RESUMEN
BACKGROUND: Neurofilament light chain (NfL), a neuronal cytoskeletal protein that is released upon neuroaxonal injury, is associated with multiple sclerosis (MS) relapsing activity and has demonstrated some prognostic ability for future relapse-related disease progression, yet its value in assessing non-relapsing disease progression remains unclear. METHODS: We examined baseline and longitudinal blood NfL levels in 1421 persons with relapsing MS (RMS) and 596 persons with primary progressive MS (PPMS) from the pivotal ocrelizumab MS trials. NfL treatment-response and risk for disease worsening (including disability progression into the open-label extension period and slowly expanding lesions [SELs] on brain MRI) at baseline and following treatment with ocrelizumab were evaluated using time-to-event analysis and linear regression models. FINDINGS: In persons from the RMS control arms without acute disease activity and in the entire PPMS control arm, higher baseline NfL was prognostic for greater whole brain and thalamic atrophy, greater volume expansion of SELs, and clinical progression. Ocrelizumab reduced NfL levels vs. controls in persons with RMS and those with PPMS, and abrogated the prognostic value of baseline NfL on disability progression. Following effective suppression of relapse activity by ocrelizumab, NfL levels at weeks 24 and 48 were significantly associated with long-term risk for disability progression, including up to 9 years of observation in RMS and PPMS. INTERPRETATION: Highly elevated NfL from acute MS disease activity may mask a more subtle NfL abnormality that reflects underlying non-relapsing progressive biology. Ocrelizumab significantly reduced NfL levels, consistent with its effects on acute disease activity and disability progression. Persistently elevated NfL levels, observed in a subgroup of persons under ocrelizumab treatment, demonstrate potential clinical utility as a predictive biomarker of increased risk for clinical progression. Suppression of relapsing biology with high-efficacy immunotherapy provides a window into the relationship between NfL levels and future non-relapsing progression. FUNDING: F. Hoffmann-La Roche Ltd.
Asunto(s)
Esclerosis Múltiple Crónica Progresiva , Esclerosis Múltiple Recurrente-Remitente , Esclerosis Múltiple , Humanos , Enfermedad Aguda , Progresión de la Enfermedad , Filamentos Intermedios , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple Crónica Progresiva/diagnóstico , Esclerosis Múltiple Crónica Progresiva/tratamiento farmacológico , Esclerosis Múltiple Recurrente-Remitente/diagnóstico por imagen , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , RecurrenciaRESUMEN
BACKGROUND AND OBJECTIVES: The B cell-depleting anti-CD20 antibody ocrelizumab (OCR) effectively reduces MS disease activity and slows disability progression. Given the role of B cells as antigen-presenting cells, the primary goal of this study was to evaluate the effect of OCR on the T-cell receptor repertoire diversity. METHODS: To examine whether OCR substantially alters the molecular diversity of the T-cell receptor repertoire, deep immune repertoire sequencing (RepSeq) of CD4+ and CD8+ T-cell receptor ß-chain variable regions was performed on longitudinal blood samples. The IgM and IgG heavy chain variable region repertoire was also analyzed to characterize the residual B-cell repertoire under OCR treatment. RESULTS: Peripheral blood samples for RepSeq were obtained from 8 patients with relapsing MS enrolled in the OPERA I trial over a period of up to 39 months. Four patients each were treated with OCR or interferon ß1-a during the double-blind period of OPERA I. All patients received OCR during the open-label extension. The diversity of the CD4+/CD8+ T-cell repertoires remained unaffected in OCR-treated patients. The expected OCR-associated B-cell depletion was mirrored by reduced B-cell receptor diversity in peripheral blood and a shift in immunoglobulin gene usage. Despite deep B-cell depletion, longitudinal persistence of clonally related B-cells was observed. DISCUSSION: Our data illustrate that the diversity of CD4+/CD8+ T-cell receptor repertoires remained unaltered in OCR-treated patients with relapsing MS. Persistence of a highly diverse T-cell repertoire suggests that aspects of adaptive immunity remain intact despite extended anti-CD20 therapy. TRIAL REGISTRATION INFORMATION: This is a substudy (BE29353) of the OPERA I (WA21092; NCT01247324) trial. Date of registration, November 23, 2010; first patient enrollment, August 31, 2011.
Asunto(s)
Esclerosis Múltiple , Humanos , Factores Inmunológicos/farmacología , Anticuerpos Monoclonales Humanizados/farmacología , Recurrencia , Receptores de Antígenos de Linfocitos TRESUMEN
Targeted B-cell depletion is a useful therapy for many diseases, including autoimmune disorders and certain cancers. We developed a sensitive blood B-cell depletion assay, MRB 1.1, compared its performance with the T-cell/B-cell/NK-cell (TBNK) assay, and assessed B-cell depletion with different therapies. The empirically defined lower limit of quantification (LLOQ) for CD19+ cells in the TBNK assay was 10 cells/µL, and 0.441 cells/µL for the MRB 1.1 assay. The TBNK LLOQ was used to compare differences between B-cell depletion in similar lupus nephritis patient populations who received rituximab (LUNAR), ocrelizumab (BELONG), or obinutuzumab (NOBILITY). After 4 weeks, 10% of patients treated with rituximab retained detectable B cells vs 1.8% with ocrelizumab and 1.7% for obinutuzumab; at 24 weeks 93% of patients who received obinutuzumab remained below LLOQ vs 63% for rituximab. More-sensitive measurements of B cells may reveal differences in potency among anti-CD20 agents, which may associate with clinical outcomes.
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Enfermedades Autoinmunes , Linfocitos B , Humanos , Rituximab/uso terapéutico , Enfermedades Autoinmunes/tratamiento farmacológico , Células Asesinas NaturalesRESUMEN
Lupus nephritis (LN) is a severe clinical manifestation of systemic lupus erythematosus (SLE) associated with significant morbidity and mortality. Assessment of severity and activity of renal involvement in SLE requires a kidney biopsy, an invasive procedure with limited prognostic value. Noninvasive biomarkers are needed to inform treatment decisions and to monitor disease activity. Proteinuria is associated with disease progression in LN; however, the composition of the LN urinary proteome remains incompletely characterized. To address this, we profiled LN urine samples using complementary mass spectrometry-based methods: protein gel fractionation, chemical labeling using tandem mass tags, and data-independent acquisition. Combining results from these approaches yielded quantitative information on 2573 unique proteins in urine from LN patients. A multiple-reaction monitoring (MRM) method was established to confirm eight proteins in an independent cohort of LN patients, and seven proteins (transferrin, α-2-macroglobulin, haptoglobin, afamin, α-1-antitrypsin, vimentin, and ceruloplasmin) were confirmed to be elevated in LN urine compared to healthy controls. In this study, we demonstrate that deep mass spectrometry profiling of a small number of patient samples can identify high-quality biomarkers that replicate in an independent LN disease cohort. These biomarkers are being used to inform clinical biomarker strategies to support longitudinal and interventional studies focused on evaluating disease progression and treatment efficacy of novel LN therapeutics.
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Biomarcadores/orina , Lupus Eritematoso Sistémico/orina , Nefritis Lúpica/orina , Proteoma/genética , Adolescente , Adulto , Anciano , Biopsia , Proteínas Portadoras/orina , Ceruloplasmina/orina , Femenino , Glicoproteínas/orina , Haptoglobinas/orina , Humanos , Riñón/metabolismo , Riñón/patología , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/patología , Nefritis Lúpica/genética , Nefritis Lúpica/patología , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Pronóstico , Albúmina Sérica Humana/orina , Transferrina/orina , Vimentina/orina , Adulto Joven , alfa 1-Antitripsina/orina , alfa-Macroglobulinas/orinaRESUMEN
GDC-0853 is a small molecule inhibitor of Bruton's tyrosine kinase (BTK) that is highly selective and noncovalent, leading to reversible binding. In double-blind, randomized, and placebo-controlled phase I healthy volunteer studies, GDC-0853 was well tolerated, with no dose-limiting adverse events (AEs) or serious AEs. The maximum tolerated dose was not reached during dose escalation (≤600 mg, single ascending dose (SAD) study; ≤250 mg twice daily (b.i.d.) and ≤500 mg once daily, 14-day multiple ascending dose (MAD) study). Plasma concentrations peaked 1-3 hours after oral administration and declined thereafter, with a steady-state half-life ranging from 4.2-9.9 hours. Independent assays demonstrated dose-dependent BTK target engagement. Based on pharmacokinetic/pharmacodynamic (PK/PD) simulations, a once-daily dosing regimen (e.g., 100 mg, q.d.) is expected to maintain a high level of BTK inhibition over the dosing interval. Taken together, the safety and PK/PD data support GDC-0853 evaluation in rheumatoid arthritis, lupus, and other autoimmune or inflammatory indications.
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Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridonas/farmacología , Adolescente , Adulto , Área Bajo la Curva , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Femenino , Semivida , Humanos , Masculino , Dosis Máxima Tolerada , Tasa de Depuración Metabólica , Persona de Mediana Edad , Piperazinas/administración & dosificación , Piperazinas/efectos adversos , Piperazinas/farmacocinética , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/efectos adversos , Inhibidores de Proteínas Quinasas/farmacocinética , Piridonas/administración & dosificación , Piridonas/efectos adversos , Piridonas/farmacocinética , Adulto JovenRESUMEN
Insufficient pancreatic ß-cell mass or function results in diabetes mellitus. While significant progress has been made in regulating insulin secretion from ß-cells in diabetic patients, no pharmacological agents have been described that increase ß-cell replication in humans. Here we report aminopyrazine compounds that stimulate robust ß-cell proliferation in adult primary islets, most likely as a result of combined inhibition of DYRK1A and GSK3B. Aminopyrazine-treated human islets retain functionality in vitro and after transplantation into diabetic mice. Oral dosing of these compounds in diabetic mice induces ß-cell proliferation, increases ß-cell mass and insulin content, and improves glycaemic control. Biochemical, genetic and cell biology data point to Dyrk1a as the key molecular target. This study supports the feasibility of treating diabetes with an oral therapy to restore ß-cell mass, and highlights a tractable pathway for future drug discovery efforts.
Asunto(s)
Proliferación Celular , Glucógeno Sintasa Quinasa 3/genética , Células Secretoras de Insulina/citología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Animales , División Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatología , Regulación hacia Abajo/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/enzimología , Masculino , Ratones , Ratones Transgénicos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Piridazinas/farmacología , Quinasas DyrKRESUMEN
Adjuvants increase vaccine potency largely by activating innate immunity and promoting inflammation. Limiting the side effects of this inflammation is a major hurdle for adjuvant use in vaccines for humans. It has been difficult to improve on adjuvant safety because of a poor understanding of adjuvant mechanism and the empirical nature of adjuvant discovery and development historically. We describe new principles for the rational optimization of small-molecule immune potentiators (SMIPs) targeting Toll-like receptor 7 as adjuvants with a predicted increase in their therapeutic indices. Unlike traditional drugs, SMIP-based adjuvants need to have limited bioavailability and remain localized for optimal efficacy. These features also lead to temporally and spatially restricted inflammation that should decrease side effects. Through medicinal and formulation chemistry and extensive immunopharmacology, we show that in vivo potency can be increased with little to no systemic exposure, localized innate immune activation and short in vivo residence times of SMIP-based adjuvants. This work provides a systematic and generalizable approach to engineering small molecules for use as vaccine adjuvants.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Diseño de Fármacos , Vacunas/administración & dosificación , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/farmacocinética , Disponibilidad BiológicaRESUMEN
AIM/HYPOTHESIS: Recent studies indicate that tyrosine kinase inhibitors, including imatinib, can reverse hyperglycemia in non-obese diabetic (NOD) mice, a model of type 1 diabetes (T1D). Imatinib inhibits c-Abl, c-Kit, and PDGFRs. Next-generation tyrosine kinase inhibitors for T1D treatment should maintain activities required for efficacy while sparing inhibition of targets that might otherwise lead to adverse events. In this study, we investigated the contribution of c-Kit inhibition by imatinib in reversal of hyperglycemia in NOD mice. METHODS: The T670I mutation in c-Kit, which confers imatinib resistance, was engineered into the mouse genome and bred onto the NOD background. Hematopoietic stem cells (HSCs) from NOD.c-Kit(T670I) mice and NOD.c-Kit(wt) littermates were expanded in the presence or absence of imatinib to verify imatinib resistance of the c-Kit(T670I) allele. Diabetic mice were treated with imatinib at the onset of hyperglycemia for three weeks, and blood glucose was monitored. RESULTS: In vitro expansion of HSCs from NOD.c-Kit(wt) mice was sensitive to imatinib, while expansion of HSCs from NOD.c-Kit(T670I) mice was insensitive to imatinib. However, in vivo treatment with imatinib lowered blood glucose levels in both strains of mice. CONCLUSIONS/INTERPRETATION: The HSC experiment confirmed that, in NOD.c-Kit(T670I) mice, c-Kit is resistant to imatinib. As both NOD.c-Kit(T670I) and NOD.c-Kit(wt) mice responded comparably to imatinib, c-Kit inhibition does not substantially contribute to the efficacy of imatinib in T1D. Thus, we conclude that inhibition of c-Kit is not required in next-generation tyrosine kinase inhibitors for T1D treatment, and may be selected against to improve the safety profile.
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Benzamidas/uso terapéutico , Hiperglucemia/tratamiento farmacológico , Piperazinas/uso terapéutico , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Pirimidinas/uso terapéutico , Animales , Femenino , Hiperglucemia/fisiopatología , Mesilato de Imatinib , Ratones , Ratones Endogámicos NOD , Mutación , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/fisiologíaRESUMEN
Type-1 diabetes (T1D) is an autoimmune disease targeting insulin-producing beta cells, resulting in dependence on exogenous insulin. To date, significant efforts have been invested to develop immune-modulatory therapies for T1D treatment. Previously, IL-2 immunotherapy was demonstrated to prevent and reverse T1D at onset in the non-obese diabetic (NOD) mouse model, revealing potential as a therapy in early disease stage in humans. In the NOD model, IL-2 deficiency contributes to a loss of regulatory T cell function. This deficiency can be augmented with IL-2 or antibody bound to IL-2 (Ab/IL-2) therapy, resulting in regulatory T cell expansion and potentiation. However, an understanding of the mechanism by which reconstituted regulatory T cell function allows for reversal of diabetes after onset is not clearly understood. Here, we describe that Ab/IL-2 immunotherapy treatment, given at the time of diabetes onset in NOD mice, not only correlated with reversal of diabetes and expansion of Treg cells, but also demonstrated the ability to significantly increase beta cell proliferation. Proliferation appeared specific to Ab/IL-2 immunotherapy, as anti-CD3 therapy did not have a similar effect. Furthermore, to assess the effect of Ab/IL-2 immunotherapy well after the development of diabetes, we tested the effect of delaying treatment for 4 weeks after diabetes onset, when beta cells were virtually absent. At this late stage after diabetes onset, Ab/IL-2 treatment was not sufficient to reverse hyperglycemia. However, it did promote survival in the absence of exogenous insulin. Proliferation of beta cells could not account for this improvement as few beta cells remained. Rather, abnormal insulin and glucagon dual-expressing cells were the only insulin-expressing cells observed in islets from mice with established disease. Thus, these data suggest that in diabetic NOD mice, beta cells have an innate capacity for regeneration both early and late in disease, which is revealed through IL-2 immunotherapy.
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Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/terapia , Células Secretoras de Insulina/inmunología , Interleucina-2/inmunología , Animales , Diabetes Mellitus Tipo 1/metabolismo , Modelos Animales de Enfermedad , Femenino , Glucagón/metabolismo , Inmunoterapia/métodos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Interleucina-2/metabolismo , Ratones , Ratones Endogámicos NOD , Regeneración/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismoRESUMEN
The identification of factors that promote ß cell proliferation could ultimately move type 1 diabetes treatment away from insulin injection therapy and toward a cure. We have performed high-throughput, cell-based screens using rodent ß cell lines to identify molecules that induce proliferation of ß cells. Herein we report the discovery and characterization of WS6, a novel small molecule that promotes ß cell proliferation in rodent and human primary islets. In the RIP-DTA mouse model of ß cell ablation, WS6 normalized blood glucose and induced concomitant increases in ß cell proliferation and ß cell number. Affinity pulldown and kinase profiling studies implicate Erb3 binding protein-1 and the IκB kinase pathway in the mechanism of action of WS6.
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Ensayos Analíticos de Alto Rendimiento , Islotes Pancreáticos/efectos de los fármacos , Urea/farmacología , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Humanos , Islotes Pancreáticos/citología , Ratones , Estructura Molecular , Peso Molecular , Relación Estructura-Actividad , Urea/análogos & derivados , Urea/químicaRESUMEN
Complement-mediated bactericidal activity has long been regarded as the serological correlate of protective immunity against Neisseria meningitidis. This was affirmed in 2005 at a WHO-sponsored meningococcal serology standardization workshop. The assay currently employed by most laboratories involves determining surviving bacterial colony counts on agar as a readout which is labor-intensive, time-consuming, and not amendable to rapid data analysis for clinical trials. Consequently, there is an acute need to develop a sensitive, high-throughput bactericidal assay to enable a rapid and robust assessment of the effectiveness of vaccine candidates. To this end, we have developed an automated, kinetic assay based on the fluorescent respiration product of resazurin which reduces assay volume, shortens assay time, and facilitates automation of data analysis. We demonstrate proof of concept for applicability of this high-throughput system with multiple meningococcal strains and utilizing different lots of human complement. The assay is robust and highly reproducible. Titers obtained by the fluorescence readout method are strongly correlated with the data obtained using the conventional, agar plate-based assay. These results demonstrate that the detection of bacteria that have survived the bactericidal reaction by measuring metabolic activity using a fluorescent dye as an alternative readout is a promising approach for the development of a high-throughput bactericidal assay.
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Anticuerpos Antibacterianos/inmunología , Automatización/métodos , Actividad Bactericida de la Sangre , Ensayos Analíticos de Alto Rendimiento/métodos , Viabilidad Microbiana/inmunología , Neisseria meningitidis/inmunología , Neisseria meningitidis/metabolismo , Colorantes Fluorescentes/metabolismo , Humanos , Inmunoensayo/métodos , Vacunas Meningococicas/inmunología , Oxazinas/metabolismo , Reproducibilidad de los Resultados , Coloración y Etiquetado/métodos , Xantenos/metabolismoRESUMEN
Foxp3 is required for the generation and activity of CD4(+)CD25(+) regulatory T (T reg) cells, which are important controllers of autoimmunity, including type-1 diabetes. To determine where T reg cells affect the diabetogenic cascade, we crossed the Foxp3 scurfy mutation, which eliminates T reg cells, with the BDC2.5 T cell receptor (TCR) transgenic mouse line. In this model, the absence of T reg cells did not augment the initial activation or phenotypic characteristics of effector T cells in the draining lymph nodes, nor accelerate the onset of T cell infiltration of the pancreatic islets. However, this insulitis was immediately destructive, causing a dramatic progression to overt diabetes. Microarray analysis revealed that T reg cells in the insulitic lesion adopted a gene expression program different from that in lymph nodes, whereas T reg cells in draining or irrelevant lymph nodes appeared very similar. Thus, T reg cells primarily impinge on autoimmune diabetes by reining in destructive T cells inside the islets, more than during the initial activation in the draining lymph nodes.
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Diabetes Mellitus Tipo 1/inmunología , Receptores de Interleucina-2/biosíntesis , Linfocitos T Reguladores/inmunología , Animales , Diabetes Mellitus Tipo 1/patología , Factores de Transcripción Forkhead/deficiencia , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/fisiología , Ganglios Linfáticos/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Páncreas/inmunologíaRESUMEN
Lysosomal proteases generate peptides presented by class II MHC molecules to CD4+ T cells. To determine whether specific lysosomal proteases might influence the outcome of a CD4+ T cell-dependent autoimmune response, we generated mice that lack cathepsin L (Cat L) on the autoimmune diabetes-prone NOD inbred background. The absence of Cat L affords strong protection from disease at the stage of pancreatic infiltration. The numbers of I-A(g7)-restricted CD4+ T cells are diminished in Cat L-deficient mice, although a potentially diabetogenic T cell repertoire persists. Within the CD4+ T cell compartments of Cat L-deficient mice, there is an increased proportion of regulatory T cells compared with that in Cat L-sufficient littermates. We suggest that it is this displaced balance of regulatory versus aggressive CD4+ T cells that protects Cat L-deficient mice from autoimmune disease. Our results identify Cat L as an enzyme whose activity is essential for the development of type I diabetes in the NOD mouse.
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Linfocitos T CD4-Positivos/inmunología , Catepsinas/inmunología , Cisteína Endopeptidasas/inmunología , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Tipo 1/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Linfocitos T CD4-Positivos/patología , Catepsina L , Catepsinas/genética , Cisteína Endopeptidasas/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patología , Lisosomas/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Noqueados , Páncreas/inmunología , Páncreas/patología , Subgrupos de Linfocitos T/patologíaRESUMEN
CD4+CD25+ T regulatory cells (Tregs) prevent autoimmune disease, yet little is known about precisely where they exert their influence naturally in a spontaneous autoimmune disorder. Here, we report that Tregs and T effector cells (Teffs) coexist within the pancreatic lesion before type 1 diabetes onset. We find that BDC2.5 T cell receptor transgenic animals contain a small subset of FoxP3 positive CD4+CD25+CD69- cells in the pancreas, actively turning over, expressing the clonotypic receptor, and containing functional regulatory activity. Gene expression profiling confirms that the CD4+CD25+CD69- cells in pancreatic tissue express transcripts diagnostic of regulatory cells, but with significantly higher levels of interleukin 10 and inducible costimulator (ICOS) than their lymph node counterparts. Blockade of ICOS rapidly converts early insulitis to diabetes, which disrupts the balance of Teffs and Tregs and promotes a very broad shift in the expression of the T regulatory-specific profile. Thus, CD4+CD25+69- Tregs operate directly in the autoimmune lesion and are dependent on ICOS to keep it in a nondestructive state.