RESUMEN
African swine fever virus causes a lethal hemorrhagic disease in domestic swine and wild boar for which currently licensed commercial vaccines are only available in Vietnam. Development of subunit vaccines is complicated by the lack of information on protective antigens as well as suitable delivery systems. Our previous work showed that a pool of eight African swine fever virus genes vectored using an adenovirus prime and modified vaccinia virus boost could prevent fatal disease after challenge with a virulent genotype I isolate of the virus. Here, we identify antigens within this pool of eight that are essential for the observed protection and demonstrate that adenovirus-prime followed by adenovirus-boost can also induce protective immune responses against genotype I African swine fever virus. Immunization with a pool of adenoviruses expressing individual African swine fever virus genes partially tailored to genotype II virus did not protect against challenge with genotype II Georgia 2007/1 strain, suggesting that different antigens may be required to induce cross-protection for genetically distinct viruses. IMPORTANCE: African swine fever virus causes a lethal hemorrhagic disease in domestic pigs and has killed millions of animals across Europe and Asia since 2007. Development of safe and effective subunit vaccines against African swine fever has been problematic due to the complexity of the virus and a poor understanding of protective immunity. In a previous study, we demonstrated that a complex combination of eight different virus genes delivered using two different viral vector vaccine platforms protected domestic pigs from fatal disease. In this study, we show that three of the eight genes are required for protection and that one viral vector is sufficient, significantly reducing the complexity of the vaccine. Unfortunately, this combination did not protect against the current outbreak strain of African swine fever virus, suggesting that more work to identify immunogenic and protective viral proteins is required to develop a truly effective African swine fever vaccine.
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Adenoviridae , Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Vectores Genéticos , Genotipo , Vacunas Virales , Animales , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/inmunología , Fiebre Porcina Africana/prevención & control , Fiebre Porcina Africana/virología , Fiebre Porcina Africana/inmunología , Porcinos , Vacunas Virales/inmunología , Vacunas Virales/genética , Vacunas Virales/administración & dosificación , Vectores Genéticos/genética , Adenoviridae/genética , Adenoviridae/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/genética , Antígenos Virales/inmunología , Antígenos Virales/genéticaRESUMEN
Vitronectin is a glycoprotein present in plasma and the extracellular matrix that is implicated in cell migration. The high amount of vitronectin found in neuroblastoma biopsies has been associated with poor prognosis. Moreover, increased vitronectin levels have been described in the plasma of patients with different cancers. Our aim was to assess vitronectin as a potential circulating biomarker of neuroblastoma prognosis. Vitronectin concentration was quantified using ELISA in culture media of four neuroblastoma cell lines grown in a monolayer and in 3D models, and in the plasma of 114 neuroblastoma patients. Three of the neuroblastoma cell lines secreted vitronectin to culture media when cultured in a monolayer and 3D models. Vitronectin release was higher by neuroblastoma cells cultured in 3D models than in the monolayer and was still elevated when cells were grown in 3D scaffolds with cross-linked vitronectin. Vitronectin secretion occurred independently of cell numbers in cultures. Its concentration in the plasma of neuroblastoma patients ranged between 52.4 and 870 µg/mL (median, 218 µg/mL). A ROC curve was used to establish a cutoff of 361 µg/mL, above which patients over 18 months old had worse prognosis (p = 0.0018). Vitronectin could be considered a new plasma prognostic biomarker in neuroblastoma and warrants confirmation in collaborative studies. Drugs inhibiting vitronectin interactions with cells and/or the extracellular matrix could represent a significant improvement in survival for neuroblastoma patients.
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Biomarcadores de Tumor , Neuroblastoma , Vitronectina , Humanos , Vitronectina/sangre , Vitronectina/metabolismo , Neuroblastoma/sangre , Neuroblastoma/patología , Neuroblastoma/metabolismo , Biomarcadores de Tumor/sangre , Pronóstico , Femenino , Línea Celular Tumoral , Lactante , Masculino , Preescolar , Medios de Cultivo/química , NiñoRESUMEN
African swine fever virus (ASFV) has a major global economic impact. With a case fatality in domestic pigs approaching 100%, it currently presents the largest threat to animal farming. Although genomic differences between attenuated and highly virulent ASFV strains have been identified, the molecular determinants for virulence at the level of gene expression have remained opaque. Here, we characterize the transcriptome of ASFV genotype II Georgia 2007/1 (GRG) during infection of the physiologically relevant host cells, porcine macrophages. In this study, we applied cap analysis gene expression sequencing (CAGE-seq) to map th0e 5' ends of viral mRNAs at 5 and 16 h postinfection. A bioinformatics analysis of the sequence context surrounding the transcription start sites (TSSs) enabled us to characterize the global early and late promoter landscape of GRG. We compared transcriptome maps of the GRG isolate and the lab-attenuated BA71V strain that highlighted GRG virulence-specific transcripts belonging to multigene families, including two predicted MGF 100 genes, I7L and I8L. In parallel, we monitored transcriptome changes in the infected host macrophage cells. Of the 9,384 macrophage genes studied, transcripts for 652 host genes were differentially regulated between 5 and 16 h postinfection compared with only 25 between uninfected cells and 5 h postinfection. NF-κB activated genes and lysosome components such as S100 were upregulated, and chemokines such as CCL24, CXCL2, CXCL5, and CXCL8 were downregulated. IMPORTANCE African swine fever virus (ASFV) causes hemorrhagic fever in domestic pigs, with case fatality rates approaching 100% and no approved vaccines or antivirals. The highly virulent ASFV Georgia 2007/1 strain (GRG) was the first isolated when ASFV spread from Africa to the Caucasus region in 2007, then spreading through Eastern Europe and, more recently, across Asia. We used an RNA-based next-generation sequencing technique called CAGE-seq to map the starts of viral genes across the GRG DNA genome. This has allowed us to investigate which viral genes are expressed during early or late stages of infection and how this is controlled, comparing their expression to the nonvirulent ASFV-BA71V strain to identify key genes that play a role in virulence. In parallel, we investigated how host cells respond to infection, which revealed how the ASFV suppresses components of the host immune response to ultimately win the arms race against its porcine host.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Interacciones Microbiota-Huesped , Macrófagos , Proteínas Virales , Fiebre Porcina Africana/inmunología , Fiebre Porcina Africana/virología , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/inmunología , Animales , Perfilación de la Expresión Génica , Georgia (República) , Interacciones Microbiota-Huesped/inmunología , Macrófagos/inmunología , Macrófagos/virología , Sus scrofa , Porcinos , Transcriptoma , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
A 40-year-old male with no previous medical history presented to emergency department with a 2-week history of progressive dyspnea. He also described night sweats and weight loss (15 kg) during the last 3 months. Thoraco-abdominal computed tomography showed multiple bilateral lung nodules associated with supra-clavicular, hilar and peri-esophageal lymphadenopathies and gastric parietal thickening. These imaging features were suggestive of primary gastric cancer with lung and lymph node metastases. Therefore, he undergone upper digestive endoscopy that showed a large ulcerated protruding lesion at the greater curvature of the body suggestive of malignancy. Gastric biopsies of the lesion confirmed a solid neoplasia constituted by solid nests and sheets of highly pleomorphic, bizarre cells with cytotrophoblastic and syncytiotrophoblastic differentiation that, on immunohistochemistry, stained positive for ß-HCG, SALL-4 and glypican-3. CT-guided biopsy of lung nodules revealed malignant cells with similar histopathological and immunohistochemical features. Elevated serum alpha-fetoprotein and ß-HCG were also detected. Clinical and ultrasound examination were negative for testicular masses. These findings were consistent with a primary gastric choriocarcinoma presenting with lung and lymph node metastases (stage IV). Although chemotherapy was started, the patient evolved unfavorably and died after 9 months. Primary gastric choriocarcinoma is a rare and aggressive gastrointestinal malignancy. This case demonstrates its rapid growth rate and high metastatic potential that may lead to symptoms from secondary involvement of distant organs.
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Coriocarcinoma , Neoplasias Gástricas , Adulto , Humanos , Masculino , Coriocarcinoma/diagnóstico por imagen , Coriocarcinoma/patología , Metástasis Linfática , Neoplasias Gástricas/diagnóstico por imagen , Neoplasias Gástricas/patología , Tomografía Computarizada por Rayos XRESUMEN
African swine fever virus (ASFV) causes hemorrhagic fever in domestic pigs, presenting the biggest global threat to animal farming in recorded history. Despite the importance of ASFV, little is known about the mechanisms and regulation of ASFV transcription. Using RNA sequencing methods, we have determined total RNA abundance, transcription start sites, and transcription termination sites at single-nucleotide resolution. This allowed us to characterize DNA consensus motifs of early and late ASFV core promoters, as well as a polythymidylate sequence determinant for transcription termination. Our results demonstrate that ASFV utilizes alternative transcription start sites between early and late stages of infection and that ASFV RNA polymerase (RNAP) undergoes promoter-proximal transcript slippage at 5' ends of transcription units, adding quasitemplated AU- and AUAU-5' extensions to mRNAs. Here, we present the first much-needed genome-wide transcriptome study that provides unique insight into ASFV transcription and serves as a resource to aid future functional analyses of ASFV genes which are essential to combat this devastating disease.IMPORTANCE African swine fever virus (ASFV) causes incurable and often lethal hemorrhagic fever in domestic pigs. In 2020, ASF presents an acute and global animal health emergency that has the potential to devastate entire national economies as effective vaccines or antiviral drugs are not currently available (according to the Food and Agriculture Organization of the United Nations). With major outbreaks ongoing in Eastern Europe and Asia, urgent action is needed to advance our knowledge about the fundamental biology of ASFV, including the mechanisms and temporal control of gene expression. A thorough understanding of RNAP and transcription factor function, and of the sequence context of their promoter motifs, as well as accurate knowledge of which genes are expressed when and the amino acid sequence of the encoded proteins, is direly needed for the development of antiviral drugs and vaccines.
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Virus de la Fiebre Porcina Africana/genética , Fiebre Porcina Africana/prevención & control , Transcripción Genética/genética , Secuencia de Aminoácidos , Animales , Genoma Viral , Fiebres Hemorrágicas Virales/virología , Sus scrofa/virología , Porcinos/virología , Terminación de la Transcripción Genética , Activación Transcripcional/genética , Transcriptoma/genética , Proteínas Virales/genéticaRESUMEN
BACKGROUND: Pegylated liposomal doxorubicin (PLD) is among the most active therapies for recurrent/progressive ovarian cancer (OC). Low-density lipoprotein receptor-related protein 1B (LRP1B) is one of the 10 most significantly deleted genes in human cancers. It mediates endocytosis of several factors from the cellular environment including liposomes. Although the LRP1B role in cancer has not been fully disclosed, its contribution to resistance to liposomal therapies has been hypothesized. This study aimed to evaluate the impact of LRP1B protein as a possible marker of response to PLD in patients with OC. METHODS: LRP1B expression and response to PLD were analyzed in OC cell lines by qRT-PCR and PrestoBlue viability assay, respectively. LRP1B protein expression was evaluated for the first time, in tumor samples from PLD-treated patients and controls (other chemotherapies) by immunohistochemistry. Association of LRP1B staining score (determined based on intensity and percentage of positively stained cells) with clinicopathological features, response to therapy and survival outcomes was evaluated. RESULTS: OC cells with increased expression of LRP1B were more sensitive to PLD. LRP1B staining score was associated with clinicopathological features, response to therapy, and survival outcomes. Higher LRP1B levels were associated with prolonged progression-free survival. This association was more evident in patients treated with PLD and in responders to PLD. CONCLUSION: Our results support a possible role of LRP1B as a predictor of response to PLD in patients with OC.
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Doxorrubicina , Neoplasias Ováricas , Protocolos de Quimioterapia Combinada Antineoplásica , Carcinoma Epitelial de Ovario , Doxorrubicina/análogos & derivados , Doxorrubicina/uso terapéutico , Humanos , Recurrencia Local de Neoplasia , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Polietilenglicoles/uso terapéutico , Receptores de LDL/uso terapéuticoRESUMEN
PURPOSE: Patients with Fanconi anaemia (FA), a rare DNA repair genetic disease, exhibit chromosome fragility, bone marrow failure, malformations and cancer susceptibility. FA molecular diagnosis is challenging since FA is caused by point mutations and large deletions in 22 genes following three heritability patterns. To optimise FA patients' characterisation, we developed a simplified but effective methodology based on whole exome sequencing (WES) and functional studies. METHODS: 68 patients with FA were analysed by commercial WES services. Copy number variations were evaluated by sequencing data analysis with RStudio. To test FANCA missense variants, wt FANCA cDNA was cloned and variants were introduced by site-directed mutagenesis. Vectors were then tested for their ability to complement DNA repair defects of a FANCA-KO human cell line generated by TALEN technologies. RESULTS: We identified 93.3% of mutated alleles including large deletions. We determined the pathogenicity of three FANCA missense variants and demonstrated that two FANCA variants reported in mutations databases as 'affecting functions' are SNPs. Deep analysis of sequencing data revealed patients' true mutations, highlighting the importance of functional analysis. In one patient, no pathogenic variant could be identified in any of the 22 known FA genes, and in seven patients, only one deleterious variant could be identified (three patients each with FANCA and FANCD2 and one patient with FANCE mutations) CONCLUSION: WES and proper bioinformatics analysis are sufficient to effectively characterise patients with FA regardless of the rarity of their complementation group, type of mutations, mosaic condition and DNA source.
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Secuenciación del Exoma , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Anemia de Fanconi/genética , Predisposición Genética a la Enfermedad , Línea Celular , Variaciones en el Número de Copia de ADN/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Anemia de Fanconi/patología , Femenino , Técnicas de Inactivación de Genes , Humanos , Masculino , Mutación Missense/genética , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Von Hippel-Lindau (VHL) syndrome is an autosomal dominant genetic disorder caused by germline mutation of the VHL gene. It is associated with multiple neoplasias including hemangioblastoma, clear cell renal cell carcinoma, pheochromocytoma, and neuroendocrine tumors. Ovarian tumors are extremely rare in this syndrome. We describe the case of a 16-yr-old girl with a previous diagnosis of bilateral pheochromocytoma and several pancreatic neuroendocrine tumors in VHL syndrome context. Follow-up abdominal-pelvic magnetic resonance imaging revealed a 33 mm, well-circumscribed nodule in the right ovary. The patient was submitted to laparoscopic right salpingo-oophorectomy. Microscopically, the tumor consisted of polygonal cells with abundant microvacuolized clear cytoplasm arranged in a solid pattern. The neoplastic cells were immunohistochemically positive for inhibin and calretinin. A diagnosis of ovarian steroid cell tumor was made. Only 4 cases with this association have been reported to date. Of the previously described cases, only one concerns a child; the others were all adult women. All of them had a previous diagnosis of VHL syndrome and presented with secondary amenorrhea and/or hirsutism due to testosterone-secreting ovarian steroid cell tumors. Although extremely rare, the association between VHL syndrome and ovarian steroid cell tumor has been reported, and our case suggests there is a link between the 2 entities.
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Tumores Neuroendocrinos/diagnóstico por imagen , Neoplasias Ováricas/diagnóstico por imagen , Feocromocitoma/diagnóstico por imagen , Tumores de los Cordones Sexuales y Estroma de las Gónadas/diagnóstico por imagen , Enfermedad de von Hippel-Lindau/diagnóstico por imagen , Adolescente , Amenorrea/complicaciones , Femenino , Mutación de Línea Germinal , Hirsutismo/complicaciones , Humanos , Imagen por Resonancia Magnética , Tumores Neuroendocrinos/complicaciones , Tumores Neuroendocrinos/patología , Neoplasias Ováricas/complicaciones , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Feocromocitoma/complicaciones , Feocromocitoma/patología , Tumores de los Cordones Sexuales y Estroma de las Gónadas/complicaciones , Tumores de los Cordones Sexuales y Estroma de las Gónadas/genética , Tumores de los Cordones Sexuales y Estroma de las Gónadas/patología , Enfermedad de von Hippel-Lindau/complicaciones , Enfermedad de von Hippel-Lindau/genética , Enfermedad de von Hippel-Lindau/patologíaRESUMEN
BACKGROUND: Massive rotator cuff tears (MRCTs) are very large tears that are often associated with an uncertain prognosis. Indeed, some MRCTs even without osteoarthritis are considered irreparable, and nonanatomic solutions are needed to improve the patient's symptoms. Reverse shoulder arthroplasty (RSA) is an option that can provide a more predictable pain relief and recovery of function. Nonetheless, outcomes after RSA for irreparable MRCTs have not been well defined. The aim of this study was to quantitatively aggregate the findings associated with the use of RSA in this subset of patients and analyze the effect on patient functional status and pain. METHODS: A comprehensive search was performed until October 2015 using MEDLINE, Scopus, Cochrane Database of Systematic Reviews, and Central Register of Controlled Trials databases. Studies that assessed the outcomes of RSA in patients with irreparable MRCT without osteoarthritis (with at least 2 years of follow-up) were included. If the results of MRCT without osteoarthritis were not possible to subgroup, the study was excluded. Methodologic quality was assessed using the Coleman Methodology Score. RESULTS: Included were 6 studies (266 shoulders) with a follow-up ranging from 24 to 61.4 months. The mean Coleman Methodology Score was 58.2 ± 11.8 points. There was an overall improvement from preoperative to postoperative assessments of the clinical score (Cohen d = 1.35, P < .001), forward flexion (d = 0.50, P = .009), external rotation (d = 0.40, P < .001), function (d = 1.04, P < .001), and pain (d = -0.89, P < .001). CONCLUSION: Patients with irreparable MRCT without presence of osteoarthritis have a high likelihood of achieving a painless shoulder and functional improvements after RSA.
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Artroplastía de Reemplazo de Hombro , Lesiones del Manguito de los Rotadores/cirugía , Humanos , Rango del Movimiento Articular , Resultado del TratamientoRESUMEN
Two strains of African swine fever virus (ASFV), the high-virulence Lisboa60 (L60) and the low-virulence NH/P68 (NHV), which have previously been used in effective immunization/protection studies, were sequenced. Both were isolated in Portugal during the 11-year period after the introduction of ASFV to the European Continent in 1957. The predicted proteins coded by both strains were compared, and where differences were found these were also compared to other strains of known virulence. This highlighted several genes with significant alterations in low-virulence strains of ASFV that may constitute virulence factors, several of which are still uncharacterized regarding their function. Phylogenetic analysis grouped L60 and NHV closest to other P72 genotype I ASFV strains from Europe and West Africa, consistent with the assumed West African origin of all European strains. Interestingly, a relatively lower genomic identity exists between L60 and NHV, both isolated in a similar geographical location 8 years apart, than with other European and west African strains isolated subsequently and in more distant locations. This may reflect the intensive passage in tissue culture, during the early 1960s, of a Portuguese isolate to obtain an attenuated vaccine, which may have led to NHV. This study contributes to a better understanding of the evolution of ASFV, and defines additional potential virulence genes for future studies of pathogenesis towards the development of effective vaccines.
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Virus de la Fiebre Porcina Africana/aislamiento & purificación , Virus de la Fiebre Porcina Africana/fisiología , Genoma Viral , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/crecimiento & desarrollo , Animales , Análisis por Conglomerados , ADN Viral/genética , Evolución Molecular , Datos de Secuencia Molecular , Filogenia , Portugal , Análisis de Secuencia de ADN , Homología de Secuencia , Porcinos , Proteínas Virales/genética , Virulencia , Factores de Virulencia/genéticaRESUMEN
Manipulation of African swine fever virus (ASFV) genomes, in particular those from field strains, is still a challenge. We have shown recently that generation of a green-fluorescent-protein-expressing, thymidine-kinase-negative (TK-) mutant of the low-pathogenic African swine fever virus field strain NHV was supported by a TK- Vero cell line. Since NHV, like other ASFV field strains, does not replicate well in Vero cells, a bromodeoxyuridine (BrdU)- resistant cell line derived from wild boar lung (WSL) cells, named WSL-Bu, was selected. WSL cells were used because they are suitable for productive replication of NHV and other ASFV field strains. Here, we show that WSL-Bu cells enable positive selection of both TK- and TK+ ASFV recombinants, which allows for novel strategies for construction of ASFV mutants. We further demonstrate for a low-pathogenic ASFV strain that TK expression is required for infectious replication in macrophages infected at low multiplicity and that vaccinia TK fully complements ASFV TK in this respect.
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Virus de la Fiebre Porcina Africana/crecimiento & desarrollo , Virus de la Fiebre Porcina Africana/genética , Recombinación Genética , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Virus de la Fiebre Porcina Africana/fisiología , Animales , Línea Celular , Pulmón , Selección Genética , Sus scrofa , Timidina Quinasa/metabolismo , Cultivo de Virus/métodos , Replicación ViralRESUMEN
Introduction: African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that encodes its own host-like RNA polymerase (RNAP) and factors required to produce mature mRNA. The formation of accurate mRNA 3' ends by ASFV RNAP depends on transcription termination, likely enabled by a combination of sequence motifs and transcription factors, although these are poorly understood. The termination of any RNAP is rarely 100% efficient, and the transcriptional "readthrough" at terminators can generate long mRNAs which may interfere with the expression of downstream genes. ASFV transcriptome analyses reveal a landscape of heterogeneous mRNA 3' termini, likely a combination of bona fide termination sites and the result of mRNA degradation and processing. While short-read sequencing (SRS) like 3' RNA-seq indicates an accumulation of mRNA 3' ends at specific sites, it cannot inform about which promoters and transcription start sites (TSSs) directed their synthesis, i.e., information about the complete and unprocessed mRNAs at nucleotide resolution. Methods: Here, we report a rigorous analysis of full-length ASFV transcripts using long-read sequencing (LRS). We systematically compared transcription termination sites predicted from SRS 3' RNA-seq with 3' ends mapped by LRS during early and late infection. Results: Using in-vitro transcription assays, we show that recombinant ASFV RNAP terminates transcription at polyT stretches in the non-template strand, similar to the archaeal RNAP or eukaryotic RNAPIII, unaided by secondary RNA structures or predicted viral termination factors. Our results cement this T-rich motif (U-rich in the RNA) as a universal transcription termination signal in ASFV. Many genes share the usage of the same terminators, while genes can also use a range of terminators to generate transcript isoforms varying enormously in length. A key factor in the latter phenomenon is the highly abundant terminator readthrough we observed, which is more prevalent during late compared with early infection. Discussion: This indicates that ASFV mRNAs under the control of late gene promoters utilize different termination mechanisms and factors to early promoters and/or that cellular factors influence the viral transcriptome landscape differently during the late stages of infection.
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Virus de la Fiebre Porcina Africana , Porcinos , Animales , Virus de la Fiebre Porcina Africana/genética , Transcripción Genética , ARN Polimerasas Dirigidas por ADN , ARN Mensajero/genética , ARNRESUMEN
The enzyme-linked immunospot (ELISpot) assay is a technique of unparalleled sensitivity to determine the frequency of antigen-specific immune cells secreting an immunomodulatory mediator upon recall antigen stimulation, making it a valuable tool in vaccine research. Typically done in multi-well microplate format, it also allows a high-throughput analysis of numerous immune cell samples, e.g., from different donor subjects, especially with the help of automated plate readers and specialized software that currently exist in most laboratories. IFN-γ is a hallmark cytokine secreted especially by T-cell subsets in recall response to pathogens, and consequently the IFN-γ ELISpot assay is one of the most widely used. The cellular arm of the immune response is known to be fundamental in protection against virulent ASFV, and therefore this assay is frequently employed in ASFV vaccine research to evaluate the results from immunization experiments.The technique involves the use of plates with wells that have a membrane for base with a strong binding capacity for amino acids that thus can be densely coated with an antibody for IFN-γ. Upon adding cells and specific antigen or other control stimuli, responding cells will release IFN-γ that is captured by the antibody in close proximity and revealed using a second antibody (sandwich method) through either chromogenic or fluorescent methods, leading to the detection of a "spot" on the membrane for each positive cell. Here we detail our protocol to detect the frequency of ASFV antigen-specific IFN-γ-producing cells in immunized pig lymphocytes and give an example of a typical result using the technique.
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Virus de la Fiebre Porcina Africana , Ensayo de Immunospot Ligado a Enzimas , Interferón gamma , Virus de la Fiebre Porcina Africana/inmunología , Virus de la Fiebre Porcina Africana/metabolismo , Animales , Antígenos , Citocinas , Ensayo de Immunospot Ligado a Enzimas/métodos , Humanos , Interferón gamma/química , Interferón gamma/metabolismo , Porcinos , Vacunas/metabolismoRESUMEN
Endometriosis-associated ovarian cancer represents the most common form of malignancy associated with this benign disease. It has a better prognosis than most types of ovarian cancer, with endometrioid adenocarcinoma and clear cell carcinoma as the main histological types. Clinical presentation is usually nonspecific and tumor biomarkers can be misleading, since they can also be elevated in the presence of benign ovarian endometriosis. We report a case of a 52-year-old woman with known ovarian and deep pelvic endometriosis, who developed ovarian clear cell carcinoma within a large endometrioma. The imaging findings highlight the key role of magnetic resonance imaging in detecting suspicious features such as loss of the "T2 shading" sign, loss of high T1 signal of an endometrioma, or the presence of mural nodules. Early detection of these malignancies is fundamental for adequate surgical treatment and overall outcome.
RESUMEN
Granuloma faciale (GF) is an uncommon inflammatory dermatosis that usually presents on the face as reddish brown or violaceous papules and nodules that may coalesce into plaques. Eosinophilic angiocentric fibrosis (EAF) is a rare fibrosing condition that shares many histologic characteristics with GF and is regarded by some authors as a mucosal counterpart of GF. We present a case of GF with concurrent EAF in a subglottic location with excellent response to an intralesional corticosteroid on the GF lesion, CO2 laser on the EAF lesion, and oral dapsone treatment.
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Eosinofilia/patología , Dermatosis Facial/patología , Granuloma/patología , Anciano , Dapsona/uso terapéutico , Eosinofilia/diagnóstico , Eosinofilia/terapia , Dermatosis Facial/diagnóstico , Dermatosis Facial/terapia , Fibrosis , Glucocorticoides/administración & dosificación , Glucocorticoides/uso terapéutico , Granuloma/diagnóstico , Granuloma/terapia , Humanos , Inyecciones Intralesiones , Laringe/patología , Láseres de Gas/uso terapéutico , Masculino , Nariz/patologíaRESUMEN
BACKGROUND: The clinical characteristics and complications of Cushing syndrome (CS) are well known and described in the literature. Nevertheless, rare, atypical presentations may go unnoticed. Osteonecrosis is a well-documented complication of glucocorticoid therapy. However, endogenous hypercortisolism is a rare, but relevant, cause of bone avascular necrosis. We describe the case of a woman with CS undiagnosed for 2 years after presenting with femoral avascular necrosis. CASE PRESENTATION: A 38-year-old Caucasian woman was referred for evaluation of secondary amenorrhea, associated with oral contraception withdrawal in the context of deep venous thrombosis (DVT). She had a previous right hip arthroplasty for treatment of avascular necrosis of the femoral head, diagnosed after 3 years of progressive right hip pain and limited mobility. She also had high blood pressure (HBP) of 5 years' duration, and reported weight gain (4 kg in 2 years). There was no history of infertility (gravida 2, para 2). Physical examination revealed buffalo hump, truncal obesity, facial plethora, muscular atrophy and proximal myopathy, and easy bruising (under anticoagulant treatment for DVT). Workup showed abnormal overnight dexamethasone suppression test (DST) (serum cortisol 21.5 µg/dL; normal < 1.8 µg/dL), elevated 24-hour urinary free cortisol (UFC) (728.9 µg/day; reference range 36.0-137.0 µg/day), and suppressed plasma adrenocorticotropic hormone (ACTH) (< 1.0 pg/mL), findings consistent with ACTH-independent CS. Urinary metanephrines and catecholamines were normal, and the remaining analytical study showed no major changes, apart from glycated hemoglobin (HbA1c) of 6.8%. Adrenal computed tomography (CT) scan showed a 25 mm lesion in the left adrenal gland, with density non-suggestive of adenoma. The patient underwent unilateral adrenalectomy and started steroid replacement. Histology revealed an adrenal cortex adenoma. Three months after surgery the patient presented with resolution of HBP and hypercortisolism (UFC 37.4 µg/day; reference range 36.0-137.0 µg/day). CONCLUSION: In some cases, CS signs may go unnoticed and the diagnosis postponed. Avascular necrosis is a rare presenting feature of endogenous hypercortisolism, and, if left untreated, complete collapse of the femoral head may ensue, rendering the need for hip replacement in up to 70% of patients. Suspicion and recognition of atypical features is therefore important in avoiding complications and delay in treatment of CS.
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Síndrome de Cushing , Osteonecrosis , Glándulas Suprarrenales , Adrenalectomía , Hormona Adrenocorticotrópica , Síndrome de Cushing/inducido químicamente , Síndrome de Cushing/diagnóstico , Femenino , Humanos , HidrocortisonaRESUMEN
Leydig cell tumors are rare ovarian neoplasms. Affected individuals typically present with amenorrhea/oligomenorrhea and rapidly progressive features of virilization. Erythrocytosis can also occur as a result of high testosterone levels.