Adenovirus Group Research Articles

Isolation and characterization of a novel chicken astrovirus in China

Chicken astrovirus (CAstV) is associated with kidney disease and visceral gout, runting and stunting syndrome, and white chick hatchery disease, causing economic losses to the poultry industry worldwide. In this study, 55.6% of 36 clinical samples from Guangdong province in China were positive for CAstV, but negative for other common enteric viruses, including avian nephritis virus, infectious bronchitis virus, fowl adenovirus Group I, Newcastle disease virus, chicken parvovirus, reovirus, and rotavirus by PCRs and RT-PCRs. A CAstV strain, named GD202013, was isolated from Guangdong province in south China, and was identified by CAstV RT-PCR. A whole genome sequence analysis demonstrated that GD202013 shares 76.0 to 88.1% identity with 24 reference strains in GenBank. Phylogenetic analysis, based on whole genome and capsid protein, showed that GD202013 is more closely related to 2 US strains (GA2011/US/2011 and 4175/US/2011) belonging to subgroup Bii. Recombination analysis indicated that GD202013 is a recombinant strain formed by 3 strains: a major parent strain CkP5/US/2016, and 2 minor parent strains (GA2011/US/2011 and G059/PL/2014). In addition, the chicken embryo infection experiment demonstrated that GD202013 causes hatchability reduction, growth depression, and death of embryos. Macroscopic and microscopic lesions in the liver, kidney and small intestine were observed in the dead-in-shell embryos. This is the first report of the novel CAstV infection in China.

Human species D adenovirus hexon capsid protein mediates cell entry through a direct interaction with CD46

Human adenovirus species D (HAdV-D) types are currently being explored as vaccine vectors for coronavirus disease 2019 (COVID-19) and other severe infectious diseases. The efficacy of such vector-based vaccines depends on functional interactions with receptors on host cells. Adenoviruses of different species are assumed to enter host cells mainly by interactions between the knob domain of the protruding fiber capsid protein and cellular receptors. Using a cell-based receptor-screening assay, we identified CD46 as a receptor for HAdV-D56. The function of CD46 was validated in infection experiments using cells lacking and overexpressing CD46, and by competition infection experiments using soluble CD46. Remarkably, unlike HAdV-B types that engage CD46 through interactions with the knob domain of the fiber protein, HAdV-D types infect host cells through a direct interaction between CD46 and the hexon protein. Soluble hexon proteins (but not fiber knob) inhibited HAdV-D56 infection, and surface plasmon analyses demonstrated that CD46 binds to HAdV-D hexon (but not fiber knob) proteins. Cryoelectron microscopy analysis of the HAdV-D56 virion-CD46 complex confirmed the interaction and showed that CD46 binds to the central cavity of hexon trimers. Finally, soluble CD46 inhibited infection by 16 out of 17 investigated HAdV-D types, suggesting that CD46 is an important receptor for a large group of adenoviruses. In conclusion, this study identifies a noncanonical entry mechanism used by human adenoviruses, which adds to the knowledge of adenovirus biology and can also be useful for development of adenovirus-based vaccine vectors.

Ubc9 Attenuates Myocardial Ischemic Injury Through Accelerating Autophagic Flux.

AimsSUMOylation is a post-translational modification that plays a crucial role in the cellular stress response. We aimed to demonstrate whether and how the SUMO E2 conjugation enzyme Ubc9 affects acute myocardial ischemic (MI) injury.Methods and ResultsAdenovirus expressing Ubc9 was administrated by multipoint injection in the border zone of heart immediately after MI in C57BL/6 mice. Neonatal rat cardiomyocytes (NRCMs) were also infected, followed by oxygen and glucose deprivation (OGD). In vivo, Ubc9 adenovirus-injected mice showed decreased cardiomyocyte apoptosis, reduced myocardial fibrosis, and improved cardiac function post-MI. In vitro, overexpression of Ubc9 decreased cardiomyocyte apoptosis, whereas silence of Ubc9 showed the opposite results during OGD. We next found that Ubc9 significantly decreased the accumulation of autophagy marker p62/SQSTM, while the LC3 II level hardly changed. When in the presence of bafilomycin A1 (BAF), the Ubc9 adenovirus plus OGD group presented a higher level of LC3 II and GFP-LC3 puncta than the OGD group. Moreover, the Ubc9 adenovirus group displayed increased numbers of yellow plus red puncta and a rising ratio of red to yellow puncta on the mRFP-GFP-LC3 fluorescence assay, indicating that Ubc9 induces an acceleration of autophagic flux from activation to degradation. Mechanistically, Ubc9 upregulated SUMOylation of the core proteins Vps34 and Beclin1 in the class III phosphatidylinositol 3-kinase (PI3K-III) complexes and boosted the protein assembly of PI3K-III complex I and II under OGD. Moreover, the colocalization of Vps34 with autophagosome marker LC3 or lysosome marker Lamp1 was augmented after Ubc9 overexpression, indicating a positive effect of Ubc9-boosted protein assembly of the PI3K-III complexes on autophagic flux enhancement.ConclusionsWe uncovered a novel role of Ubc9 in protecting cardiomyocytes from ischemic stress via Ubc9-induced SUMOylation, leading to increased PI3K-III complex assembly and autophagy-positioning. These findings may indicate a potential therapeutic target, Ubc9, for treatment of myocardial ischemia.

Prevalence, risk factors, and clinical characteristics of rotavirus and adenovirus among Lebanese hospitalized children with acute gastroenteritis

BackgroundAcute gastroenteritis is a very common infectious disease facing all age groups worldwide, especially the pediatric population. Viruses, bacteria, and parasites are all possible causes of infectious gastroenteritis; however, viruses have become more frequently identified with the advances in the ability to diagnose viral infections, particularly rotavirus and adenovirus. We aimed in our study to compare between the prevalence, risk factors, and clinical characteristics of rotavirus and adenovirus among children with viral gastroenteritis in Lebanon. Materials and methodsA 12-months retrospective study was performed between January 1st and December 31st, 2018 including 308 children aged 1 month to 12 years, who were admitted to three tertiary healthcare centers in South Lebanon. Medical data were retrieved from patients’ files, including clinical and laboratory information. ResultsRotavirus was found in stool of 204 patients (66.23 %), followed by adenovirus in 78 cases (25.32 %), and mixed group (rotavirus and adenovirus) in 26 cases (8.44%). The highest prevalence of rotavirus in our present study was seen among children between 12 and 23 months old, whereas patients infected with adenovirus were mainly aged between 24-35 months or 4–11 months. Majority of patients in the adenovirus and mixed groups had high-grade fever compared to the rotavirus group. Laboratory findings presented significantly higher average of white blood cells (WBCs), absolute neutrophil count (ANC), and C-reactive protein (CRP) in the mixed group compared to the two other groups. Monthly distribution of rotavirus and adenovirus infection revealed a biennial pattern of rotavirus incidence during January and July–August while frequency of adenovirus infection was highest during July–August. ConclusionDue to the high prevalence of viral diarrhea among the pediatric age group in our region, particularly rotavirus and adenovirus, along with the associated non-specific signs and symptoms, we highly recommend that medical laboratories be equipped for virus detection. Also, vaccination against rotavirus should be considered as a prevention strategy.

Quantitative Detection of Human Adenovirus and Human Rotavirus Group A in Wastewater and El-Rahawy Drainage Canal Influencing River Nile in the North of Giza, Egypt.

Environmental monitoring is critical in a developing country like Egypt where there is an insufficient framework for recording and tracking outbreaks. In this study, the prevalence of human adenovirus (HAdV), rotavirus group A (RVA) was determined in urban sewage, activated sludge, drainage water, drainage sediment, Nile water, and Nile sediment, using quantitative polymerase chain reaction (qPCR) analysis. HAdV was detected in 50% of urban sewage with viral concentrations ranging from 103 to 107 genome copies/liter (GC/L), 33% of activated sludge with viral concentrations ranging from 103 to 107 GC/kilogram (GC/kg), 95% of drainage water with viral concentrations ranging from 103 to 107 GC/L, 75% of drainage sediment with viral concentrations ranging from 103 to 107 GC/L, 50% of Nile water with viral concentrations ranging from 103 to 107 GC/L, and 45% of Nile sediment with viral concentrations ranging from 103 to 107 GC/kg. RVA was detected in 50% of urban sewage with viral concentrations ranging from 103 to 107 GC/L, 75% of activated sludge with viral concentrations ranging from 103 to 107 GC/L, 58% of drainage water with viral concentrations ranging from 103 to 107 GC/L, 50% of drainage sediment with viral concentrations ranging from 103 to 107 GC/L, and 45% of Nile water with viral concentrations ranging from 103 to 107 GC/kg. In conclusion, Abu-Rawash WWTP acts as a source of HAdV and RVA, releasing them into El-Rahawy drain then to the River Nile Rosetta branch.

Sirtuin (Sirt) 3 Overexpression Prevents Retinopathy in Streptozotocin-Induced Diabetic Rats.

BackgroundSirtuin (Sirt) 3 could promote autophagy by downregulating the expression of genes related to neovascularization in retinal endothelial cells. In this study, we aimed to investigate the effect of Sirt3 overexpression on retinopathy in streptozotocin (STZ)-induced diabetic rats, and to assess its mechanisms.Material/MethodsNtraperitoneal injection of STZ in rats was used to produce a diabetic model. The study rats were divided into 4 groups (n=6 for each group): a control group; a model group; a model+scrambled adenovirus group; and a model+Sirt3 overexpression group. Hematoxylin and eosin (H&E) staining determined the pathological changes of retina tissues. Immunohistochemistry, fluorescence quantitative polymerase chain reaction, and western blotting were used to detect the expression of Sirt3, vascular endothelial growth factor (VEGF), and microtubule-associated protein 1A/1B-light chain 3 (LC3).ResultsIn the model group, the inner limiting membrane was swollen, uneven and thickened, and the capillary endothelial cells occasionally protruded into the inner limiting membrane. These abnormalities were prevented by Sirt3 overexpression. Compared with the control group, the expression of Sirt3 at both mRNA and protein levels in the model group was significantly reduced, while the expression of VEGF was increased versus the control group (P<0.05). The expression of LC3 at both mRNA and protein levels was not different between the model group and the control group. Compared with the model group, the expression of Sirt3, LC3, and LC3-II was significantly increased, while the expression of VEGF was significantly decreased in the model+Sirt3 overexpression group (P<0.05).ConclusionsSirt3 overexpression has a preventive effect on diabetic retinopathy likely through promoting the expression of autophagy-related proteins and downregulating the expression of VEGF.

Diagnosis of Acute Gastroenteritis with Immunochromatography and Effectiveness of Rotavirus Vaccine in a Japanese Clinic.

Despite the well known effectiveness of two licensed live attenuated oral rotavirus (RV)-vaccines, Rotarix and RotaTeq, constant monitoring of vaccine effectiveness (VE) is essential considering the evolving power and reassortment capability of RVs. In this study, we detected RV, norovirus (NV) and adenovirus (AV) infections using immunochromatography (IC)-based kits in children with acute gastroenteritis (AGE) who attended a pediatric clinic in Kiryu city, Gunma, Japan during June, 2014–September, 2018. VEs were determined using a test-negative study design. Among 1658 AGE-children, RV, NV and AV were detected in 96 (5.8 %), 146 (8.8 %) and 46 (2.8 %) children, respectively. Interestingly, the distributions of infections were found to be associated with age and sex. Namely, RV infections were significantly higher in female (P=0.02) and in the 19–30 month age group children, while NV and AV infections predominated in the 13–24 month and 7–18 month age groups, respectively. The disease severity for RV and NV infections remained similar and significantly higher than that of AV infections. The VE of RV-vaccines was 49.8 % (95 % CI: 22.7 to 67.3 %) against all RV infections, which was increased up to 67.2 % (95 % CI: 35.3 to 83.4 %) against severe RV infections. RV-vaccinated children experienced less severe symptoms in RV-infections while non-RV AGE remained less serious for both RV-vaccinated and unvaccinated children. Finally, the prevalence of RV infection remained minimized (≤5.4 %) in this population since 2015. Thus, this study provided important information on distribution of major AGEs in young children and exhibited the effective role of RV vaccines in post-vaccine era.

1773. Development and Evaluation of an Automated Adenovirus Quantitative Assay Using the Luminex ARIES® System

BackgroundThe quantification of circulating human adenovirus (HAdV) DNA is the recommended diagnostic method to predict disseminated disease. Most current HAdV quantitative assays are manually performed and lack the flexibility needed to provide rapid answers when required.The purpose of this study was to evaluate the application of MultiCode® HAdV PCR assay for use with the Luminex ARIES® system (Luminex Corporation, Austin, TX) as an automated and random access test for the quantitative detection of HAdV DNA in plasma.MethodsAnalytical performance characteristics including assay limit of detection/quantitation (LoD/LoQ), accuracy, and inter-, intra-reproducibility were studied using commercial panels (Exact Diagnostics, Fort Worth, TX). Assay specificity was determined usingHAdV reference strains obtained from the American Type Culture Collection (Manassas, VA) and plasma spiked with related Herpes viruses and pathogens commonly found in the blood. Assay accuracy was verified with analysis of 30 plasma samples spiked with different concentrations of control material that covered the full range of HAdV DNA levels. We also prospectively analyzed 180 plasma samples collected from 102 patients. DNA from all samples were extracted, amplified and detected on a single automated Luminex ARIES® system.ResultsThe assay has a wide linear range from 2.55 to 9.4 log10 HAdV DNA copies/mL (coefficient of determination; R2 = 0.995) with a detection limit of 1.82 log10 (95% positivity rate), and a limit of quantification of 2.55 log10 copies/mL. The assay detected HAdV DNA from Adenovirus groups A-F, although slight shifts in Tm peaks were observed. Inter- and intra-assay reproducibility was evaluated using 6 panels of commercial standards, producing variation coefficients of 5% and 2%, respectively. Assay accuracy results reflected a good correlation with a mean difference of 0.10 log10 copies/mL. The results of the prospective study showed 6/102 (5.8%) patients had HAdV viremia, including 4 (3.9%) patients with a viral load ≥4 log10/mL, which might necessitate therapy.ConclusionThe HAdV quantitative assay using the Luminex ARIES® system provides excellent performance for routine testing with the additional advantage of random access capabilities for urgent testing to identify patients at risk for disseminated disease.Disclosures All authors: No reported disclosures.

Adenovirus vector‑mediated invivo gene transfer of nuclear factor erythroid‑2p45‑related factor 2 promotes functional recovery following spinal cord contusion.

The aim of the present study was to investigate whether nuclear factor erythroid 2p45‑related factor2(Nrf2) overexpression by gene transfer may protect neurons/glial cells and the association between neurons/glial cells and axons in spinal cord injury (SCI). In the present study, Nrf2 recombinant adenovirus (Ad) vectors were constructed. The protein levels of Nrf2 in the nucleus and of the Nrf2‑regulated gene products heme oxygenase‑1 (HO‑1) and NAD (P)H‑quinone oxidoreductase‑1 (NQO1), were detected using western blot analysis in PC12 cells following 48 h of transfection. Furthermore, the expression of Nrf2 was localized using an immunofluorescence experiment, and the expression of Nrf2, HO‑1 and NQO1 were detected using an immunohistochemical experiment in the grey matter of spinal cord in rats. Post‑injury motor behavior was assessed via the Basso, Beattie and Bresnahan (BBB) locomotor scale method. In PC12 cells, subsequent to Ad‑Nrf2 transfection, nuclear Nrf2, HO‑1 and NQO1 levels were significantly increased compared with the control (P<0.01). There was statistically significant changes in the PC12‑Ad‑Nrf2 group [Nrf2 (1.146±0.095), HO‑1 (1.816±0.095) and NQO1 (1.421±0.138)] compared with the PC12‑control group [Nrf2 (0.717±0.055), HO‑1 (1.264±0.081) and NQO1 (0.921±0.088)] and PC12‑Ad‑green fluorescent protein group [Nrf2 (0.714±0.111), HO‑1 (1.238±0.053) and NQO1 (0.987±0.045); P<0.01]. The BBB scores of the rats indicated that they had improved functional recovery following the local injection of Ad‑Nrf2. On the third day following the operation, BBB scores in the adenovirus groups (0.167±0.408) were significantly decreased compared with the SCI group (1±0.894; P<0.05). In the injured section of the spinal cord in the rats, the number of positive cells expressing Nrf2, HO‑1 and NQO1 were raised compared with the control and SCI groups, indicating that the adenovirus vector‑mediated gene transfer of Nrf2 promotes functional recovery following spinal cord contusion in rats.

Genome Analysis of A Novel Recombinant Human Adenovirus Type 1 in China

Human adenovirus (HAdV) group C are the common etiologic in infants with severe acute respiratory infections (SARI). In the study, we report that a novel recombinant HAdV-C group strain (SH2016) was isolated from an infant with SARI in Shanghai in Feb. 4, 2016. The whole-genome sequence of SH2016 strain was generated and compared to other HAdV genomes publicly available. The strain SH2016 genome contains 35,946 nucleotides and coded 40 putative proteins, which was divided into 11 regions. RDP and phylogenetic analyses of the complete genome showed that the SH2016 strain was arranged into a novel subtype and might be recombined with HAdV-1 and HAdV-2. Our finding indicated that the frequent recombination among the HAdV-C group played an important role in driving force for polymorphism of human HAdV-C group prevalent in Shanghai, China. Further epidemiological surveillance of HAdV-C group is necessary to explore whether the novel HAdV-C group will maintain long-term stability. And the pathogenicity and clinical characteristics of the novel HAdV-C group member should be done more.

Construction and immunological evaluation of recombinant adenovirus vaccines co-expressing GP3 and GP5 of EU-type porcine reproductive and respiratory syndrome virus in pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) keeps causing economic damages in the swine sector across the globe. There has been emergence of the European (EU) genotype of porcine reproductive and respiratory syndrome virus (Genotype-I PRRSV) in China in recent years. The presently available vaccines cannot unable to provide safeguard against PRRSV infection completely. This study was aimed to construct recombinant adenovirus expressing the ORF3 and ORF5 genes of the EU-type PRRSV strain. Then, the recombinant adenovirus vaccines for EU-type PRRSV (rAd-E3518, rAd-E35, rAd-E3 and rAd-E5) which we constructed and evaluated were constructed and identified by western blot and PCR. All recombinant adenovirus vaccines were evaluated for humoral and cellular responses and EU-type PRRSV challenge in pigs. The results showed that the group of rAd-E3518+Quil A developed higher GP3 and GP5 specific antibody responses compared to the group of rAd-E3518. The majority of the neutralizing antibody titers were higher than 1:16 (P<0.05), the fusion of IL-18 has increased significantly PRRSV-stimulated secretion of IFN-γ and IL-4 in porcine serum, the group of rAd-E3518+Quil A produced highest T-lymphocytes (CD3+CD4+ and CD3+CD8+ T cells) proliferative in peripheral blood of pigs. The animals were challenged with the EU-type PRRSV strain and the viral load was detected in the several tissues, the viral load of rAd-E3518 and rAd-E3518+Quil A were lower than the wild-type adenovirus group. Our findings provide evidence to confirm that the recombinant adenovirus vaccine can protect pigs from EU-PRRSV infection.

A recombinant adenovirus expressing the E protein of duck Tembusu virus induces protective immunity in duck.

Duck Tembusu virus disease, caused by the duck Tembusu virus (DTMUV), can lead to a severe reduction in egg production and growth retardation in laying ducks and ducklings, respectively. In this study, we engineered a novel recombinant adenovirus expressing the E protein of DTMUV (rAd-E) in AAV-293 cells (analyzed by western blot and indirect immunofluorescence assays). Intramuscular immunization of Cherry Valley ducks with rAd-E was performed to evaluate host cellular and humoral immune responses. Compared to the phosphate-buffered saline administered group and the negative control wild-type adenovirus (wtAd) group, the rAd-E vaccinated group showed increased cellular and humoral responses. The results from the cytokine release and lymphocyte proliferation assays showed that rAd-E induced a stronger cellular immune response than the control group (P<0.01), 4 weeks after primary immunization. The results of enzyme-linked immunosorbent and virus neutralization assays showed that rAd-E induced higher titers of specific neutralizing antibodies, 2 weeks after primary immunization. The DTMUV challenge experiment showed a higher survival rate (80%) of ducks in the rAd-E group, when challenged with 0.5 ml (ELD50=10−2.67/0.2 ml) of the DTMUV strain AH-F10. These results indicate that rAd-E effectively protects ducks against DTMUV infection. Therefore, rAd-E could be a vaccine candidate to provide an effective and safe method for prevention and control of DTMUV infection.

Rosuvastatin stabilizes atherosclerotic plaques by reducing CD40L overexpression-induced downregulation of P4Hα1 in ApoE-/- mice.

Background Cluster of differentiation 40 ligand (CD40L) and rosuvastatin (RSV) affect atherosclerotic plaque stability, but little is known about their roles in extracellular matrix (ECM) production. We investigated the effects of CD40L and RSV on pre-existing advanced plaques. Methods and results Pre-existing advanced plaques were induced in apolipoprotein E-knockout (ApoE-/-) mice by the surgical placement of carotid constrictive silastic collars. Two weeks after surgery, mice were divided into the following treatment groups: control, empty adenovirus, CD40L adenovirus, CD40L adenovirus + RSV, and RSV. Mice received adenovirus via two tail-vein injections (2 × 109 pfu each) and/or RSV via intragastric administration (5 mg/kg; daily for 4 weeks). Mice in the CD40L adenovirus group exhibited increased plaque disruption rates, increased relative plaque macrophage and lipid content, reduced plaque collagen content, and increased local inflammation compared to the other treatment groups, but no significant differences in plaque area were observed among the groups. Notably, in the atherosclerotic plaques of the CD40L adenovirus group, both the mRNA and protein expression of prolyl-4-hydroxylase alpha 1 (P4Hα1) was significantly decreased, leading to a consequent decrease in the protein expression of collagen types I and III. Treatment with RSV decreased the serum levels of CD40L in a lipid-independent fashion and attenuated the effects of CD40L overexpression, particularly with respect to P4Hα1 downregulation. Conclusions CD40L destabilized advanced plaques in the carotid arteries of ApoE-/- mice, in part by decreasing P4Hα1 expression, and consequently collagen expression. These destabilizing effects were attenuated by RSV.

Caracterización molecular del adenovirus aviar en pollos comerciales del Ecuador

El Adenovirus Aviar del grupo I (FAdV-I), es considerado como un patógeno de mucha importancia en la industria avícola en el Ecuador y a nivel mundial. La Hepatitis por Cuerpos de Inclusión (IBH) y el Síndrome del Hidropericardio (HPS), son las principales enfermedades asociadas a este virus, y a pesar de ser un agente etiológico que por lo general se encuentra relacionado a enfermedades inmunodepresoras como Anemia Infecciosa de las Aves o la enfermedad de Gumboro, recientes estudios han demostrado la actividad patológica del FAdV-I como responsable primario de estas enfermedades. Desde la década de 1990, se reportaron varios brotes importantes de IBH y HPS en muchos países de América Latina incluyendo el Ecuador, logrando identificar principalmente al serotipo 4 del FAdV-I como agente causal de estos brotes. El objetivo de este estudio fue determinar por métodos moleculares como la Reacción en Cadena de la Polimerasa (PCR), la presencia de los serotipos del FAdV-I que se encuentran circulando en granjas de aves comerciales en Ecuador. Fueron colectados diferentes órganos de aves con problemas entéricos en la zona norte del Ecuador, y posteriormente impregnados en tarjetas FTA para su transporte y procesamiento. 4/13 (30,8%) muestras fueron positivas a FAdV-I y mediante el secuenciamiento y análisis bioinformático del ADN amplificado por la PCR, se caracterizaron los serotipos 6 y 11 del FAdV-I. Estos datos nos demuestran la variedad de serotipos presentes en granjas avícolas del Ecuador y que podrían estar influenciando en el estado sanitario de la industria avícola del país.

New Adenovirus Groups in Western Palaearctic Bats.

In the context of long-term screening for viruses on Western Palaearctic bats, we tested for the presence of adenovirus 1392 oropharyngeal swabs and 325 stool samples taken from 27 bat species. Adenoviruses were detected in 12 species of the Vespertilionidae and the Rhinolophidae families. Fifty positive respiratory and 26 positive stool samples were studied. Phylogenetic analyses of partial hexon protein and partial DNA-dependent DNA polymerase genes indicate that all these bat adenoviruses belong to the genus Mastadenovirus but without constituting a monophyletic cluster. According to genetic identities, the new groups are distinct to the previously described Bat mastadenovirus A and B species and contribute with potentially new members. Our data support that diversity of bat mastadenovirus is host-dependent and increase the knowledge of potentially pathogenic virus from bats. Due to the active role of bats as viral reservoirs, the characterization of these viruses is relevant for Public Health.

Abstract 5096: Pre- and early postnatal loss of tsc1/2 induces kidney hybrid oncocytic/chromophobe tumors (HOCT) via c-JUN

Abstract Renal cell carcinoma (RCC) occurs in up to 4% of tuberous sclerosis complex (TSC) patients, often affecting children and young adults. The mechanisms of TSC-associated RCC are not well understood. We explored the pathogenesis of TSC-associated RCC using three mouse models. First, loss of Tsc2 was induced by i.p. tamoxifen (TM) injection (10mg/kg) at embryonic day 17.5 (E17.5) in Tsc2flox/floxROSA26-CreER+ mice (n=24), examined at age 6 m. Second, loss of Tsc1 was induced by injecting Cre-encoding adenovirus directly into the left kidney of Tsc1flox/flox mice at P1 (n=19), examined at age 1 year. Third, Tsc2 was knock out through 5 consecutive times of i.p. tamoxifen injection (1mg/mouse/day) when the Tsc2flox/floxROSA26-CreER+ mice were 6 weeks old (n=7), examined at age 6 month. We found the following: In the E17.5 TM injection mouse model, each kidney had more than 200 tumor lesions, which almost occupied the whole kidney cortex and medulla. Extensive TSC-associated kidney papillary RCC (PRCC) and hybrid oncocytic/chromophobe tumors (HOCT) were seen in every kidney. The same types of tumor but fewer tumor lesions (~40/kidney) were found in the Cre-encoding adenovirus group. All the females in the P6 week group had the same phenotype as E17.5 TM injection group, but only single cyst was found in the male kidneys. So, the earlier those mice lost TSC1/2, the more severe their tumor burdens. IHC was performed to further validate the diagnosis. All the PRCC showed strong and diffuse expression of phospho-S6-S240/244 (pS6), CK7, and carbonic anhydrase-IX, with negative vimentin staining, while all the HOCT were positive for pS6, S-100 (a diagnostic marker used for human HOCT) and vimentin. The single cysts in the P6 week TM injection male kidneys only showed pS6 and CK7 staining. To further explore the mechanism of tumorigenesis, a phospho-RTK screen was done using lysates from kidney tumors (PRCC and HOCT) and normal kidneys. The pAXL level was 2.8-fold higher in comparison to normal kidney. Immunoblotting revealed high levels of p-AXL and total AXL in kidney tumor lysates compared to normal kidney. AXL mRNA, measured by real-time RT-PCR, was increased 2-fold (p=0.011). c-Jun, a known driver of AXL expression, was also highly expressed in those tumors but negative in normal kidneys as shown by immunoblot. Immunoblot analysis of a cell line established from the kidney tumors showed TSC-dependent c-Jun and AXL expression. These novel mouse models of TSC-related RCC closely resemble those found in TSC patients and elucidate distinct mechanisms of disease development and potential therapeutic targets. Citation Format: Heng Du, Heng-jia Liu, Damir Khabibullin, Mahsa Zarei, John Dreier, Chin-Lee Wu, Elizabeth Henske, David Kwiatkowski. Pre- and early postnatal loss of tsc1/2 induces kidney hybrid oncocytic/chromophobe tumors (HOCT) via c-JUN [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5096.

Exogenous Tissue Inhibitor of Metalloproteinase-2 Affects Matrix Metalloproteinase-2 Expression in Conjunctival Filtering Blebs and Bleb Scarring in Rats.

Objective To examine the effect of tissue inhibitor of metalloproteinase-2 (TIMP-2) on conjunctival filtering bleb scarring. Methods A model of conjunctival filtering bleb was established whereby rats were injected with saline, blank adenoviral vector, or adenoviral vector carrying TIMP-2 into the bleb. Filtration bleb formation and matrix metalloproteinase-2 (MMP-2) expression were examined. Results All operated eyes formed obvious elevated blebs on day 1. In the normal saline group, empty plasmid group, and gene transfection group maintenance time of filtrating blebs was 5–14, 5–14, and 6–16 days, and average survival time was 8.24, 8.16, and 9.44 days, respectively. MMP-2 expression increased slightly in the gene transfection group at 3 and 5 days after surgery, reached a peak after 14 days, and then gradually decreased. MMP-2 expression was weakly positive in the normal conjunctival epithelium, but was hardly detected in the lamina propria. Seven days after surgery, the epithelium and lamina propria of the conjunctival filtering bleb exhibited strong positive expression in the empty plasmid group but only weak expression in the adenovirus group. Conclusion Exogenous TIMP-2 interfered with local MMP-2 expression, delaying peak expression of MMP-2 and slowing the scarring of filtering blebs during wound healing of subconjunctival tissue.

Prevalence of Family History of Autoimmune Disorders in Juvenile Systemic Lupus Erythematosus

One of the causes of infants' hospitalizations is bronchiolitis, while different viral agents could be causative agents. As there is little information regarding the common agents of bronchiolitis in Iranian infants, we designed this study to determine which agents were responsible for hospitalization due to bronchiolitis among infants in an Iranian tertiary center. Two hundred and three infants with bronchiolitis who were hospitalized in Bahrami hospital were enrolled. Data regarding age, sex, duration of hospitalization, exposure to smoking, previous antibiotic usage and fever were collected for all enrolled cases. Throat sample by means of soap was collected and rapid test with immunochromatography (IC) test was performed. Rapid test was positive in 59 (29%) cases and three cases had concomitant infection with two viruses. The most common viral agent was RSV (Respiratory Syncytial Virus). Mean age was significantly lower in cases with RSV or RSV+ adenovirus infectious in comparison with other two groups (adenovirus or influenza only), while mean duration of hospitalization was significantly longer in RSV/RSV+ adenovirus group. RSV is the most common viral etiology of bronchiolitis in Iranian infants less than one year old, which is related with younger age and longer duration of hospitalization. Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease. Near 10% of affected children have a relative with SLE. Autoimmune diseases are more common in relatives of children with SLE. As there is no study regarding the prevalence of autoimmune disease in cases with pediatric SLE, we designed this study to evaluate the prevalence of autoimmune disease in children with SLE. In this cross sectional study, 50 children with SLE and 50 healthy children were enrolled. A structured questionnaire was used to collect data regarding the presence of autoimmune diseases in relatives. One thousand eight hundred and thirty two relatives were evaluated in the case group and 1699 in the control group. The number of relatives with autoimmune diseases was significantly higher in the case group (26 vs 10). The most common autoimmune diseases were lupus, followed by thyroid diseases among cases, and thyroid diseases and rheumatoid arthritis in controls. According to the results of this study, the prevalence of autoimmune disorders is more common in relatives of children with SLE than in those of controls.

Viral Aetiology of Bronchiolitis in Hospitalised Children in a Tertiary Center in Tehran

One of the causes of infants' hospitalizations is bronchiolitis, while different viral agents could be causative agents. As there is little information regarding the common agents of bronchiolitis in Iranian infants, we designed this study to determine which agents were responsible for hospitalization due to bronchiolitis among infants in an Iranian tertiary center. Two hundred and three infants with bronchiolitis who were hospitalized in Bahrami hospital were enrolled. Data regarding age, sex, duration of hospitalization, exposure to smoking, previous antibiotic usage and fever were collected for all enrolled cases. Throat sample by means of soap was collected and rapid test with immunochromatography (IC) test was performed. Rapid test was positive in 59 (29%) cases and three cases had concomitant infection with two viruses. The most common viral agent was RSV (Respiratory Syncytial Virus). Mean age was significantly lower in cases with RSV or RSV+ adenovirus infectious in comparison with other two groups (adenovirus or influenza only), while mean duration of hospitalization was significantly longer in RSV/RSV+ adenovirus group. RSV is the most common viral etiology of bronchiolitis in Iranian infants less than one year old, which is related with younger age and longer duration of hospitalization.

Role of GSK-3β in lipid emulsion-induced inhibition of bupivacaine-induced apoptosis in cardiomyocytes of rats: evaluation using RNA interference adenovirus infection method

Objective To evaluate the role of glycogen synthase kinase 3 beta (GSK-3β) in lipid emulsion-induced inhibition of bupivacaine-induced apoptosis in cardiomyocytes of rats using RNA interference (RNAi) adenovirus infection method. Methods H9C2 cells were transferred into 96-well cell plates at a density of 1×105 cells/ml after culture and then divided into 8 groups (n=10 each) using a random number table: control group (group C), bupivacaine group (group B), lipid emulsion group (group LE), bupivacaine plus lipid emulsion group (group B+ LE), control plus GSK-3βRNAi adenovirus (GSK-3βi) group (group C+ GSK-3βi), bupivacaine plus GSK-3βi group (group B+ GSK-3βi), lipid emulsion plus GSK-3βi group (group LE+ GSK-3βi) and bupivacaine plus lipid emulsion plus GSK-3βi group (group B+ LE+ GSK-3βi). In B, LE and B+ LE groups, the cells were incubated with culture medium containing 1 mmol/L bupivacaine, 1% lipid emulsion and 1 mmol/L bupivacaine plus 1% lipid emulsion, respectively.In C+ GSK-3βi, B+ GSK-3βi, LE+ GSK-3βi and B+ LE+ GSK-3βi groups, the cells were incubated with the drugs mentioned above on 2nd day after being infected by adenovirus.At 24 h after incubation with drugs, the expression of Bax and Bcl-2 was determined by Western blot, and the apoptosis rate was calculated using DAPI staining. Results Compared with group C, the expression of Bax was significantly up-regulated, the expression of Bcl-2 was down-regulated, and the apoptosis rate was increased in group B (P<0.05). Compared with group B, the expression of Bax was significantly down-regulated, the expression of Bcl-2 was up-regulated, and the apoptosis rate was decreased in group B+ LE (P<0.05). Compared with group B+ LE, the expression of Bax was significantly up-regulated, the expression of Bcl-2 was down-regulated and the apoptosis rate was increased in group B+ LE+ GSK-3βi (P<0.05). Conclusion The mechanism by which lipid emulsion inhibits bupivacaine-induced apoptosis in cardiomyocytes of rats is associated with GSK-3β. Key words: Fat emulsions, intravenous; Bupivacaine; Apoptosis; Myocytes, cardiac