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Oral fluids provide ready detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host responses. This study sought to evaluate relationships between oral virus, oral and systemic anti-SARS-CoV-2-specific antibodies, and symptoms. Oral fluids (saliva/throat wash (saliva/TW)) and serum were collected from asymptomatic and symptomatic, nasopharyngeal (NP) SARS-CoV-2 RT-qPCR+ human participants (n = 45). SARS-CoV-2 RT-qPCR and N-antigen detection by immunoblot and lateral flow assay (LFA) were performed. RT-qPCR for subgenomic RNA (sgRNA) was sequence confirmed. SARS-CoV-2-anti-S protein RBD LFA and ELISA assessed IgM and IgG responses. Structural analysis identified host salivary molecules analogous to SARS-CoV-2-N-antigen. At time of enrollment (baseline, BL), LFA-detected N-antigen in 86% of TW and was immunoblot-confirmed. However, only 3/17 were saliva/TW qPCR+ . Sixty percent of saliva and 83% of TW demonstrated persistent N-antigen at 4 weeks. N-antigen LFA signal in three anti-spike sero-negative participants suggested potential cross-detection of 4 structurally analogous salivary RNA binding proteins (alignment 19-29aa, RMSD 1-1.5 Angstroms). At enrollment, symptomatic participants demonstrated replication-associated sgRNA junctions, were IgG+ (94%/100% in saliva/TW), and IgM+ (63%/54%). At 4 weeks, SARS-CoV-2 IgG (100%/83%) and IgM (80%/67%) persisted. Oral and serum IgG correlated 100% with NP+ PCR status. Cough and fatigue severity (p = 0.010 and 0.018 respectively), and presence of weakness, nausea, and composite upper respiratory symptoms (p = 0.037, 0.005, and 0.017, respectively) were negatively associated with saliva IgM but not TW or serum IgM. Throat wash IgM levels were higher in women compared to men, although the association did not reach statistical significance (median: 290 (female) versus 0.697, p = 0.056). Important to transmission and disease course, oral viral replication and persistence showed clear relationships with select symptoms and early oral IgM responses during early infection. N-antigen cross-reactivity may reflect mimicry of structurally analogous host proteins.
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Anticuerpos Antivirales , COVID-19 , SARS-CoV-2 , Saliva , Humanos , COVID-19/inmunología , COVID-19/virología , COVID-19/diagnóstico , SARS-CoV-2/inmunología , Saliva/virología , Saliva/inmunología , Femenino , Masculino , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Adulto , Persona de Mediana Edad , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Fosfoproteínas/inmunología , ARN Viral , Nasofaringe/virología , Proteínas de la Nucleocápside de Coronavirus/inmunología , AncianoRESUMEN
Objectives: Oral fluids provide ready detection of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host responses. This study sought to determine relationships between oral virus, oral anti-SARS-CoV-2-specific antibodies, and symptoms. Methods: Saliva/throat wash (saliva/TW) were collected from asymptomatic and symptomatic, nasopharyngeal (NP) SARS-CoV-2 RT-qPCR+, subjects (n=47). SARS-CoV-2 RT-qPCR, N-antigen detection by immunoblot and lateral flow assay (LFA) were performed. RT-qPCR targeting viral subgenomic RNA (sgRNA) was sequence confirmed. SARS-CoV-2-anti-S protein RBD LFA assessed IgM and IgG responses. Structural analysis identified host salivary molecules analogous to SARS-CoV-2-N-antigen. Statistical analyses were performed. Results: At baseline, LFA-detected N-antigen was immunoblot-confirmed in 82% of TW. However, only 3/17 were saliva/TW qPCR+. Sixty percent of saliva and 83% of TW demonstrated persistent N-antigen at 4 weeks. N-antigen LFA signal in three negative subjects suggested potential cross-detection of 4 structurally analogous salivary RNA binding proteins (alignment 19-29aa, RMSD 1-1.5 Angstroms). At entry, symptomatic subjects demonstrated replication-associated sgRNA junctions, were IgG+ (94%/100% in saliva/TW), and IgM+ (75%/63%). At 4 weeks, SARS-CoV-2 IgG (100%/83%) and IgM (80%/67%) persisted. Oral IgG correlated 100% with NP+PCR status. Cough and fatigue severity (p=0.0008 and 0.016), and presence of nausea, weakness, and composite upper respiratory symptoms (p=0.005, 0.037 and 0.017) were negatively associated with oral IgM. Female oral IgM levels were higher than male (p=0.056). Conclusion: Important to transmission and disease course, oral viral replication and persistence showed clear relationships with select symptoms, early Ig responses, and gender during early infection. N-antigen cross-reactivity may reflect mimicry of structurally analogous host proteins.
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Objectives: Oral fluids provide ready detection of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host responses. This study sought to determine relationships between oral virus, oral anti-SARS-CoV-2-specific antibodies, and symptoms. Methods: Saliva/throat wash (saliva/TW) were collected from asymptomatic and symptomatic, nasopharyngeal (NP) SARS-CoV-2 RT-qPCR+, subjects (n=47). SARS-CoV-2 RT-qPCR, N-antigen detection by immunoblot and lateral flow assay (LFA) were performed. RT-qPCR targeting viral subgenomic RNA (sgRNA) was sequence confirmed. SARS-CoV-2-anti-S protein RBD LFA assessed IgM and IgG responses. Structural analysis identified host salivary molecules analogous to SARS-CoV-2-N-antigen. Statistical analyses were performed. Results: At baseline, LFA-detected N-antigen was immunoblot-confirmed in 82% of TW. However, only 3/17 were saliva/TW qPCR+. Sixty percent of saliva and 83% of TW demonstrated persistent N-antigen at 4 weeks. N-antigen LFA signal in three negative subjects suggested potential cross-detection of 4 structurally analogous salivary RNA binding proteins (alignment 19-29aa, RMSD 1-1.5 Angstroms). At entry, symptomatic subjects demonstrated replication-associated sgRNA junctions, were IgG+ (94%/100% in saliva/TW), and IgM+ (75%/63%). At 4 weeks, SARS-CoV-2 IgG (100%/83%) and IgM (80%/67%) persisted. Oral IgG correlated 100% with NP+PCR status. Cough and fatigue severity (p=0.0008 and 0.016), and presence of nausea, weakness, and composite upper respiratory symptoms (p=0.005, 0.037 and 0.017) were negatively associated with oral IgM. Female oral IgM levels were higher than male (p=0.056). Conclusion: Important to transmission and disease course, oral viral replication and persistence showed clear relationships with select symptoms, early Ig responses, and gender during early infection. N-antigen cross-reactivity may reflect mimicry of structurally analogous host proteins.
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Human Papillomavirus (HPV)-associated oral disease has increased during the era of HIV antiretroviral therapy. HPV and HIV proteins may be co-present at mucosal surfaces. Recent published studies have determined that HIVtat is secreted in the saliva and has been detected in oral mucosa even in the context of antiretroviral therapy. We hypothesized that HIVtat promoted oral HPV pathogenesis. Clinical HPV16 cloned episomes were introduced into differentiated oral epithelial cells (OKF6tert1). HIVtat mediated transactivation, DNA damage, oxidative stress, and effects on cellular differentiation were assessed. Detection of keratin 10 and of loricrin confirmed terminal differentiation. Sodium butyrate-treated (NaB) cells demonstrated an eight-fold increase in cross-linked involucrin, suggesting full terminal differentiation. HIVtat modulated this differentiation both in the presence and absence of NaB. Later viral events, including E6* and E1^E4 gene expression were assessed. HIVtat mediated relief of repressed L1 expression that mapped to a known inhibitory region (nucleotides 5561-6820). Viruses from HIVtat co-expressing cells exhibited robust de novo HPV16 infection. In conclusion, a novel oral keratinocyte monolayer system supported replication of an HPV16 clinical isolate where direct HIVtat and oral HPV interactions enhanced HPV de novo infection.
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Human Papillomavirus (HPV) associated oral disease continues to increase, both in the context of immune competence and of immune suppression. There are few models of oral HPV infection and current models are laborious. We hypothesized that differentiated oral epithelial cells could support the HPV life cycle. Clinical HPV16 cloned episomes were introduced into differentiated oral epithelial cells (OKF6tert1). Viral and cellular gene expression was assessed in the presence or absence of sodium butyrate, a differentiating agent that moved the cells to full terminal differentiation. Detection of keratin 10, cross-linked involucrin, and loricrin in the presence and absence of sodium butyrate confirmed terminal differentiation. Increasing sodium butyrate concentrations in the absence of HPV, were associated with decreased suprabasal markers and increased terminal differentiation markers. However, in the presence of HPV and of increasing sodium butyrate concentrations, both mitotic and suprabasal markers were increased and the terminal differentiation marker, loricrin, decreased. In this unique differentiated state, early and late viral gene products were detected including spliced mRNAs for E6*, E1^E4, and L1. E7 and L1 proteins were detected. The ratio of late (E1^E4) to early (E6/E7) transcripts in HPV16+ OKF6tert1 cells was distinct compared to HPV16+ C33a cells. Consistent with permissive HPV replication, DNA damage responses (phospho-chk2, gamma-H2AX), HPV E2-dependent LCR transactivation, and DNase-resistant particles were detected and visualized by transmission electron microscopy. In sum, monolayers of differentiated immortalized oral epithelial cells supported the full HPV life cycle. HPV may optimize the differentiation state of oral epithelial cells to facilitate its replication.
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BACKGROUND: Periodontal destruction can be the result of different known and yet-to-be-discovered biological pathways. Recent human genetic association studies have implicated interferon-gamma inducible protein 16 (IFI16) and absent in melanoma 2 (AIM2) with high periodontal interleukin (IL)-1ß levels and more destructive disease, but mechanistic evidence is lacking. Here, we sought to experimentally validate these observational associations and better understand IFI16 and AIM2's roles in periodontitis. METHODS: Periodontitis was induced in Ifi204-/- (IFI16 murine homolog) and Aim2-/- mice using the ligature model. Chimeric mice were created to identify the main source cells of Ifi204 in the periodontium. IFI16-silenced human endothelial cells were treated with periodontal pathogens in vitro. Periodontal tissues from Ifi204-/- mice were evaluated for alveolar bone (micro-CT), cell inflammatory infiltration (MPO+ staining), Il1b (qRT-PCR), and osteoclast numbers (cathepsin K+ staining). RESULTS: Ifi204-deficient mice> exhibited >20% higher alveolar bone loss than wild-type (WT) (P < 0.05), while no significant difference was found in Aim2-/- mice. Ifi204's effect on bone loss was primarily mediated by a nonbone marrow source and was independent of Aim2. Ifi204-deficient mice had greater neutrophil/macrophage trafficking into gingival tissues regardless of periodontitis development compared to WT. In human endothelial cells, IFI16 decreased the chemokine response to periodontal pathogens. In murine periodontitis, Ifi204 depletion elevated gingival Il1b and increased osteoclast numbers at diseased sites (P < 0.05). CONCLUSIONS: These findings support IFI16's role as a novel regulator of inflammatory cell trafficking to the periodontium that protects against bone loss and offers potential targets for the development of new periodontal disease biomarkers and therapeutics.
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Pérdida de Hueso Alveolar , Proteínas Nucleares , Periodontitis , Fosfoproteínas , Pérdida de Hueso Alveolar/genética , Pérdida de Hueso Alveolar/metabolismo , Pérdida de Hueso Alveolar/prevención & control , Animales , Biomarcadores/metabolismo , Catepsina K , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Interferón gamma/metabolismo , Interferones/metabolismo , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Periodontitis/genética , Periodontitis/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismoRESUMEN
There is no agnostic GWAS evidence for the genetic control of IL-1ß expression in periodontal disease. Here we report a GWAS for "high" gingival crevicular fluid IL-1ß expression among 4910 European-American adults and identify association signals in the IL37 locus. rs3811046 at this locus (p = 3.3 × 10-22) is associated with severe chronic periodontitis (OR = 1.50; 95% CI = 1.12-2.00), 10-year incident tooth loss (≥3 teeth: RR = 1.33; 95% CI = 1.09-1.62) and aggressive periodontitis (OR = 1.12; 95% CI = 1.01-1.26) in an independent sample of 4927 German/Dutch adults. The minor allele at rs3811046 is associated with increased expression of IL-1ß in periodontal tissue. In RAW macrophages, PBMCs and transgenic mice, the IL37 variant increases expression of IL-1ß and IL-6, inducing more severe periodontal disease, while IL-37 protein production is impaired and shows reduced cleavage by caspase-1. A second variant in the IL37 locus (rs2708943, p = 4.2 × 10-7) associates with attenuated IL37 mRNA expression. Overall, we demonstrate that IL37 variants modulate the inflammatory cascade in periodontal disease.
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Variación Genética , Estudio de Asociación del Genoma Completo , Líquido del Surco Gingival/metabolismo , Inflamación/metabolismo , Inflamación/patología , Interleucina-1/genética , Interleucina-1beta/metabolismo , Periodoncio/patología , Secuencia de Aminoácidos , Animales , Periodontitis Crónica/sangre , Periodontitis Crónica/genética , Periodontitis Crónica/patología , Modelos Animales de Enfermedad , Femenino , Sitios Genéticos , Células HEK293 , Haplotipos/genética , Humanos , Inflamación/sangre , Interleucina-1/metabolismo , Interleucina-1beta/sangre , Interleucina-1beta/genética , Leucocitos Mononucleares/metabolismo , Ratones Transgénicos , Polimorfismo de Nucleótido Simple/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Accidente Cerebrovascular/genética , Pérdida de Diente/genéticaRESUMEN
BACKGROUND: The single nucleotide polymorphism (SNP) context of a previously identified periodontitis-associated locus is investigated, and its association with microbial, biologic, and periodontal disease clinical parameters is examined. METHODS: A 200-kb spanning region of 1q12 previously highlighted in a genome-wide association scan among 4,766 European American individuals (SNP rs1633266) was annotated. Two haplotype blocks were selected. Association of these polymorphisms with data on microbial plaque composition, gingival crevicular fluid (GCF)-interleukin (IL)-1ß levels, and clinical parameters of periodontal disease were examined. Descriptive analysis of IFI16 and AIM2 protein expression in gingival tissues from healthy individuals (n = 2) and individuals with chronic periodontitis (n = 2) was done via immunohistochemistry. RESULTS: The highlighted locus is a 100-kb region containing the interferon γ-inducible protein 16 (IFI16) and absent in melanoma 2 (AIM2) genes. Two haplotype blocks, rs6940 and rs1057028, were significantly associated with increased extent bleeding on probing and levels of microorganisms Porphyromonas gingivalis, Tannerella forsythia, and Campylobacter rectus (P ≤0.05). Haplotype block rs1057028 was also significantly associated with pathogens Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans, increased GCF-IL-1ß levels, and extent of probing depth ≥4 mm (P ≤0.05). Prevalence of severe periodontitis (biofilm-gingival interface P3 classification) was positively associated with haplotype block rs1057028. Similar trends were observed for haplotype block rs1057028. IFI16 and AIM2 protein expression was observed in multiple cell types of gingival tissues, including inflammatory cells. CONCLUSION: This study found IFI16 and AIM2 SNPs associated with higher levels of periodontal microorganisms and an increased percentage of periodontal disease clinical parameters, suggesting the need for functional studies and additional fine-mapping of variants in the 1q12-locus.
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Periodontitis Crónica/genética , Proteínas de Unión al ADN/genética , Encía/metabolismo , Proteínas Nucleares/genética , Fosfoproteínas/genética , Polimorfismo de Nucleótido Simple , Anciano , Aggregatibacter actinomycetemcomitans/aislamiento & purificación , Periodontitis Crónica/metabolismo , Periodontitis Crónica/microbiología , Proteínas de Unión al ADN/metabolismo , Placa Dental/microbiología , Femenino , Fusobacterium nucleatum/aislamiento & purificación , Estudio de Asociación del Genoma Completo , Encía/microbiología , Humanos , Masculino , Persona de Mediana Edad , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Población BlancaRESUMEN
HIV-associated Salivary Gland Disease (HIVSGD) is among the most common salivary gland-associated complications in HIV positive individuals and was associated with the small DNA tumorvirus BK polyomavirus (BKPyV). The BKPyV non-coding control region (NCCR) is the main determinant of viral replication and rearranges readily. This study analyzed the BKPyV NCCR architecture and viral loads of 35 immunosuppressed individuals. Throatwash samples from subjects diagnosed with HIVSGD and urine samples from transplant patients were BKPyV positive and yielded BKPyV NCCR sequences. 94.7% of the BKPyV HIVSGD NCCRs carried a rearranged OPQPQQS block arrangement, suggesting a distinct architecture among this sample set. BKPyV from HIV positive individuals without HIVSGD harbored NCCR block sequences that were distinct from OPQPQQS. Cloned HIVSGD BKPyV isolates displayed active promoters and efficient replication capability in human salivary gland cells. The unique HIVSGD NCCR architecture may represent a potentially significant oral-tropic BKPyV substrain.
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Virus BK/genética , Infecciones por VIH/virología , Faringe/virología , Infecciones por Polyomavirus/virología , Regiones Promotoras Genéticas , Enfermedades de las Glándulas Salivales/virología , Infecciones Tumorales por Virus/virología , Adulto , Líquidos Corporales/virología , ADN Viral , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Oncoviruses cause tremendous global cancer burden. For several DNA tumor viruses, human genome integration is consistently associated with cancer development. However, genomic features associated with tumor viral integration are poorly understood. We sought to define genomic determinants for 1897 loci prone to hosting human papillomavirus (HPV), hepatitis B virus (HBV) or Merkel cell polyomavirus (MCPyV). These were compared to HIV, whose enzyme-mediated integration is well understood. A comprehensive catalog of integration sites was constructed from the literature and experimentally-determined HPV integration sites. Features were scored in eight categories (genes, expression, open chromatin, histone modifications, methylation, protein binding, chromatin segmentation and repeats) and compared to random loci. Random forest models determined loci classification and feature selection. HPV and HBV integrants were not fragile site associated. MCPyV preferred integration near sensory perception genes. Unique signatures of integration-associated predictive genomic features were detected. Importantly, repeats, actively-transcribed regions and histone modifications were common tumor viral integration signatures.
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Antiasmáticos/uso terapéutico , Asma/tratamiento farmacológico , Investigación Biomédica/economía , Conducta Cooperativa , Descubrimiento de Drogas/economía , Comunicación Interdisciplinaria , Apoyo a la Investigación como Asunto , Antiasmáticos/economía , Asma/economía , Difusión de Innovaciones , Descubrimiento de Drogas/organización & administración , Humanos , Terapia Molecular Dirigida/economía , Apoyo a la Investigación como Asunto/organización & administraciónRESUMEN
Although metastasis-associated lung adenocarcinoma transcript (MALAT)-1 is known to be consistently upregulated in several epithelial malignancies, little is known about its function or regulation. We therefore examined the relationship between MALAT-1 expression and candidate modulators such as DNA tumor virus oncoproteins human papillomavirus (HPV)-16 E6 and E7, BK virus T antigen (BKVTAg), mouse polyoma virus middle T antigen (MPVmTAg) and tumor suppressor genes p53 and pRb. Using suppressive subtractive hybridization (SSH) and real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays, MALAT-1 was shown to be increased in viral oncongene-expressing salivary gland biopsies from humans and mice. The results also indicated that MALAT-1 transcripts and promoter activity were increased in vitro when viral oncongene-expressing plasmids were introduced into different cell types. These same viral oncogenes in addition to increasing MALAT-1 transcription have also been shown to inhibit p53 and/or pRb function. In p53 mutant or inactive cell lines MALAT-1 was also shown to be highly upregulated. We hypothesize that there is a correlation between MALAT-1 over-expression and p53 deregulation. In conclusion, we show that disruption of p53, by both polyoma and papilloma oncoproteins appear to play an important role in the up-regulation of MALAT-1. MALAT-1 might therefore represent a biomarker for p53 deregulation within malignancies.
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Traumatic brain injury (TBI) elicits innate inflammatory responses that can lead to secondary brain injury. To better understand the mechanisms involved in TBI-induced inflammation, we examined the nature of macrophages responding to TBI in mice. In this model, brain macrophages were increased >20-fold the day after injury and >77-fold 4 days after injury in the ipsilateral hemisphere compared with sham controls. TBI macrophage subsets were identified by using a reporter mouse strain (YARG) that expresses eYFP from an internal ribosome entry site (IRES) inserted at the 3' end of the gene for arginase-1 (Arg1), a hallmark of alternatively activated (M2) macrophages. One day after TBI, 21 ± 1.5% of ipsilateral brain macrophages expressed relatively high levels of Arg1 as detected by yellow fluorescent protein, and this subpopulation declined thereafter. Arg1(+) cells localized with macrophages near the TBI lesion. Gene expression analysis of sorted Arg1(+) and Arg1(-) brain macrophages revealed that both populations had profiles that included features of conventional M2 macrophages and classically activated (M1) macrophages. The Arg1(+) cells differed from Arg1(-) cells in multiple aspects, most notably in their chemokine repertoires. Thus, the macrophage response to TBI initially involves heterogeneous polarization toward at least two major subsets.
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Arginasa/metabolismo , Lesiones Encefálicas/inmunología , Encéfalo/inmunología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Animales , Arginasa/genética , Proteínas Bacterianas/genética , Movimiento Celular , Quimiocinas/biosíntesis , Perfilación de la Expresión Génica , Inflamación/inmunología , Proteínas Luminiscentes/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
BACKGROUND: The aim of this study was to examine biomolecular profiles in a cohort of young adults with squamous cell cancers (SCCs) of the oral tongue. METHODS: We identified all patients aged 18 to 39 years diagnosed with SCC of the oral tongue at our institution. Immunohistochemical (IHC) staining was performed for p16(INK4a) , epidermal growth factor receptor (EGFR), phosphorylated-EGFR (pEGFR), p53, and ERCC1. Human papillomavirus (HPV) testing was performed using in situ hybridization (ISH) and polymerase chain reaction (PCR). Biomarker expression and HPV status were correlated with outcomes. RESULTS: We identified 25 patients with sufficient tumor samples. Median age at diagnosis was 30 years (range, 20-39 years). p16(INK4a) overexpression was observed in 11 of 25 patients, whereas HPV-16 positivity was observed in none of the tumor samples by ISH and 2 of the tumor samples by PCR. p16(INK4a) positivity was correlated with improved relapse-free survival (hazard ratio [HR] = 0.23, p = .01) and overall survival (HR = 0.28, p = .05). Neither EGFR, pEGFR, p53, nor excision repair cross-complementing rodent repair deficiency, complementation group 1 (ERCC1) expression correlated with outcome on univariate analysis. CONCLUSIONS: p16(INK4a) overexpression was common and was a marker of favorable prognosis. p16(INK4a) overexpression was not a reliable predictor of HPV positivity in our cohort.
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Carcinoma de Células Escamosas/genética , Genes p16 , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/complicaciones , Neoplasias de la Lengua/genética , Adolescente , Adulto , Análisis de Varianza , Biomarcadores de Tumor/análisis , Biopsia con Aguja , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/terapia , Estudios de Cohortes , ADN Viral/análisis , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/mortalidad , Recurrencia Local de Neoplasia/patología , Estadificación de Neoplasias , Papillomaviridae/genética , Infecciones por Papillomavirus/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Pronóstico , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Medición de Riesgo , Pruebas Serológicas , Estadísticas no Paramétricas , Análisis de Supervivencia , Neoplasias de la Lengua/mortalidad , Neoplasias de la Lengua/patología , Neoplasias de la Lengua/terapia , Adulto JovenRESUMEN
Ferritin binds specifically and saturably to a variety of cell types, and recently several ferritin receptors have been cloned. TIM-2 is a specific receptor for H ferritin (HFt) in the mouse. TIM-2 is a member of the T cell immunoglobulin and mucin domain containing (TIM) protein family and plays an important role in immunity. The expression of TIM-2 outside of the immune system indicates that this receptor may have broader roles. We tested whether ferritin binding to TIM-2 can serve as an iron delivery mechanism. TIM-2 was transfected into normal (TCMK-1) mouse kidney cells, where it was appropriately expressed on the cell surface. HFt was labeled with (55)Fe and (55)Fe-HFt was incubated with TIM-2 positive cells or controls. (55)Fe-HFt uptake was observed only in TIM-2 positive cells. HFt uptake was also seen in A20 B cells, which express endogenous TIM-2. TIM-2 levels were not increased by iron chelation. Uptake of (55)Fe-HFt was specific and temperature-dependent. HFt taken up by TIM-2 positive cells transited through the endosome and eventually entered a lysosomal compartment, distinguishing the HFt pathway from that of transferrin, the classical vehicle for cellular iron delivery. Iron delivered following binding of HFt to TIM-2 entered the cytosol and became metabolically available, resulting in increased levels of endogenous intracellular ferritin. We conclude that TIM-2 can function as an iron uptake pathway.
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Apoferritinas/metabolismo , Hierro/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Transporte Biológico , Endosomas/metabolismo , Riñón/metabolismo , Lisosomas/metabolismo , Proteínas de la Membrana/administración & dosificación , Proteínas de la Membrana/genética , Ratones , Unión Proteica , TransfecciónRESUMEN
Cross-talk between Gα(i)- and Gα(q)-linked G-protein-coupled receptors yields synergistic Ca(2+) responses in a variety of cell types. Prior studies have shown that synergistic Ca(2+) responses from macrophage G-protein-coupled receptors are primarily dependent on phospholipase Cß3 (PLCß3), with a possible contribution of PLCß2, whereas signaling through PLCß4 interferes with synergy. We here show that synergy can be induced by the combination of Gßγ and Gα(q) activation of a single PLCß isoform. Synergy was absent in macrophages lacking both PLCß2 and PLCß3, but it was fully reconstituted following transduction with PLCß3 alone. Mechanisms of PLCß-mediated synergy were further explored in NIH-3T3 cells, which express little if any PLCß2. RNAi-mediated knockdown of endogenous PLCßs demonstrated that synergy in these cells was dependent on PLCß3, but PLCß1 and PLCß4 did not contribute, and overexpression of either isoform inhibited Ca(2+) synergy. When synergy was blocked by RNAi of endogenous PLCß3, it could be reconstituted by expression of either human PLCß3 or mouse PLCß2. In contrast, it could not be reconstituted by human PLCß3 with a mutation of the Y box, which disrupted activation by Gßγ, and it was only partially restored by human PLCß3 with a mutation of the C terminus, which partly disrupted activation by Gα(q). Thus, both Gßγ and Gα(q) contribute to activation of PLCß3 in cells for Ca(2+) synergy. We conclude that Ca(2+) synergy between Gα(i)-coupled and Gα(q)-coupled receptors requires the direct action of both Gßγ and Gα(q) on PLCß and is mediated primarily by PLCß3, although PLCß2 is also competent.
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Señalización del Calcio/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Fosfolipasa C beta/metabolismo , Animales , Complemento C5a/metabolismo , Humanos , Macrófagos/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Mutagénesis , Células 3T3 NIH , Fosfolipasa C beta/genética , ARN Interferente Pequeño , Receptores Purinérgicos P2/metabolismo , Uridina Difosfato/metabolismoRESUMEN
BACKGROUND: Human papillomaviruses (HPVs) remain a serious world health problem due to their association with anogenital/oral cancers and warts. While over 100 HPV types have been identified, a subset is associated with malignancy. HPV16 and 18 are the most prevalent oncogenic types, while HPV6 and 11 are most commonly responsible for anogenital warts. While other quantitative PCR (qPCR) assays detect oncogenic HPV, there is no single tube assay distinguishing the most frequent oncogenic types and the most common types found in warts. RESULTS: A Sybr Green-based qPCR assay was developed utilizing degenerate primers to the highly conserved HPV E1 theoretically detecting any HPV type. A single tube multiplex qPCR assay was also developed using type-specific primer pairs and TaqMan probes that allowed for detection and quantitation of HPV6,11,16,18. Each HPV type was detected over a range from 2 x 10(1) to 2 x 10(6)copies/reaction providing a reliable method of quantitating type-specific HPV in 140 anogenital/cutaneous/oral benign and malignant specimens. 35 oncogenic and low risk alpha genus HPV types were detected. Concordance was detected in previously typed specimens. Comparisons to the gold standard detected an overall sensitivity of 89% (95% CI: 77% - 96%) and specificity of 90% (95%CI: 52% - 98%). CONCLUSION: There was good agreement between the ability of the qPCR assays described here to identify HPV types in malignancies previously typed using standard methods. These novel qPCR assays will allow rapid detection and quantitation of HPVs to assess their role in viral pathogenesis.
Asunto(s)
Líquidos Corporales/virología , Genitales/virología , Boca/virología , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Virología/métodos , Benzotiazoles , Cartilla de ADN/genética , Diaminas , Femenino , Humanos , Masculino , Compuestos Orgánicos/metabolismo , Papillomaviridae/clasificación , Papillomaviridae/genética , Infecciones por Papillomavirus/virología , Quinolinas , Sensibilidad y Especificidad , Coloración y Etiquetado/métodos , Proteínas Virales/genéticaRESUMEN
Clostridium difficile toxins cause acute colitis by disrupting the enterocyte barrier and promoting inflammation. ToxB from C. difficile inactivates Rho family GTPases and causes release of cytokines and eicosanoids by macrophages. We studied the effects of ToxB on GPCR signaling in murine RAW264.7 macrophages and found that ToxB elevated Ca(2+) responses to Galphai-linked receptors, including the C5aR, but reduced responses to Galphaq-linked receptors, including the UDP receptors. Other Rho inhibitors also reduced UDP Ca(2+) responses, but they did not affect C5a responses, suggesting that ToxB inhibited UDP responses by inhibiting Rho but enhanced C5a responses by other mechanisms. By using PLCbeta isoform-deficient BMDM, we found that ToxB inhibited Ca(2+) signaling through PLCbeta4 but enhanced signaling through PLCbeta3. Effects of ToxB on GPCR Ca(2+) responses correlated with GPCR use of PLCbeta3 versus PLCbeta4. ToxB inhibited UDP Ca(2+) signaling without reducing InsP3 production or the sensitivity of cellular Ca(2+) stores to exogenous InsP3, suggesting that ToxB impairs UDP signaling at the level of InsP3/Ca(2+)coupling. In contrast, ToxB elevated InsP3 production by C5a, and the enhancement of Ca(2+) signaling by C5a was prevented by inhibition of PLA(2) or 5-LOX but not COX, implicating LTs but not prostanoids in the mechanism. In sum, ToxB has opposing, independently regulated effects on Ca(2+) signaling by different GPCR-linked PLCbeta isoforms in macrophages.
Asunto(s)
Proteínas Bacterianas/farmacología , Toxinas Bacterianas/farmacología , Calcio/metabolismo , Macrófagos/efectos de los fármacos , Fosfolipasa C beta/fisiología , Fosfolipasas A2/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Western Blotting , Células Cultivadas , Complemento C5a/farmacología , Citoesqueleto/metabolismo , Femenino , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositoles/metabolismo , Isoformas de Proteínas , Transducción de Señal , Uridina Difosfato/farmacología , Proteínas de Unión al GTP rho/genéticaRESUMEN
Ferritin is a spherical molecule composed of 24 subunits of two types, ferritin H chain (FHC) and ferritin L chain (FLC). Ferritin stores iron within cells, but it also circulates and binds specifically and saturably to a variety of cell types. For most cell types, this binding can be mediated by ferritin composed only of FHC (HFt) but not by ferritin composed only of FLC (LFt), indicating that binding of ferritin to cells is mediated by FHC but not FLC. By using expression cloning, we identified human transferrin receptor-1 (TfR1) as an important receptor for HFt with little or no binding to LFt. In vitro, HFt can be precipitated by soluble TfR1, showing that this interaction is not dependent on other proteins. Binding of HFt to TfR1 is partially inhibited by diferric transferrin, but it is hindered little, if at all, by HFE. After binding of HFt to TfR1 on the cell surface, HFt enters both endosomes and lysosomes. TfR1 accounts for most, if not all, of the binding of HFt to mitogen-activated T and B cells, circulating reticulocytes, and all cell lines that we have studied. The demonstration that TfR1 can bind HFt as well as Tf raises the possibility that this dual receptor function may coordinate the processing and use of iron by these iron-binding molecules.