RESUMO
High-risk human papillomaviruses (hr-HPVs) are the key risk factors implicated in the development of a significant proportion of head and neck squamous cell carcinomas (HNSCCs). We aimed to investigate the distribution of hr-HPV types and HPV16 lineages in a sample of patients with HNSCC and the possible association between HPV status and the expression of P16INK4A and NF-κB in Iranian HNSCC patients. We examined 108 formalin-fixed, paraffin-embedded (FFPE) histologically confirmed primary SCC tissue specimens of different head and neck anatomical sites. HPV types and HPV16 lineages were determined by nested PCR and overlapping nested PCR assays, respectively, followed by gene sequencing and phylogenetic analysis. The expression of p16INK4a and NF-κB was evaluated by immunohistochemistry. Twenty-five (23.1%) HNSCC tissue specimens were tested positive for HPV infection. The most prevalent HPV type was HPV-16, followed by HPV18 and HPV11. HPV16 variants belonged to the lineage A and lineage D which were further sorted into sublineages A1, A2, and D2. A significant association between HPV status and p16INK4a immunoreactivity was observed in more than 76% of the HPV-related HNSCCs (P < 0.0001). The overexpression of p16INK4a and cytoplasmic NF-κB was more common in low-grade HNSCC tumors. Our data highlights that HPV16, in particular the A2 sublineage, followed by A1 and D2 sublineages are the major agents associated with HNSCCs in Iran. Based on HPV16 predominance and its lineage distribution pattern, it seems that the prophylactic vaccines developed for cervical cancer prevention could also be applicable for the prevention of HPV-related HNSCCs in our population.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Neoplasias de Cabeça e Pescoço/genética , Papillomavirus Humano 16/isolamento & purificação , NF-kappa B/genética , Infecções por Papillomavirus/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/virologia , Papillomavirus Humano 16/classificação , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Humanos , Irã (Geográfico) , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , Filogenia , Estudos Retrospectivos , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia , Adulto JovemRESUMO
BACKGROUND: Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell in the commercial-scale production of biopharmaceutical proteins. Modification of genes involved in apoptosis may improve the productivity of CHO cells. Executive caspases, including caspases 3 and 7, play critical roles in apoptosis. The effects of the ablation of the caspase 7 gene on proliferation and viability of CHO cells remains unknown. In this study, we applied clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) to target caspase 7 gene of CHO K1 cell via all in one and homology targeted integration strategies. Consequently, the effect of caspase 7 deficiency on cell proliferation, viability, and apoptosis was studied by MTT assay and flow cytometry. RESULTS: Findings of gel electrophoresis, western blotting, and sequencing confirmed the caspase 7 gene silencing in CHO cells (CHO-KO). Proliferation assay revealed that caspase 7 deficiency in CHO cells resulted in the reduction of proliferation in various CHO-KO clones. Besides, the disruption of caspase 7 had negative effects on cell viability in exposure with NaBu which confirmed by MTT assay. Results of flow cytometry using Anexin V/PI demonstrated that Nabu treatment (11 mM) declined the percentage of live CHO-K1 and CHO-KO cells to 70.3% and 5.79%. These results verified that the CHO-K1 cells were more resistant to apoptosis than CHO-KO, however most of CHO-KO cells undergone early apoptosis (91.9%) which seems to be a fascinating finding. CONCLUSION: These results reveal that caspase 7 may be involved in the cell cycle progression of CHO cells. Furthermore, it seems that targeting caspase 7 is not the ideal route as it had previously been imagined within the prevention of apoptosis but the relation between caspase 7 deficiency, cell cycle arrest, and the occurrence of early apoptosis will require more investigation.
Assuntos
Apoptose , Caspase 7/deficiência , Proliferação de Células , Sobrevivência Celular , Animais , Células CHO , Caspase 7/genética , Cricetinae , CricetulusRESUMO
Early diagnosis and genotyping of high-risk human papillomavirus (HR-HPV) in cervical tissue specimens is significant for cervical cancer prevention. A sensitive microplate fluorometric hybridization assay (MFHA) was designed for the detection of HPV DNA 16 and 18 in cervical tissue. Following optimization and validation of the method, 60 formalin-fixed and paraffin-embedded cervical samples representing different cervical intraepithelial neoplasia grades of HPV-associated lesions were tested to determine the sensitivity and specificity of the assay. Using consensus GP5+/6+ biotin-labeled primers to amplify a conserved region within the L1 gene, the amplicons were added to the microplate wells coated with specific probes for the hybridization of HPV 16 and 18 individually. Final detection was performed with streptavidin-AlexaFluor488 conjugated. The results were then compared with type-specific nested polymerase chain reaction (PCR) and colorimetric microplate assay. While the agreement between the results obtained by the type-specific nested PCR and fluorometric assay for the detection of both HR-HPV types was 100%, this agreement for the detection of HPV type 16 and 18 using microplate colorimetric assay was 94.2% and 85% respectively. Overall, the results of the fluorometric and colorimetric assays are promising for detecting both HR-HPV types in a large number of cervical tissue samples with the higher MFHA assay sensitivity.
Assuntos
Colo do Útero/virologia , DNA Viral/genética , Fluorometria/métodos , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Hibridização de Ácido Nucleico/métodos , Adulto , Bancos de Espécimes Biológicos , Primers do DNA , Feminino , Corantes Fluorescentes , Genótipo , Técnicas de Genotipagem , Humanos , Pessoa de Meia-Idade , Inclusão em Parafina , Sensibilidade e Especificidade , Neoplasias do Colo do Útero/virologia , Adulto JovemRESUMO
ABSTRACT Background: Hypothyroidism due to Hashimoto's thyroiditis (HT) is the commonest autoimmune endocrine illness in which antibodies against thyroid organ result in inflammation. The disease has a complex etiology that involves genetic and environmental influences. Viral infections may be involved in triggering of the disease as their molecular mimicry enhance autoimmune responses. Human herpesvirus-6 (HHV-6) is recognized for its contribution to some autoimmune diseases. Objective: In the current study, the prevalence of HHV-6 active infection in patients with HT and with non-autoimmune thyroid disorders were compared with patients with euthyroidism. In addition, a correlation between presence of HHV-6 infections and HT was investigated. Methods: A total of 151 patients with clinically and laboratory confirmed HT, 59 patients with non-autoimmune thyroid disorders, and 32 patients with normal thyroid function were included in the study. For further confirmation of HT disease, all the precipitants were tested for anti-thyroid peroxidase (TPO), and anti-thyroglobulin (TG) antibodies. For detection of both HHV-6 types A and B, nested PCR and restriction enzyme digestion were used. HHV-6 DNA positive samples were further investigated by DNA sequencing analysis. Results: HHV-6A DNA was found in serum sample of 57 out of 151 patients (38%) with HT, which was significantly more often than in patients with non-autoimmune thyroid disorders (p = 0.001). However, HHV-6 DNA was not detected in serum samples of euthyroid subjects. Conclusions: The results support a possible role for active HHV-6A infection, demonstrated by the presence of HHV-6 DNA in sera, in the development of HT.
Assuntos
Humanos , Masculino , Feminino , Adolescente , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Adulto Jovem , Herpesvirus Humano 6/genética , Infecções por Roseolovirus/virologia , Doença de Hashimoto/virologia , Glândula Tireoide/virologia , DNA Viral/análise , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: Hypothyroidism due to Hashimoto's thyroiditis (HT) is the commonest autoimmune endocrine illness in which antibodies against thyroid organ result in inflammation. The disease has a complex etiology that involves genetic and environmental influences. Viral infections may be involved in triggering of the disease as their molecular mimicry enhance autoimmune responses. Human herpesvirus-6 (HHV-6) is recognized for its contribution to some autoimmune diseases. OBJECTIVE: In the current study, the prevalence of HHV-6 active infection in patients with HT and with non-autoimmune thyroid disorders were compared with patients with euthyroidism. In addition, a correlation between presence of HHV-6 infections and HT was investigated. METHODS: A total of 151 patients with clinically and laboratory confirmed HT, 59 patients with non-autoimmune thyroid disorders, and 32 patients with normal thyroid function were included in the study. For further confirmation of HT disease, all the precipitants were tested for anti-thyroid peroxidase (TPO), and anti-thyroglobulin (TG) antibodies. For detection of both HHV-6 types A and B, nested PCR and restriction enzyme digestion were used. HHV-6 DNA positive samples were further investigated by DNA sequencing analysis. RESULTS: HHV-6A DNA was found in serum sample of 57 out of 151 patients (38%) with HT, which was significantly more often than in patients with non-autoimmune thyroid disorders (p=0.001). However, HHV-6 DNA was not detected in serum samples of euthyroid subjects. CONCLUSIONS: The results support a possible role for active HHV-6A infection, demonstrated by the presence of HHV-6 DNA in sera, in the development of HT.
Assuntos
Doença de Hashimoto/virologia , Herpesvirus Humano 6/genética , Infecções por Roseolovirus/virologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , DNA Viral/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Glândula Tireoide/virologia , Adulto JovemRESUMO
OBJECTIVES: To investigate the prevalence of human polyomavirus (BK and JC viruses) infection in peripheral blood mononuclear cells of healthy blood donors. METHODS: The study included 250 healthy blood donors. Five-milliliter blood was drawn into sterile EDTA tubes and PBMCs were isolated from whole blood. The isolated PBMCs were counted and stored at -70°C for future investigation. DNA was extracted and subjected to simple, sensitive and specific semi-nested PCR as well as QPCR using both general and specific primers for different assays. RESULTS: Of 250 blood samples, 66 (26.4%) were positive for BKV DNA (146-34,514 copies/106 cells). JC DNA was found in 45 (18%) blood samples (65-21,250 copies/106 cells). Co-infection with these viruses were found in 11 (4.4%) out of 250 blood samples. DISCUSSION: Our study provides important data on polyomavirus infection in peripheral blood mononuclear leukocytes in immunocompetent individuals. These data indicate significant differences between the prevalence of BKV and JCV infection in healthy blood donors. The prevalence of BK and JC virus infection is higher in the age range 30-39 years compared to other age ranges.
Assuntos
Vírus BK/isolamento & purificação , Doadores de Sangue , Vírus JC/isolamento & purificação , Leucócitos Mononucleares/virologia , Infecções por Polyomavirus/virologia , Infecções Tumorais por Vírus/virologia , Adulto , Distribuição por Idade , Vírus BK/genética , DNA Viral/isolamento & purificação , Feminino , Humanos , Irã (Geográfico)/epidemiologia , Vírus JC/genética , Masculino , Pessoa de Meia-Idade , Infecções por Polyomavirus/sangue , Infecções por Polyomavirus/epidemiologia , Prevalência , Reação em Cadeia da Polimerase em Tempo Real , Infecções Tumorais por Vírus/sangue , Infecções Tumorais por Vírus/epidemiologia , Carga Viral , Adulto JovemRESUMO
ABSTRACT Objectives: To investigate the prevalence of human polyomavirus (BK and JC viruses) infection in peripheral blood mononuclear cells of healthy blood donors. Methods: The study included 250 healthy blood donors. Five-milliliter blood was drawn into sterile EDTA tubes and PBMCs were isolated from whole blood. The isolated PBMCs were counted and stored at −70 °C for future investigation. DNA was extracted and subjected to simple, sensitive and specific semi-nested PCR as well as QPCR using both general and specific primers for different assays. Results: Of 250 blood samples, 66 (26.4%) were positive for BKV DNA (146-34,514 copies/106 cells). JC DNA was found in 45 (18%) blood samples (65-21,250 copies/106 cells). Co-infection with these viruses were found in 11 (4.4%) out of 250 blood samples. Discussion: Our study provides important data on polyomavirus infection in peripheral blood mononuclear leukocytes in immunocompetent individuals. These data indicate significant differences between the prevalence of BKV and JCV infection in healthy blood donors. The prevalence of BK and JC virus infection is higher in the age range 30-39 years compared to other age ranges.