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1.
Article in Chinese | WPRIM | ID: wpr-941034

ABSTRACT

OBJECTIVE@#To construct an adenovirus vector expressing artificial splicing factor capable of regulating alternative splicing of Yap1 in cardiomyocytes.@*METHODS@#The splicing factors with different sequences were constructed against Exon6 of YAP1 based on the sequence specificity of Pumilio1. The PCR fragment of the artificially synthesized PUF-SR or wild-type PUFSR was cloned into pAd-Track plasmid, and the recombinant plasmids were transformed into E. coli DH5α for plasmid amplification. The amplified plasmids were digested with Pac I and transfected into 293A cells for packaging to obtain the adenovirus vectors. Cultured neonatal rat cardiomyocytes were transfected with the adenoviral vectors, and alternative splicing of YAP1 was detected using quantitative and semi-quantitative PCR; Western blotting was performed to detect the signal of the fusion protein Flag.@*RESULTS@#The transfection efficiency of the adenovirus vectors was close to 100% in rat cardiomyocytes, and no fluorescent protein was detected in the cells with plasmid transfection. The results of Western blotting showed that both the negative control and Flag-SR-NLS-PUF targeting the YAPExon6XULIE sequence were capable of detecting the expression of the protein fused to Flag. The results of reverse transcription-PCR and PCR demonstrated that the artificial splicing factor constructed based on the 4th target sequence of YAP1 effectively regulated the splicing of YAP1 Exon6 in the cardiomyocytes (P < 0.05).@*CONCLUSION@#We successfully constructed adenovirus vectors capable of regulating YAP1 alternative splicing rat cardiomyocytes.


Subject(s)
Adenoviridae/metabolism , Alternative Splicing , Animals , Animals, Newborn , Escherichia coli/metabolism , Genetic Vectors , Myocytes, Cardiac/metabolism , Plasmids , RNA Splicing Factors/metabolism , Rats , Transfection
2.
Chinese Journal of Burns ; (6): 119-129, 2022.
Article in Chinese | WPRIM | ID: wpr-935986

ABSTRACT

Objective: To explore the effects of P311 on the angiogenesis ability of human microvascular endothelial cell 1 (HMEC-1) in vitro and the potential molecular mechanism. Methods: The experimental research method was used. HMEC-1 was collected and divided into P311 adenovirus group and empty adenovirus group according to the random number table (the same grouping method below), which were transfected correspondingly for 48 h. The cell proliferation activity was detected using the cell counting kit 8 on 1, 3, and 5 days of culture. The residual scratch area of cells at post scratch hour 6 and 11 was detected by scratch test, and the percentage of the residual scratch area was calculated. The blood vessel formation of cells at 8 h of culture was observed by angiogenesis experiment in vitro, and the number of nodes and total length of the tubular structure were measured. The protein expressions of vascular endothelial growth factor receptor 2 (VEGFR2), phosphorylated VEGFR2 (p-VEGFR2), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylated ERK1/2 (p-ERK1/2) in cells were detected by Western blotting. HMEC-1 was collected and divided into P311 adenovirus+small interfering RNA (siRNA) negative control group, empty adenovirus+siRNA negative control group, P311 adenovirus+siRNA-VEGFR2 group, and empty adenovirus+siRNA-VEGFG2 group, which were treated correspondingly. The protein expressions of VEGFR2, p-VEGFR2, ERK1/2, and p-ERK1/2 in cells were detected by Western blotting at 24 h of transfection. The blood vessel formation of cells at 24 h of transfection was observed by angiogenesis experiment in vitro, and the number of nodes and total length of the tubular structure were measured. HMEC-1 was collected and divided into P311 adenovirus+dimethylsulfoxide (DMSO) group, empty adenovirus+DMSO group, P311 adenovirus+ERK1/2 inhibitor group, and empty adenovirus+ERK1/2 inhibitor group, which were treated correspondingly. The protein expressions of ERK1/2 and p-ERK1/2 in cells were detected by Western blotting at 2 h of treatment. The blood vessel formation of cells at 2 h of treatment was observed by angiogenesis experiment in vitro, and the number of nodes and total length of the tubular structure were measured. The sample number at each time point in each group was 6. Data were statistically analyzed with independent sample t test, analysis of variance for repeated measurement, one-way analysis of variance, and least significant difference test. Results: Compared with that of empty adenovirus group, the proliferation activity of cells in P311 adenovirus group did not show significant difference on 1, 3, and 5 days of culture (with t values of -0.23, -1.30, and -1.52, respectively, P>0.05). The residual scratch area percentages of cells in P311 adenovirus group were significantly reduced at post scratch hour 6 and 11 compared with those of empty adenovirus group (with t values of -2.47 and -2.62, respectively, P<0.05). At 8 h of culture, compared with those of empty adenovirus group, the number of nodes and total length of the tubular structure of cells in P311 adenovirus group were significantly increased (with t values of 4.49 and 4.78, respectively, P<0.01). At 48 h of transfection, compared with those of empty adenovirus group, the protein expressions of VEGFR2 and ERK1/2 of cells in P311 adenovirus group showed no obvious changes (P>0.05), and the protein expressions of p-VEGFR2 and p-ERK1/2 of cells in P311 adenovirus group were significantly increased (with t values of 17.27 and 16.08, P<0.01). At 24 h of transfection, the protein expressions of p-VEGFR2 and p-ERK1/2 of cells in P311 adenovirus+siRNA negative control group were significantly higher than those in empty adenovirus+siRNA negative control group (P<0.01). The protein expressions of VEGFR2, p-VEGFR2, and p-ERK1/2 of cells in P311 adenovirus+siRNA negative control group were significantly higher than those in P311 adenovirus+siRNA-VEGFR2 group (P<0.01). The protein expressions of VEGFR2 and p-ERK1/2 of cells in empty adenovirus+siRNA negative control group were significantly higher than those in empty adenovirus+siRNA-VEGFR2 group (P<0.05 or P<0.01). At 24 h of transfection, the number of nodes of the tubular structure in cells of P311 adenovirus+siRNA negative control group was 720±62, which was significantly more than 428±38 in empty adenovirus+siRNA negative control group and 364±57 in P311 adenovirus+siRNA-VEGFR2 group (with P values both <0.01). The total length of the tubular structure of cells in P311 adenovirus+siRNA negative control group was (21 241±1 139) μm, which was significantly longer than (17 005±1 156) μm in empty adenovirus+siRNA negative control group and (13 494±2 465) μm in P311 adenovirus+siRNA-VEGFR2 group (with P values both <0.01). The number of nodes of the tubular structure in cells of empty adenovirus+siRNA negative control group was significantly more than 310±75 in empty adenovirus+siRNA-VEGFR2 group (P<0.01), and the total length of the tubular structure of cells in empty adenovirus+siRNA negative control group was significantly longer than (11 600±2 776) μm in empty adenovirus+siRNA-VEGFR2 group (P<0.01). At 2 h of treatment, the protein expression of p-ERK1/2 of cells in P311 adenovirus+DMSO group was significantly higher than that in empty adenovirus+DMSO group and P311 adenovirus+ERK1/2 inhibitor group (with P values both <0.01), and the protein expression of p-ERK1/2 of cells in empty adenovirus+DMSO group was significantly higher than that in empty adenovirus+ERK1/2 inhibitor group (P<0.05). At 2 h of treatment, the number of nodes of the tubular structure in cells of P311 adenovirus+DMSO group was 726±72, which was significantly more than 421±39 in empty adenovirus+DMSO group and 365±41 in P311 adenovirus+ERK1/2 inhibitor group (with P values both <0.01). The total length of the tubular structure of cells in P311 adenovirus+DMSO group was (20 318±1 433) μm, which was significantly longer than (16 846±1 464) μm in empty adenovirus+DMSO group and (15 114±1 950) μm in P311 adenovirus+ERK1/2 inhibitor group (with P values both <0.01). The number of nodes of the tubular structure in cells of empty adenovirus+DMSO group was significantly more than 317±67 in empty adenovirus+ERK1/2 inhibitor group (P<0.01), and the total length of the tubular structure of cells in empty adenovirus+DMSO group was significantly longer than (13 188±2 306) μm in empty adenovirus+ERK1/2 inhibitor group (P<0.01). Conclusions: P311 can enhance the angiogenesis ability of HMEC-1 by activating the VEGFR2/ERK1/2 signaling pathway.


Subject(s)
Adenoviridae/genetics , Cell Line , Endothelial Cells , Endothelium, Vascular , Humans , Neovascularization, Physiologic , Nerve Tissue Proteins , Oncogene Proteins , Signal Transduction , Transfection , Vascular Endothelial Growth Factor A
3.
Chinese Journal of Hepatology ; (12): 38-44, 2022.
Article in Chinese | WPRIM | ID: wpr-935906

ABSTRACT

Objective: To investigate the effect of adenovirus-mediated shRNA down-regulating phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression on vinculin, filamin A, and cortactin in activated hepatic stellate cells (HSCs). Methods: Activated rats hepatic stellate cell line (HSC-T6) was cultured in vitro. Recombinant adenovirus Ad-shRNA/PTEN carrying PTEN targeted RNA interference sequence [short hairpin RNA (shRNA)] and empty control virus Ad-GFP were transfected into HSCs. The PTEN mRNA and protein expression of HSCs in each group were detected by real-time fluorescence quantitative PCR and Western blot. The expressional change of vinculin, filamin A and cortactin in HSCs of each group were detected by confocal laser scanning immunofluorescence microscope. Image-pro plus 6.0 software was used for image analysis and processing. The integrated optical density (IOD) of the fluorescence protein expression was measured. The experiment was divided into three groups: control group (DMEM instead of adenovirus solution in the adenovirus transfection step), Ad-GFP group (transfected with empty virus Ad-GFP only expressing green fluorescent protein), and Ad-shRNA/PTEN group (recombinant adenovirus Ad-shRNA/PTEN carrying shRNA targeting PTEN and expressing green fluorescent protein). One-way analysis of variance was used for comparison of mean value among the three groups, and LSD-test was used for comparison between the groups. Results: shRNA targeted PTEN was successfully transfected and the expression of PTEN mRNA and protein in HSC (P < 0.05) was significantly down-regulated. HSCs vinculin was mainly expressed in the cytoplasm. HSCs vinculin fluorescence IOD in the Ad-shRNA/PTEN group (19 758.83 ± 1 520.60) was higher than control (7 737.16 ± 279.93) and Ad-GFP group (7 725.50 ± 373.03) (P < 0.05), but there was no statistically significant difference between control group and Ad-GFP group (P > 0.05). There was no statistically significant difference in the fluorescence IOD of Filamin A among the three groups (P > 0.05), but the subcellular distribution of Filamin A among the three groups were changed. Filamin A in the Ad-shrNA /PTEN HSC group was mainly distributed in the cytoplasm. Filamin A HSC was mainly located in the nucleus.The filamin A HSC in the control group and Ad-GFP group was mainly located in the nucleus. The nucleocytoplasmic ratio of Filamin A in the AD-shrNA /PTEN group (0.60 ± 0.15) was significantly lower than control group (1.20 ± 0.15) and Ad-GFP group (1.08 ± 0.23), P < 0.05. but there was no statistically significant difference in filamin A nucleocytoplasmic ratio of HSC between the control group and the Ad-GFP group (P > 0.05). Cortactin HSCs in the three groups was mainly distributed in the cytoplasm. The cortactin fluorescence IOD of HSCs in the Ad-shRNA/PTEN group was significantly higher than control group (22 959.94 ± 1 710.42) and the Ad-GFP group (22 547.11 ± 1 588.72 ) (P < 0.05), while there was no statistically significant difference in the IOD of cortactin fluorescence in HSCs between the control group and the Ad-GFP group (P > 0.05). Conclusion: The down-regulation of PTEN expression raises the expression of microfilament-binding protein vinculin and cortactin, and changes the subcellular distribution of another microfilament binding protein filamin A, that is, translocation from nucleus to the cytoplasm in activated HSC in vitro.


Subject(s)
Adenoviridae/metabolism , Animals , Carrier Proteins , Cell Proliferation , Cortactin , Filamins/genetics , Hepatic Stellate Cells/metabolism , PTEN Phosphohydrolase/metabolism , RNA, Small Interfering/genetics , Rats , Vinculin/genetics
4.
Chinese Journal of Biotechnology ; (12): 1824-1836, 2022.
Article in Chinese | WPRIM | ID: wpr-927820

ABSTRACT

In order to construct a recombinant replication deficient human type 5 adenovirus (Ad5) expressing a foot-and-mouth disease virus (FMDV) capsid protein, specific primers for P12A and 3B3C genes of FMDV-OZK93 were synthesized. The P12A and 3B3C genes were then amplified and connected by fusion PCR, and a recombinant shuttle plasmid pDC316-mCMV-EGFP-P12A3B3C expressing the FMDV-OZK93 capsid protein precursor P12A and 3B3C protease were obtained by inserting the P12A3B3C gene into the pDC316-mCMV-EGFP plasmid. The recombinant adenovirus rAdv-P12A3B3C-OZK93 was subsequently packaged, characterized and amplified using AdMaxTM adenovirus packaging system, and the expression was verified by infecting human embryonic kidney cell HEK-293. The humoral and cellular immunity levels of well-expressed and purified recombinant adenovirus immunized mice were evaluated. The results showed that rAdv-P12A3B3C-OZK93 could be stably passaged and the maximum virus titer reached 1×109.1 TCID50/mL. Western blotting and indirect immunofluorescence showed that rAdv-P12A3B3C-OZK93 expressed the FMDV-specific proteins P12A and VP1 in HEK-293 cells. In addition, the PK cell infection experiment confirmed that rAdv-P12A3B3C-OZK93 could infect porcine cells, which is essential for vaccination in pigs. Comparing with the inactivated vaccine group, the recombinant adenovirus could induce higher FMDV-specific IgG antibodies, γ-IFN and IL-10. This indicates that the recombinant adenovirus has good immunity for animal, which is very important for the subsequent development of foot-and-mouth disease vaccine.


Subject(s)
Adenoviridae/genetics , Adenoviruses, Human/genetics , Animals , Antibodies, Viral , Capsid/metabolism , Capsid Proteins , Foot-and-Mouth Disease/prevention & control , Foot-and-Mouth Disease Virus/genetics , HEK293 Cells , Humans , Mice , Recombinant Proteins/genetics , Serogroup , Swine , Viral Proteins , Viral Vaccines/genetics
5.
Chinese Journal of Biotechnology ; (12): 1086-1095, 2022.
Article in Chinese | WPRIM | ID: wpr-927765

ABSTRACT

ERα-36 is a novel subtype of estrogen receptor α which promotes tumor cell proliferation, invasion and drug resistance, and it serves as a therapeutic target. However, only small-molecule compounds targeting ERα-36 are under development as anticancer drugs at present. Gene therapy approach targeting ERα-36 can be explored using recombinant adenovirus armed with decoy receptor. The recombinant shuttle plasmid pDC316-Ig κ-ERα-36-Fc-GFP was constructed via genetic engineering to express an Ig κ-signaling peptide-leading secretory recombinant fusion protein ERα-36-Fc. The recombinant adenovirus Ad-ERα-36-Fc-GFP was subsequently packaged, characterized and amplified using AdMaxTM adenovirus packaging system. The expression of fusion protein and functional outcome of Ad-ERα-36-Fc-GFP transduction were further analyzed with triple-negative breast cancer MDA-MB-231 cells. Results showed that the recombinant adenovirus Ad-ERα-36-Fc-GFP was successfully generated. The virus effectively infected MDA-MB-231 cells which resulted in expression and secretion of the recombinant fusion protein ERα-36-Fc, leading to significant inhibition of EGFR/ERK signaling pathway. Preparation of the recombinant adenovirus Ad-ERα-36-Fc-GFP provides a basis for further investigation on cancer gene therapy targeting ERα-36.


Subject(s)
Adenoviridae/genetics , Cell Proliferation , Estrogen Receptor alpha/metabolism , Recombinant Proteins , Transfection
6.
Med.lab ; 26(4): 383-389, 2022. Tabs, ilus
Article in Spanish | LILACS | ID: biblio-1412540

ABSTRACT

La enfermedad por coronavirus SARS-CoV-2 que surgió en el año 2019 (COVID-19), ha obligado al rápido desarrollo de vacunas para prevenir su propagación e intentar controlar la pandemia. Dentro de las vacunas desarrolladas, las primeras en ser aprobadas con una tecnología nueva en el campo de la vacunación, fueron las vacunas basadas en ARNm (ácido ribonucleico mensajero), que lograron tasas de efectividad cercanas al 95 % para la prevención de la enfermedad COVID-19 grave. Los eventos adversos comunes son reacciones locales leves, pero ha habido varios informes de pacientes que desarrollaron tiroiditis subaguda y disfunción tiroidea después de recibir la vacuna contra SARS-CoV-2. Este artículo presenta dos casos de tiroiditis subaguda poco después de recibir la vacuna contra COVID-19


The SARS-CoV-2 coronavirus disease which emerged in 2019 (COVID-19), has forced the rapid development of vaccines to prevent the spread of infection and attempt to control the pandemic. Among the vaccines developed, one of the first to be approved with a new technology in the field of vaccination, was the mRNA (messenger ribonucleic acid) vaccine, with rates of effectiveness close to 95% for the prevention of severe COVID-19 disease. Common adverse events are mild local reactions, but there have been some reports of patients developing sub-acute thyroiditis and thyroid dysfunction after receiving the SARS-CoV-2 vaccine. This article presents two case reports of subacute thyroiditis shortly after receiving the COVID-19 vaccine


Subject(s)
Humans , COVID-19 , Thyroiditis , RNA, Messenger , Vaccines , Adenoviridae , Goiter
7.
J. pediatr. (Rio J.) ; 97(4): 420-425, July-Aug. 2021. tab
Article in English | LILACS | ID: biblio-1287045

ABSTRACT

Abstract Objective This study aimed to investigate human adenovirus 36 (Adv36) as an associated factor for adiposity in children and adolescents aged 9-12 years. Methods This was a case-control study comparing overweight (cases) and eutrophic (controls) children and adolescents aged 9-12 years based on their body mass index in relation to human adenovirus 36 serology. Human adenovirus 36-specific neutralizing antibodies were assessed using the serum neutralization assay, and a questionnaire regarding the subjects' personal backgrounds, breastfeed history, age of starting daycare, and eating and exercise habits was also applied. Results A total of 101 (51, eutrophic; 50, overweight) children were included in the study. The Adv36 seropositivity rate was of 15.8%, which increased the chance of being overweight by 3.17 times (p = 0.049). Enrollment in a full-time daycare center before the age of 24 months increased the chance of being overweight by 2.78 times (p = 0.027). Metabolic parameters (total cholesterol and blood glucose) were insignificantly different among children who were seropositive or seronegative for human adenovirus 36. Conclusion This study concluded that excessive weight was positively associated with seropositivity for human adenovirus 36. Early enrollment in a full-time daycare was also an associated factor for obesity. Such data, confirmed in new studies, reinforces the role of human adenovirus 36 in the increase of childhood adiposity.


Subject(s)
Humans , Child, Preschool , Child , Adolescent , Adenoviruses, Human , Pediatric Obesity , Body Mass Index , Case-Control Studies , Adenoviridae , Adiposity
8.
Article in Chinese | WPRIM | ID: wpr-888337

ABSTRACT

OBJECTIVE@#To construct and identify adenovirus vector co-expressing hBMP2 and hVEGF165 fusion protein which labeled with green fluorescence protein, and laying the foundtion of the effect of hBMP2 and hVEGF165 gene inducing BMMSCs differentiation to osteoblast and bone defect repaired in the body.@*METHODS@#BMP2 and VEGF165 gene was amplified from cDNA library by PCR and inserted to the polyclonal site of adenovirus shuttle plasmid pAd-MCMV-GFP. Ad-BMP2- VEGF165 was recombinated and propagated in HEK293 cells by co-transfecting with the constructed recombinant shuttle plasmid pAd-MCMV-BMP2-VEGF165 and adenovirus helper plasmid pBHGloxΔ E1, 3Cre. The recombinant adenovirus was purified and virustiter was determined, and then to research GFP expression and to calculate the adenovirus transfection rate in rabbit BMMSCs.@*RESULTS@#The recombinant adenovirus vector Ad-BMP2-VEGF165 was successfully constructed by the methods of gene analyzing, colony PCR, Western blotting and observing GFP expression, and the titer of the adenovirus was 1×10@*CONCLUSION@#Recombinant adenovirus vector containing hBMP2 and hVEGF165 gene was successfully constructed and its high titer was obtained.


Subject(s)
Adenoviridae/genetics , Animals , Bone Marrow Cells , Genetic Vectors/genetics , HEK293 Cells , Humans , Mesenchymal Stem Cells , Rabbits , Transfection
9.
Metro cienc ; 28(4): 36-41, 2020/10/29. tab
Article in Spanish | LILACS | ID: biblio-1151650

ABSTRACT

RESUMEN Objetivo: Evaluar el uso de los biomarcadores proteína C reactiva (PCR) y procalcitonina (PCT) para la valoración de severidad clínica en pacientes pediá-tricos diagnosticados con infección por adenovirus. Método: Se estudiaron 100 pacientes mayores de 28 días y menores a 15 años con diagnóstico confir-mado de infección por adenovirus, en el Hospital Vozandes Quito, Hospital Metropolitano y Hospital de los Valles en el periodo enero 2010 a mayo 2019, se recolectó información demográfica y clínica de forma retrospectiva. las características clínicas y biomarcadores se compararon de acuerdo a la severidad de la patología respiratoria presentada, cuya clasificación por requerimientos terapéuticos la definió como leve, moderada y grave. Resultados: El 89% de los pacientes presentó severidad clínica leve, el 11% moderada y ninguno fue clasificado como severa. El 35% de los pacientes presentaron coinfección con al menos un virus adicional, estos presentaron severidad clínica moderada en el 63,3%. Los valores promedio de PCR fueron 61,3 ± 54,06 mg/L y PCT 0,65 ± 0,8ng/mL. Entre los pacientes con severidad clínica leve y moderada vemos que PCR y PCT fueron superiores en pacientes con severidad clínica leve 66,07±55,09 mg/L vs 22,71±19,60 mg/L, p=0,003 y 0,70±0,83 ng/mL vs 0,23±0,26 ng/mL, p=0.005 respectivamente. Al momento de predecir la severidad clínica de los pacientes pediátricos diagnosticados con adenovirus el área bajo la curva encontrada en estos dos parámetros fue 0,241 y 0,224 para PCT y PCR respectivamente, con valores de sensibilidad y especificidad bajo el 50%. Conclusión: Los biomarcadores PCT y PCR presentan limitada utilidad al momento de predecir la severidad clínica de los pacientes pediátricos diagnosticados con infección por adenovirus.


ABSTRACT Objective: To evaluate the use of C-reactive protein (CRP) and procalcitonin (PCT) biomarkers for the assessment of clinical severity in pediatric patients diag-nosed with adenovirus infection. Method: 100 patients older than 28 days and younger than 15 years with a confirmed diagnosis of adenovirus infection were studied at the Hospital Vozandes Quito, Hospital Metropolitano and Hospital de los Valles in the period January 2010 to May 2019, demographic information was collected and retrospectively. the clinical characteristics and biomarkers were compared according to the severity of the respiratory pathology presented, whose classification by therapeutic requirements defined it as mild, moderate and severe. Results: 89% of the patients presented mild clinical severity, 11% moderate and none were classified as severe. 35% of the patients presented coinfection with at least one additional virus, these presented moderate clinical severity in 63.3%. Mean CRP values were 61.3 ± 54.06 mg / L and PCT 0.65 ± 0.8ng / mL. Among patients with mild and moderate clinical severity, we see that CRP and PCT were higher in patients with mild clinical severity 66.07 ± 55.09 mg / L vs 22.71 ± 19.60 mg / L, p = 0.003 and 0, 70 ± 0.83 ng / mL vs 0.23 ± 0.26 ng / mL, p = 0.005 respectively. When predicting the clinical severity of pediatric patients diagnosed with adenovirus, the area under the curve found in these two parameters was 0.241 and 0.224 for PCT and CRP, respectively, with sensitivity and specificity values below 50%. Conclusion: PCT and CRP biomarkers have limited usefulness when predicting the clinical severity of pediatric patients diagnosed with adenovirus infection


Subject(s)
Humans , Male , Female , Infant , Child, Preschool , Child , Adolescent , C-Reactive Protein , Sensitivity and Specificity , Adenoviridae Infections , Pathology , Therapeutics , Biomarkers , Adenoviridae
10.
J. med. virol ; 92(10): 1-6, Aug. 2, 2020. tab
Article in English | LILACS, ColecionaSUS, CONASS, SES-RS | ID: biblio-1120884

ABSTRACT

Respiratory viral infection can cause severe disease and hospitalization, especially among children, the elderly, and patients with comorbidities. In Brazil, the official surveillance system of severe acute respiratory infection (SARI) investigates influenza A (IAV) and B (IBV) viruses, respiratory syncytial virus (RSV), adenovirus (HAdV), and parainfluenza viruses (hPIV 1­3). In Rio Grande do Sul (RS), Brazil, many fatalities associated with SARI between 2013 and 2017 occurred among patients without underlying diseases and for whom the causative agent had not been identified using official protocols. This cross­sectional study analyzed the presence of coronaviruses (HCoV), bocavirus (HBoV), metapneumovirus (hMPV), and rhinovirus in patients who died of SARI despite not having comorbidities, and that were negative for IAV, IBV, RSV, HAdV, and hPIV. Nasopharyngeal aspirates/swabs from patients were used for nucleic acid extraction. The presence of HCoVs OC43, HKU1, NL63, and 229E; HBoV; hMPV; and rhinovirus was assessed by quantitative reverse transcription­polymerase chain reaction. Clinical data were also analyzed. Between 2013 and 2017, 16 225 cases of SARI were reported in RS; 9.8% of the patients died; 20% of all fatal cases were patients without comorbidities and for whom no pathogen was detected using standard protocols. Analysis of 271 of these cases identified HCoV in nine cases; HBoV, hMPV, and rhinovirus were detected in 3, 3, and 10 cases, respectively. Of note, patients infected with HCoV were adults. Results reinforce the importance of including coronaviruses in diagnostic panels used by official surveillance systems because besides their pandemic potential, endemic HCoVs are associated to severe disease in healthy adults.


Subject(s)
Humans , Male , Female , Child , Adolescent , Adult , Middle Aged , Aged , Aged, 80 and over , Young Adult , Respiratory System , Coronavirus , Epidemiological Monitoring , Infections , Patients , Rhinovirus , Viruses , Virus Diseases , Adenoviridae , Disease , Severe Acute Respiratory Syndrome , Influenza, Human , Bocavirus
11.
Article in Chinese | WPRIM | ID: wpr-828727

ABSTRACT

OBJECTIVE@#To study the clinical features of severe type 7 adenovirus pneumonia in children.@*METHODS@#A retrospective analysis was performed for the clinical data of children who were diagnosed with severe type 7 adenovirus pneumonia from February to June, 2019.@*RESULTS@#Among the 45 children, the male/female ratio was 3:2 and the median age was 14 months. All children had repeated fever, cough, and pulmonary moist rales, and the mean duration of fever was 14±4 days. The median time from fever to dyspnea was 8 days, and the time from fever to mechanical ventilation was 11.6±2.5 d. There was no significant increase in white blood cell count, with neutrophils as the main type. There were slight reductions in hemoglobin and albumin, while platelet and fibrinogen remained normal. There were increases in aspartate aminotransferase, lactate dehydrogenase, procalcitonin, and C-reaction protein. The detection rate of mixed pathogens was 84%. Effusion in both lungs was the major change on chest imaging (64%). Bronchoscopic manifestations were endobronchitis, tracheomalacia, and plastic bronchitis. The incidence rate of respiratory complications was 100%, and extrapulmonary complications mainly involved the circulatory system (47%), digestive system (36%), and nervous system (31%). Among the 45 children, 16 were administered with 400 mg/kg intravenous immunoglobulin (IVIG) daily for 5 days, with a mean duration of fever of 16±5 days, and 29 were administered with 1 g/kg IVIG daily for 2 days, with a mean duration of fever of 13±4 days; there was a significant difference in the mean duration of fever between the two groups (P=0.046). The overall mortality rate was 11%.@*CONCLUSIONS@#Severe type 7 adenovirus pneumonia in children has severe conditions, with a high incidence rate of complications and a high mortality rate, so it should be diagnosed and treated as early as possible.


Subject(s)
Adenoviridae , Bronchitis , Female , Fever , Humans , Infant , Male , Pneumonia, Viral , Retrospective Studies
12.
Article in Chinese | WPRIM | ID: wpr-828673

ABSTRACT

OBJECTIVE@#To study the clinical features of children with severe adenovirus pneumonia (SAP) and hemophagocytic syndrome (HPS).@*METHODS@#A retrospective analysis was performed from the chart review data of 30 children with SAP and HPS who were admitted from January 2014 to June 2019. According to the prognosis, the children were divided into a good prognosis group (n=18) and a poor prognosis group (n=12).@*RESULTS@#Among the 30 children with SAP and HPS, the ratio of male to female was 2:1. The median age of onset was 1 year and 3 months (range 3 months to 5 years), and the mean course of fever was 19±7 d. Of the 30 children, 28 (93%) experienced disease onset in January to June. High-throughput gene detection of serum pathogens showed that 16 (53%) children were positive for human adenovirus type 7 (HAdV-7), and the other 14 (47%) children were positive for HAdV antigen based on immunofluorescence assay for throat swab, with unknown type. Of all 30 children, 29 (97%) had respiratory complications, 24 (80%) had cardiovascular complications, 16 (53%) had gastrointestinal complications, and 9 (30%) had toxic encephalopathy. Eighteen children (60%) improved or recovered and 12 (40%) did not recover (3 died). Compared with the good prognosis group, the poor prognosis group had a significantly longer course from onset to diagnosis of HPS (P<0.05), significantly higher levels of fibrinogen and tumor necrosis factor-α (P<0.05), and a significantly lower level of interferon-γ (P<0.05). The mean follow-up time was 6±2 months; 11 (41%) children recovered, 1 (4%) experienced recurrence of HPS, and 15 (56%) had the sequela of post-infectious bronchiolitis obliterans (PIBO).@*CONCLUSIONS@#HPS may be observed in children with SAP, and PIBO is the most common sequela of SAP.


Subject(s)
Adenoviridae , Adenoviridae Infections , Child, Preschool , Female , Humans , Infant , Lymphohistiocytosis, Hemophagocytic , Male , Pneumonia, Viral , Retrospective Studies
13.
Article in Chinese | WPRIM | ID: wpr-828672

ABSTRACT

OBJECTIVE@#To study the clinical features of children with adenovirus pneumonia and hemophagocytic lymphohistiocytosis (HLH).@*METHODS@#A retrospective analysis was performed on the mediacal data of 7 children with adenovirus pneumonia and HLH from March to September, 2019.@*RESULTS@#The age of these children ranged from 11 months to 5 years, and among these children, 5 were aged <2 years and 5 were boys. None of these children had underlying diseases. All children were hospitalized due to persistent high fever and cough, and the peak temperature of fever was 39°C to 41°C. With disease progression, 7 children developed hepatomegaly and 6 developed splenomegaly. Routine blood test results showed reductions in two or three lineages of blood cells, with increases in serum ferritin (SF), C-reactive protein (CRP), procalcitonin (PCT), and lactate dehydrogenase (LDH). Phagocytosis of blood cells was observed in 6 children. Radiological examination of lungs showed pneumonia changes. All 7 children were diagnosed with human adenovirus type 7 infection based on pathogenic metagenome detection. No abnormality was found by HLH gene detection and the children were diagnosed with secondary HLH. All children received intravenous immunoglobulin. Among these children, 4 received dexamethasone and etoposide chemotherapy, 3 received dexamethasone alone, and 4 received plasma exchange. Of the 7 children, 2 died and 5 were recovered. Compared with those who survived, the children who died had significantly greater reductions in the three lineages of blood cells and significantly greater increases in serum levels of CRP, PCT, SF, and LDH.@*CONCLUSIONS@#The children with adenovirus pneumonia and HLH have main clinical features of persistent high fever, progressive reductions in two or three lineages of peripheral blood cells, and involvement of other organ systems, including hepatosplenomegaly. Significant increases in serum levels of CRP, PCT, SF, and LDH may suggest a poor prognosis.


Subject(s)
Adenoviridae , Child, Preschool , Etoposide , Female , Humans , Immunoglobulins, Intravenous , Infant , Lymphohistiocytosis, Hemophagocytic , Male , Retrospective Studies
14.
Article in Chinese | WPRIM | ID: wpr-827180

ABSTRACT

OBJECTIVE@#To transinfect SD adipose tissue-derived stem cell (ADSC) in vitro with a recombinant adenoviral vector containing human B-domain-deleted FVIII (BDDhFⅧ), so as to lay the foundation for the treatment of hemophilia A by using ADSC combined with BDDhFⅧ gene.@*METHODS@#ADSCs were isolated from the inguinal adipose tissue of SD rats and passed to third passage for identification. Third passage ADSCs were transfected in vitro with recombinant adenovirus vector Ad-BDDhFⅧ-GFP. The experiments were divided into Ad-BDDhFⅧ-GFP-transfected ADSCs group (A), Ad-GFP-transfected ADSC group (B), and untransfected ADSC group (C). CCK-8 method was used to detect the proliferation of transfected cells in three groups, and the expression level of hFⅧ antigen in cell supernatant was detected by ELISA. RT-PCR and Western blot respectively were used to detect the mRNA and protein expression of BDDhFⅧ in the three groups after transfection.@*RESULTS@#The growth curve of third passage cells isolated and cultured showed an inverted "S" shap; the flow cytometry detection showed the positive expression of CD29, CD90, CD44, and the negative expression of CD45 in third passage cells. After the adipogenic and osteogenic induction, the cells could transformed to adipogenic and osteogenic directions. CCK-8 detection showed that the proliferation of cells in 3 groups not was influenced. ELISA showed that the expression of hFⅧAg in group A was significantly higher than that in group B and C (P<0.05). RT-PCR showed that compared with group A, there was no target band in B and C groups, and BDDhFⅧ gene was not expressed. The results in group A were consistent with the length of amplified fragments, and BDDhFⅧ target gene was expressed. Western blot analysis showed that the expression of hFⅧ protein in group A was significantly higher than that in group B and C. (P<0.05).@*CONCLUSION@#Recombinant adenovirus Ad-BDDhFⅧ-GFP can effectively transfect rat ADSC in vitro, which lays an experimental foundation for gene therapy of hemophilia A.


Subject(s)
Adenoviridae , Adipose Tissue , Animals , Cell Differentiation , Cells, Cultured , Factor VIII , Humans , Rats , Rats, Sprague-Dawley , Stem Cells , Transfection
15.
Chinese Journal of Biotechnology ; (12): 763-771, 2020.
Article in Chinese | WPRIM | ID: wpr-826900

ABSTRACT

The recombinant adenoviruses expressing miR-22 (Ad-miR-22) was constructed and the effect of Ad-miR-22 on insulin signal pathway and glucose uptake in HepG2 cells was analyzed. MiR-22 gene was amplified by PCR from human hepatocytes and cloned into the pAdTrack-CMV vector to generate the shuttle plasmid pAdT-22. The positive colonies were confirmed by PCR and sequencing. The resultant shuttle plasmid was linearized with Pme I, followed by co-transformation into competent BJ5183 cells containing an adenoviral backbone plasmid (pAdEasy-1) to create the recombinant plasmid pAd-miR-22. After digested with Pac I, the linearized pAd-miR-22 was transfected into 293A packaging cell line to generate recombinant adenoviruses Ad-miR-22. HepG2 cells were infected with Ad-miR-22 or control Ad-GFP (adenoviruses expressing green fluorescent protein), and then the miR-22 expression levels were analyzed by qPCR. The result shows that adenovirus-mediated overexpression of miR-22 significantly decreased insulin-induced glucose uptake in HepG2 cells. Moreover, overexpression of miR-22 markedly decreased insulin-induced phosphorylation of GSK-3β. miR-22 also increased the mRNA levels of gluconeogenic genes in HepG2 cells. Furthermore, Western blotting results indicate that the protein expression of SIRT1 decreased in Ad-miR-22 infected HepG2 cells as compared with Ad-GFP infected HepG2 cells. In summary, overexpressing of miR-22 significantly increased gluconeogenesis while decreased glucose uptake in HepG2 cells. The effect of miR-22 on glucose metabolism may be mediated by SIRT1.


Subject(s)
Adenoviridae , Genetics , Glucose , Metabolism , Glycogen Synthase Kinase 3 beta , Metabolism , Hep G2 Cells , Humans , MicroRNAs , Genetics , Metabolism , Signal Transduction , Genetics , Transfection
16.
Autops. Case Rep ; 10(4): e2020191, 2020. graf
Article in English | LILACS | ID: biblio-1131851

ABSTRACT

Illustrative cases of diseases that are difficult to suspect and diagnose can serve as useful reminders. Invasive pulmonary aspergillosis and adenovirus hepatitis are two such diseases, both revealed by autopsy in this case of Hodgkin lymphoma refractory to chemotherapy treated with allogeneic hematopoietic stem cell transplantation complicated by these two fatal infections. This patient was cured of Hodgkin lymphoma, Clostridioides difficile colitis and thrombotic thrombocytopenic purpura using the marvels of modern medicine. This case illustrates many features of aspergillosis and adenovirus hepatitis, shows the value of autopsy in revealing diagnoses, and illustrates the limits of modern medicine, which should serve as a mental spur in our efforts to advance medical science, to try to defeat the numerous demons of disease, who seem to keep outwitting us.


Subject(s)
Humans , Male , Adult , Hodgkin Disease/complications , Adenoviridae , Invasive Pulmonary Aspergillosis/pathology , Hepatitis , Autopsy
18.
Article in English | LILACS | ID: biblio-1092153

ABSTRACT

ABSTRACT Objective: To report the case of a child who developed acute respiratory distress syndrome (ARDS) from a pulmonary infection by adenovirus. Case description: A female patient aged 2 years and 6 months, weighting 10,295 grams developed fever, productive cough and vomiting, later on progressing to ARDS despite initial therapy in accordance with the institutional protocol for ARDS treatment. The child evolved to refractory hypoxemia and hypercapnia, requiring high parameters of mechanical pulmonary ventilation and use of vasoactive agents. In the treatment escalation, the patient received steroids, inhaled nitric oxide (iNO), was submitted to the prone position, started oscillatory high-frequency ventilation (HFOV) and extracorporeal membrane oxygenation (ECMO) was indicated due to severe refractory hypoxemia. During this time, the patient's clinical response was favorable to HFOV, improving oxygenation index and hypercapnia, allowing the reduction of vasoactive medications and mechanical ventilation parameters, and then the indication of ECMO was suspended. The patient was discharged after 26 days of hospital stay without respiratory or neurological sequelae. Comments: Adenovirus infections occur mainly in infants and children under 5 years of age and represent 2 to 5% of respiratory diseases among pediatric patients. Although most children with adenovirus develop a mild upper respiratory tract disease, more severe cases can occur. ARDS is a serious pulmonary inflammatory process with alveolar damage and hypoxemic respiratory failure; Adenovirus pneumonia in children may manifest as severe pulmonary morbidity and respiratory failure that may require prolonged mechanical ventilation. Exclusive pulmonary recruitment and HFOV are advantageous therapeutic options.


RESUMO Objetivo: Descrever paciente que evoluiu com síndrome do desconforto respiratório agudo (SDRA) a partir de infecção pulmonar por adenovírus. Descrição do caso: Paciente de dois anos e seis meses, sexo feminino, peso de 10295 g, que apresentou com quadro de febre, tosse produtiva e vômitos, evoluindo para SDRA. Apesar da terapêutica inicial em conformidade com o protocolo institucional de tratamento da SDRA, a criança evoluiu para hipoxemia e hipercapnia refratárias, necessitando de elevados parâmetros de ventilação pulmonar mecânica e utilização de agentes vasoativos. No escalonamento da terapêutica, a paciente recebeu terapias adjuvantes, foi iniciada ventilação oscilatória de alta frequência (VOAF) e indicada oxigenação por membrana extracorpórea (OMEC) pela hipoxemia grave refratária. Nesse ínterim, a paciente apresentou resposta clínica favorável à VOAF, melhorando do quadro ventilatório e possibilitando a redução das medicações vasoativas e dos parâmetros de ventilação mecânica. A paciente recebeu alta hospitalar após 26 dias de internação, sem sequelas respiratórias ou neurológicas. Comentários: As infecções por adenovírus ocorrem principalmente em lactentes e crianças com menos de cinco anos de idade e representam de 2 a 5% das doenças respiratórias entre os pacientes pediátricos. Embora a maioria das crianças com infecção por adenovírus desenvolva doença leve do trato respiratório superior, casos mais graves podem ocorrer com comprometimento do trato respiratório inferior. A pneumonia por adenovírus em crianças pode se manifestar com morbidade pulmonar grave e insuficiência respiratória com risco de vida, o que resulta na necessidade de suporte mecânico prolongado. O recrutamento pulmonar exclusivo pela VOAF pode ser uma opção terapêutica útil.


Subject(s)
Humans , Female , Child, Preschool , Pneumonia, Viral/complications , Respiratory Distress Syndrome, Newborn/etiology , Respiratory Distress Syndrome, Newborn/therapy , High-Frequency Ventilation/methods , Extracorporeal Membrane Oxygenation/methods , Adenovirus Infections, Human/complications , Pneumonia, Viral/diagnostic imaging , Methylprednisolone/therapeutic use , Echocardiography , Adenoviridae/isolation & purification , Prone Position , Intubation, Intratracheal , Anti-Inflammatory Agents/therapeutic use
19.
Rev. méd. Chile ; 147(2): 256-260, Feb. 2019. graf
Article in English | LILACS | ID: biblio-1004341

ABSTRACT

ABSTRACT Adenovirus (ADV) is a recognized cause of severe disease among immunocompromised patients. We report a previously healthy 39-year-old female, admitted with influenza pneumonia and evolving with lung hemorrhage and acute renal failure requiring mechanical ventilation and hemodialysis. She received high corticosteroid doses due to an initial suspicion of alveolar hemorrhage. Lymphopenia already present before steroid use (567/μL), was maintained during the whole hospital stay (mean 782/μL). From the second week of admission she presented a high-volume diarrhea (mean 2.5 L/day) associated to intermittent bloody stools. An ulcerative enterocolitis was confirmed by CT images and colonoscopy. ADV was detected in a colonic tissue sample by real time PCR but not by a commercial filmarray test. Cidofovir-probenecid and racecadotril therapy were indicated without changing the clinical course of diarrhea and the patient finally died.


Adenovirus (ADV) es una causa reconocida de enfermedades graves en pacientes inmunocomprometidos. Informamos el caso de una mujer de 39 años, previamente sana, que ingresó por neumonía grave por influenza, evolucionando con hemorragia pulmonar y falla renal aguda, requiriendo ventilación mecánica y hemodiálisis. Recibió altas dosis de corticoides por la sospecha inicial de una hemorragia alveolar. Tuvo linfopenia durante toda su estadía (promedio 782/μL), la que ya estaba presente antes del uso de los corticoides (567/μL). Desde la segunda semana de hospitalización, presentó una diarrea de alto volumen (promedio 2,5 L/día) asociada a la presencia de sangre en deposiciones en forma intermitente. Se confirmó una enterocolitis ulcerativa por tomografía computada y colonoscopía. Se detectó ADV en muestras de biopsia colónica por PCR en tiempo real pero no por un test de PCR múltiples automatizado comercial. Fue tratada con cidofovir-probenecid y racecadrotrilo sin impacto clínico y la paciente finalmente falleció.


Subject(s)
Humans , Female , Adult , Cross Infection/etiology , Immunocompromised Host , Adenoviridae Infections/complications , Enterocolitis/etiology , Gastrointestinal Hemorrhage/etiology , Adenoviridae/isolation & purification , Cross Infection/diagnosis , Cross Infection/immunology , Fatal Outcome , Adenoviridae Infections/microbiology , Diarrhea/complications , Enterocolitis/diagnosis , Enterocolitis/immunology , Gastrointestinal Hemorrhage/diagnosis , Gastrointestinal Hemorrhage/immunology
20.
Article in Chinese | WPRIM | ID: wpr-819045

ABSTRACT

Severe adenovirus pneumonia has a high mortality and incidence of sequelae. Fever and cough are the main symptoms of children's severe adenovirus pneumonia, but such clinical manifestations are lack of specificity. For children with persistent high fever who are in the epidemic age and season, the adenovirus etiology detection, blood routine, cytokines, T cell subsets and imaging examinations are suggested. Children with early manifestations of infiltration of lung segment and lobar parenchyma, obvious emphysema, interstitial pneumonia or a large amount of pleural effusion should be alerted to have severe adenovirus pneumonia. This article reviews the epidemiological characteristics and risk factors of adenovirus pneumonia in different seasons, regions and serology, and the laboratory findings and imaging features of severe adenovirus pneumonia, which would be helpful for the early identification of the disease.


Subject(s)
Adenoviridae , Adenoviridae Infections , Diagnosis , Fever , Humans , Pleural Effusion , Pneumonia, Viral , Diagnosis , Research
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