RESUMEN
Postmortem examination results of a patient with Guillain-Barré syndrome and confirmed Zika virus infection revealed demyelination of the sciatic and cranial IV nerves, providing evidence of the acute demyelinating inflammatory polyneuropathy Guillain-Barré syndrome variant. Lack of evidence of Zika virus in nervous tissue suggests that pathophysiology was antibody mediated without neurotropism.
Asunto(s)
Autopsia , Coinfección/virología , Síndrome de Guillain-Barré/complicaciones , Infección por el Virus Zika/complicaciones , Anciano , Coinfección/patología , Síndrome de Guillain-Barré/patología , Síndrome de Guillain-Barré/virología , Humanos , Masculino , Puerto Rico , Infección por el Virus Zika/patología , Infección por el Virus Zika/virologíaRESUMEN
Gene expression analyses based on messenger RNA (mRNA) expression require accurate data normalization. When using endogenous reference genes, these should be carefully validated. Validated reference genes vary greatly depending on tissue, cell subsets and experimental context. This study was aimed to identify reference genes that have more stable mRNA levels among individuals in peripheral blood mononuclear cells (PBMC); fresh skin biopsies; lung and brain autopsies as well as, skin biopsies formalin-fixed paraffin-embedded (FFPE). Therefore, 6 endogenous reference genes were evaluated by quantitative real-time polymerase chain reaction: 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box-binding protein (TBP), beta-2-microbolin (B2M), ubiquitin C (UBC) and mitochondrially encoded ATP synthase 6 (MT-ATP6). Furthermore, validation of their stabilities and performance as reference genes was determined by geNorm and NormFinder programs. The results show that the most stable genes for PBMC and fresh skin biopsies were TBP and UBC; in FFPE lung autopsies and skin biopsies were GAPDH and B2M; and in FFPE brain autopsies were GAPDH and UBC. In addition, 18S rRNA was the least stable of all genes analyzed. These data concluded that even genes constitutively expressed have transcript level variations in different tissues as well as storage and experimental conditions. These observations suggest that suitable reference genes should be selected for normalization of gene expression data analysis.
As análises de expressão gênica baseadas na expressão do RNA mensageiro (mRNA) requerem normalização precisa dos dados. Ao usar genes de referência endógenos, estes devem ser cuidadosamente validados. Os genes de referência validados variam muito, dependendo do tecido, subconjuntos de células e contexto experimental. Este estudo teve como objetivo identificar genes de referência que apresentam níveis de mRNA mais estáveis ââentre indivíduos em células mononucleares do sangue periférico (PBMC); biópsias de pele fresca; autópsias pulmonares e cerebrais, bem como biópsias de pele fixadas em formalina e embebidas em parafina (FFPE). Portanto, 6 genes de referência endógenos foram avaliados por reação quantitativa em cadeia da polimerase em tempo real: rRNA 18S, gliceraldeído-3-fosfato desidrogenase (GAPDH), proteína de ligação à caixa TATA (TBP), beta-2-microbolina (B2M), ubiquitina C (UBC) e ATP sintase 6 mitocondrialmente codificada (MT-ATP6). Além disso, a validação de suas estabilidades e desempenho como genes de referência foi determinada pelos programas geNorm e NormFinder. Os resultados mostram que os genes mais estáveis ââpara PBMC e biópsias de pele fresca foram TBP e UBC; nas autópsias pulmonares de FFPE e biópsias de pele foram GAPDH e B2M; e nas autópsias cerebrais de FFPE foram GAPDH e UBC. Além disso, o 18S rRNA foi o menos estável de todos os genes analisados. Esses dados concluíram que mesmo os genes expressos constitutivamente apresentam variações no nível de transcrição em diferentes tecidos, bem como condições experimentais e de armazenamento. Essas observações sugerem que genes de referência adequados devem ser selecionados para normalização da análise dos dados de expressão gênica.
Asunto(s)
Enfermedades Parasitarias , Humanos , ARN MensajeroRESUMEN
BACKGROUND: Zika virus is an arthropod-borne virus that is a member of the family Flaviviridae transmitted mainly by mosquitoes of the genus Aedes. Although usually asymptomatic, infection can result in a mild and self-limiting illness characterised by fever, rash, arthralgia, and conjunctivitis. An increase in the number of children born with microcephaly was noted in 2015 in regions of Brazil with high transmission of Zika virus. More recently, evidence has been accumulating supporting a link between Zika virus and microcephaly. Here, we describe findings from three fatal cases and two spontaneous abortions associated with Zika virus infection. METHODS: In this case series, formalin-fixed paraffin-embedded tissue samples from five cases, including two newborn babies with microcephaly and severe arthrogryposis who died shortly after birth, one 2-month-old baby, and two placentas from spontaneous abortions, from Brazil were submitted to the Infectious Diseases Pathology Branch at the US Centers for Disease Control and Prevention (Atlanta, GA, USA) between December, 2015, and March, 2016. Specimens were assessed by histopathological examination, immunohistochemical assays using a mouse anti-Zika virus antibody, and RT-PCR assays targeting the NS5 and envelope genes. Amplicons of RT-PCR positive cases were sequenced for characterisation of strains. FINDINGS: Viral antigens were localised to glial cells and neurons and associated with microcalcifications in all three fatal cases with microcephaly. Antigens were also seen in chorionic villi of one of the first trimester placentas. Tissues from all five cases were positive for Zika virus RNA by RT-PCR, and sequence analyses showed highest identities with Zika virus strains isolated from Brazil during 2015. INTERPRETATION: These findings provide strong evidence of a link between Zika virus infection and different congenital central nervous system malformations, including microcephaly as well as arthrogryposis and spontaneous abortions. FUNDING: None.
Asunto(s)
Encéfalo/patología , Encéfalo/virología , Deformidades Congénitas de las Extremidades/virología , Microcefalia/virología , Complicaciones Infecciosas del Embarazo/patología , Complicaciones Infecciosas del Embarazo/virología , Primer Trimestre del Embarazo , Infección por el Virus Zika/congénito , Infección por el Virus Zika/patología , Virus Zika/aislamiento & purificación , Aborto Espontáneo/virología , Adulto , Antígenos Virales/aislamiento & purificación , Autopsia , Brasil , Resultado Fatal , Femenino , Humanos , Inmunohistoquímica/métodos , Lactante , Deformidades Congénitas de las Extremidades/diagnóstico por imagen , Masculino , Microcefalia/patología , Neuroglía/patología , Neuroglía/virología , Placenta/patología , Placenta/virología , Embarazo , Segundo Trimestre del Embarazo , Tercer Trimestre del Embarazo , ARN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Síndrome , Ultrasonografía Prenatal , Virus Zika/inmunologíaRESUMEN
This study investigated the genetic features of Toxoplasma gondii isolated directly in autopsies of HIV-infected patients who died with severe disseminated toxoplasmosis. This retrospective analysis was conducted in a cohort of 15 HIV-infected patients with clinical and laboratory data. They had previous cerebral toxoplasmosis at least 6 months before the disseminated toxoplasmosis episode. The hypothesis was that they were infected with highly virulent parasites due to the condition in which they died. T. gondii genotyping was done directly in DNA extracted from 30 autopsy brain and lung samples (2 per patient) and mutilocus PCR-RFLP genotyping was done using 12 molecular markers. The 30 clinical samples were genotyped successfully in 8 or more loci and six suggestive genotypes were identified. One of them was Toxo DB #11, previously identified in different domestic animals and virulent in experimental animals. The other five suggestive genotypes identified in 14 patients were not described. TgHuDis1 was the most frequent and was determined in 8 patients. TgHuDis3 and TgHuDis5 were identified in two patients each. TgHuDis2 and TgHuDis4 have been identified in one patient each. These suggestive genotypes could be considered as virulent, since they caused severe tissue damage and had similar characteristics as Toxo # DB 11
Asunto(s)
Humanos , Toxoplasma , Toxoplasmosis , Síndrome de Inmunodeficiencia Adquirida , Infecciones Oportunistas Relacionadas con el SIDARESUMEN
This study investigated the genetic features of Toxoplasma gondii isolated directly in autopsies of HIV-infected patients who died with severe disseminated toxoplasmosis. This retrospective analysis was conducted in a cohort of 15 HIV-infected patients with clinical and laboratory data. They had previous cerebral toxoplasmosis at least 6 months before the disseminated toxoplasmosis episode. The hypothesis was that they were infected with highly virulent parasites due to the condition in which they died. T. gondii genotyping was done directly in DNA extracted from 30 autopsy brain and lung samples (2 per patient) and mutilocus PCR-RFLP genotyping was done using 12 molecular markers. The 30 clinical samples were genotyped successfully in 8 or more loci and six suggestive genotypes were identified. One of them was Toxo DB #11, previously identified in different domestic animals and virulent in experimental animals. The other five suggestive genotypes identified in 14 patients were not described. TgHuDis1 was the most frequent and was determined in 8 patients. TgHuDis3 and TgHuDis5 were identified in two patients each. TgHuDis2 and TgHuDis4 have been identified in one patient each. These suggestive genotypes could be considered as virulent, since they caused severe tissue damage and had similar characteristics as Toxo # DB 11.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/complicaciones , Toxoplasma/clasificación , Toxoplasmosis/mortalidad , Síndrome de Inmunodeficiencia Adquirida/mortalidad , Síndrome de Inmunodeficiencia Adquirida/patología , Adulto , Autopsia , Encéfalo/parasitología , Encéfalo/patología , Brasil , Estudios de Cohortes , ADN Protozoario/química , ADN Protozoario/aislamiento & purificación , Femenino , Fijadores , Formaldehído , Técnicas de Genotipaje , Humanos , Inmunohistoquímica , Pulmón/parasitología , Pulmón/patología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Estudios Retrospectivos , Toxoplasma/genética , Toxoplasmosis/parasitología , Toxoplasmosis/patología , Adulto JovenRESUMEN
Zika virus is a mosquito-borne flavivirus that is related to dengue virus and transmitted primarily by Aedes aegypti mosquitoes, with humans acting as the principal amplifying host during outbreaks. Zika virus was first reported in Brazil in May 2015 (1). By February 9, 2016, local transmission of infection had been reported in 26 countries or territories in the Americas.* Infection is usually asymptomatic, and, when symptoms are present, typically results in mild and self-limited illness with symptoms including fever, rash, arthralgia, and conjunctivitis. However, a surge in the number of children born with microcephaly was noted in regions of Brazil with a high prevalence of suspected Zika virus disease cases. More than 4,700 suspected cases of microcephaly were reported from mid-2015 through January 2016, although additional investigations might eventually result in a revised lower number (2). In response, the Brazil Ministry of Health established a task force to further investigate possible connections between the virus and brain anomalies in infants (3).
Asunto(s)
Encéfalo/virología , Placenta/virología , Infección por el Virus Zika/diagnóstico , Virus Zika/aislamiento & purificación , Aborto Espontáneo/virología , Antígenos Virales/aislamiento & purificación , Brasil/epidemiología , Femenino , Humanos , Recién Nacido , Embarazo , ARN Viral/aislamiento & purificación , Virus Zika/inmunología , Infección por el Virus Zika/congénitoRESUMEN
This study investigated the genetic features of T. gondii isolated directly in autopsies of HIV-infected patients who died with severe disseminated toxoplasmosis. This retrospective analysis was conducted in a cohort of 15 HIV-infected patients with clinical and laboratory data. They had previous cerebral toxoplasmosis at least 6 months before the disseminated toxoplasmosis episode. The hypothesis was that they were infected with highly virulent parasites due to the condition in which they died. T. gondii genotyping was done directly in DNA extracted from 30 autopsy brain and lung samples (2 per patient) and mutilocus PCR-RFLP genotyping was done using 12 molecular markers. The 30 clinical samples were genotyped successfully in 8 or more loci and six suggestive genotypes were identified. One of them was Toxo DB #11, previously identified in different domestic animals and virulent in experimental animals. The other five suggestive genotypes...(AU)
Asunto(s)
Toxoplasma , Síndrome de Inmunodeficiencia Adquirida , ADN de Forma ARESUMEN
Gene expression analyses based on messenger RNA (mRNA) expression require accurate datanormalization. When using endogenous reference genes, these have to be carefully validated. Validated reference genes vary greatly depending on tissue, cell subsets and experimental context.The aim of this study was to identify reference genes that present more stable mRNA levels amongindividuals in peripheral blood mononuclear cells (PBMC); fresh skin biopsies; lung and brainautopsies as well as, skin biopsies formalin-fixed paraffin-embedded (FFPE). Therefore, 6endogenous reference genes were evaluated by quantitative real-time polymerase chain reaction: 18SrRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box-binding protein (TBP),beta-2-microbolin (B2M), ubiquitin C (UBC) and mitochondrially encoded ATP synthase 6 (MTATP6).Furthermore, validation of their stability and suitability as reference genes was determined bythe geNorm program. The results show that in PBMC and fresh skin biopsies, TBP and UBC wereidentified as the most stable, while in FFPE lung autopsies and skin biopsies, GAPDH and B2M, andin FFPE brain autopsies, GAPDH and UBC turned out to be the most stable...
Asunto(s)
Expresión Génica , Genes , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
PURPOSE: To analyze the effects of application of 1% and 3% insulin-like growth factor I (IGF-1) cream on the process of wound healing in induced skin lesions in diabetic and non-diabetic rats and evaluate its effect on expression of myofibroblasts. METHODS: Ninety-six Wistar adult male rats were divided into six groups, with 16 rats in each group, as follows: group 1: non-diabetic, untreated; group 2: non-diabetic, treated with 1% IGF-1 cream; group 3: non-diabetic, treated with 3% IGF-1 cream; group 4: diabetic, untreated; group 5: diabetic, treated with 1% IGF-1 cream; and group 6: diabetic, treated with 3% IGF-1 cream. In groups 4, 5, and 6, diabetes was induced by intravenous injection of alloxan. After diabetes had been induced, animals were mantained for 3 months. The experimental procedure consisted of the creation of a circular incision of 0.9 mm in diameter using a metal punch. Following this, wounds were treated daily according to the assigned treatment regimen. Groups 2 and 5 were treated with 1% IGF-1 cream, groups 3 and 6 with 3% IGF-1 cream, and groups 1 and 4 and the untreated groups with 0.9% saline solution. From each group, samples from 4 rats were taken at three, seven, 14, and 21 days after the injury. Samples were fixed in 10% formalin to prepare slides for histological analysis. Slides stained with hematoxylin-eosin (H&E) and Masson were observed vascular proliferation, mononuclear cells, polymorphonuclear cells, fibroblast proliferation, re-epithelialization, and collagen fibers. This study analyzed the expression of α-smooth muscle actin using specific antibodies to correlate the temporal expression of α-smooth muscle-specific actin (α-SM actin), a molecular marker for myofibroblast transformation. RESULTS: Macroscopic observation of wounds showed a more rapid re-epithelialization of wounds treated with IGF. Regarding acute inflammatory reactions, the results of the analysis of vascular proliferation and polymorphonuclear and mononuclear cells showed no statistically significant differences in any of the periods studied (according to the results of a Mann-Whitney test). The initial immunohistochemical analysis of tissue samples conducted to compare the expression of α-smooth muscle actin between groups showed a relevant response in the expression of myofibroblasts. Data were analyzed using ANOVA and were found to be statistically significant. CONCLUSION: The topical application of 1% and 3% IGF-1 creams increases the expression of myofibroblasts in the process of wound healing in rats.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Factor I del Crecimiento Similar a la Insulina/uso terapéutico , Miofibroblastos/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Actinas/análisis , Administración Tópica , Aloxano , Animales , Proliferación Celular , Diabetes Mellitus Experimental/inducido químicamente , Modelos Animales de Enfermedad , Inmunohistoquímica , Masculino , Ratas Wistar , Reproducibilidad de los Resultados , Crema para la Piel/uso terapéutico , Factores de Tiempo , Resultado del Tratamiento , Cicatrización de Heridas/fisiologíaRESUMEN
To analyze the effects of application of 1% and 3% insulin-like growth factor I (IGF-1) cream on the process of wound healing in induced skin lesions in diabetic and non-diabetic rats and evaluate its effect on expression of myofibroblasts. METHODS: Ninety-six Wistar adult male rats were divided into six groups, with 16 rats in each group, as follows: group 1: non-diabetic, untreated; group 2: non-diabetic, treated with 1% IGF-1 cream; group 3: non-diabetic, treated with 3% IGF-1 cream; group 4: diabetic, untreated; group 5: diabetic, treated with 1% IGF-1 cream; and group 6: diabetic, treated with 3% IGF-1 cream. In groups 4, 5, and 6, diabetes was induced by intravenous injection of alloxan. After diabetes had been induced, animals were mantained for 3 months. The experimental procedure consisted of the creation of a circular incision of 0.9 mm in diameter using a metal punch. Following this, wounds were treated daily according to the assigned treatment regimen. Groups 2 and 5 were treated with 1% IGF-1 cream, groups 3 and 6 with 3% IGF-1 cream, and groups 1 and 4 and the untreated groups with 0.9% saline solution. From each group, samples from 4 rats were taken at three, seven, 14, and 21 days after the injury. Samples were fixed in 10% formalin to prepare slides for histological analysis. Slides stained with hematoxylin-eosin (H&E) and Masson were observed vascular proliferation, mononuclear cells, polymorphonuclear cells, fibroblast proliferation, re-epithelialization, and collagen fibers. This study analyzed the expression of α-smooth muscle actin using specific antibodies to correlate the temporal expression of α-smooth muscle-specific actin (α-SM actin), a molecular marker for myofibroblast transformation. RESULTS: Macroscopic observation of wounds showed a more rapid re-epithelialization of wounds treated with IGF. Regarding acute inflammatory reactions, the results of the analysis of vascular.(AU)
Asunto(s)
Animales , Ratas , Insulina/farmacología , Complicaciones de la Diabetes , Crecimiento/fisiología , Ratas/clasificaciónRESUMEN
To analyze the effects of application of 1% and 3% insulin-like growth factor I (IGF-1) cream on the process of wound healing in induced skin lesions in diabetic and non-diabetic rats and evaluate its effect on expression of myofibroblasts. METHODS: Ninety-six Wistar adult male rats were divided into six groups, with 16 rats in each group, as follows: group 1: non-diabetic, untreated; group 2: non-diabetic, treated with 1% IGF-1 cream; group 3: non-diabetic, treated with 3% IGF-1 cream; group 4: diabetic, untreated; group 5: diabetic, treated with 1% IGF-1 cream; and group 6: diabetic, treated with 3% IGF-1 cream. In groups 4, 5, and 6, diabetes was induced by intravenous injection of alloxan. After diabetes had been induced, animals were mantained for 3 months. The experimental procedure consisted of the creation of a circular incision of 0.9 mm in diameter using a metal punch. Following this, wounds were treated daily according to the assigned treatment regimen. Groups 2 and 5 were treated with 1% IGF-1 cream, groups 3 and 6 with 3% IGF-1 cream, and groups 1 and 4 and the untreated groups with 0.9% saline solution. From each group, samples from 4 rats were taken at three, seven, 14, and 21 days after the injury. Samples were fixed in 10% formalin to prepare slides for histological analysis. Slides stained with hematoxylin-eosin (H&E) and Masson were observed vascular proliferation, mononuclear cells, polymorphonuclear cells, fibroblast proliferation, re-epithelialization, and collagen fibers. This study analyzed the expression of α-smooth muscle actin using specific antibodies to correlate the temporal expression of α-smooth muscle-specific actin (α-SM actin), a molecular marker for myofibroblast transformation. RESULTS: Macroscopic observation of wounds showed a more rapid re-epithelialization of wounds treated with IGF. Regarding acute inflammatory reactions, the results of the analysis of vascular.
Asunto(s)
Animales , Ratas , Crecimiento/fisiología , Complicaciones de la Diabetes , Insulina/farmacología , Ratas/clasificaciónRESUMEN
BACKGROUND: Dengue is a disease whose clinical manifestations range from asymptomatic infections to a severe disease. There have been some previous reports of myocardial involvement in dengue, but this association has not been completely established. METHODS: From January to July of 2011, patients hospitalized with dengue, confirmed through dengue nonstructural protein 1 and/or immunoglobulin M detection, were included in this study and troponin I and N terminal fragment of B-type natriuretic peptide levels were determined. Patients with abnormal biomarkers underwent echocardiography and when any abnormality was detected, they underwent cardiac magnetic resonance imaging. RESULTS: Eighty-one patients were evaluated and 12 patients (15%) presented with elevated biomarker levels. Compared to controls, they had higher leukocyte (P < .001) and platelet counts (P = .005); higher C-reactive protein (P = .02), and a lower viral load (P = .03). There was no difference according to clinical dengue classification; dengue hemorrhagic fever/dengue shock syndrome severity; duration of symptoms; or prevalence of secondary infection between the 2 groups. Two patients died secondary to cardiogenic shock before imaging studies. Necroscopic findings were compatible to myocarditis in both, and immunohistochemistry for dengue virus showed increased staining on mononuclear cells located in the myocardial tissue. Of the 10 patients who underwent echocardiography, depressed left ventricular ejection fraction (LVEF) was identified in 1, left ventricular segmental abnormalities with preserved LVEF in 2, and an important pericardial effusion with tamponade in another. Cardiac involvement was confirmed by CMR in these 4 patients. CONCLUSIONS: Dengue viruses were shown to cause cardiac disease with clinical manifestations ranging from mild elevation of biomarkers to myocarditis and/or pericarditis.