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1.
Br J Dermatol ; 189(4): 447-458, 2023 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-37243544

RESUMO

BACKGROUND: Treatment for the debilitating disease hidradenitis suppurativa (HS) is inadequate in many patients. Despite an incidence of approximately 1%, HS is often under-recognized and underdiagnosed, and is associated with a high morbidity and poor quality of life. OBJECTIVES: To gain a better understanding of the pathogenesis of HS, in order to design new therapeutic strategies. METHODS: We employed single-cell RNA sequencing to analyse gene expression in immune cells isolated from involved HS skin vs. healthy skin. Flow cytometry was used to quantify the absolute numbers of the main immune populations. The secretion of inflammatory mediators from skin explant cultures was measured using multiplex and enzyme-linked immunosorbent assays. RESULTS: Single-cell RNA sequencing analysis identified a significant enrichment in the frequency of plasma cells, T helper (Th) 17 cells and dendritic cell subsets in HS skin, and the immune transcriptome was distinct and more heterogeneous than healthy skin. Flow cytometry revealed significantly increased numbers of T cells, B cells, neutrophils, dermal macrophages and dendritic cells in HS skin. Genes and pathways associated with Th17 cells, interleukin (IL)-17, IL-1ß and the NLRP3 inflammasome were enhanced in HS skin, particularly in samples with a high inflammatory load. Inflammasome constituent genes principally mapped to Langerhans cells and a subpopulation of dendritic cells. The secretome of HS skin explants contained significantly increased concentrations of inflammatory mediators, including IL-1ß and IL-17A, and culture with an NLRP3 inflammasome inhibitor significantly reduced the secretion of these, as well as other, key mediators of inflammation. CONCLUSIONS: These data provide a rationale for targeting the NLRP3 inflammasome in HS using small-molecule inhibitors that are currently being tested for other indications.


Assuntos
Hidradenite Supurativa , Humanos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Qualidade de Vida , Pele/patologia , Inflamação , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/uso terapêutico
3.
Exp Dermatol ; 31(7): 1036-1047, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35213752

RESUMO

Psoriasis vulgaris is an inflammatory skin disease that affects 2%-3% of the population worldwide. One of the major challenges in discovering novel therapies is the poor translatability of animal models to human disease. Therefore, it is imperative to develop human preclinical models of psoriasis that are amenable to pharmacological intervention. Here, we report a 3-D reconstituted human epidermis (RHE) culture system treated with cytokines commonly associated with psoriasis (TNFα, IL-17A and IL-22) that reproduced some key features of the human disease. The effects on epidermal morphology, gene transcription and cytokine production, which are dysregulated in psoriasis were assessed. Certain morphological features of psoriatic epidermis were evident in cytokine-stimulated RHEs, including hypogranulosis and parakeratosis. In addition, RHEs responded to a cytokine mix in a dose-dependent manner by expressing genes and proteins associated with impaired keratinocyte differentiation (keratin 10/K10, loricrin), innate immune responses (S100A7, DEFB4, elafin) and inflammation (IL-1α, IL-6, IL-8, IL-10, IL-12/23p40, IL-36γ, GM-CSF and IFNγ) typical of psoriasis. These disease-relevant changes in morphology, gene transcription and cytokine production were robustly attenuated by pharmacologically blocking TNFα/IL-17A-induced NF-κB activation with IKK-2 inhibitor IV. Conversely, inhibition of IL-22-induced JAK1 signalling with ABT-317 strongly attenuated morphological features of the disease but had no effect on NFκB-dependent cytokine production, suggesting distinct mechanisms of action by the cytokines driving psoriasis. These data support the use of cytokine-induced RHE models for identifying and targeting keratinocyte signalling pathways important for disease progression and may provide translational insights into novel keratinocyte mechanisms for novel psoriasis therapies.


Assuntos
Interleucina-17 , Psoríase , Animais , Humanos , Interleucina-17/metabolismo , Queratinócitos/metabolismo , NF-kappa B/metabolismo , Psoríase/metabolismo , Pele/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
4.
MAbs ; 13(1): 1964420, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34460338

RESUMO

Four antibodies that inhibit interleukin (IL)-23 are approved for the treatment of moderate-to-severe plaque psoriasis. Here, we present non-clinical data comparing ustekinumab, guselkumab, tildrakizumab and risankizumab with regard to thermostability, IL-23 binding affinity, inhibitory-binding mode, in vitro potency and in vivo efficacy. Risankizumab and guselkumab exhibited 5-fold higher affinity for IL-23 and showed more potent inhibition of IL-23 signaling than ustekinumab and tildrakizumab. Risankizumab and guselkumab completely blocked the binding of IL-23 to IL-23Rα as expected, whereas tildrakizumab did not. In vitro, risankizumab and guselkumab blocked the terminal differentiation of TH17 cells in a similar manner, while tildrakizumab had minimal impact on TH17 differentiation. In a human IL-23-induced ear-swelling mouse model, risankizumab and guselkumab were more effective than ustekinumab and tildrakizumab at reducing IL-17, IL-22, and keratinocyte gene expression. Our results indicate that the four clinically approved antibodies targeting IL-23 differ in affinity and binding epitope. These attributes contribute to differences in in vitro potency, receptor interaction inhibition mode and in vivo efficacy in preclinical studies as described in this report, and similarly may affect the clinical performance of these drugs.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais/farmacologia , Interleucina-23/antagonistas & inibidores , Psoríase/tratamento farmacológico , Ustekinumab/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/metabolismo , Afinidade de Anticorpos , Sítios de Ligação de Anticorpos , Células Cultivadas , Modelos Animais de Doenças , Estabilidade de Medicamentos , Epitopos , Feminino , Temperatura Alta , Humanos , Interleucina-23/imunologia , Interleucina-23/metabolismo , Camundongos Endogâmicos C57BL , Desnaturação Proteica , Estabilidade Proteica , Psoríase/imunologia , Psoríase/metabolismo , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th17/metabolismo , Ustekinumab/imunologia , Ustekinumab/metabolismo
5.
Life Sci ; 271: 119195, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33581125

RESUMO

AIMS: Ulcerative colitis and Crohn's disease, collectively known as inflammatory bowel disease (IBD), are chronic inflammatory disorders of the intestine for which key elements in disease initiation and perpetuation are defects in epithelial barrier integrity. Achieving mucosal healing is essential to ameliorate disease outcome and so new therapies leading to epithelial homeostasis and repair are under investigation. This study was designed to determine the mechanisms by which IL-22 regulates intestinal epithelial cell function. MAIN METHODS: Human intestinal organoids and resections, as well as mice were used to evaluate the effect of IL-22 on stem cell expansion, proliferation and expression of mucus components. IL-22 effect on barrier function was assessed in polarized T-84 cell monolayers. Butyrate co-treatments and organoid co-cultures with immune cells were performed to monitor the impact of microbial-derived metabolites and inflammatory environments on IL-22 responses. KEY FINDINGS: IL-22 led to epithelial stem cell expansion, proliferation, barrier dysfunction and anti-microbial peptide production in human and mouse models evaluated. IL-22 also altered the mucus layer by inducing an increase in membrane mucus but a decrease in secreted mucus and goblet cell content. IL-22 had the same effect on anti-microbial peptides and membrane mucus in both healthy and IBD human samples. In contrast, this IL-22-associated epithelial phenotype was different when treatments were performed in presence of butyrate and organoids co-cultured with immune cells. SIGNIFICANCE: Our data indicate that IL-22 promotes epithelial regeneration, innate defense and membrane mucus production, strongly supporting the potential clinical utility of IL-22 as a mucosal healing therapy in IBD.


Assuntos
Células Epiteliais/fisiologia , Homeostase/fisiologia , Interleucinas/fisiologia , Interleucinas/uso terapêutico , Mucosa Intestinal/fisiologia , Animais , Linhagem Celular , Técnicas de Cocultura , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/patologia , Células Epiteliais/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Interleucinas/farmacologia , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Organoides/efeitos dos fármacos , Organoides/fisiologia , Interleucina 22
6.
Exp Dermatol ; 30(6): 820-830, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33377546

RESUMO

Since first recognized in 1839, the pathogenesis of acne inversa (AI) has undergone repeated revisions. Although there is agreement that AI involves occlusion of hair follicles with subsequent inflammation and the formation of tracts, the histologic progression of this disease still requires refinement. The objective of this study was to examine the histologic progression of AI based on the examination of a large cohort of punch biopsies and excisional samples that were examined first by hematoxylin and eosin staining. The most informative of these samples were step-sectioned and stained by immunohistochemistry for epithelial and inflammatory markers. Based on this examination, the following observations were made: 1) AI arises from the epithelium of the infundibulum of terminal and vellus hairs; 2) These form cysts and epithelial tendrils that extend into soft tissue; 3) Immunohistochemical staining demonstrates the epithelium of AI is disordered with infundibular and isthmic differentiation and de novo expression of stem cell markers; 4) The inflammatory response in AI is heterogeneous and largely due to cyst rupture. The conclusions of this investigation were that AI is an epithelial-driven disease caused by infiltrative, cyst forming tendrils and most of the inflammation is due to cyst rupture and release of cornified debris and bacteria. Cyst rupture often occurs below the depths of punch biopsy samples indicating their use for analysis may give an incomplete picture of the disease. Finally, our data suggest that unless therapies inhibit tendril development, it is unlikely they will cause prolonged treatment-induced remission in AI.


Assuntos
Acne Vulgar/patologia , Progressão da Doença , Hidradenite Supurativa/patologia , Folículo Piloso/patologia , Humanos , Inflamação/patologia
7.
JCI Insight ; 5(19)2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-32841223

RESUMO

Hidradenitis suppurativa (HS) is a highly prevalent, morbid inflammatory skin disease with limited treatment options. The major cell types and inflammatory pathways in skin of patients with HS are poorly understood, and which patients will respond to TNF-α blockade is currently unknown. We discovered that clinically and histologically healthy appearing skin (i.e., nonlesional skin) is dysfunctional in patients with HS with a relative loss of immune regulatory pathways. HS skin lesions were characterized by quantitative and qualitative dysfunction of type 2 conventional dendritic cells, relatively reduced regulatory T cells, an influx of memory B cells, and a plasma cell/plasmablast infiltrate predominantly in end-stage fibrotic skin. At the molecular level, there was a relative bias toward the IL-1 pathway and type 1 T cell responses when compared with both healthy skin and psoriatic patient skin. Anti-TNF-α therapy markedly attenuated B cell activation with minimal effect on other inflammatory pathways. Finally, we identified an immune activation signature in skin before anti-TNF-α treatment that correlated with subsequent lack of response to this modality. Our results reveal the fundamental immunopathogenesis of HS and provide a molecular foundation for future studies focused on stratifying patients based on likelihood of clinical response to TNF-α blockade.


Assuntos
Biomarcadores/análise , Regulação da Expressão Gênica , Hidradenite Supurativa/tratamento farmacológico , Linfócitos T Reguladores/imunologia , Transcriptoma/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Estudos de Casos e Controles , Redes Reguladoras de Genes , Hidradenite Supurativa/imunologia , Hidradenite Supurativa/patologia , Humanos , Transdução de Sinais , Análise de Célula Única/métodos , Linfócitos T Reguladores/efeitos dos fármacos
8.
PLoS One ; 15(3): e0228221, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32155151

RESUMO

Inflammatory bowel diseases (IBD) are complex, multifactorial disorders characterized by chronic relapsing intestinal inflammation. IBD is diagnosed around 1 in 1000 individuals in Western countries with globally increasing incident rates. Association studies have identified hundreds of genes that are linked to IBD and potentially regulate its pathology. The further dissection of the genetic network underlining IBD pathogenesis and pathophysiology is hindered by the limited capacity to functionally characterize each genetic association, including generating knockout animal models for every associated gene. Cutting-edge CRISPR/Cas9-based technology may transform the field of IBD research by efficiently and effectively introducing genetic alterations. In the present study, we used CRISPR/Cas9-based technologies to genetically modify hematopoietic stem cells. Through cell sorting and bone marrow transplantation, we established a system to knock out target gene expression by over 90% in the immune system of reconstituted animals. Using a CD40-mediated colitis model, we further validated our CRISPR/Cas9-based platform for investigating gene function in experimental IBD. In doing so, we developed a model system that delivers genetically modified mice in a manner much faster than conventional methodology, significantly reducing the time from target identification to in vivo target validation and expediting drug development.


Assuntos
Antígenos CD40/imunologia , Sistemas CRISPR-Cas/genética , Células-Tronco Hematopoéticas/metabolismo , Animais , Antígenos CD40/metabolismo , Colite/imunologia , Colite/terapia , Regulação da Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Camundongos
9.
Front Immunol ; 11: 547102, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33643277

RESUMO

Background & Aims: Diligent side-by-side comparisons of how different methodologies affect growth efficiency and quality of intestinal colonoids have not been performed leaving a gap in our current knowledge. Here, we summarize our efforts to optimize culture conditions for improved growth and functional differentiation of mouse and human colon organoids. Methods: Mouse and human colon organoids were grown in four different media. Media-dependent long-term growth was measured by quantifying surviving organoids via imaging and a cell viability readout over five passages. The impact of diverse media on differentiation was assessed by quantifying the number of epithelial cell types using markers for enterocytes, stem cells, Goblet cells, and enteroendocrine cells by qPCR and histology upon removal of growth factors. Results: In contrast to Wnt3a-conditioned media, media supplemented with recombinant Wnt3a alone did not support long-term survival of human or mouse colon organoids. Mechanistically, this observation can be attributed to the fact that recombinant Wnt3a did not support stem cell survival or proliferation as demonstrated by decreased LGR5 and Ki67 expression. When monitoring expression of markers for epithelial cell types, the highest level of organoid differentiation was observed after combined removal of Wnt3a, Noggin, and R-spondin from Wnta3a-conditioned media cultures. Conclusion: Our study defined Wnt3a-containing conditioned media as optimal for growth and survival of human and mouse organoids. Furthermore, we established that the combined removal of Wnt3a, Noggin, and R-spondin results in optimal differentiation. This study provides a step forward in optimizing conditions for intestinal organoid growth to improve standardization and reproducibility of this model platform.


Assuntos
Técnicas de Cultura de Células , Colo/citologia , Organoides/citologia , Técnicas de Cultura de Tecidos , Animais , Biomarcadores , Proteínas de Transporte/metabolismo , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/metabolismo , Humanos , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Necroptose , Transdução de Sinais , Células-Tronco/metabolismo , Proteína Wnt3/metabolismo
10.
J Dermatol ; 46(6): 482-497, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31062408

RESUMO

The interleukin (IL)-23/IL-17 axis plays a central role in the pathogenesis of psoriasis and is elevated in lesional psoriatic skin. Different murine models have been developed to mimic this pathophysiology each carrying specific merits and limitations. In an attempt to address some of these limitations, B10.RIII mice received a single hydrodynamic injection of IL-23 minicircles (MC) to induce hepatic transcription and the endogenous production of IL-23. Plasma and ear IL-23 levels were dose-dependently (0.3-3 µg) increased in MC injected mice and were sustained over the 14-day study duration. Beginning on day 7 post-injection, mice developed dose-related ear inflammation, histologically confirmed increases in epidermal and dermal area, as well as enhanced neutrophil and macrophage content. Flow cytometry demonstrated increased levels of granulocytes, T cells and monocytes/macrophages in the ear skin, with T cells identified as the main cellular source of IL-17A. Evaluation of mRNA and protein showed time-dependent, increased levels of the IL-23/IL-17 pathway and inflammatory/microbial cytokines/chemokines in the ear which differed kinetically from circulating levels. An anti-IL-23p40 antibody was assessed following both prophylactic administration and administration once the disease was established. Prophylactic dosing completely prevented the development of the ear phenotype across endpoints. Treatment administration showed a dose-related response, with a maximum inhibition of 64-94%, depending on endpoint. These data demonstrate that the IL-23 MC model is a useful approach to study IL-23/IL-17-driven skin inflammation and may facilitate preclinical assessment of novel therapies.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Interleucina-17/imunologia , Interleucina-23/imunologia , Psoríase/imunologia , Animais , DNA Circular/administração & dosagem , DNA Circular/genética , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Técnicas de Transferência de Genes , Humanos , Interleucina-17/metabolismo , Interleucina-23/antagonistas & inibidores , Interleucina-23/genética , Masculino , Camundongos , Psoríase/sangue , Psoríase/tratamento farmacológico , Psoríase/patologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Pele/imunologia , Pele/patologia , Resultado do Tratamento
11.
J Histochem Cytochem ; 67(3): 203-219, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30501365

RESUMO

Myelin is composed primarily of lipids and diseases affecting myelin are associated with alterations in its lipid composition. However, correlation of the spatial (in situ) distribution of lipids with the disease-associated compositional and morphological changes is not well defined. Herein we applied high resolution matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS), immunohistochemistry (IHC), and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) to evaluate brain lipid alterations in the dysmyelinating shiverer (Shi) mouse and cuprizone (Cz) mouse model of reversible demyelination. MALDI-IMS revealed a decrease in the spatial distribution of sulfatide (SHexCer) species, SHexCer (d42:2), and a phosphatidylcholine (PC) species, PC (36:1), in white matter regions like corpus callosum (CC) both in the Shi mouse and Cz mouse model. Changes in these lipid species were restored albeit not entirely upon spontaneous remyelination after demyelination in the Cz mouse model. Lipid distribution changes correlated with the local morphological changes as confirmed by IHC. LC-ESI-MS analyses of CC extracts confirmed the MALDI-IMS derived reductions in SHexCer and PC species. These findings highlight the role of SHexCer and PC in preserving the normal myelin architecture and our experimental approaches provide a morphological basis to define lipid abnormalities relevant to myelin diseases.


Assuntos
Ceramidas/metabolismo , Doenças Desmielinizantes/metabolismo , Bainha de Mielina/metabolismo , Fosfatidilcolinas/metabolismo , Sulfoglicoesfingolipídeos/metabolismo , Animais , Corpo Caloso/metabolismo , Corpo Caloso/ultraestrutura , Cuprizona/administração & dosagem , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Imuno-Histoquímica , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Transgênicos , Bainha de Mielina/ultraestrutura , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Substância Branca/metabolismo , Substância Branca/ultraestrutura
12.
Toxicol Pathol ; 45(7): 983-1003, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-29162012

RESUMO

Quantitative image analysis (IA) is a rapidly evolving area of digital pathology. Although not a new concept, the quantification of histological features on photomicrographs used to be cumbersome, resource-intensive, and limited to specialists and specialized laboratories. Recent technological advances like highly efficient automated whole slide digitizer (scanner) systems, innovative IA platforms, and the emergence of pathologist-friendly image annotation and analysis systems mean that quantification of features on histological digital images will become increasingly prominent in pathologists' daily professional lives. The added value of quantitative IA in pathology includes confirmation of equivocal findings noted by a pathologist, increasing the sensitivity of feature detection, quantification of signal intensity, and improving efficiency. There is no denying that quantitative IA is part of the future of pathology; however, there are also several potential pitfalls when trying to estimate volumetric features from limited 2-dimensional sections. This continuing education session on quantitative IA offered a broad overview of the field; a hands-on toxicologic pathologist experience with IA principles, tools, and workflows; a discussion on how to apply basic stereology principles in order to minimize bias in IA; and finally, a reflection on the future of IA in the toxicologic pathology field.


Assuntos
Processamento de Imagem Assistida por Computador , Patologia/métodos , Algoritmos , Animais , Estudos de Avaliação como Assunto , Humanos , Aprendizado de Máquina , Ratos
14.
PLoS One ; 11(10): e0164423, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27760209

RESUMO

Mouse models lupus nephritis (LN) have provided important insights into disease pathogenesis, although none have been able to recapitulate all features of the human disease. Using comprehensive longitudinal analyses, we characterized a novel accelerated mouse model of lupus using pristane treatment in SNF1 (SWR X NZB F1) lupus prone mice (pristane-SNF1 mice). Pristane treatment in SNF1 mice accelerated the onset and progression of proteinuria, autoantibody production, immune complex deposition and development of renal lesions. At week 14, the pristane-SNF1 model recapitulated kidney disease parameters and molecular signatures seen in spontaneous disease in 36 week-old SNF1 mice and in a traditional IFNα-accelerated NZB X NZW F1 (BWF1) model. Blood transcriptome analysis revealed interferon, plasma cell, neutrophil, T-cell and protein synthesis signatures in the pristane-SNF1 model, all known to be present in the human disease. The pristane-SNF1 model appears to be particularly useful for preclinical research, robustly exhibiting many characteristics reminiscent of human disease. These include i) a stronger upregulation of the cytosolic nucleic acid sensing pathway, which is thought to be key component of the pathogenesis of the human disease, and ii) more prominent kidney interstitial inflammation and fibrosis, which have been both associated with poor prognosis in human LN. To our knowledge, this is the only accelerated model of LN that exhibits a robust tubulointerstitial inflammatory and fibrosis response. Taken together our data show that the pristane-SNF1 model is a novel accelerated model of LN with key features similar to human disease.


Assuntos
Túbulos Renais/efeitos dos fármacos , Túbulos Renais/patologia , Nefrite Lúpica/patologia , Terpenos/farmacologia , Animais , Autoanticorpos/biossíntese , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Fibrose , Glomerulonefrite/induzido quimicamente , Glomerulonefrite/complicações , Humanos , Hipergamaglobulinemia/induzido quimicamente , Hipergamaglobulinemia/complicações , Inflamação/induzido quimicamente , Inflamação/complicações , Nefrite Lúpica/complicações , Nefrite Lúpica/imunologia , Nefrite Lúpica/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
15.
Nature ; 537(7618): 50-6, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27582220

RESUMO

Alzheimer's disease (AD) is characterized by deposition of amyloid-ß (Aß) plaques and neurofibrillary tangles in the brain, accompanied by synaptic dysfunction and neurodegeneration. Antibody-based immunotherapy against Aß to trigger its clearance or mitigate its neurotoxicity has so far been unsuccessful. Here we report the generation of aducanumab, a human monoclonal antibody that selectively targets aggregated Aß. In a transgenic mouse model of AD, aducanumab is shown to enter the brain, bind parenchymal Aß, and reduce soluble and insoluble Aß in a dose-dependent manner. In patients with prodromal or mild AD, one year of monthly intravenous infusions of aducanumab reduces brain Aß in a dose- and time-dependent manner. This is accompanied by a slowing of clinical decline measured by Clinical Dementia Rating-Sum of Boxes and Mini Mental State Examination scores. The main safety and tolerability findings are amyloid-related imaging abnormalities. These results justify further development of aducanumab for the treatment of AD. Should the slowing of clinical decline be confirmed in ongoing phase 3 clinical trials, it would provide compelling support for the amyloid hypothesis.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/psicologia , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Anticorpos Monoclonais Humanizados/uso terapêutico , Placa Amiloide/tratamento farmacológico , Placa Amiloide/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Amiloide/efeitos dos fármacos , Amiloide/metabolismo , Peptídeos beta-Amiloides/química , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/farmacocinética , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Ensaios Clínicos Fase III como Assunto , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Modelos Biológicos , Placa Amiloide/patologia , Agregação Patológica de Proteínas/tratamento farmacológico , Solubilidade
16.
PLoS One ; 11(5): e0155897, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27191595

RESUMO

Over half of adults are seropositive for JC polyomavirus (JCV), but rare individuals develop progressive multifocal leukoencephalopathy (PML), a demyelinating JCV infection of the central nervous system. Previously, PML was primarily seen in immunosuppressed patients with AIDS or certain cancers, but it has recently emerged as a drug safety issue through its association with diverse immunomodulatory therapies. To better understand the relationship between the JCV life cycle and PML pathology, we studied autopsy brain tissue from a 70-year-old psoriasis patient on the integrin alpha-L inhibitor efalizumab following a ~2 month clinical course of PML. Sequence analysis of lesional brain tissue identified PML-associated viral mutations in regulatory (non-coding control region) DNA, capsid protein VP1, and the regulatory agnoprotein, as well as 9 novel mutations in capsid protein VP2, indicating rampant viral evolution. Nine samples, including three gross PML lesions and normal-appearing adjacent tissues, were characterized by histopathology and subject to quantitative genomic, proteomic, and molecular localization analyses. We observed a striking correlation between the spatial extent of demyelination, axonal destruction, and dispersion of JCV along white matter myelin sheath. Our observations in this case, as well as in a case of PML-like disease in an immunocompromised rhesus macaque, suggest that long-range spread of polyomavirus and axonal destruction in PML might involve extracellular association between virus and the white matter myelin sheath.


Assuntos
Encéfalo/virologia , Vírus JC/patogenicidade , Leucoencefalopatia Multifocal Progressiva/virologia , Bainha de Mielina/metabolismo , Replicação Viral , Idoso , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Feminino , Humanos , Vírus JC/genética , Vírus JC/fisiologia , Macaca mulatta , Masculino , Mutação , Bainha de Mielina/patologia , Bainha de Mielina/virologia , Proteínas Virais de Fusão/genética , Proteínas Virais Reguladoras e Acessórias/genética , Virulência/genética
17.
Immunology ; 147(4): 399-413, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26694902

RESUMO

Retinoic acid receptor-related orphan nuclear receptor γ (RORγ) orchestrates a pro-inflammatory gene expression programme in multiple lymphocyte lineages including T helper type 17 (Th17) cells, γδ T cells, innate lymphoid cells and lymphoid tissue inducer cells. There is compelling evidence that RORγ-expressing cells are relevant targets for therapeutic intervention in the treatment of autoimmune and inflammatory diseases. Unlike Th17 cells, where RORγ expression is induced under specific pro-inflammatory conditions, γδ T cells and other innate-like immune cells express RORγ in the steady state. Small molecule mediated disruption of RORγ function in cells with pre-existing RORγ transcriptional complexes represents a significant and challenging pharmacological hurdle. We present data demonstrating that a novel, selective and potent small molecule RORγ inhibitor can block the RORγ-dependent gene expression programme in both Th17 cells and RORγ-expressing γδ T cells as well as a disease-relevant subset of human RORγ-expressing memory T cells. Importantly, systemic administration of this inhibitor in vivo limits pathology in an innate lymphocyte-driven mouse model of psoriasis.


Assuntos
Doenças Autoimunes/etiologia , Doenças Autoimunes/metabolismo , Benzamidas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Piridinas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Dermatite/tratamento farmacológico , Dermatite/imunologia , Dermatite/metabolismo , Dermatite/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Memória Imunológica/efeitos dos fármacos , Interleucina-17/metabolismo , Camundongos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Células Th17/citologia , Células Th17/imunologia , Células Th17/metabolismo
18.
Neuropharmacology ; 103: 57-68, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26690893

RESUMO

Reducing the production of larger aggregation-prone amyloid ß-peptides (Aß) remains an untested therapeutic approach for reducing the appearance and growth of Aß plaques in the brain, which are a hallmark pathological feature of Alzheimer's disease. γ-Secretase modulators (GSMs) are therapeutics that impact γ-secretase-dependent cleavage of amyloid precursor protein to promote the production of shorter Aß peptides that are less prone to aggregation and plaque deposition. This is accomplished without inhibiting overall γ-secretase function and cleavage of other substrates, which is believed to be a source of deleterious side effects. Here, we report the pharmacokinetic and pharmacodynamic properties of BIIB042, a novel bioavailable and brain-penetrant GSM. In cell-based assays, BIIB042 reduced the levels of Aß42, increased the levels of Aß38 and had little effect on the levels of Aß40, the most abundant Aß species. Similar pharmacodynamic properties were confirmed in the central nervous system and in plasma of mice and rats, and also in plasma of cynomolgus monkeys after a single oral dose of BIIB042. BIIB042 reduced Aß42 levels and Aß plaque burden in Tg2576 mice, which overexpress human amyloid precursor protein and serve as a model system for Alzheimer's disease. BIIB042 did not inhibit cleavage of other γ-secretase substrates in cell-based and in vivo signaling and cleavage assays. The pharmacodynamic effects of lowering Aß42 in the central nervous system coupled with demonstrated efficacy in reducing plaque pathology suggests modulation of γ-secretase, with molecules like BIIB042, is a compelling therapeutic approach for the treatment of Alzheimer's disease.


Assuntos
Aldeídos/farmacocinética , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Aldeídos/administração & dosagem , Peptídeos beta-Amiloides/sangue , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Macaca fascicularis , Masculino , Camundongos , Placa Amiloide/metabolismo , Isoformas de Proteínas/sangue , Ratos , Ratos Endogâmicos F344
19.
Toxicol Pathol ; 39(6): 988-1002, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21971934

RESUMO

A morphology-based assay such as immunohistochemistry (IHC) should be a highly effective means to define the expression of a target molecule of interest, especially if the target is a protein. However, over the past decade, IHC as a platform for biomarkers has been challenged by more quantitative molecular assays with reference standards but that lack morphologic context. For IHC to be considered a "top-tier" biomarker assay, it must provide truly quantitative data on par with non-morphologic assays, which means it needs to be run with reference standards. However, creating such standards for IHC will require optimizing all aspects of tissue collection, fixation, section thickness, morphologic criteria for assessment, staining processes, digitization of images, and image analysis. This will also require anatomic pathology to evolve from a discipline that is descriptive to one that is quantitative. A major step in this transformation will be replacing traditional ocular microscopes with computer monitors and whole slide images, for without digitization, there can be no accurate quantitation; without quantitation, there can be no standardization; and without standardization, the value of morphology-based IHC assays will not be realized.


Assuntos
Biomarcadores/análise , Imuno-Histoquímica/métodos , Animais , Expressão Gênica/fisiologia , Técnicas de Preparação Histocitológica , Humanos , Processamento de Imagem Assistida por Computador/métodos , Imuno-Histoquímica/normas , Microscopia , Padrões de Referência , Reprodutibilidade dos Testes
20.
Aust N Z J Public Health ; 32(4): 354-60, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18782399

RESUMO

OBJECTIVES: To assess the risk to the Australian blood supply posed by emerging or re-emerging viral infections. METHOD: A review was undertaken of the English-speaking literature on the potential for emerging viral threats to human health in Australia, the future implications of virus ecology, climate change and population movement and the implications for blood transfusion. RESULTS: Published data confirm that Australia's blood supply is among the safest in the world for currently screened viral pathogens as a result of rigorous surveillance, donor selection and state-of-the-art processing and laboratory testing. However, Australia has a number of other viral pathogens with the potential to threaten the safety of the blood supply such as the Ross River, Barmah Forrest, Kunjin, Japanese Encephalitis, Murray Valley Encephalitis and dengue viruses. Of these, dengue is currently of most concern to blood safety because; it can cause fatalities, there are regular seasonal outbreaks in Northern Australia and, in contrast to other viruses mentioned above an overseas case of transfusion transmission has already been documented. Notably, despite the lack of a suitable dengue screening test the ARCBS already implements supplementary measures to protect the blood supply during outbreaks. CONCLUSION: Current interventions have proven extremely effective in minimising transfusion transmission in Australia of recognised viral pathogens. The threat posed by emerging viral pathogens to the safety of blood transfusion emphasises the need for global collaboration and consideration of further intervention strategies on a country by country basis including options such as nucleic acid testing and pathogen reduction technologies.


Assuntos
Bancos de Sangue/tendências , Transfusão de Sangue , Segurança , Viroses/transmissão , Infecções por Arbovirus/sangue , Infecções por Arbovirus/transmissão , Austrália , Bancos de Sangue/normas , Humanos , Medição de Risco , Fatores de Risco , Viroses/sangue
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