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1.
J Immunol ; 204(4): 775-787, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31900335

RESUMO

Immunogenic cell death (ICD) occurs when a dying cell releases cytokines and damage-associated molecular patterns, acting as adjuvants, and expresses Ags that induce a specific antitumor immune response. ICD is studied mainly in the context of regulated cell death pathways, especially caspase-mediated apoptosis marked by endoplasmic reticulum stress and calreticulin exposure and, more recently, also in relation to receptor-interacting protein kinase-driven necroptosis, whereas unregulated cell death like accidental necrosis is nonimmunogenic. Importantly, the murine cancer cell lines used in ICD studies often express virally derived peptides that are recognized by the immune system as tumor-associated Ags. However, it is unknown how different cell death pathways may affect neoepitope cross-presentation and Ag recognition of cancer cells. We used a prophylactic tumor vaccination model and observed that both apoptotic and necroptotic colon carcinoma CT26 cells efficiently immunized mice against challenge with a breast cancer cell line that expresses the same immunodominant tumor Ag, AH1, but only necroptotic CT26 cells would mount an immune response against CT26-specific neoepitopes. By CRISPR/Cas9 genome editing, we knocked out AH1 and saw that only necroptotic CT26 cells were still able to protect mice against tumor challenge. Hence, in this study, we show that endogenous AH1 tumor Ag expression can mask the strength of immunogenicity induced by different cell death pathways and that upon knockout of AH1, necroptosis was more immunogenic than apoptosis in a prophylactic tumor vaccination model. This work highlights necroptosis as a possible preferred ICD form over apoptosis in the treatment of cancer.


Assuntos
Antígenos de Neoplasias/imunologia , Apoptose/imunologia , Epitopos Imunodominantes/imunologia , Necroptose/imunologia , Neoplasias Experimentais/imunologia , Animais , Linhagem Celular Tumoral , Feminino , Camundongos , Camundongos Endogâmicos BALB C
2.
Biotechnol J ; 13(8): e1700747, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29436782

RESUMO

Choosing a potent selection antibiotic (SA), is a crucial success factor when creating stably transfected cell lines using an antibiotic selection marker. The selection capacity of this antibiotic is defined as its ability to kill sensitive, untransfected parental cells, while leaving resistant, transfected cells unharmed. Currently, no procedure has been described to determine this selection capacity. Therefore, a protocol to obtain a numerical value, called the "selectivity factor" (SF), that defines the selection capacity of SAs is developed. The SF is determined by using a modified MTT (3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide) assay for both sensitive and resistant cells, and applies to commonly used cell lines. To prove the concept, the SF of the SA G418 and hygromycin B (HmB) on several cell lines is determined. The SF of G418 on BHK-21 cells is very high, indicating that G418 is an ideal SA for transfected BHK-21 cells. For HeLa cells, the SF of G418 is very low suggesting G418 is not an optimal SA for selecting transfected HeLa cells. For these cells, HmB would be a better choice. These conclusions are confirmed by an independent cell death assay. The SF identifies the most optimal SA for a certain cell line, reduces the risk of selecting spontaneously resistant cell clones, and streamlines the process of generating stable cell lines. Most importantly, the method is especially time saving when obtaining stable cell lines expressing toxic genes, and reduces culture times for generating large numbers of cell lines from the same parental cell line.


Assuntos
Antibacterianos/metabolismo , Técnicas de Cultura de Células/normas , Técnicas Genéticas/normas , Vetores Genéticos/metabolismo , Transfecção/métodos , Antibacterianos/farmacologia , Contagem de Células , Vetores Genéticos/genética , Células HeLa , Humanos , Modelos Lineares , Sais de Tetrazólio/metabolismo , Tiazóis/metabolismo
3.
Nat Protoc ; 11(8): 1444-54, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27414760

RESUMO

Several cell death assays have been developed based on a single biochemical parameter such as caspase activation or plasma membrane permeabilization. Our fluorescent apoptosis/necrosis (FAN) assay directly measures cell death and distinguishes between caspase-dependent apoptosis and caspase-independent necrosis of cells grown in any multiwell plate. Cell death is monitored in standard growth medium as an increase in fluorescence intensity of a cell-impermeable dye (SYTOX Green) after plasma membrane disintegration, whereas apoptosis is detected through caspase-mediated release of a fluorophore from its quencher (DEVD-amc). The assay determines the normalized percentage of dead cells and caspase activation per condition as an end-point measurement or in real time (automated). The protocol can be applied to screen drugs, proteins or siRNAs for interference with cell death while simultaneously detecting cell death modality switching between apoptosis and necrosis. Initial preparation may take up to 5 d, but the typical hands-on time is ∼2 h.


Assuntos
Morte Celular , Fluorometria/métodos , Animais , Linhagem Celular , Humanos , Camundongos , Coloração e Rotulagem , Fatores de Tempo
4.
Cell Rep ; 15(2): 274-87, 2016 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-27050509

RESUMO

Successful immunogenic apoptosis in experimental cancer therapy depends on the induction of strong host anti-tumor responses. Given that tumors are often resistant to apoptosis, it is important to identify alternative molecular mechanisms that elicit immunogenic cell death. We have developed a genetic model in which direct dimerization of FADD combined with inducible expression of RIPK3 promotes necroptosis. We report that necroptotic cancer cells release damage-associated molecular patterns and promote maturation of dendritic cells, the cross-priming of cytotoxic T cells, and the production of IFN-γ in response to tumor antigen stimulation. Using both FADD-dependent and FADD-independent RIPK3 induction systems, we demonstrate the efficient vaccination potential of immunogenic necroptotic cells. Our study broadens the current concept of immunogenic cell death and opens doors for the development of new strategies in cancer therapy.


Assuntos
Antineoplásicos/imunologia , Apoptose , Imunidade , Neoplasias/imunologia , Vacinação , Alarminas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimiocinas/metabolismo , Apresentação Cruzada/efeitos dos fármacos , Apresentação Cruzada/imunologia , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Imunidade/efeitos dos fármacos , Ligantes , Camundongos , Modelos Biológicos , NF-kappa B/metabolismo , Necrose , Fagocitose/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Tetraciclina/farmacologia
5.
Nat Immunol ; 15(3): 248-57, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24441789

RESUMO

The role of the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in homeostasis of the immune system is incompletely understood. Here we found that dendritic cells (DCs) constitutively activated the UPR sensor IRE-1α and its target, the transcription factor XBP-1, in the absence of ER stress. Loss of XBP-1 in CD11c+ cells led to defects in phenotype, ER homeostasis and antigen presentation by CD8α+ conventional DCs, yet the closely related CD11b+ DCs were unaffected. Whereas the dysregulated ER in XBP-1-deficient DCs resulted from loss of XBP-1 transcriptional activity, the phenotypic and functional defects resulted from regulated IRE-1α-dependent degradation (RIDD) of mRNAs, including those encoding CD18 integrins and components of the major histocompatibility complex (MHC) class I machinery. Thus, a precisely regulated feedback circuit involving IRE-1α and XBP-1 controls the homeostasis of CD8α+ conventional DCs.


Assuntos
Apresentação Cruzada/imunologia , Proteínas de Ligação a DNA/imunologia , Células Dendríticas/imunologia , Endorribonucleases/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Desdobramento de Proteína , Fatores de Transcrição/imunologia , Resposta a Proteínas não Dobradas/imunologia , Animais , Apresentação de Antígeno/imunologia , Antígenos CD8/imunologia , Antígenos CD8/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Dendríticas/metabolismo , Retículo Endoplasmático/imunologia , Endorribonucleases/metabolismo , Retroalimentação Fisiológica/fisiologia , Homeostase/imunologia , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição de Fator Regulador X , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação a X-Box
6.
Expert Rev Vaccines ; 11(6): 695-719, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22873127

RESUMO

The aim of this review is to make researchers aware of the benefits of an efficient quality control system for prediction of a developed vaccine's efficacy. Two major goals should be addressed when inactivating a virus for vaccine purposes: first, the infectious virus should be inactivated completely in order to be safe, and second, the viral epitopes important for the induction of protective immunity should be conserved after inactivation in order to have an antigen of high quality. Therefore, some problems associated with the virus inactivation process, such as virus aggregate formation, protein crosslinking, protein denaturation and degradation should be addressed before testing an inactivated vaccine in vivo.


Assuntos
Tecnologia Farmacêutica/métodos , Vacinas Virais/química , Vacinas Virais/imunologia , Viroses/prevenção & controle , Inativação de Vírus , Animais , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Humanos , Controle de Qualidade , Tecnologia Farmacêutica/normas , Vacinas de Produtos Inativados/efeitos adversos , Vacinas de Produtos Inativados/química , Vacinas de Produtos Inativados/imunologia , Vacinas Virais/efeitos adversos , Viroses/imunologia , Vírus/imunologia , Vírus/patogenicidade
7.
PLoS One ; 6(2): e16827, 2011 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-21359217

RESUMO

Sialoadhesin is exclusively expressed on specific subpopulations of macrophages. Since sialoadhesin-positive macrophages are involved in inflammatory autoimmune diseases, such as multiple sclerosis, and potentially in the generation of immune responses, targeted delivery of drugs, toxins or antigens via sialoadhesin-specific immunoconjugates may prove a useful therapeutic strategy. Originally, sialoadhesin was characterized as a lymphocyte adhesion molecule, though recently its involvement in internalization of sialic acid carrying pathogens was shown, suggesting that sialoadhesin is an endocytic receptor. In this report, we show that porcine sialoadhesin-specific antibodies and F(ab')2 fragments trigger sialoadhesin internalization, both in primary porcine macrophages and in cells expressing recombinant porcine sialoadhesin. Using chemical inhibitors, double immunofluorescence stainings and dominant-negative constructs, porcine sialoadhesin internalization was shown to be clathrin- and Eps15-dependent and to result in targeting to early endosomes but not lysosomes. Besides characterizing the sialoadhesin endocytosis mechanism, two sialoadhesin-specific immunoconjugates were evaluated. We observed that porcine sialoadhesin-specific immunotoxins efficiently kill sialoadhesin-expressing macrophages. Furthermore, porcine sialoadhesin-specific albumin immunoconjugates were shown to be internalized in macrophages and immunization with these immunoconjugates resulted in a rapid and robust induction of albumin-specific antibodies, this compared to immunization with albumin alone. Together, these data expand sialoadhesin functionality and show that it can function as an endocytic receptor, a feature that cannot only be misused by sialic acid carrying pathogens, but that may also be used for specific targeting of toxins or antigens to sialoadhesin-expressing macrophages.


Assuntos
Antígenos/metabolismo , Macrófagos Alveolares/metabolismo , Glicoproteínas de Membrana/fisiologia , Receptores Imunológicos/fisiologia , Toxinas Biológicas/metabolismo , Vesículas Transportadoras/metabolismo , Animais , Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Endocitose/fisiologia , Imunotoxinas/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/imunologia , Glicoproteínas de Membrana/metabolismo , Transporte Proteico/fisiologia , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/metabolismo , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico , Suínos
8.
BMC Biotechnol ; 10: 48, 2010 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-20587060

RESUMO

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) causes major economic losses in the pig industry worldwide. In vivo, the virus infects a subpopulation of tissue macrophages. In vitro, PRRSV only replicates in primary pig macrophages and African green monkey kidney derived cells, such as Marc-145. The latter is currently used for vaccine production. However, since virus entry in Marc-145 cells is different compared to entry in primary macrophages, specific epitopes associated with virus entry could potentially alter upon growth on Marc-145 cells. To avoid this, we constructed CHO and PK15 cell lines recombinantly expressing the PRRSV receptors involved in virus entry into macrophages, sialoadhesin (Sn) and CD163 (CHOSn-CD163 and PK15Sn-CD163) and evaluated their potential for production of PRRSV. RESULTS: Detailed analysis of PRRSV infection revealed that LV and VR-2332 virus particles could attach to and internalize into the CHOSn-CD163 and PK15Sn-CD163 cells. Initially, this occurred less efficiently for macrophage grown virus than for Marc-145 grown virus. Upon internalization, disassembly of the virus particles was observed. The two cell lines could be infected with PRRSV strains LV and VR-2332. However, it was observed that Marc-145 grown virus infected the cells more efficiently than macrophage grown virus. If the cells were treated with neuraminidase to remove cis-acting sialic acids that hinder the interaction of the virus with Sn, the amount of infected cells with macrophage grown virus increased. Comparison of both cell lines showed that the PK15Sn-CD163 cell line gave in general better results than the CHOSn-CD163 cell line. Only 2 out of 5 PRRSV strains replicated well in CHOSn-CD163 cells. Furthermore, the virus titer of all 5 PRRSV strains produced after passaging in PK15Sn-CD163 cells was similar to the virus titer of those strains produced in Marc-145 cells. Analysis of the sequence of the structural proteins of original virus and virus grown for 5 passages on PK15Sn-CD163 cells showed either no amino acid (aa) changes (VR-2332 and 07V063), one aa (LV), two aa (08V194) or three aa (08V204) changes. None of these changes are situated in known neutralizing epitopes. CONCLUSIONS: A PRRSV susceptible cell line was constructed that can grow virus to similar levels compared to currently available cell lines. Mutations induced by growth on this cell lines were either absent or minimal and located outside known neutralizing epitopes. Together, the results show that this cell line can be used to produce vaccine virus and for PRRSV virus isolation.


Assuntos
Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/genética , Linhagem Celular , Glicoproteínas de Membrana/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Receptores de Superfície Celular/genética , Receptores Imunológicos/genética , Cultura de Vírus , Animais , Células CHO , Técnicas de Cultura de Células , Cricetinae , Cricetulus , Macrófagos/virologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico , Suínos , Transfecção
9.
Vet Res ; 40(6): 62, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19674538

RESUMO

Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe economic losses in the pig industry worldwide. Currently, vaccines based on inactivated PRRSV provide limited protection of pigs against infection, most likely because viral epitopes associated with the induction of neutralizing antibodies are not or poorly conserved during inactivation. To analyze the effect of inactivation procedures on the interaction of PRRSV with receptors involved in virus entry, a new assay was set up in this study. Viral entry-associated domains are most likely important for the induction of neutralizing antibodies, since neutralizing antibodies block interaction of PRRSV with cellular receptors. To investigate the interaction of PRRSV with the cellular receptors upon different inactivation procedures, attachment to and internalization of inactivated PRRSV into macrophages were monitored. AT-2 could not inactivate PRRSV completely and is therefore not useful for vaccine development. PRRSV inactivated with ultraviolet light, binary ethyleneimine and gamma irradiation, which all mainly have an effect at the genomic level, showed no difference compared to control live virus at all levels of virus entry, whereas PRRSV treated with formaldehyde, glutaraldehyde and pH changes, which all have a modifying effect on proteins, was not able to internalize into macrophages anymore. These results suggest that inactivation with methods with a main effect on the viral genome preserve PRRSV entry-associated domains and are useful for future development of an effective inactivated vaccine against PRRSV. Although PRRSV incubation at 37 degrees C can completely inactivate PRRSV with preservation of entry-associated domains, this method is not recommended for vaccine development, since the mechanism is yet unknown.


Assuntos
Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Vacinas de Produtos Inativados/imunologia , Proteínas Virais/genética , Vacinas Virais/imunologia , Inativação de Vírus , Internalização do Vírus , Animais , Linhagem Celular , Macrófagos/virologia , Estrutura Terciária de Proteína , Suínos , Proteínas Virais/química , Replicação Viral
10.
Vet Res ; 40(6): 63, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19674539

RESUMO

Porcine reproductive and respiratory syndrome virus (PRRSV) can induce reproductive disorders and is involved in the porcine respiratory disease complex, causing tremendous economic losses to the swine industry. Inactivated PRRSV vaccines are preferred over attenuated vaccines because of their safety and flexibility towards emerging virus strains, but the efficacy of current inactivated PRRSV vaccines is questionable. In this study, experimental inactivated PRRSV vaccines were developed, based on two formerly optimized inactivation procedures: UV irradiation and treatment with binary ethylenimine (BEI). In a first experiment, it was shown that vaccination with UV- or BEI-inactivated virus in combination with Incomplete Freund's Adjuvant induced virus-specific antibodies and strongly primed the virus-neutralizing (VN) antibody response. Subsequently, the influence of adjuvants on the immunogenicity of neutralizing epitopes on the inactivated virus was investigated. It was shown that vaccination with BEI-inactivated virus in combination with a commercial oil-in-water adjuvant induced high titers (3.4 log(2)) of VN antibodies in 6/6 pigs, instead of only priming the neutralizing antibody response. After challenge, neutralizing antibody titers in these vaccinated animals rose to a mean value of 5.5 log(2), and the duration of the viremia was reduced to an average of 1 week. This study shows that, by the use of an optimized inactivation procedure and a suitable adjuvant, inactivated PRRSV vaccines can be developed that induce VN antibodies and offer partial protection upon challenge.


Assuntos
Anticorpos Antivirais/sangue , Síndrome Respiratória e Reprodutiva Suína/prevenção & controle , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Síndrome Respiratória e Reprodutiva Suína/sangue , Síndrome Respiratória e Reprodutiva Suína/imunologia , Suínos , Fatores de Tempo , Vacinas Virais/administração & dosagem , Viremia
11.
J Virol ; 81(17): 9546-50, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17567703

RESUMO

The sialic acid-binding lectin sialoadhesin (Sn) is a macrophage-restricted receptor for porcine reproductive and respiratory syndrome virus (PRRSV). To investigate the importance of pSn sialic acid-binding activity for PRRSV infection, an R(116)-to-E mutation was introduced in the predicted sialic acid-binding domain of pSn, resulting in a mutant, pSn(RE), that could not bind sialic acids. PSn, but not pSn(RE), allowed PRRSV binding and internalization. These data show that the sialic acid-binding activity of pSn is essential for PRRSV attachment to pSn and thus identifies the variable, N-terminal domain of Sn as a PRRSV binding domain.


Assuntos
Macrófagos/virologia , Glicoproteínas de Membrana/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Receptores Imunológicos/metabolismo , Receptores Virais/metabolismo , Ligação Viral , Substituição de Aminoácidos/genética , Animais , Sítios de Ligação/genética , Células CHO , Cricetinae , Cricetulus , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Virais/genética , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico , Internalização do Vírus
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