside uncontrolled proliferation. Macrophages infiltrate these tumours in large numbers and engage in the phagocytic clearance of apoptotic cells in situ. Here we tested the hypothesis that apoptosis of BL cells may provide a mechanism for recruitment of macrophages to these tumours. We show that monocytes and macrophages, but not neutrophils, preferentially migrated to apoptotic BL cells in vitro. Transfection of BL cells with the anti-apoptotic gene bcl-2 both prevented apoptosis and abolished macrophage chemotaxis. Macrophage migration to BL populations correlated well with the number of apoptotic BL cells present (the Pearson correlation r = 0.81, p<0.0001). Chemoattraction of murine macrophages to apoptotic human BL cells demonstrated that the mechanism was conserved across these species. In an attempt to identify the macrophage receptors involved in this process, we investigated whether CD14 and CD36, two receptors important in the phagocytic clearance of apoptotic cells, were also involved in the chemotactic macrophage response. We found that bone marrow-derived macrophages from CD14-/- and CD36-/- mice moved as well as wild-type macrophages in chemotaxis assays towards apoptotic BL cells. Migrating macrophages were found to be up-regulated in their expression of CD14, however, suggesting that, although this receptor does not appear to be required for 'sensing' apoptotic cells, it may be up-regulated on the surface of the migrating macrophage in readiness for apoptotic corpse clearance.