RESUMO
Conventional histopathology involves expensive and labor-intensive processes that often consume tissue samples, rendering them unavailable for other analyses. We present a novel end-to-end workflow for pathology powered by hyperspectral microscopy and deep learning. First, we developed a custom hyperspectral microscope to nondestructively image the autofluorescence of unstained tissue sections. We then trained a deep learning model to use autofluorescence to generate virtual histologic stains, which avoids the cost and variability of chemical staining procedures and conserves tissue samples. We showed that the virtual images reproduce the histologic features present in the real-stained images using a randomized nonalcoholic steatohepatitis (NASH) scoring comparison study, where both real and virtual stains are scored by pathologists (D.T., A.D.B., R.K.P.). The test showed moderate-to-good concordance between pathologists' scoring on corresponding real and virtual stains. Finally, we developed deep learning-based models for automated NASH Clinical Research Network score prediction. We showed that the end-to-end automated pathology platform is comparable with an independent panel of pathologists for NASH Clinical Research Network scoring when evaluated against the expert pathologist consensus scores. This study provides proof of concept for this virtual staining strategy, which could improve cost, efficiency, and reliability in pathology and enable novel approaches to spatial biology research.
Assuntos
Aprendizado Profundo , Hepatopatia Gordurosa não Alcoólica , Humanos , Microscopia , Reprodutibilidade dos Testes , PatologistasRESUMO
BACKGROUND & AIMS: The development of accurate non-invasive tests to detect and measure the extent of fibrosis and disease activity in patients with non-alcoholic steatohepatitis (NASH) - the progressive phenotype of non-alcoholic fatty liver disease (NAFLD) - is of great clinical importance. Herein, we aimed to validate the performance of PRO-C3 and ADAPT for the detection of moderate/severe fibrosis within the CENTAUR screening population. METHODS: PRO-C3 was assessed in plasma from the screening population of the phase IIb CENTAUR study (NCT02217475) in adults with NASH and liver fibrosis. The relation between PRO-C3 and histologic features of NASH was evaluated, as well as the demographics of patients with high and low levels of PRO-C3. The diagnostic ability of PRO-C3, as a standalone marker or incorporated into ADAPT, to identify patients with F≥2 and NASH was estimated using receiver-operating characteristic analysis and logistic regression models. RESULTS: A total of 517 individuals with matched biopsy and PRO-C3 measurements were included. Patients with PRO-C3 levels ≥20.2 ng/ml showed increased levels of insulin, HOMA-IR, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, and platelet count compared to patients with low PRO-C3 (p <0.05). PRO-C3 increased stepwise with increasing liver fibrosis, lobular inflammation, hepatocyte ballooning, steatosis, and NAFLD activity score (p <0.05), and could distinguish between NAFL and NASH (p <0.0001). PRO-C3 was independently associated with fibrosis and NASH when adjusted for clinical confounders. ADAPT outperformed Fibrosis-4, AST-to-platelet ratio index, and AST/ALT ratio as a predictor of advanced fibrosis and NASH (p <0.001). CONCLUSION: PRO-C3 was associated with NAFLD activity score and fibrosis. ADAPT outperformed other non-invasive scores for detecting NASH. These data support the use of PRO-C3 and ADAPT as diagnostic tools to identify patients with NASH eligible for inclusion in clinical trials. CLINICAL TRIAL NUMBER: NCT02217475 LAY SUMMARY: PRO-C3 is a serological biomarker associated with liver disease activity and fibrosis. Its performance for the detection of disease activity and fibrosis is improved when it is incorporated into the ADAPT score. Herein, we showed that ADAPT was better at selecting patients with non-alcoholic steatohepatitis for inclusion in clinical trials than other non-invasive scores.
Assuntos
Biomarcadores/análise , Cirrose Hepática/diagnóstico , Área Sob a Curva , Biomarcadores/sangue , Biópsia/métodos , Biópsia/estatística & dados numéricos , Complemento C3/análise , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/estatística & dados numéricos , Feminino , Humanos , Fígado/patologia , Cirrose Hepática/sangue , Cirrose Hepática/fisiopatologia , Modelos Logísticos , Masculino , Programas de Rastreamento/métodos , Programas de Rastreamento/estatística & dados numéricos , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Curva ROC , Estudos Retrospectivos , Índice de Gravidade de DoençaRESUMO
Primary sclerosing cholangitis (PSC) is a chronic cholestatic disease with no approved treatments. C-C chemokine receptor types 2 and 5 (CCR2/CCR5) play an important role in inflammation and fibrosis and are potential therapeutic targets for PSC. We evaluated the efficacy and safety of cenicriviroc (CVC), a dual antagonist of CCR2 and CCR5, for the treatment of PSC. This was a single-arm, open-label, exploratory study of CVC in adults with a clinical diagnosis of PSC, serum alkaline phosphatase (ALP) ≥1.5 times the upper limit of normal (ULN), with or without inflammatory bowel disease, across eight sites in the United States and Canada. The primary endpoint was percent change in ALP over 24 weeks; key secondary efficacy endpoints were proportion of participants who achieved ALP normalization and overall response (decrease to <1.5 times the ULN or 50% decrease). Of the 24 participants, 20 completed the study. The mean age was 43 years, 50% were female, and the mean body mass index was 25 kg/m2. From a median ALP baseline of 369 U/L (range: 173, 1,377 U/L), a median absolute reduction of 49.5 U/L (range: -460, 416 U/L) was achieved at week 24, corresponding to a median reduction of 18.0% (range: -46%, 89%). No participant achieved ALP normalization or a 50% decrease; 2 participants (10%) achieved a reduction in ALP to < 1.5 times the ULN, and 4 had ≥25% increase. Twenty participants (83.3%) reported at least one adverse event; most were mild to moderate in severity. The most frequent events were rash, fatigue, and dizziness. Conclusion: After 24 weeks of CVC treatment, adults with PSC achieved a modest reduction (median 18%) in the surrogate endpoint of ALP. CVC was well tolerated, and no new safety signals were observed. ClinicalTrials.gov identifier: NCT02653625.
Assuntos
Colangite Esclerosante/tratamento farmacológico , Imidazóis/uso terapêutico , Sulfóxidos/uso terapêutico , Adolescente , Adulto , Idoso , Fosfatase Alcalina/sangue , Biomarcadores/sangue , Canadá , Colangite Esclerosante/sangue , Feminino , Humanos , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Estados Unidos , Adulto JovemRESUMO
BACKGROUND: Chronic alcohol consumption is associated with neuroinflammation, neuronal damage, and behavioral alterations including addiction. Alcohol-induced neuroinflammation is characterized by increased expression of proinflammatory cytokines (including TNFα, IL-1ß, and CCL2) and microglial activation. We hypothesized chronic alcohol consumption results in peripheral immune cell infiltration to the CNS. Since chemotaxis through the CCL2-CCR2 signaling axis is critical for macrophage recruitment peripherally and centrally, we further hypothesized that blockade of CCL2 signaling using the dual CCR2/5 inhibitor cenicriviroc (CVC) would prevent alcohol-induced CNS infiltration of peripheral macrophages and alter the neuroinflammatory state in the brain after chronic alcohol consumption. METHODS: C57BL/6J female mice were fed an isocaloric or 5% (v/v) ethanol Lieber DeCarli diet for 6 weeks. Some mice received daily injections of CVC. Microglia and infiltrating macrophages were characterized and quantified by flow cytometry and visualized using CX3CR1eGFP/+ CCR2RFP/+ reporter mice. The effect of ethanol and CVC treatment on the expression of inflammatory genes was evaluated in various regions of the brain, using a Nanostring nCounter inflammation panel. Microglia activation was analyzed by immunofluorescence. CVC-treated and untreated mice were presented with the two-bottle choice test. RESULTS: Chronic alcohol consumption induced microglia activation and peripheral macrophage infiltration in the CNS, particularly in the hippocampus. Treatment with CVC abrogated ethanol-induced recruitment of peripheral macrophages and partially reversed microglia activation. Furthermore, the expression of proinflammatory markers was upregulated by chronic alcohol consumption in various regions of the brain, including the cortex, hippocampus, and cerebellum. Inhibition of CCR2/5 decreased alcohol-mediated expression of inflammatory markers. Finally, microglia function was impaired by chronic alcohol consumption and restored by CVC treatment. CVC treatment did not change the ethanol consumption or preference of mice in the two-bottle choice test. CONCLUSIONS: Together, our data establish that chronic alcohol consumption promotes the recruitment of peripheral macrophages into the CNS and microglia alterations through the CCR2/5 axis. Therefore, further exploration of the CCR2/5 axis as a modulator of neuroinflammation may offer a potential therapeutic approach for the treatment of alcohol-associated neuroinflammation.
Assuntos
Encéfalo/metabolismo , Etanol/toxicidade , Macrófagos/metabolismo , Microglia/metabolismo , Receptores CCR2/metabolismo , Receptores CCR5/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Antagonistas dos Receptores CCR5/farmacologia , Etanol/administração & dosagem , Feminino , Imidazóis/farmacologia , Mediadores da Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Receptores CCR2/antagonistas & inibidores , Sulfóxidos/farmacologiaRESUMO
BACKGROUND AND AIMS: Cenicriviroc (CVC) is a C-C chemokine receptors type 2 and 5 dual antagonist under evaluation for treating liver fibrosis in adults with nonalcoholic steatohepatitis (NASH). Year 1 primary analysis of the 2-year CENTAUR study showed that CVC had an antifibrotic effect without impacting steatohepatitis. Herein, we report the final data from year 2 exploratory analyses. APPROACH AND RESULTS: This was a randomized, controlled study of adults with NASH, nonalcoholic fatty liver disease activity score ≥4, and NASH Clinical Research Network stage 1-3 fibrosis. Participants in arms A and C received CVC 150 mg or placebo, respectively, for 2 years; arm B received placebo in year 1 and switched to CVC in year 2. Liver biopsy was performed at baseline, year 1, and year 2. Of 289 randomized participants, 242 entered year 2. At year 2, 24% of patients who switched to CVC and 17% who remained on placebo achieved ≥1-stage fibrosis improvement and no worsening of NASH (P = 0.37). Twice the proportion on CVC who achieved fibrosis response at year 1 maintained benefit at year 2 (60% arm A versus 30% arm C), including 86% on CVC who had stage 3 fibrosis at baseline. Over 2 years, a similar proportion on CVC or placebo achieved ≥1-stage fibrosis improvement and no worsening of NASH (15% arm A versus 17% arm C). In patients with fibrosis responses, we observed consistent reductions in levels of N-terminal type 3 collagen propeptide and enhanced liver fibrosis scores, while increases in aspartate aminotransferase-to-platelet ratio index and Fibrosis-4 scores were consistently observed in nonresponders. Safety profile was comparable across groups. CONCLUSIONS: CVC was well tolerated, and year 2 data corroborate antifibrotic findings from year 1. The majority on CVC who achieved fibrosis response at year 1 maintained it at year 2, with greater effect in advanced fibrosis. ClinicalTrials.gov number, NCT02217475 (CENTAUR).
Assuntos
Aspartato Aminotransferases/sangue , Imidazóis , Cirrose Hepática , Fígado/patologia , Hepatopatia Gordurosa não Alcoólica , Contagem de Plaquetas/métodos , Sulfóxidos , Biópsia/métodos , Antagonistas dos Receptores CCR5/administração & dosagem , Antagonistas dos Receptores CCR5/efeitos adversos , Progressão da Doença , Monitoramento de Medicamentos/métodos , Feminino , Humanos , Imidazóis/administração & dosagem , Imidazóis/efeitos adversos , Cirrose Hepática/diagnóstico , Cirrose Hepática/etiologia , Cirrose Hepática/prevenção & controle , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/diagnóstico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Gravidade do Paciente , Receptores CCR2/metabolismo , Sulfóxidos/administração & dosagem , Sulfóxidos/efeitos adversos , Resultado do TratamentoRESUMO
INTRODUCTION: Nonalcoholic steatohepatitis (NASH) is a sub-classification of nonalcoholic fatty liver disease (NAFLD) characterized by increased risk of progressive liver fibrosis. Cenicriviroc (CVC) is a novel, orally administered, potent chemokine 2 and 5 receptor antagonist currently in development for the treatment of liver fibrosis in adults with NASH. METHODS AND ANALYSIS: Efficacy and safety of CVC will be comprehensively evaluated in a global, Phase 3, multicenter, randomized, double-blind, placebo-controlled study (AURORA, NCT03028740) of subjects with NASH and Stage F2 or F3 fibrosis. Approximately 2000 adults (Part 1, 1200 subjects; Part 2, 800 additional subjects) aged 18-75 years with histological evidence of NASH with Stage F2 or F3 fibrosis (NASH Clinical Research Network classification system) will be randomized 2:1 to CVC 150 mg or placebo orally once daily. Primary efficacy endpoints will include the proportion of subjects with ≥1-stage improvement in liver fibrosis and no worsening of steatohepatitis at Month 12 relative to screening (Part 1), and time to first occurrence of any adjudicated event: death; histopathologic progression to cirrhosis; liver transplant; Model of End-Stage Liver Disease score ≥ 15; ascites; hospitalization due to liver decompensation (Part 2). Patient-reported outcomes will assess changes in health outcomes from baseline (Chronic Liver Disease Questionnaire - NAFLD; Work Productivity and Activity Impairment in NASH; 36-Item Short Form Health Survey version 2). Adverse events will be assessed throughout the study. As there are currently no approved treatments indicated for NASH, the AURORA CVC Phase 3 study addresses an unmet medical need.
Assuntos
Imidazóis/uso terapêutico , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/etiologia , Hepatopatia Gordurosa não Alcoólica/complicações , Receptores CCR/antagonistas & inibidores , Sulfóxidos/uso terapêutico , Adolescente , Adulto , Idoso , Progressão da Doença , Método Duplo-Cego , Feminino , Humanos , Cirrose Hepática/patologia , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/patologia , Projetos de Pesquisa , Índice de Gravidade de Doença , Adulto JovemRESUMO
BACKGROUND: Nonalcoholic steatohepatitis (NASH) is a multifactorial disease involving different contributing mechanisms, with no approved therapies so far. Tropifexor (TXR), a farnesoid X receptor agonist, and cenicriviroc (CVC), a chemokine receptor types 2/5 antagonist, target the steatotic, inflammatory, and/or fibrotic pathways involved in NASH. DESIGN: TANDEM (CLJC242A2201J; NCT03517540) is a 48-week, phase 2b, randomized, double-blind, multicenter study in 200 adult patients with biopsy-proven NASH and liver fibrosis. Patients will be randomized in a 1:1:1:1 ratio to receive either TXR 140⯵g once daily (qd), CVC 150â¯mg qd, TXR 140⯵gâ¯+â¯CVC 150â¯mg qd, or TXR 90⯵gâ¯+â¯CVC 150â¯mg qd. The study comprises a 48-week treatment period and 4â¯weeks of follow-up. The key inclusion criterion is presence of NASH with fibrosis stage F2/F3 as seen on screening liver biopsy or on historical liver biopsy performed within 6â¯months prior to screening. OBJECTIVES: The primary objective is evaluation of the safety and tolerability of combination therapy compared with the monotherapies over 48â¯weeks. The secondary objective is to evaluate efficacy as assessed by ≥1-point improvement in liver fibrosis versus baseline or resolution of steatohepatitis after 48â¯weeks. SUMMARY: TANDEM will evaluate the combination of TXR and CVC with respect to safety and efficacy outcomes related to improvement in fibrosis or resolution of steatohepatitis. Given the effects of TXR and CVC in multiple pathophysiological pathways associated with NASH, combination therapy is likely to show additional benefits compared with monotherapy.
Assuntos
Benzotiazóis/uso terapêutico , Antagonistas dos Receptores CCR5/uso terapêutico , Imidazóis/uso terapêutico , Isoxazóis/uso terapêutico , Cirrose Hepática/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Receptores Citoplasmáticos e Nucleares/agonistas , Sulfóxidos/uso terapêutico , Ensaios Clínicos Fase II como Assunto , Método Duplo-Cego , Quimioterapia Combinada , Humanos , Cirrose Hepática/patologia , Estudos Multicêntricos como Assunto , Hepatopatia Gordurosa não Alcoólica/patologia , Ensaios Clínicos Controlados Aleatórios como Assunto , Receptores CCR2/antagonistas & inibidores , Resultado do TratamentoRESUMO
[This corrects the article DOI: 10.3389/fimmu.2018.02558.].
RESUMO
Metacyclic Leishmania promastigotes are transmitted by sand flies that inject parasites and saliva into the host's skin. Previous studies have demonstrated that DNA plasmids encoding Lutzomyia longipalpis salivary proteins LJM17 and LJL143, when used to immunize dogs, resulted in a systemic and local Th1 cell-mediated immunity that interfered in parasite survival in vitro. Here we evaluated the ability of these same salivary antigens to induce anti-Leishmania immunity and to confer protection by immunizing dogs using a novel vaccination strategy more suitable for use in the field. The strategy consisted of a single dose of plasmid followed by two doses of recombinant Canarypoxvirus (rCanarypoxvirus) expressing L. longipalpis salivary proteins (LJM17 or LJL143). Thirty days after the final immunization, dogs were intradermally challenged with 107Leishmania infantum promastigotes in the presence of L. longipalpis saliva. We followed the experimentally infected dogs for 10 months to characterize clinical, parasitological, and immunological parameters. Upon vaccination, all immunized dogs presented strong and specific humoral responses with increased serum concentrations of IFN-γ, TNF, IL-7, and IL-15. The serum of dogs immunized with LJM17 also exhibited high levels of IL-2, IL-6, and IL-18. L. infantum infection was established in all experimental groups as evidenced by the presence of anti-Leishmania IgG, and by parasite detection in the spleen and skin. Dogs immunized with LJM17-based vaccines presented higher circulating levels of IFN-γ, IL-2, IL-6, IL-7, IL-15, IL-18, TNF, CXCL10, and GM-CSF post-infection when compared with controls. Results demonstrated that relevant Leishmania-specific immune responses were induced following vaccination of dogs with L. longipalpis salivary antigen LJM17 administered in a single priming dose of plasmid DNA, followed by two booster doses of recombinant Canarypox vector. Importantly, a significant increase in pro-inflammatory cytokines and chemokines known to be relevant for protection against leishmaniasis was evidenced after challenging LJM17-vaccinated dogs as compared to controls. Although similar results were observed following immunization with LJL143, the pro-inflammatory response observed after immunization was attenuated following infection. Collectively, these data suggest that the LJM17-based vaccine induced an immune profile consistent with the expected protective immunity against canine leishmaniosis. These results clearly support the need for further evaluation of the LJM17 antigen, using a heterologous prime-boost vaccination strategy against canine visceral leishmaniosis (CVL).
Assuntos
Proteínas de Insetos/imunologia , Leishmania infantum/fisiologia , Leishmaniose Visceral/imunologia , Proteínas e Peptídeos Salivares/imunologia , Vacinas de DNA/imunologia , Animais , Vírus da Varíola dos Canários/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Cães , Vetores Genéticos , Humanos , Imunidade Humoral , Imunização , Mediadores da Inflamação/metabolismo , Proteínas de Insetos/genética , Psychodidae/imunologia , Proteínas Recombinantes/genética , Proteínas e Peptídeos Salivares/genéticaRESUMO
OBJECTIVE: To evaluate changes in neuropsychological (NP) performance and in plasma and cell surface markers of peripheral monocyte activation/migration after treatment with cenicriviroc (CVC), a dual C-C chemokine receptor type 2 (CCR2) and type 5 (CCR5) antagonist, in treatment-experienced, HIV-infected individuals. SETTING: Single-arm, 24-week, open-label clinical trial. METHODS: HIV-infected individuals on antiretroviral therapy ≥1 year with plasma HIV RNA ≤50 copies per milliliter and below-normal cognitive performance [defined as age-, sex-, and education-adjusted NP performance (NPZ) <-0.5 in a single cognitive domain or in global performance] were enrolled. Changes over 24 weeks were assessed for global and domain-specific NPZ scores, plasma markers of monocyte/macrophage activation [neopterin, soluble (s)CD14, and sCD163] quantified by ELISA, and CCR2 and CCR5 expression on monocytes, and T cells measured by flow cytometry. RESULTS: Seventeen of 20 enrolled participants completed the study. Improvements over 24 weeks were observed in global NPZ [median change (Δ) = 0.24; P = 0.008], and in cognitive domains of attention (Δ0.23; P = 0.011) and working memory (Δ0.44; P = 0.017). Plasma levels of sCD163, sCD14 and neopterin decreased significantly (P's < 0.01). CCR2 and CCR5 monocyte expression remained unchanged; however, CCR5 levels on CD4 and CD8 T cells and CCR2 expression on CD4 T cells increased (P's < 0.01). CONCLUSIONS: CVC given over 24 weeks was associated with improved NP test performance and decreased plasma markers of monocyte immune activation in virally suppressed, HIV-infected participants. These data potentially link changes in monocyte activation to cognitive performance. Further study of CVC for HIV cognitive impairment in a randomized controlled study is warranted.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Cognição , Infecções por HIV/tratamento farmacológico , Monócitos/imunologia , Receptores CCR2/antagonistas & inibidores , Receptores CCR5/efeitos dos fármacos , Carga Viral , Feminino , Infecções por HIV/imunologia , Infecções por HIV/psicologia , Infecções por HIV/virologia , Humanos , Contagem de Leucócitos , Ativação de Macrófagos , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Projetos PilotoRESUMO
The aim of this study was to evaluate cenicriviroc (CVC), a dual antagonist of CC chemokine receptor types 2 and 5, for treatment of nonalcoholic steatohepatitis (NASH) with liver fibrosis (LF). A randomized, double-blind, multinational phase 2b study enrolled subjects with NASH, a nonalcoholic fatty liver disease activity score (NAS) ≥4, and LF (stages 1-3, NASH Clinical Research Network) at 81 clinical sites. Subjects (N = 289) were randomly assigned CVC 150 mg or placebo. Primary outcome was ≥2-point improvement in NAS and no worsening of fibrosis at year 1. Key secondary outcomes were: resolution of steatohepatitis (SH) and no worsening of fibrosis; improvement in fibrosis by ≥1 stage and no worsening of SH. Biomarkers of inflammation and adverse events were assessed. Full study recruitment was achieved. The primary endpoint of NAS improvement in the intent-to-treat population and resolution of SH was achieved in a similar proportion of subjects on CVC (N = 145) and placebo (N = 144; 16% vs. 19%, P = 0.52 and 8% vs. 6%, P = 0.49, respectively). However, the fibrosis endpoint was met in significantly more subjects on CVC than placebo (20% vs. 10%; P = 0.02). Treatment benefits were greater in those with higher disease activity and fibrosis stage at baseline. Biomarkers of systemic inflammation were reduced with CVC. Safety and tolerability of CVC were comparable to placebo. CONCLUSION: After 1 year of CVC treatment, twice as many subjects achieved improvement in fibrosis and no worsening of SH compared with placebo. Given the urgent need to develop antifibrotic therapies in NASH, these findings warrant phase 3 evaluation. (Hepatology 2018;67:1754-1767).
Assuntos
Antagonistas dos Receptores CCR5/uso terapêutico , Imidazóis/uso terapêutico , Cirrose Hepática/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Adulto , Idoso , Biomarcadores/sangue , Antagonistas dos Receptores CCR5/efeitos adversos , Método Duplo-Cego , Feminino , Humanos , Imidazóis/efeitos adversos , Fígado/patologia , Cirrose Hepática/complicações , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/complicações , Sulfóxidos , Resultado do TratamentoRESUMO
BACKGROUND: Non-alcoholic steatohepatitis (NASH) is often accompanied by liver fibrosis, which can progress to cirrhosis; C-C chemokine receptors type 2 and 5 (CCR2/CCR5), which mediate interactions driving inflammation and fibrosis, are promising treatment targets. Cenicriviroc (CVC), a dual-CCR2/CCR5 antagonist, has potent anti-inflammatory and antifibrotic activity in animal models; in HIV-positive subjects it reduced soluble CD14 levels, aspartate aminotransferase-to-platelet count ratio index, and non-invasive hepatic fibrosis risk scores; favorable tolerability was demonstrated in ~600 subjects. Efficacy and safety of CVC 150 mg for treating NASH with liver fibrosis are being evaluated over 2 years (primary endpoint at Year 1 [Y1]). DESIGN: Phase 2b, randomized, double-blind, placebo-controlled, multinational study (CENTAUR; NCT02217475). Adults with histological evidence of NASH, non-alcoholic fatty liver disease activity score (NAS) ≥ 4, and liver fibrosis (stages 1-3 NASH clinical research network system) enrolled. Subjects have increased risk of progression to cirrhosis due to ≥1 characteristic: type 2 diabetes; body mass index > 25 kg/m(2) with ≥1 feature of metabolic syndrome; bridging fibrosis and/or NAS ≥ 5. Liver biopsy evaluation at Screening, Y1, and Year 2 (Y2). OBJECTIVES: Assess histologic improvement (≥2-point in NAS with ≥1-point improvement in >1 category) without worsening of fibrosis at Y1 (primary); evaluate complete NASH resolution without worsening of fibrosis at Y2 (key secondary). DISCUSSION: CENTAUR is the first prospective study evaluating an oral agent exclusively enrolling subjects with NASH and liver fibrosis, with increased risk of developing cirrhosis. It will compare shorter versus longer CVC treatment and assess correlations between decreased inflammation and fibrosis.
Assuntos
Antagonistas dos Receptores CCR5/uso terapêutico , Imidazóis/uso terapêutico , Cirrose Hepática/complicações , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Administração Oral , Adolescente , Adulto , Idoso , Protocolos Clínicos , Método Duplo-Cego , Esquema de Medicação , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/complicações , Estudos Prospectivos , Projetos de Pesquisa , Sulfóxidos , Resultado do Tratamento , Adulto JovemRESUMO
Canine leishmaniasis (CanL) is a chronic fatal disease of dogs and a major source of human infection through propagation of parasites in vectors. Here, we infected 8 beagles through multiple experimental vector transmissions with Leishmania infantum-infected Lutzomyia longipalpis. CanL clinical signs varied, although live parasites were recovered from all dog spleens. Splenic parasite burdens correlated positively with Leishmania-specific interleukin 10 levels, negatively with Leishmania-specific interferon γ and interleukin 2 levels, and negatively with Leishmania skin test reactivity. A key finding was parasite persistence for 6 months in lesions observed at the bite sites in all dogs. These recrudesced following a second transmission performed at a distal site. Notably, sand flies efficiently acquired parasites after feeding on lesions at the primary bite site. In this study, controlled vector transmissions identify a potentially unappreciated role for skin at infectious bite sites in dogs with CanL, providing a new perspective regarding the mechanism of Leishmania transmissibility to vector sand flies.
Assuntos
Doenças do Cão/parasitologia , Insetos Vetores/parasitologia , Leishmania infantum , Leishmaniose Visceral/veterinária , Psychodidae/parasitologia , Animais , Reservatórios de Doenças/veterinária , Doenças do Cão/imunologia , Doenças do Cão/patologia , Doenças do Cão/transmissão , Cães , Feminino , Humanos , Imunidade Celular , Imunidade Humoral , Mordeduras e Picadas de Insetos/parasitologia , Mordeduras e Picadas de Insetos/patologia , Mordeduras e Picadas de Insetos/veterinária , Interferon gama/metabolismo , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/patologia , Leishmaniose Visceral/transmissão , Pele/parasitologia , Baço/parasitologiaRESUMO
BACKGROUND: Visceral leishmaniasis (VL) is transmitted by sand flies. Protection of needle-challenged vaccinated mice was abrogated in vector-initiated cutaneous leishmaniasis, highlighting the importance of developing natural transmission models for VL. METHODS: We used Lutzomyia longipalpis to transmit Leishmania infantum or Leishmania donovani to hamsters. Vector-initiated infections were monitored and compared with intracardiac infections. Body weights were recorded weekly. Organ parasite loads and parasite pick-up by flies were assessed in sick hamsters. RESULTS: Vector-transmitted L. infantum and L. donovani caused ≥5-fold increase in spleen weight compared with uninfected organs and had geometric mean parasite loads (GMPL) comparable to intracardiac inoculation of 10(7)-10(8) parasites, although vector-initiated disease progression was slower and weight loss was greater. Only vector-initiated L. infantum infections caused cutaneous lesions at transmission and distal sites. Importantly, 45.6%, 50.0%, and 33.3% of sand flies feeding on ear, mouth, and testicular lesions, respectively, were parasite-positive. Successful transmission was associated with a high mean percent of metacyclics (66%-82%) rather than total GMPL (2.0 × 10(4)-8.0 × 10(4)) per midgut. CONCLUSIONS: This model provides an improved platform to study initial immune events at the bite site, parasite tropism, and pathogenesis and to test drugs and vaccines against naturally acquired VL.
Assuntos
Modelos Animais de Doenças , Mordeduras e Picadas de Insetos/parasitologia , Insetos Vetores/parasitologia , Leishmaniose Visceral/patologia , Psychodidae/parasitologia , Animais , Peso Corporal , Cricetinae , Progressão da Doença , Leishmania donovani/patogenicidade , Leishmania infantum/patogenicidade , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Leishmaniose Cutânea/transmissão , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/transmissão , Masculino , Tamanho do Órgão , Carga Parasitária , Baço/parasitologia , Baço/patologiaRESUMO
Immunization with xenogeneic DNA is a promising cancer treatment to overcome tolerance to self-antigens. Heat shock protein 70 (HSP70) is over-expressed in various kinds of tumors and is believed to be involved in tumor progression. This study tested a xenogeneic chicken HSP70 (chHSP70) DNA vaccine in an experimental canine transmissible venereal tumor (CTVT) model. Three vaccination strategies were compared: the first (PE) was designed to evaluate the prophylactic efficacy of chHSP70 DNA vaccination by delivering the vaccine before tumor inoculation in a prime boost setting, the second (T) was designed to evaluate the therapeutic efficacy of the same prime boost vaccine by vaccinating the dogs after tumor inoculation; the third (PT) was similar to the first strategy (PE), with the exception that the electroporation booster injection was replaced with a transdermal needle-free injection. Tumor growth was notably inhibited only in the PE dogs, in which the vaccination program triggered tumor regression significantly sooner than in control dogs (NT). The CD4(+) subpopulation of tumor-infiltrating lymphocytes and canine HSP70 (caHSP70)-specific IFN-γ-secreting lymphocytes were significantly increased during tumor regression in the PE dogs as compared to control dogs, demonstrating that specific tolerance to caHSP70 has been overcome. In contrast, no benefit of the therapeutic strategy (T) could be noticed and the (PT) strategy only led to partial control of tumor growth. In summary, antitumor prophylactic activity was demonstrated using the chHSP70 DNA vaccine including a boost via electroporation. Our data stressed the importance of DNA electroporation as a booster to get the full benefit of DNA vaccination but also of cancer immunotherapy initiation as early as possible. Xenogeneic chHSP70 DNA vaccination including an electroporation boost is a potential vaccine to HSP70-expressing tumors, although further research is still required to better understand true clinical potential.
Assuntos
Vacinas Anticâncer/imunologia , Proteínas de Choque Térmico HSP70/imunologia , Vacinas de DNA/imunologia , Tumores Venéreos Veterinários/prevenção & controle , Sequência de Aminoácidos , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Galinhas , Cães , Eletroporação , Interferon gama/imunologia , Células Matadoras Naturais/imunologia , Dados de Sequência Molecular , Alinhamento de Sequência , Vacinação , Tumores Venéreos Veterinários/imunologiaRESUMO
Immunity to a sand fly salivary protein protects against visceral leishmaniasis (VL) in hamsters. This protection was associated with the development of cellular immunity in the form of a delayed-type hypersensitivity response and the presence of IFN-gamma at the site of sand fly bites. To date, there are no data available regarding the cellular immune response to sand fly saliva in dogs, the main reservoirs of VL in Latin America, and its role in protection from this fatal disease. Two of 35 salivary proteins from the vector sand fly Lutzomyia longipalpis, identified using a novel approach termed reverse antigen screening, elicited strong cellular immunity in dogs. Immunization with either molecule induced high IgG(2) antibody levels and significant IFN-gamma production following in vitro stimulation of PBMC with salivary gland homogenate (SGH). Upon challenge with uninfected or infected flies, immunized dogs developed a cellular response at the bite site characterized by lymphocytic infiltration and IFN-gamma and IL-12 expression. Additionally, SGH-stimulated lymphocytes from immunized dogs efficiently killed Leishmania infantum chagasi within autologous macrophages. Certain sand fly salivary proteins are potent immunogens obligatorily co-deposited with Leishmania parasites during transmission. Their inclusion in an anti-Leishmania vaccine would exploit anti-saliva immunity following an infective sand fly bite and set the stage for a protective anti-Leishmania immune response.
Assuntos
Proteínas de Insetos/imunologia , Leishmania infantum/imunologia , Leishmaniose Visceral/imunologia , Proteínas e Peptídeos Salivares/imunologia , Animais , Formação de Anticorpos , Citocinas/genética , Citocinas/metabolismo , Interpretação Estatística de Dados , Cães , Feminino , Expressão Gênica , Hipersensibilidade Tardia , Imunidade Celular , Mordeduras e Picadas de Insetos/imunologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Insetos Vetores/genética , Insetos Vetores/imunologia , Insetos Vetores/parasitologia , Leishmania infantum/fisiologia , Linfócitos/imunologia , Psychodidae/genética , Psychodidae/imunologia , Psychodidae/parasitologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/metabolismo , Pele/imunologiaRESUMO
Delayed-type hypersensitivity (DTH) response to arthropod vector salivary proteins is associated with protection against pathogen transmission. Massive cDNA sequencing, high-throughput DNA plasmid construction and DNA immunisation were used to identify twelve DTH inducing proteins isolated from a Phlebotomus ariasi salivary gland cDNA library. Additionally, nine P. ariasi DNA plasmids produced specific anti-saliva antibodies, four of these showed a Th1 immune response while the other two exhibited a Th2 profile as determined by IgG2a and IgG1 isotype switching, respectively. In order to validate the specificity of sand fly DNA plasmids, mice previously exposed to sand fly saliva were intradermally injected once with selected P. ariasi plasmids and a specific DTH response consisting of infiltration of mononuclear cells in varying proportions was observed at 24 and 48 h. This approach can help to identify DTH inducing proteins that may be related to host protection against vector-borne diseases or other disease agents where cellular immune response is protective.
Assuntos
DNA Complementar/imunologia , Avaliação Pré-Clínica de Medicamentos/métodos , Hipersensibilidade Tardia/imunologia , Phlebotomus/imunologia , Glândulas Salivares/metabolismo , Proteínas e Peptídeos Salivares/imunologia , Sequência de Aminoácidos , Animais , Formação de Anticorpos/imunologia , Biologia Computacional , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Biblioteca Gênica , Imunoglobulina A/biossíntese , Imunoglobulina A/genética , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Insetos Vetores , Camundongos , Dados de Sequência Molecular , Proteínas e Peptídeos Salivares/genética , Análise de Sequência de Proteína , Vacinas/imunologia , Vacinas de DNA/imunologiaRESUMO
As of today, most DNA vaccination trials have been performed with plasmid preparations highly enriched in supercoiled molecules (sc) and the importance of supercoiled versus open circular (oc) plasmid isoforms for vaccine immunogenicity has only received limited attention. This study demonstrated that a single rabies DNA vaccination fully protected cats against a lethal rabies challenge as early as 3 weeks post vaccination provided that the proportion of supercoiled isoform in the vaccinal solution is at least 48%. In contrast, vaccination with a plasmid containing only 20% of supercoiled molecules induced significant but only partial protection. Further, a single rabies DNA vaccination with plasmids containing at least 70% of supercoiled molecules triggered statistically significant specific antibody titers and specific Th-1 oriented cell-based immunity as early as 2 and 3 weeks post vaccination, respectively. It is concluded that the oc isoforms are less efficient than supercoiled isoforms at inducing a complete profile of immune responses. Therefore, it is proposed that the target threshold of supercoiling that must be met by a rabies DNA vaccine to guarantee optimal immune responses and protection, be set at 70% of supercoiled molecules in the vaccine solution.
Assuntos
DNA Super-Helicoidal/genética , DNA Viral/genética , Plasmídeos/genética , Vacina Antirrábica/genética , Vacina Antirrábica/imunologia , Raiva/prevenção & controle , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/biossíntese , Gatos , Esquemas de Imunização , Interferon gama/genética , Interferon gama/imunologia , Raiva/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia , Vacinas de DNA/genética , Vacinas de DNA/imunologiaRESUMO
The increasing international movement of horses combined with the relaxation of veterinary regulations has resulted in an increased incidence of equine infectious diseases. Vaccination, along with management measures, has become the primary method for the effective control of these diseases. Traditionally modified live and inactivated vaccines have been used and these vaccines have proven to be very successful in preventing disease. However, there are a number of equine infectious diseases for which conventional technology has shown its limitations. The advent of recombinant technology has stimulated the development of second generation vaccines, including gene deleted mutants, live vectored vaccines and DNA vaccines. These vaccines have in common that protective antigens are endogenously processed and presented along the molecules of the MHC I and MHC II complex, resulting in the stimulation of both humoral and cell-mediated immune responses similar to natural infection. The present paper provides a review of the vaccines being employed today against the most important equine viral diseases followed by a summary of new developments that are expected to bring improved vaccines to the market in the foreseeable future.
Assuntos
Doenças dos Cavalos/prevenção & controle , Doenças dos Cavalos/virologia , Vacinação/veterinária , Vacinas Virais , Animais , Arterite/veterinária , Encefalomielite Equina , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/imunologia , Cavalos , Infecções por Orthomyxoviridae/veterinária , Febre do Nilo Ocidental/veterináriaRESUMO
In order for DNA vaccines to become a practical alternative to conventional vaccines their ability to induce antibody responses in large mammals needs to be improved. We used DNA vaccination against rabies in the horse as a model to test the potential of two different strategies to enhance antibody responses in a large mammalian species. The administration of the DNA vaccine in the presence of aluminum phosphate improved both the onset and the intensity of serological responses but was not potent enough to achieve seroconversion in all vaccinated ponies. However, when the DNA vaccine was formulated with the cationic lipid DMRIE-DOPE instead of aluminum phosphate, a very strong impact on both onset and intensity of serological responses was observed. This latter strategy ensured excellent seroconversion in all vaccinated ponies after a primary course of two injections, demonstrating a clear improvement of the homogeneity of the induced responses. These data indicate that rabies DNA vaccination is feasible in horses and further suggests that properly formulated DNA vaccines can generate immune responses in large veterinary species at a level comparable to the responses achieved with conventional vaccines.