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1.
J Immunol ; 188(11): 5210-20, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22611248

RESUMO

The cytokine-induced activation cascade of NF-kappaB in mammals and the activation of the morphogen dorsal in Drosophila embryos show striking structural and functional similarities (Toll/IL-1, Cactus/I-kappaB, and dorsal/NF-kappaB). Here we demonstrate that these parallels extend to the immune response of Drosophila. In particular, the intracellular components of the dorsoventral signaling pathway (except for dorsal) and the extracellular Toll ligand, spätzle regulatory gene cassette, control expression of the antifungal peptide gene drosomycin in adults. We also show that mutations in the Toll signaling pathway dramatically reduce survival after fungal infection. Antibacterial genes are induced either by a distinct pathway involving the immune deficiency gene (imd) or by combined activation of both imd and dorsoventral pathways.


Assuntos
Antifúngicos/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/imunologia , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Micoses/imunologia , Micoses/prevenção & controle , Fosfoproteínas/fisiologia , Receptores Toll-Like/fisiologia , Animais , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , História do Século XX , Família Multigênica/genética , Micoses/metabolismo
2.
MAbs ; 16(1): 2324801, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38441119

RESUMO

Biologics have revolutionized disease management in many therapeutic areas by addressing unmet medical needs and overcoming resistance to standard-of-care treatment in numerous patients. However, the development of unwanted immune responses directed against these drugs, humoral and/or cellular, can hinder their efficacy and have safety consequences with various degrees of severity. Health authorities ask that a thorough immunogenicity risk assessment be conducted during drug development to incorporate an appropriate monitoring and mitigation plan in clinical studies. With the rapid diversification and complexification of biologics, which today include modalities such as multi-domain antibodies, cell-based products, AAV delivery vectors, and nucleic acids, developers are faced with the challenge of establishing a risk assessment strategy sometimes in the absence of specific regulatory guidelines. The European Immunogenicity Platform (EIP) Open Symposium on Immunogenicity of Biopharmaceuticals and its one-day training course gives experts and newcomers across academia, industry, and regulatory agencies an opportunity to share experience and knowledge to overcome these challenges. Here, we report the discussions that took place at the EIP's 14th Symposium, held in April 2023. The topics covered included immunogenicity monitoring and clinical relevance, non-clinical immunogenicity risk assessment, regulatory aspects of immunogenicity assessment and reporting, and the challenges associated with new modalities, which were discussed in a dedicated session.


Assuntos
Produtos Biológicos , Humanos , Anticorpos , Desenvolvimento de Medicamentos , Medição de Risco
3.
AAPS J ; 24(3): 68, 2022 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-35554731

RESUMO

The determination of a tailored anti-drug antibody (ADA) testing strategy is based on the immunogenicity risk assessment to allow a correlation of ADAs with changes to pharmacokinetics, efficacy, and safety. The clinical impact of ADA formation refines the immunogenicity risk assessment and defines appropriate risk mitigation strategies. Health agencies request for high-risk biotherapeutics to extend ADA monitoring for patients that developed an ADA response to the drug until ADAs return to baseline levels. However, there is no common understanding in which cases an extension of ADA follow-up sampling beyond the end of study (EOS) defined in the clinical study protocol is required. Here, the Immunogenicity Strategy Working Group of the European Immunogenicity Platform (EIP) provides recommendations on requirements for an extension of ADA follow-up sampling in clinical studies where there is a high risk of serious consequences from ADAs. The importance of ADA evaluation during a treatment-free period is recognized but the decision whether to extend ADA monitoring at a predefined EOS should be based on evaluation of ADA data in the context of corresponding clinical signals. If the clinical data set shows that safety consequences are minor, mitigated, or resolved, further ADA monitoring may not be required despite potentially detectable ADAs above baseline. Extended ADA monitoring should be centered on individual patient benefit.


Assuntos
Anticorpos , Humanos
4.
Dev Growth Differ ; 53(9): 982-93, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22150153

RESUMO

We present an evolutionary approach to dissecting conserved developmental mechanisms. We reason that important mechanisms for making the bodyplan will act early, to generate the major features of the body and that they will be conserved in evolution across many metazoa, and thus, that they will be available in very different animals. This led to our specific approach of microarrays to screen for very early conserved developmental regulators in parallel in an insect, Drosophila and a vertebrate, Xenopus. We screened for the earliest conserved targets of the ectopically expressed hox gene Hoxc6/Antennapedia in both species and followed these targets up, using in situ hybridization, in the Xenopus system. The results indicate that relatively few of the early Hox target genes are conserved: these are mainly involved in the specification of the antero-posterior body axis and in gastrulation.


Assuntos
Proteína do Homeodomínio de Antennapedia/genética , Proteína do Homeodomínio de Antennapedia/metabolismo , Evolução Biológica , Genes Homeobox , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Animais , Proteína do Homeodomínio de Antennapedia/biossíntese , Padronização Corporal/genética , Drosophila/embriologia , Drosophila/genética , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Genômica/métodos , Proteínas de Homeodomínio/biossíntese , Xenopus/embriologia , Xenopus/genética , Proteínas de Xenopus/biossíntese
5.
Curr Biol ; 16(12): 1224-31, 2006 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-16782014

RESUMO

The development of the complex network of epithelial tubes that ultimately forms the Drosophila tracheal system relies on cell migration, cell shape changes, cell rearrangements, cell differentiation, and branch fusion . Most of these events are controlled by a combination of distinct transcription factors and cell-cell signaling molecules, but few proteins that do not fall within these two functional classes have been associated with tracheal development. We show that the MAGUK protein Polychaetoid (Pyd/ZO-1), the Drosophila homolog of the junctional protein ZO-1 , plays a dual role in the formation of tracheal tubes. pyd/ZO-1 mutant embryos display branch fusion defects due to the lack of reliable determination of the fusion cell fate. In addition, pyd/ZO-1 mutant embryos show impaired cell intercalation in thin tracheal branches. Pyd/ZO-1 localizes to the adherens junctions (AJs) in tracheal cells and might thus play a direct role in the regulation of the dynamic state of the AJ during epithelial remodeling. Our study suggests that MAGUK proteins might play important roles during AJ remodeling in epithelial morphogenesis.


Assuntos
Proteínas de Drosophila/fisiologia , Drosophila/embriologia , Proteínas de Membrana/fisiologia , Morfogênese , Fosfoproteínas/fisiologia , Traqueia/embriologia , Junções Aderentes/metabolismo , Animais , Diferenciação Celular , Drosophila/citologia , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Proteínas de Fluorescência Verde/análise , Larva/citologia , Larva/genética , Larva/crescimento & desenvolvimento , Proteínas de Membrana/genética , Morfogênese/genética , Mutação , Fosfoproteínas/genética , Proteínas de Junções Íntimas , Traqueia/citologia , Fatores de Transcrição/metabolismo , Proteína da Zônula de Oclusão-1
6.
Mol Ther Methods Clin Dev ; 8: 105-120, 2018 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-29359172

RESUMO

Retinitis pigmentosa is a form of retinal degeneration usually caused by genetic mutations affecting key functional proteins. We have previously demonstrated efficacy in a mouse model of RLBP1 deficiency with a self-complementary AAV8 vector carrying the gene for human RLBP1 under control of a short RLBP1 promoter (CPK850).1 In this article, we describe the nonclinical safety profile of this construct as well as updated efficacy data in the intended clinical formulation. In Rlbp1-/- mice dosed at a range of CPK850 levels, a minimum efficacious dose of 3 × 107 vg in a volume of 1 µL was observed. For safety assessment in these and Rlbp1+/+ mice, optical coherence tomography (OCT) and histopathological analysis indicated retinal thinning that appeared to be dose-dependent for both Rlbp1 genotypes, with no qualitative difference noted between Rlbp1+/+ and Rlbp1-/- mice. In a non-human primate study, RLBP1 mRNA expression was detected and dose dependent intraocular inflammation and retinal thinning were observed. Inflammation resolved slowly over time and did not appear to be exacerbated in the presence of anti-AAV8 antibodies. Biodistribution was evaluated in rats and satellite animals in the non-human primate study. The vector was largely detected in ocular tissues and low levels in the optic nerve, superior colliculus, and lateral geniculate nucleus, with limited distribution outside of these tissues. These data suggest that an initial subretinal dose of ∼3 × 107 vg/µL CPK850 can safely be used in clinical trials.

7.
FASEB J ; 20(12): 2036-49, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17012256

RESUMO

RPE65 is the retinal isomerase essential for conversion of all-trans-retinyl ester to 11-cis-retinol in the visual cycle. Leber's congenital amaurosis (LCA), an autosomal recessive form of RP resulting in blindness, is commonly caused by mutations in the Rpe65 gene. Whereas the molecular mechanisms by which these mutations contribute to retinal disease remain largely unresolved, affected patients show marked RPE damage and photoreceptor degeneration. We evaluated gene expression in Rpe65-/- mouse model of LCA before and at the onset of photoreceptor cell death in 2, 4, and 6 month old animals. Microarray analysis demonstrates altered expression of genes involved in phototransduction, apoptosis regulation, cytoskeleton organization, and extracellular matrix (ECM) constituents. Cone-specific phototransduction genes are strongly decreased, reflecting early loss of cones. In addition, remaining rods show modified expression of genes encoding components of the cytoskeleton and ECM. This may affect rod physiology and interaction with the adjacent RPE and lead to loss of survival signals, as reflected by the alteration of apoptosis-related genes Together, these results suggest that RPE65 defect triggers an overall remodeling of the neurosensitive retina that may, in turn, disrupt photoreceptor homeostasis and induce apoptosis signaling cascade toward retinal cell death.


Assuntos
Cegueira/genética , Proteínas do Olho/genética , Regulação da Expressão Gênica , Animais , Apoptose/genética , Cegueira/etiologia , Cegueira/patologia , Proteínas de Transporte , Proteínas do Citoesqueleto/genética , Modelos Animais de Doenças , Progressão da Doença , Proteínas da Matriz Extracelular/genética , Perfilação da Expressão Gênica , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Células Fotorreceptoras/patologia , Visão Ocular/genética , cis-trans-Isomerases/genética
8.
Bioanalysis ; 6(10): 1395-407, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24958123

RESUMO

BACKGROUND: To generate exhaustive data on the stability of human anti-immunotherapeutic antibodies. MATERIALS & METHODS: Samples collected from over 100 different subjects at various timepoints were analyzed shortly after serum collection using specific ELISAs and re-analyzed after long-term storage or multiple cycles of freeze-thaw. The general acceptance criteria for incurred sample reanalysis for ligand-binding assays were applied, as well as alternative stricter acceptance criteria promoted by various white papers. RESULTS: Anti-immunotherapeutic antibodies are stable in undiluted serum samples stored at -80°C for at least 3.5 years and 3-12 freeze-thaw cycles. CONCLUSION: Samples were selected to cover the heterogeneity of the polyclonal human immune response, therefore this stability data can be extended to all anti-vaccine and anti-drug antibodies.


Assuntos
Anticorpos/sangue , Ensaio de Imunoadsorção Enzimática , Anticorpos/imunologia , Anticorpos/metabolismo , Congelamento , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Serviços Terceirizados , Estabilidade Proteica , Temperatura , Fatores de Tempo , Vacinação , Vacinas/imunologia
9.
Bioanalysis ; 6(10): 1409-13, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24958124

RESUMO

Long- and short-term stability testing of the analyte is one of the key parameters in bioanalytical method validation in support of pharmacokinetics. However, for immunogenicity testing, the scientific rationale for long- and short-term stability testing on quality control samples most often spiked with polyclonal antibody raised in a different species should be questioned. Therefore, the European Bioanalysis Forum (EBF) formed a Topic Team to discuss the scientific rationale for stability testing of anti-drug antibodies (ADAs). A review of EBF member companies' experience on ADA stability and on anti-vaccine antibodies from vaccine projects was the basis of this discussion. EBF recommends to perform short-term stability testing of the positive control, but not to perform long-term stability testing of ADAs in nonclinical and clinical studies.


Assuntos
Anticorpos/sangue , Vacinas/imunologia , Anticorpos/metabolismo , Europa (Continente) , Humanos , Estabilidade Proteica , Controle de Qualidade , Temperatura , Fatores de Tempo
11.
Proc Natl Acad Sci U S A ; 103(39): 14417-22, 2006 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-16971484

RESUMO

Mushroom bodies (MBs) are the centers for olfactory associative learning and elementary cognitive functions in the Drosophila brain. As a way to systematically elucidate genes preferentially expressed in MBs, we have analyzed genome-wide alterations in transcript profiles associated with MB ablation by hydroxyurea. We selected 100 genes based on microarray data and examined their expression patterns in the brain by in situ hybridization. Seventy genes were found to be expressed in the posterodorsal cortex, which harbors the MB cell bodies. These genes encode proteins of diverse functions, including transcription, signaling, cell adhesion, channels, and transporters. Moreover, we have examined developmental functions of 40 of the microarray-identified genes by transgenic RNA interference; 8 genes were found to cause mild-to-strong MB defects when suppressed with a MB-Gal4 driver. These results provide important information not only on the repertoire of genes that control MB development but also on the repertoire of neural factors that may have important physiological functions in MB plasticity.


Assuntos
Drosophila melanogaster/genética , Hidroxiureia/farmacologia , Análise em Microsséries/métodos , Corpos Pedunculados/efeitos dos fármacos , RNA Mensageiro/genética , Animais , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos/genética , Corpos Pedunculados/anormalidades , Corpos Pedunculados/citologia , Interferência de RNA
12.
Proc Natl Acad Sci U S A ; 100(7): 4024-9, 2003 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-12655063

RESUMO

Pax-6 genes encode evolutionarily conserved transcription factors capable of activating the gene-expression program required to build an eye. When ectopically expressed in Drosophila imaginal discs, Pax-6 genes induce the eye formation on the corresponding appendages of the adult fly. We used two different Drosophila full-genome DNA microarrays to compare gene expression in wild-type leg discs versus leg discs where eyeless, one of the two Drosophila Pax-6 genes, was ectopically expressed. We validated these data by analyzing the endogenous expression of selected genes in eye discs and identified 371 genes that are expressed in the eye imaginal discs and up-regulated when an eye morphogenetic field is ectopically induced in the leg discs. These genes mainly encode transcription factors involved in photoreceptor specification, signal transducers, cell adhesion molecules, and proteins involved in cell division. As expected, genes already known to act downstream of eyeless during eye development were identified, together with a group of genes that were not yet associated with eye formation.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Olho/embriologia , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Embrião não Mamífero/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Análise de Sequência com Séries de Oligonucleotídeos/métodos
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