RESUMO
Cervical cancer represents a global concern with 604,000 new cases and 342,000 deaths reported annually, with the vast majority diagnosed in low income countries. Despite high-risk Human Papillomavirus (HR HPV)-induced cervical cancer has become highly preventable through prophylactic vaccines, screening programs are critical in the control of cervical carcinogenesis in populations with limited access to vaccination and in older generations of women who have already been exposed to HR HPV infection. The surge of HPV molecular tests has provided a more sensitive and accurate diagnostic alternative to cytology screening. Given that HPV DNA testing presents a low positive predicted value, leading to unnecessary treatment, the E6 oncoprotein from HR HPV types arises as a promising diagnostic marker for its overexpression in transformed HPV-positive cancer cells. For these reasons, this study aimed at obtaining monoclonal antibodies (mAbs) against the E6 oncoprotein of one of the most prevalent HR HPV types worldwide, HPV18, in order to develop a highly specific and sensitive indirect competitive ELISA (icELISA). The production of hybridomas secreting HPV18 E6 mAbs was carried out through a combined tolerization and immunization strategy, in order to avoid cross-reactivity with the E6 protein from low-risk HPV types 6 and 11. We selected the 7D2 hybridoma clone, which recognized HPV18 E6 and showed some cross-reactivity against the HR HPV45 E6 oncoprotein. The 7D2 mAb enabled the development of a sensitive, reliable and reproducible icELISA to detect and quantify small amounts of HPV18 E6 biomarker for cervical cancer progression. The present study establishes a valid 7D2-based icELISA that constitutes a promising bioanalytical method for the early detection and quantification of HPV18 E6 oncoprotein in cervical swab samples and cancer prevention.
Assuntos
Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Neoplasias do Colo do Útero , Feminino , Humanos , Idoso , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/metabolismo , Papillomavirus Humano , Proteínas Repressoras/genética , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Papillomaviridae/metabolismo , Ensaio de Imunoadsorção EnzimáticaRESUMO
Zika virus (ZIKV) is a flavivirus that has emerged as a global health threat after the 2015 outbreak in the Americas, where devastating congenital defects were documented. There are currently no vaccines to prevent ZIKV infections nor commercially available clinical diagnostic tests demonstrated to identify ZIKV without cross-reactive interference of related flaviviruses. Early diagnosis is critical when treating symptomatic patients and in preventing ZIKV transmission. In this context, the development of sensitive and accurate diagnostic methods are urgently needed for the detection of ZIKV acute infection. The aim of this study consisted of obtaining monoclonal antibodies (mAbs) against denatured monomeric ZIKV Nonstructural protein 1 (ZNS1), a useful diagnostic marker for flavivirus early detection, in order to develop a highly specific and sensitive ZNS1 indirect competitive ELISA (icELISA). The production of hybridomas secreting ZNS1 mAbs was carried out through immunizations with denatured monomeric ZNS1. We selected 1F5 and 6E2 hybridoma clones, which recognized the heat-denatured ZNS1 hexameric form by indirect ELISA. Cross-reaction studies indicated that these mAbs specifically bind to a ZNS1 linear epitope, and that they do not cross-react with the NS1 protein from other related flaviviruses. The 1F5 mAb enabled the development of a sensitive and reproducible icELISA to detect and quantify small amounts of ZNS1 disease marker in heat-denatured human sera. Here, we establish a reliable 1F5 based-icELISA that constitutes a promising diagnostic tool for control strategies and the prevention of ZIKV propagation.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Antivirais/química , Antígenos Virais/genética , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas não Estruturais Virais/genética , Infecção por Zika virus/diagnóstico , Zika virus/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/isolamento & purificação , Antígenos Virais/administração & dosagem , Antígenos Virais/imunologia , Ligação Competitiva , Clonagem Molecular , Diagnóstico Precoce , Ensaio de Imunoadsorção Enzimática/normas , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Hibridomas/química , Hibridomas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Multimerização Proteica , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Proteínas não Estruturais Virais/administração & dosagem , Proteínas não Estruturais Virais/imunologia , Zika virus/genética , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologiaRESUMO
Sensitive, accurate and cost-effective diagnostic tests are urgently needed to detect Zika virus (ZIKV) infection. Nonstructural 1 (NS1) glycoprotein is an excellent diagnostic marker since it is released in a hexameric conformation from infected cells into the patient's bloodstream early in the course of the infection. We established a stable rZNS1-His-expression system in HEK293 cells through lentiviral transduction. A novel optimization approach to enhance rZNS1-His protein secretion in the mammalian expression system was accomplished through 50â¯nM rapamycin incubation followed by serum-free media incubation for 9 days, reaching protein yields of â¼10â¯mg/l of culture medium. Purified rZNS1-His hexamer was recognized by anti-NS1 antibodies in ZIKV patient's serum, and showed the ability to induce a humoral response in immunized mice. The obtained recombinant protein is a reliable biological tool that can potentially be applied in the development of diagnostic tests to detect ZIKV in infected patients during the acute phase.
RESUMO
Shiga toxin-producing Escherichia coli (STEC) is the major etiologic agent of hemolytic-uremic syndrome (HUS). The high rate of HUS emphasizes the urgency for the implementation of primary prevention strategies to reduce its public health impact. Argentina shows the highest rate of HUS worldwide, being E. coli O157 the predominant STEC-associated HUS serogroup (>70%), followed by E. coli O145 (>9%). To specifically detect these serogroups we aimed at developing highly specific monoclonal antibodies (mAbs) against the O-polysaccharide (O-PS) section of the lipopolysaccharide (LPS) of the dominant STEC-associated HUS serogroups in Argentina. The development of hybridomas secreting mAbs against O157 or O145 was carried out through a combined immunization strategy, involving adjuvated-bacterial immunizations followed by immunizations with recombinant O-PS-protein conjugates. We selected hybridoma clones that specifically recognized the engineered O-PS-protein conjugates of O157 or O145 serogroups. Indirect ELISA of heat-killed bacteria showed specific binding to O157 or O145 serogroups, respectively, while no cross-reactivity with other epidemiological important STEC strains, Brucella abortus, Salmonella group N or Yersinia enterocolitica O9 was observed. Western blot analysis showed specific recognition of the sought O-PS section of the LPS by all mAbs. Finally, the ability of the developed mAbs to bind the surface of whole bacteria cells was confirmed by flow cytometry, confocal microscopy and agglutination assays, indicating that these mAbs present an exceptional degree of specificity and relative affinity in the detection and identification of E. coli O157 and O145 serogroups. These mAbs may be of significant value for clinical diagnosis and food quality control applications. Thus, engineered O-PS specific moieties contained in the recombinant glycoconjugates used for combined immunization and hybridoma selection are an invaluable resource for the development of highly specific mAbs.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Síndrome Hemolítico-Urêmica/tratamento farmacológico , Síndrome Hemolítico-Urêmica/microbiologia , Escherichia coli Shiga Toxigênica/imunologia , Ensaio de Imunoadsorção Enzimática , Escherichia coli O157/imunologia , Hibridomas , Antígenos O/imunologia , Sorogrupo , SorotipagemRESUMO
Cow milk protein allergy (CMPA) is the most common childhood food allergy, which can sometimes persist or can newly develop in adulthood with severe symptoms. CMPA's treatment is complete dietary avoidance of milk proteins. To achieve this task, patients have to be aware of milk proteins found as "hidden allergens" in food commodities. In regard to milk proteins, it has been reported that allergenicity of caseins remains unaffected upon heat treatment. For these reasons, we aimed to obtain monoclonal antibodies (mAbs) against native and denatured ß-casein, one of the most abundant and antigenic caseins, in order to develop an indirect competitive ELISA (icELISA) to detect and quantify traces of this milk allergen in raw and processed foodstuffs. We developed two specific hybridoma clones, 1H3 and 6A12, which recognized ß-casein in its denatured and native conformations by indirect ELISA (iELISA). Cross-reaction analysis by Western blot and iELISA indicated that these mAbs specifically recognized ß-casein from bovine and goat milk extracts, while they did not cross-react with proteins present in other food matrixes. These highly specific mAbs enabled the development of sensitive, reliable and reproducible icELISAs to detect and quantify this milk protein allergen in food commodities. The extraction of ß-casein from foodstuff was efficiently carried out at 60°C for 15 minutes, using an extraction buffer containing 1% SDS. The present study establishes a valid 1H3 based-icELISA, which allows the detection and quantification -0.29 ppm and 0.80 ppm, respectively- of small amounts of ß-casein in raw and processed foods. Furthermore, we were able to detect milk contamination in incurred food samples with the same sensitivity as a commercial sandwich ELISA thus showing that this icELISA constitutes a reliable analytical method for control strategies in food industry and allergy prevention.
Assuntos
Alérgenos/imunologia , Caseínas/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Análise de Alimentos/métodos , Leite/imunologia , Animais , Anticorpos Monoclonais/imunologia , Bovinos , Laticínios/normas , Sensibilidade e EspecificidadeRESUMO
E2F transcription factors regulate a wide range of biological processes, including the cellular response to DNA damage. In the present study, we examined whether E2F family members are transcriptionally induced following treatment with several genotoxic agents, and have a role on the cell DNA damage response. We show a novel mechanism, conserved among diverse species, in which E2F1 and E2F2, the latter specifically in neuronal cells, are transcriptionally induced after DNA damage. This upregulation leads to increased E2F1 and E2F2 protein levels as a consequence of de novo protein synthesis. Ectopic expression of these E2Fs in neuronal cells reduces the level of DNA damage following genotoxic treatment, while ablation of E2F1 and E2F2 leads to the accumulation of DNA lesions and increased apoptotic response. Cell viability and DNA repair capability in response to DNA damage induction are also reduced by the E2F1 and E2F2 deficiencies. Finally, E2F1 and E2F2 accumulate at sites of oxidative and UV-induced DNA damage, and interact with γH2AX DNA repair factor. As previously reported for E2F1, E2F2 promotes Rad51 foci formation, interacts with GCN5 acetyltransferase and induces histone acetylation following genotoxic insult. The results presented here unveil a new mechanism involving E2F1 and E2F2 in the maintenance of genomic stability in response to DNA damage in neuronal cells.
Assuntos
Dano ao DNA , Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F2/metabolismo , Instabilidade Genômica , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Cicloeximida/toxicidade , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Dactinomicina/toxicidade , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F2/genética , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/efeitos da radiação , Células HEK293 , Histonas/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , MAP Quinase Quinase Quinases/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Inibidores da Síntese de Proteínas/toxicidade , Rad51 Recombinase/metabolismo , Raios Ultravioleta , Regulação para Cima/efeitos dos fármacos , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
DNA is continuously exposed to damaging agents that can lead to changes in the genetic information with adverse consequences. Nonetheless, eukaryotic cells have mechanisms such as the DNA damage response (DDR) to prevent genomic instability. The DNA of eukaryotic cells is packaged into nucleosomes, which fold the genome into highly condensed chromatin, but relatively little is known about the role of chromatin accessibility in DNA repair. p19INK4d, a cyclin-dependent kinase inhibitor, plays an important role in cell cycle regulation and cellular DDR. Extensive data indicate that p19INK4d is a critical factor in the maintenance of genomic integrity and cell survival. p19INK4d is upregulated by various genotoxics, improving the repair efficiency for a variety of DNA lesions. The evidence of p19INK4d translocation into the nucleus and its low sequence specificity in its interaction with DNA prompted us to hypothesize that p19INK4d plays a role at an early stage of cellular DDR. In the present study, we demonstrate that upon oxidative DNA damage, p19INK4d strongly binds to and relaxes chromatin. Furthermore, in vitro accessibility assays show that DNA is more accessible to a restriction enzyme when a chromatinized plasmid is incubated in the presence of a protein extract with high levels of p19INK4d. Nuclear protein extracts from cells overexpressing p19INK4d are better able to repair a chromatinized and damaged plasmid. These observations support the notion that p19INK4d would act as a chromatin accessibility factor that allows the access of the repair machinery to the DNA damage site.
Assuntos
Cromatina/metabolismo , Inibidor de Quinase Dependente de Ciclina p19/metabolismo , Dano ao DNA , Estresse Oxidativo , Transporte Ativo do Núcleo Celular , Animais , Northern Blotting , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Cromatina/genética , Inibidor de Quinase Dependente de Ciclina p19/genética , Reparo do DNA , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Microscopia Confocal , Ligação ProteicaRESUMO
BACKGROUND: During evolution, organisms with renewable tissues have developed mechanisms to prevent tumorigenesis, including cellular senescence and apoptosis. Cellular senescence is characterized by a permanent cell cycle arrest triggered by both endogenous stress and exogenous stress. The p19INK4d, a member of the family of cyclin-dependent kinase inhibitors (INK4), plays an important role on cell cycle regulation and in the cellular DNA damage response. We hypothesize that p19INK4d is a potential factor involved in the onset and/or maintenance of the senescent state. METHODS: Senescence was confirmed by measuring the cell cycle arrest and the senescence-associated ß-galactosidase activity. Changes in p19INK4d expression and localization during senescence were determined by Western blot and immunofluorescence assays. Chromatin condensation was measured by microccocal nuclease digestion and histone salt extraction. RESULTS: The data presented here show for the first time that p19INK4d expression is up-regulated by different types of senescence. Changes in senescence-associated hallmarks were driven by modulation of p19 expression indicating a direct link between p19INK4d induction and the establishment of cellular senescence. Following a senescence stimulus, p19INK4d translocates to the nucleus and tightly associates with chromatin. Moreover, reduced levels of p19INK4d impair senescence-related global genomic heterochromatinization. Analysis of p19INK4d mRNA and protein levels in tissues from differently aged mice revealed an up-regulation of p19INK4d that correlates with age. CONCLUSION: We propose that p19INK4d participates in the cellular mechanisms that trigger senescence by contributing to chromatin compaction. GENERAL SIGNIFICANCE: This study provides novel insights into the dynamics process of cellular senescence, a central tumor suppressive mechanism.
Assuntos
Envelhecimento/genética , Senescência Celular/genética , Inibidor de Quinase Dependente de Ciclina p19/genética , Heterocromatina/genética , Animais , Carcinogênese , Pontos de Checagem do Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p19/metabolismo , Dano ao DNA/genética , Regulação da Expressão Gênica , Camundongos , beta-Galactosidase/biossínteseRESUMO
Central to the maintenance of genomic integrity is the cellular DNA damage response. Depending on the type of genotoxic stress and through the activation of multiple signaling cascades, it can lead to cell cycle arrest, DNA repair, senescence, and apoptosis. p19INK4d, a member of the INK4 family of CDK inhibitors, plays a dual role in the DNA damage response, inhibiting cell proliferation and promoting DNA repair. Consistently, p19INK4d has been reported to become upregulated in response to UV irradiation and a great variety of genotoxic agents. Here, this induction is shown to result from a transcriptional stimulatory mechanism that can occur at every phase of the cell cycle except during mitosis. Moreover, evidence is presented that demonstrates that E2F1 is involved in the induction of p19INK4d following UV treatment, as it is prevented by E2F1 protein ablation and DNA-binding inhibition. Specific inhibition of this regulation using triplex-forming oligonucleotides that target the E2F response elements present in the p19INK4d promoter also block p19INK4d upregulation and sensitize cells to DNA damage. These results constitute the first description of a mechanism for the induction of p19INK4d in response to UV irradiation and demonstrate the physiological relevance of this regulation following DNA damage.