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The CVnCoV (CureVac) mRNA vaccine for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was recently evaluated in a phase 2b/3 efficacy trial in humans1. CV2CoV is a second-generation mRNA vaccine containing non-modified nucleosides but with optimized non-coding regions and enhanced antigen expression. Here we report the results of a head-to-head comparison of the immunogenicity and protective efficacy of CVnCoV and CV2CoV in non-human primates. We immunized 18 cynomolgus macaques with two doses of 12 µg lipid nanoparticle-formulated CVnCoV or CV2CoV or with sham (n = 6 per group). Compared with CVnCoV, CV2CoV induced substantially higher titres of binding and neutralizing antibodies, memory B cell responses and T cell responses as well as more potent neutralizing antibody responses against SARS-CoV-2 variants, including the Delta variant. Moreover, CV2CoV was found to be comparably immunogenic to the BNT162b2 (Pfizer) vaccine in macaques. Although CVnCoV provided partial protection against SARS-CoV-2 challenge, CV2CoV afforded more robust protection with markedly lower viral loads in the upper and lower respiratory tracts. Binding and neutralizing antibody titres were correlated with protective efficacy. These data demonstrate that optimization of non-coding regions can greatly improve the immunogenicity and protective efficacy of a non-modified mRNA SARS-CoV-2 vaccine in non-human primates.
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Vacinas contra COVID-19/genética , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Imunogenicidade da Vacina , Nucleosídeos/química , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas de mRNA/genética , Vacinas de mRNA/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacina BNT162/imunologia , COVID-19/imunologia , COVID-19/virologia , Vacinas contra COVID-19/normas , Feminino , Macaca fascicularis/imunologia , Masculino , Células B de Memória/imunologia , Nucleosídeos/genética , Sistema Respiratório/imunologia , Sistema Respiratório/virologia , SARS-CoV-2/imunologia , Linfócitos T/imunologia , Vacinas Sintéticas/normas , Carga Viral , Vacinas de mRNA/normasRESUMO
The International Alliance for Biological Standardization and the Coalition for Epidemic Preparedness Innovations organized a joint webinar on the use of platform technologies for vaccine development. To tackle new emerging infectious diseases, including SARS-CoV-2, rapid response platforms, using the same basic components as a backbone, yet adaptable for use against different pathogens by inserting new genetic or protein sequences, are essential. Furthermore, it is evident that development of platform technologies needs to continue, due to the emerging variants of SARS-CoV-2. The objective of the meeting was to discuss techniques for platform manufacturing that have been used for COVID-19 vaccine development, with input from regulatory authorities on their experiences with, and expectations of, the platforms. Industry and regulators have been very successful in cooperating, having completed the whole process from development to licensing at an unprecedented speed. However, we should learn from the experiences, to be able to be even faster when a next pandemic of disease X occurs.
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Vacinas contra COVID-19/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , Desenvolvimento de Medicamentos , SARS-CoV-2/imunologia , Vacinas contra COVID-19/uso terapêutico , Congressos como Assunto , HumanosRESUMO
Drug-induced liver injury is the most frequent reason for market withdrawal of approved drugs, and is difficult to predict in animal models. Here, we analyzed transcriptomic data derived from short- and long-term cultured primary human hepatocytes (PHH) exposed to the well known human hepatotoxin chlorpromazine (CPZ). Samples were collected from five PHH cultures after short-term (1 and 3 days) and long-term (14 days) repeat daily treatment with 0.1 or 0.2 µM CPZ, corresponding to C(max). Two PHH cultures were additionally treated with 1 µM CPZ, and the three others with 0.02 µM CPZ. Differences in the total number of gene changes were seen between donors and throughout treatment. Specific transcriptomic hepatotoxicity signatures were created for CPZ and consisted of inflammation/hepatitis, cholestasis, and liver proliferation in all five donors, as well as fibrosis and steatosis, which were observed in four of five donors. Necrosis was present in three of five donors, and an indicative signature of cirrhosis was observed after long-term 14-day repeat treatment, also in three of five donors. The inter-donor variability in the inflammatory response to CPZ treatment was associated with variability in the strength of the response of the transcriptomic hepatotoxicity signatures, suggesting that features of inflammation could be related to the idiosyncratic hepatotoxic effects of CPZ in humans.
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Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Clorpromazina/administração & dosagem , Clorpromazina/efeitos adversos , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Transcriptoma/genética , Idoso , Células Cultivadas , Feminino , Hepatócitos/metabolismo , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Fígado/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Omicron and its subvariants (BA.2, BA.4, BA.5) represented the most commonly circulating variants of concern (VOC) in the coronavirus disease 2019 (COVID-19) pandemic in 2022. Despite high vaccination rates with approved SARS-CoV-2 vaccines encoding the ancestral spike (S) protein, these Omicron subvariants have collectively resulted in increased viral transmission and disease incidence. This necessitates the development and characterization of vaccines incorporating later emerging S proteins to enhance protection against VOC. In this context, bivalent vaccine formulations may induce broad protection against VOC and potential future SARS-CoV-2 variants. Here, we report preclinical data for a lipid nanoparticle (LNP)-formulated RNActive® N1-methylpseudouridine (N1mΨ) modified mRNA vaccine (CV0501) based on our second-generation SARS-CoV-2 vaccine CV2CoV, encoding the S protein of Omicron BA.1. The immunogenicity of CV0501, alone or in combination with a corresponding vaccine encoding the ancestral S protein (ancestral N1mΨ), was first measured in dose-response and booster immunization studies performed in Wistar rats. Both monovalent CV0501 and bivalent CV0501/ancestral N1mΨ immunization induced robust neutralizing antibody titers against the BA.1, BA.2 and BA.5 Omicron subvariants, in addition to other SARS-CoV-2 variants in a booster immunization study. The protective efficacy of monovalent CV0501 against live SARS-CoV-2 BA.2 infection was then assessed in hamsters. Monovalent CV0501 significantly reduced SARS-CoV-2 BA.2 viral loads in the airways, demonstrating protection induced by CV0501 vaccination. CV0501 has now advanced into human Phase 1 clinical trials (ClinicalTrials.gov Identifier: NCT05477186).
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Combining optimized spike (S) protein-encoding mRNA vaccines to target multiple SARS-CoV-2 variants could improve control of the COVID-19 pandemic. We compare monovalent and bivalent mRNA vaccines encoding B.1.351 (Beta) and/or B.1.617.2 (Delta) SARS-CoV-2 S-protein in a transgenic mouse and a Wistar rat model. The blended low-dose bivalent mRNA vaccine contains half the mRNA of each respective monovalent vaccine, but induces comparable neutralizing antibody titres, enrichment of lung-resident memory CD8+ T cells, antigen-specific CD4+ and CD8+ responses, and protects transgenic female mice from SARS-CoV-2 lethality. The bivalent mRNA vaccine significantly reduces viral replication in both Beta- and Delta-challenged mice. Sera from bivalent mRNA vaccine immunized female Wistar rats also contain neutralizing antibodies against the B.1.1.529 (Omicron BA.1 and BA.5) variants. These data suggest that low-dose and fit-for-purpose multivalent mRNA vaccines encoding distinct S-proteins are feasible approaches for extending the coverage of vaccines for emerging and co-circulating SARS-CoV-2 variants.
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Vacinas contra COVID-19 , COVID-19 , SARS-CoV-2 , Animais , Feminino , Camundongos , Ratos , Anticorpos Neutralizantes , Anticorpos Antivirais , Linfócitos T CD8-Positivos , COVID-19/prevenção & controle , Vacinas contra COVID-19/imunologia , Camundongos Transgênicos , Modelos Animais , Vacinas de mRNA/imunologia , Ratos Wistar , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Vacinas Combinadas/imunologiaRESUMO
More than two years after the emergence of SARS-CoV-2, 33 COVID-19 vaccines, based on different platforms, have been approved in 197 countries. Novel variants that are less efficiently neutralised by antibodies raised against ancestral SARS-CoV-2 are circulating, highlighting the need to adapt vaccination strategies. Here, we compare the immunogenicity of a first-generation mRNA vaccine candidate, CVnCoV, with a second-generation mRNA vaccine candidate, CV2CoV, in rats. Higher levels of spike (S) protein expression were observed in cell culture with the CV2CoV mRNA than with the CVnCoV mRNA. Vaccination with CV2CoV also induced higher titres of virus neutralising antibodies with accelerated kinetics in rats compared with CVnCoV. Significant cross-neutralisation of the SARS-CoV-2 variants, Alpha (B.1.1.7), Beta (B.1.351), and the 'mink' variant (B1.1.298) that were circulating at the time in early 2021 were also demonstrated. In addition, CV2CoV induced higher levels of antibodies at lower doses than CVnCoV, suggesting that dose-sparing could be possible with the next-generation SARS-CoV-2 vaccine, which could improve worldwide vaccine supply.
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A third vaccine dose is often required to achieve potent, long-lasting immune responses. We investigated the effect of three 8-µg doses of CVnCoV, CureVac's severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine candidate containing sequence-optimized unmodified mRNA encoding the spike (S) glycoprotein, administered at 0, 4, and 28 weeks, on immune responses in rhesus macaques. After the third dose, S-specific binding and neutralizing antibodies increased 50-fold compared with post-dose 2 levels, with increased responses also evident in the lower airways and against the SARS-CoV-2 B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta) variants. Enhanced binding affinity of serum antibodies after the third dose correlated with higher somatic hypermutation in S-specific B cells, corresponding with improved binding properties of monoclonal antibodies expressed from isolated B cells. Administration of low-dose mRNA led to fewer cells expressing antigen in vivo at the injection site and in the draining lymph nodes compared with a 10-fold higher dose, possibly reducing engagement of precursor cells with the antigen and resulting in the suboptimal response observed after two-dose vaccination schedules in phase IIb/III clinical trials of CVnCoV. However, when immune memory is established, a third dose efficiently boosts the immunological responses and improves antibody affinity and breadth.
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mRNA vaccines can be developed and produced quickly, making them prime candidates for immediate outbreak responses. Furthermore, clinical trials have demonstrated rapid protection following mRNA vaccination. Thus, we sought to investigate how quickly mRNA vaccines elicit antibody responses compared to other vaccine modalities. We first compared the immune kinetics of mRNA and DNA vaccines expressing SARS-CoV-2 spike in mice. We observed rapid induction of antigen-specific binding and neutralizing antibodies by day 5 following mRNA (4 µg/mouse), but not DNA (50 µg/mouse), immunization. Comparing innate responses hours post immunization, the mRNA vaccine induced increased levels of IL-5, IL-6, and MCP-1 cytokines which maybe promoting humoral responses downstream. We then evaluated the immune kinetics of an HIV-1 mRNA vaccine in comparison to DNA, protein, and rhesus adenovirus 52 (RhAd52) vaccines of the same HIV-1 envelope antigen in mice. Again, induction of envelope-specific antibodies was observed by day 5 following mRNA vaccination, whereas antibodies were detected by day 7-14 following DNA, protein, and RhAd52 vaccination. Thus, eliciting rapid humoral immunity may be a unique and advantageous property of mRNA vaccines for controlling infectious disease outbreaks.
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mRNA technologies have recently proven clinical efficacy against coronavirus disease 2019 and are among the most promising technologies to address the current pandemic. Here, we show preclinical data for our clinical candidate CVnCoV, a lipid nanoparticle-encapsulated mRNA vaccine that encodes full-length, pre-fusion stabilised severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein. In contrast to previously published approaches, CVnCoV is exclusively composed of naturally occurring nucleotides. Immunisation with CVnCoV induced strong humoral responses with high titres of virus-neutralising antibodies and robust T-cell responses. CVnCoV vaccination protected hamsters from challenge with wild-type SARS-CoV-2, demonstrated by the absence of viral replication in the lungs. Hamsters vaccinated with a suboptimal dose of CVnCoV leading to breakthrough viral replication exhibited no evidence of vaccine-enhanced disease. Overall, data presented here provide evidence that CVnCoV represents a potent and safe vaccine candidate against SARS-CoV-2.
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mRNA vaccines can be developed and produced quickly, making them attractive for immediate outbreak responses. Furthermore, clinical trials have demonstrated rapid protection following mRNA vaccination. We sought to investigate how quickly mRNA vaccines elicit antibody responses compared to other vaccine modalities. We first examined immune kinetics of mRNA and DNA vaccines expressing SARS-CoV-2 spike in mice. We observed rapid induction of antigen-specific binding and neutralizing antibodies by day 5 following mRNA, but not DNA, immunization. The mRNA vaccine also induced increased levels of IL-5, IL-6 and MCP-1. We then evaluated immune kinetics of an HIV-1 mRNA vaccine in comparison to DNA, protein, and rhesus adenovirus 52 (RhAd52) vaccines with the same HIV-1 envelope antigen in mice. Induction of envelope-specific antibodies was observed by day 5 following mRNA vaccination, whereas antibodies were detected by day 7-14 following DNA, protein, and RhAd52 vaccination. Eliciting rapid humoral immunity may be an advantageous property of mRNA vaccines for controlling infectious disease outbreaks. IMPORTANCE: mRNA vaccines can be developed and produced in record time. Here we demonstrate induction of rapid antibody responses by mRNA vaccines encoding two different viral antigens by day 5 following immunization in mice. The rapid immune kinetics of mRNA vaccines can be an advantageous property that makes them well suited for rapid control of infectious disease outbreaks.
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BACKGROUND: We used the RNActive® technology platform (CureVac N.V., Tübingen, Germany) to prepare CVnCoV, a COVID-19 vaccine containing sequence-optimized mRNA coding for a stabilized form of SARS-CoV2 spike (S) protein encapsulated in lipid nanoparticles (LNP). METHODS: This is an interim analysis of a dosage escalation phase 1 study in healthy 18-60-year-old volunteers in Hannover, Munich and Tübingen, Germany, and Ghent, Belgium. After giving 2 intramuscular doses of CVnCoV or placebo 28 days apart we assessed solicited local and systemic adverse events (AE) for 7 days and unsolicited AEs for 28 days after each vaccination. Immunogenicity was measured as enzyme-linked immunosorbent assay (ELISA) IgG antibodies to SARS-CoV2 Sprotein and receptor binding domain (RBD), and SARS-CoV2 neutralizing titers (MN50). RESULTS: In 245 volunteers who received 2 CVnCoV vaccinations (2⯵g, nâ¯= 47, 4⯵g, nâ¯= 48, 6⯵g, nâ¯= 46, 8⯵g, nâ¯= 44, 12⯵g, nâ¯= 28) or placebo (nâ¯= 32) there were no vaccine-related serious AEs. Dosage-dependent increases in frequency and severity of solicited systemic AEs, and to a lesser extent local AEs, were mainly mild or moderate and transient in duration. Dosage-dependent increases in IgG antibodies to Sprotein and RBD and MN50 were evident in all groups 2 weeks after the second dose when 100% (23/23) seroconverted to Sprotein or RBD, and 83% (19/23) seroconverted for MN50 in the 12⯵g group. Responses to 12⯵g were comparable to those observed in convalescent sera from known COVID-19 patients. CONCLUSION: In this study 2 CVnCoV doses were safe, with acceptable reactogenicity and 12⯵g dosages elicited levels of immune responses that overlapped those observed in convalescent sera.
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COVID-19 , Nanopartículas , Vacinas , Adolescente , Adulto , Anticorpos Antivirais , COVID-19/terapia , Vacinas contra COVID-19 , Método Duplo-Cego , Humanos , Imunização Passiva , Imunogenicidade da Vacina , Lipídeos , Pessoa de Meia-Idade , RNA Mensageiro , SARS-CoV-2 , Adulto Jovem , Soroterapia para COVID-19RESUMO
The ongoing SARS-CoV-2 pandemic necessitates the fast development of vaccines. Recently, viral mutants termed variants of concern (VOC) which may escape host immunity have emerged. The efficacy of spike encoding mRNA vaccines (CVnCoV and CV2CoV) against the ancestral strain and the VOC B.1.351 was tested in a K18-hACE2 transgenic mouse model. Naive mice and mice immunized with a formalin-inactivated SARS-CoV-2 preparation were used as controls. mRNA-immunized mice develop elevated SARS-CoV-2 RBD-specific antibody and neutralization titers which are readily detectable, but significantly reduced against VOC B.1.351. The mRNA vaccines fully protect from disease and mortality caused by either viral strain. SARS-CoV-2 remains undetected in swabs, lung, or brain in these groups. Despite lower neutralizing antibody titers compared to the ancestral strain BavPat1, CVnCoV and CV2CoV show complete disease protection against the novel VOC B.1.351 in our studies.
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Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/imunologia , Animais , Linhagem Celular , Chlorocebus aethiops , Genoma Viral/genética , Humanos , Camundongos , Camundongos Transgênicos , SARS-CoV-2/genética , Células VeroRESUMO
UNLABELLED: Hepatocytes in culture are a valuable tool to investigate mechanisms involved in the response of the liver to cytokines. However, it is well established that hepatocytes cultured on monolayers of dried stiff collagen dedifferentiate, losing specialized liver functions. In this study, we show that hepatocyte dedifferentiation is a reversible consequence of a specific signaling network constellation triggered by the extracellular matrix. A dried stiff collagen activates focal adhesion kinase (FAK) via Src, leading to activation of the Akt and extracellular signal-regulated kinase (ERK) 1/2 pathways. Akt causes resistance to transforming growth factor beta (TGF-beta)-induced apoptosis by antagonizing p38, whereas ERK1/2 signaling opens the route to epithelial-mesenchymal transition (EMT). Apoptosis resistance is reversible by inhibiting Akt or Src, and EMT can be abrogated by blocking the ERK1/2 pathway. In contrast to stiff collagen, a softer collagen gel does not activate FAK, keeping the hepatocytes in a state where they remain sensitive to TGF-beta-induced apoptosis and do not undergo EMT. In this culture system, inhibition of p38 as well as overexpression of constitutively active Akt causes apoptosis resistance, whereas constitutively active Ras induces EMT. Finally, we show that matrix-induced EMT is reversible by replating cells from dried stiff to soft gel collagen. Our results demonstrate that hepatocyte dedifferentiation in vitro is an active process driven by FAK-mediated Akt and ERK1/2 signaling. This leads to similar functional and morphological alterations as observed for regenerating hepatocytes in vivo and is reversible when Akt and/or ERK1/2 signaling pathways are antagonized. CONCLUSION: Hepatocytes can exist in a differentiated and a dedifferentiated state that are reversible and can be switched by manipulating the responsible key factors of the signaling network.
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Apoptose , Desdiferenciação Celular , Matriz Extracelular/fisiologia , Hepatócitos/citologia , Hepatócitos/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Células Cultivadas , Masculino , CamundongosRESUMO
Several anthropogenous and naturally occurring substances, referred to as estrogen active compounds (EACs), are able to interfere with hormone and in particular estrogen receptor signaling. EACs can either cause adverse health effects in humans and wildlife populations or have beneficial effects on estrogen-dependent diseases. The aim of this study was to examine global gene expression profiles in estrogen receptor (ER)-proficient Ishikawa plus and ER-deficient Ishikawa minus endometrial cancer cells treated with selected well-known EACs (diethylstilbestrol, genistein, zearalenone, resveratrol, bisphenol A and o,p'-DDT). We also investigated the effect of the pure antiestrogen ICI 182,780 (ICI) on the expression patterns caused by these compounds. Transcript levels were quantified 24 h after compound treatment using Illumina BeadChip Arrays. We identified 87 genes with similar expression changes in response to all EAC treatments in Ishikawa plus. ICI lowered the magnitude or reversed the expression of these genes, indicating ER dependent regulation. Apart from estrogenic gene regulation, bisphenol A, o,p'-DDT, zearalenone, genistein and resveratrol displayed similarities to ICI in their expression patterns, suggesting mixed estrogenic/antiestrogenic properties. In particular, the predominant antiestrogenic expression response of resveratrol could be clearly distinguished from the other test compounds, indicating a distinct mechanism of action. Divergent gene expression patterns of the phytoestrogens, as well as weaker estrogenic gene expression regulation determined for the anthropogenous chemicals bisphenol A and o,p'-DDT, warrants a careful assessment of potential detrimental and/or beneficial effects of EACs. The characteristic expression fingerprints and the identified subset of putative marker genes can be used for screening chemicals with an unknown mode of action and for predicting their potential to exert endocrine disrupting effects.
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Disruptores Endócrinos/farmacologia , Neoplasias do Endométrio/genética , Antagonistas de Estrogênios/farmacologia , Estrogênios/farmacologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Receptores de Estrogênio/efeitos dos fármacos , Compostos Benzidrílicos , Linhagem Celular Tumoral , Análise por Conglomerados , DDT/farmacologia , Dietilestilbestrol/farmacologia , Disruptores Endócrinos/toxicidade , Neoplasias do Endométrio/metabolismo , Estradiol/análogos & derivados , Estradiol/farmacologia , Estrogênios/toxicidade , Feminino , Fulvestranto , Perfilação da Expressão Gênica/métodos , Genisteína/farmacologia , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Fenóis/farmacologia , Fitoestrógenos/farmacologia , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Reprodutibilidade dos Testes , Resveratrol , Medição de Risco , Estilbenos/farmacologia , Fatores de Tempo , Zearalenona/farmacologiaRESUMO
Induction of drug-metabolizing enzymes (DMEs) is highly species-specific and can lead to drug-drug interaction and toxicities. In this series of studies we tested the species specificity of the antidiabetic drug development candidate and mixed peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist (S)-4-O-tolylsulfanyl-2-(4-trifluormethyl-phenoxy)-butyric acid (EMD 392949, EMD) with regard to the induction of gene expression and activities of DMEs, their regulators, and typical PPAR target genes. EMD clearly induced PPARalpha target genes in rats in vivo and in rat hepatocytes but lacked significant induction of DMEs, except for cytochrome P450 (P450) 4A. CYP2C and CYP3A were consistently induced in livers of EMD-treated monkeys. Interestingly, classic rodent peroxisomal proliferation markers were induced in monkeys after 17 weeks but not after a 4-week treatment, a fact also observed in human hepatocytes after 72 h but not 24 h of EMD treatment. In human hepatocyte cultures, EMD showed similar gene expression profiles and induction of P450 activities as in monkeys, indicating that the monkey is predictive for human P450 induction by EMD. In addition, EMD induced a similar gene expression pattern as the PPARalpha agonist fenofibrate in primary rat and human hepatocyte cultures. In conclusion, these data showed an excellent correlation of in vivo data on DME gene expression and activity levels with results generated in hepatocyte monolayer cultures, enabling a solid estimation of human P450 induction. This study also clearly highlighted major differences between primates and rodents in the regulation of major inducible P450s, with evidence of CYP3A and CYP2C inducibility by PPARalpha agonists in monkeys and humans.
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Ácido Butírico/administração & dosagem , Ácido Butírico/farmacologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Idoso , Animais , Ácido Butírico/química , Células Cultivadas , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Fígado/fisiologia , Macaca fascicularis , Masculino , Pessoa de Meia-Idade , PPAR alfa/genética , PPAR alfa/metabolismo , Ratos , Ratos Wistar , Especificidade da EspécieRESUMO
BACKGROUND: Compound failures have been emerging in later stages of pharmaceutical drug development and are in many cases not detected until the administration to humans in clinical trials or even after approval. Among the most frequent adverse effects are drug-induced liver injury generated by species-specific susceptibilities (e.g., in xenobiotic metabolism and/or the occurrence of idiosyncratic drug hepatotoxicity). OBJECTIVES: Detecting or predicting unfavorable drug effects on the liver as early as possible in drug development is crucial in making the drug development process more efficient and the application of new drugs to humans in clinical studies and medical use safer. METHODS: To achieve this goal, primary hepatocyte cultures from various species, including humans, are analyzed for morphological, functional and gene expression alterations after compound treatment. RESULTS/CONCLUSION: Primary hepatocyte cultures appear to be a promising tool for the detection of general or liver toxicity and the evaluation of species-specific drug effects.
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Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/induzido quimicamente , Hepatócitos/metabolismo , Preparações Farmacêuticas/metabolismo , Testes de Toxicidade/métodos , Animais , Doença Hepática Induzida por Substâncias e Drogas/patologia , Previsões , Humanos , Especificidade da EspécieRESUMO
Oestrogen receptors (ER)alpha and beta modify the expression of genes involved in cell growth, proliferation and differentiation through binding to oestrogen response elements (EREs) located in a number of gene promoters. Transient transfection of different luciferase reporter vectors 3xEREs-Vit, 2xEREs-tk and ERE-C3 showed that the transactivation capacity of both ER subtypes was influenced by 1) the nature of the inducer (oestradiol (E2), phyto- and anti-oestrogen (AE)), 2) the structure of the promoter (nucleotidic sequence, number of ERE, length of the promoter sequence) and 3) the cell line (containing endogenous ER (MCF-7) or in which ER was stably expressed (MDA-MB-231-HE-5 (ERalpha+) or MDA-MB-231-HERB (ERbeta+)). ER subtype did not display the same efficacy on the different constructions in the presence of E2 and of AE according to the cell (e.g. in MCF-7 cells: tk>>Vit>>C3 approximately 0 while in MDA-MB-231 cells: Vit>>tk approximately C3). E2 response was higher in MCF-7 cells, probably due to higher ER expression level (maximal at 10(-10)M instead of 10(-8)M for E2 in HE-5 cells). Finally, the same ligand could exert opposite activities on the same promoter according to the ER isoform expressed: in the MDA-MB-231 cells, AE acted as inducers of the C3 promoter via ERbeta whereas ERalpha/AE complexes down-regulated this promoter. Approximately 70% of breast tumours express ER and most tumour cells coexpress both ER isotypes. Thus, different types of ER dimers can be formed in such tumours (ERbeta or ERalpha homodimers or ERalpha/ERbeta heterodimers). We therefore studied the influence of the coexistence of the two ERs on the ligand-induced transcriptional process following transient transfection of ERalpha in ERbeta+ cells, and inversely ERbeta in ERalpha+ cells. ERbeta-transfection inhibited the E2- and genistein-induced ERalpha-dependent transcription on all promoters in all cell lines except C3 in MCF-7; this inhibitory effect was lost following transfection of ERbeta deleted of its AF-1 (ERbeta-AF-2). These results suggest that the dominant negative properties of ERbeta are mainly due to its AF-1 function. Interestingly, transfection of an ERbeta-AF-2 construct into MCF-7 cells potentiated the transcription inhibitory capacity of 4-OH-tamoxifen (OHT) on the Vit and tk promoters. Thus, (1) OHT exerts an agonistic activity through the AF-1 function of ER and (2) expression of ERbeta in breast cancer cells seems to favour the AE treatment. Contrary to ERbeta, ERalpha-transfection had little effect on ERbeta transactivation capacity in HERB cells. Finally, the ratio ERalpha/ERbeta constitutes one decisive parameters to orientate the transcriptional mechanism of a target gene in the presence of agonist as well as of antagonist ligands.
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Estradiol/farmacologia , Receptor alfa de Estrogênio/fisiologia , Receptor beta de Estrogênio/química , Receptor beta de Estrogênio/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Elementos de Resposta , Moduladores de Receptor Estrogênico/farmacologia , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Genisteína/farmacologia , Humanos , Proteínas Mutantes/genética , Isoformas de Proteínas , Estrutura Terciária de Proteína , Transdução de Sinais/genética , Transcrição Gênica/efeitos dos fármacos , Transfecção , Células Tumorais CultivadasRESUMO
We incubated primary rat hepatocytes on collagen monolayer as well as in collagen sandwich cultures with serum-containing or serum-free medium formulations. Morphological monitoring of hepatocytes revealed that hepatocytes cultured on collagen monolayer adopted their polygonal shape and started to create aggregates earlier than sandwich-cultured cells. Bile canaliculi-like structures were observed in every cell culture system but were more prominent in serum-free cultures. Hepatocytes in collagen-sandwich configuration and serum-free medium were the most viable after 72 h of culture, still displaying polygonal shape, clear cytoplasm and stable canaliculi-like structures. Differential gene expression patterns were determined for each cell culture condition using quantitative TaqMan Low Density Arrays (LDA). Gene expression analysis revealed distinct profiles in monolayer versus sandwich cultures and in particular in serum-free versus serum-containing culture medium. The hepatocytes cultured in the collagen-sandwich with serum-free medium showed the least variation in expression values over time. Importantly, stress markers were not induced in the serum-free sandwich culture, in contrast to the monolayer and the serum-containing sandwich cultures. Additionally, expression of the investigated cytochrome P450 genes was maintained in the serum-free monolayer and the sandwich cultures. In conclusion, culturing primary rat hepatocytes in a sandwich between two layers of gelled collagen and in a serum-free medium formulation, appears to be most suitable for long-term in vitro hepatotoxicity screening.
Assuntos
Técnicas de Cultura de Células , Perfilação da Expressão Gênica , Hepatócitos/citologia , Animais , Bovinos , Sobrevivência Celular , Células Cultivadas , Colágeno , Meios de Cultura , Meios de Cultura Livres de Soro , Dimetil Sulfóxido/farmacologia , Matriz Extracelular , Hepatócitos/metabolismo , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , SoroRESUMO
The androgen receptor (AR) is a member of the nuclear receptor superfamily and mediates the physiological effects of androgens. Androgens are essential for male development and disruption of androgen signaling may cause androgen-dependent developmental defects and/or tumors. Here we present a comparative analysis of various model systems for the investigation of endocrine active compounds in human cell lines. We generated reporter plasmids containing androgen response elements derived from the human secretory component or the rat probasin genes as well as the glucocorticoid consensus response element and compared their activities to that of the mouse mammary tumor virus promotor. Additionally, we generated an expression plasmid containing the AR cDNA derived from LNCaP cells. In 22Rv1 cells transiently transfected with human AR, all reporters displayed a dose-dependent, high activity when treated with androgens. Interestingly, the potency of testosterone and its metabolite dihydrotestosterone was very low in HepG2 but not in 22Rv1 cells, independent of the reporter used. The efficacies of the androgens tested were comparable in both cell lines but highly dependent on the reporter used. Based on these results, 22Rv1 cells provide a highly sensitive in vitro test system to analyze endocrine activities of xenobiotics. Furthermore, this study highlights the need to investigate the (anti-) androgenic activity of compounds in dependence of the cellular and promoter context.
Assuntos
Antagonistas de Androgênios/farmacologia , Androgênios/farmacologia , Genes Reporter/genética , Regiões Promotoras Genéticas/genética , Receptores Androgênicos/metabolismo , Animais , Ligação Competitiva , Bioensaio/métodos , Linhagem Celular Tumoral , Di-Hidrotestosterona/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Plasmídeos , Ratos , Testosterona/farmacologia , Transfecção , Xenobióticos/farmacologiaRESUMO
In MCF-7 (estrogen receptor (ER)+) and in MDA-MB-231 (ER-) cells stably transfected with either estrogen receptor alpha (ERalpha) or beta (ERbeta) subtype (MDA-MB-231 stably transfected with the mouse ERalpha cDNA (MERA) and MDA-MB-231 stably transfected with the human ERbeta cDNA (HERB), respectively) N-term heat shock protein of 90kDa (hsp90) ligands (geldanamycin and radicicol) and C-term hsp90 ligands (novobiocin) decrease the basal and estradiol (E(2))-induced transcription activity of ER on an estrogen responsive element (ERE)-LUC reporter construct concomitantly with or 1h after E(2) treatment. All hsp90 ligands induced an E(2)- and MG132-inhibited decrease of both ER cell content. However, the kinetics of these degradations are slower than those induced by the selective estrogen receptor down-regulator RU 58668 (RU). This suggests that inhibition of the hsp90 ATPase activity targets both ERs to the 26S proteasome and that hsp90 interacts with both ER subtypes. Rapamycin (Rapa) and cyclosporin A (CsA), ligands of immunophilins FK506 binding protein (FKBP52) and cyclophilin of 40kDa (CYP40) interacting in separate ER-hsp90 complexes, both induced a proteasomal-mediated degradation of ERs but not of their cognate immunophilin. Moreover, they also decrease the E(2)-induced luciferase transcription but weaker than RU and hsp90 ligands. Fluorescence activated cell sorter (FACS) analysis revealed a blockade of cell progression by RU and 4-hydroxy-tamoxifen at the G(1) phase of the cell cycle and an induction of apoptosis in MCF-7 cells. Rapa and mainly CsA (but not FK506) and hsp90 ligands promote by their own apoptosis in MCF-7, in MERA, and in HERB cells and in MDA-MB-231 ER-null cells. These data suggest that (1) hsp90, as for all steroid receptors, acts as a molecular chaperone for ERbeta; (2) ER-ligands (except tamoxifen), hsp90- and immunophilin-ligands (except FK506) target the two ER subtypes to a proteasome-mediated proteolysis via different signalling pathways; (3) hsp90- and immunophilin-ligands Rapa and CsA, alone or in association with anti-estrogens such as RU, may constitute a potential therapeutic strategy for breast cancer treatment.