The etiology and differential diagnosis of "autoimmune hepatitis-like liver disease" in children: a single-center retrospective study.

Background: Children with autoimmune hepatitis (AIH) often present with symptoms similar to those of other liver diseases. This study consists of a comparison between the clinical and histological characteristics of AIH and those of other four AIH-like liver diseases [i.e., drug-induced liver injury (DILI), gene deficiency, infectious liver disease and other etiology of liver disease], as well as an evaluation of the AIH scoring system's diagnostic performance. Methods: All children with AIH-like liver disease at our center from January 2013 to December 2022 were included. The clinical and histological characteristics of the AIH group were retrospectively analyzed and compared with those of the other four groups. Results: A total of 208 children were included and divided into AIH group (18 patients), DILI group (38 patients), gene deficiency group (44 patients), infectious liver disease group (74 patients), and other etiology group (34 patients). The antinuclear antibodies (ANA) ≥ 1:320 rate was significantly higher in the AIH compared to the other four groups after multiple testing correction (p < 0.0125), while patients with positive antibodies to liver-kidney microsomal-1 (anti-LKM1, n = 3) and smooth muscle antibodies (SMA, n = 2) were only observed in the AIH group. The positive rates of antibodies to liver cytosol type1 (anti-LC1) and Ro52 were higher than those in the other four groups. The serum immunoglobulin G (IgG) and globulin levels, as well as the proportions of portal lymphoplasmacytic infiltration, lobular hepatitis with more than moderate interface hepatitis, and lobular hepatitis with lymphoplasmacytic infiltration, were significantly higher in the AIH group than in the other four groups after multiple testing correction (p < 0.0125). The cirrhosis rate in the AIH group was higher than that in the DILI and infectious liver disease groups (p < 0.0125). Both the simplified (AUC > 0.73) and the revised systems (AUC > 0.93) for AIH have good diagnostic performance, with the latter being superior (p < 0.05). Conclusion: Positive autoantibodies (ANA ≥ 1:320 or anti-LKM1 positive, or accompanied by SMA, anti-LC1 or Ro-52 positive) and elevated serum IgG or globulin levels contribute to early recognition of AIH. The presence of lobular hepatitis with more than moderate interface hepatitis and lymphoplasmacytic infiltration contribute to the diagnosis of AIH.

A case report of intrahepatic bile duct dilatation caused by WDR19 gene mutation and presented as Caroli syndrome.

Background: Caroli syndrome or Caroli disease is characterized by focal dilation of the intrahepatic bile ducts, with or without congenital liver fibrosis. Mutations in the WDR19 gene can result in nephropathy, an autosomal recessive cystic kidney disease. However, this genetic mutation is clinically associated with Caroli syndrome or disease. We hypothesize that WDR19 gene mutations may contribute to extrarenal phenotypes such as Caroli disease or syndrome. Case Description: The outpatient department received a 1-year-old male patient with persistent dilated bile ducts for over four months. Subsequent ultrasound examination revealed liver cirrhosis, splenomegaly, and cystic dilatation of the intrahepatic bile duct. He was subsequently admitted for comprehensive diagnosis and treatment. Accordingly, we performed computed tomography (CT)-hepatic portal venography, magnetic resonance-cholangiography, and the plain liver scan, the results revealed liver cirrhosis, splenomegaly, cystic dilatation of the intrahepatic bile duct, as well as atypical hyperplasia nodules in the right posterior lobe of the liver and lymphatic hyperplasia and enlargement in the porta hepatis and the space between the liver and stomach. As the possibility of early small liver cancer could not be excluded due to the presence of nodules, surgical resection was performed followed by pathological examination and whole genome exome testing. The pathological findings revealed hepatocyte swelling, hydropic degeneration, and sporadic necrosis. Fibrous tissue hyperplasia was observed in the portal vein area, along with local pseudolobule formation. Also, numerous small bile duct hyperplasia was observed with lymphocyte infiltration, which is consistent with cirrhosis. Moreover, the hepatocytes of the small focal area showed atypical hyperplasia. Considering the above findings, Caroli syndrome was diagnosed. The genetic results showed two heterozygous mutations in the WDR19 gene, c.2290delC (p.Q764Nfs*29) and c.2401G>C (p.G801R). Therefore, the child's intrahepatic bile duct dilatation and cirrhosis were considered as the manifestations of Caroli syndrome caused by mutations in the WDR19 gene. Conclusions: Mutations in the WDR19 gene can manifest as Caroli disease or Caroli syndrome. For the definite diagnosis of liver diseases of unknown etiology, whole exome sequencing may be more conducive.

Advances in researches on long coronavirus disease in children: a narrative review.

Background and Objective: In the context of the global pandemic of coronavirus disease 2019 (COVID-19), more than 700 million infections and millions of deaths have occurred in countries around the world. Currently, two main sequelae of this disease are considered to occur in children, namely, multi-system inflammatory syndrome in children and long COVID. Among these two, the incidence of long COVID is higher and its impact on the population is more extensive, which is the focus of us. However, due to the lack of relevant studies and the limitations of most studies, the studies on sequelae of COVID-19 infection lag behind those of adults, but they have begun to attract the attention of some clinicians and researchers. We aim to summarize the current knowledge of long COVID in children, helping pediatricians and researchers to better understand this disease and providing guidance on research and clinical treatment of it. Methods: We reviewed all the studies on "long COVID", pediatric, children, adolescent, post-COVID syndrome in PubMed published after 2019. Key Content and Findings: This review summarizes the latest researches on epidemiology, pathogenesis, clinical manifestations, prevention and treatment of long COVID in children. Based on the existing research data, we summarized and analyzed the characteristics of long COVID in children, discovering the means to decipher the diagnosis of COVID-19 in children and some potential therapeutic treatments. Conclusions: We aim to summarize existing research on long COVID in children and help pediatricians and government agencies quickly understand the disease so that it can be used for clinical diagnosis, treatment and prevention in the population. In addition, providing a research basis for further researches on the cellular and even molecular level to explain the occurrence and development of diseases, and has a guiding role for future research direction.

Identification of a novel heterozygous variant in the PEX26 gene in an infant: a case report.

Background: The protein PEX26 is involved in the biogenesis and maintenance of peroxisomes, which are organelles within cells. Dysfunction of PEX26 results in peroxisome biogenesis disorders (PBDs) complementation group 8 (CG8), leading to Zellweger spectrum disorders (ZSDs). These disorders present as a syndrome with multiple congenital anomalies, varying in clinical severity. Case Description: We present the case of a 7-month-old boy who exhibited hepatic impairment with hepatomegaly, sensorineural hearing loss, developmental delay, abnormal ossification, and mild craniofacial dysmorphology. Tandem mass spectrometry analysis of plasma isolated from whole blood revealed a significant increase in the levels of very long chain fatty acids (VLCFAs) C26:0, C26:0/C22:0, and C24:0/C22:0, consistent with peroxisomal fatty acid oxidation disorder. Exome sequencing identified two variants in the PEX26 gene (c.347T>C and c.616C>T), with the latter being a suspected pathogenic variation. The variant can lead to a defect in the PEX26 gene, resulting in impaired peroxisome biogenesis, ß-oxidation of VLCFAs, and disruption of other biochemical pathways. Ultimately, this cascade of events manifests as ZSDs. Currently, symptomatic supportive treatment is the main approach for managing this condition and regular follow-up is being conducted for the patient. Conclusions: The present study introduces a novel heterozygous variant comprising two previously unidentified variants in the PEX26 gene, thereby expanding the range of known genetic alterations and highlighting the effectiveness of highly efficient exome sequencing in patients with undetermined multiple system dysfunctions.

NEXMIF pathogenic variant in a female child with epilepsy and multiple organ failure: a case report.

Background: The neurite extension and migration factor (NEXMIF) gene encodes the neurite growth-directed factor whose main function is to play a role in neurite extension and migration for nerve development. It is associated with X-linked intellectual disability 98 and X-linked dominant inheritance, and its clinical manifestations mainly include intellectual disability, autistic behavior, poor development, dysmorphic features, gastroesophageal reflux, renal infection, and early seizures. Few cases of patients with NEXMIF variants had been reported, and to date, no deaths have been reported to our knowledge. Case Description: We present a clinical report of a female child who had a history of epilepsy, and was diagnosed with multiple organ failure (MOF), sepsis, hemophagocytic lymphohistiocytosis, severe pneumonia, and pulmonary hemorrhaging. Genetic testing identified the NEXMIF variant c.937C>T (p.R313*) in this patient. Despite aggressive treatment with anti-inflammation drugs with methylprednisolone, plasma exchange, hemodialysis and mechanical ventilation, the patient died. Conclusions: We reported the first case of the NEXMIF variant in a patient with the symptom of MOF, including acute liver failure and acute kidney injury (Grade 3). In addition, some complications, such as sepsis, hemophagocytic syndrome, pneumonia, and pulmonary hemorrhage, can also occur with this disease. All of these complications may have contributed to the patient's death. This report not only broadens the phenotype for NEXMIF variants but may also help physicians involved in the care of patients with this syndrome and enhance their understanding of this variant.

Concordance of adenosine deaminase with immunoglobulins and lymphocyte subsets in EBV-related diseases.

BACKGROUND: Clinical manifestations of Epstein-Barr virus (EBV) infection are diverse. This study aimed to explore the immune response in EBV-related diseases and the correlation between immune cells and adenosine deaminase (ADA) levels. METHODS: This study was conducted at the Children's Hospital of Soochow University. In total, 104 patients with EBV-associated respiratory tract infection (EBV-RTI), 32 patients with atypical EBV infection, 54 patients with EBV-associated infectious mononucleosis (IM1, with normal alanine aminotransferase [ALT] levels), 50 patients with EBV-IM2 (with elevated ALT levels), 50 patients with acute respiratory infection (AURI, with other pathogens), and 30 healthy controls were enrolled in this study. Indicators of ADA, immunoglobulins (Igs), and lymphocyte subsets were analyzed for EBV-related diseases. RESULTS: Differences in the white blood cell, lymphocyte counts, ADA levels, IgA, IgG and IgM titers, percentage of CD3+, CD3+CD4+, CD3+CD8+, CD16+CD56+, CD3-CD19+, and CD19+CD23+ lymphocytes, and CD4+/CD8+ ratio between EBV-related disease groups were all statistically significant (P < 0.01). ADA levels in the EBV-related disease groups were significantly higher than those in the control group (P < 0.01). The lymphocyte count, ADA levels, IgA and IgG titers, and percentage of CD3+ and CD3+CD8 + lymphocytes in the atypical EBV infection, EBV-IM1, and EBV-IM2 groups were significantly higher than those in the EBV-RTI, AUTI, and control groups (P < 0.01), whereas the percentage of CD3+CD4+, CD3-CD19+, and CD19+CD23+ lymphocytes and CD4+/CD8+ ratio showed the opposite trend. ADA levels were consistent with and closely related to the viral load and cellular and humoral immunity in EBV-related diseases. CONCLUSIONS: ADA levels, humoral immunity, and cellular immunity were diverse in EBV-related diseases, and ADA was closely related to Igs and lymphocyte subsets.

Case report: Acinetobacter baumannii septic arthritis in an immunocompetent infant.

Acinetobacter baumannii is a gram-negative coccobacilli, mainly causing nosocomial infections with poor prognosis, especially in patients with prolonged hospitalization or antibiotics administration. A. baumannii pneumonia is the most common clinical form and usually occurs in critically ill patients in the intensive care unit. However, septic arthritis caused by A. baumannii is rarely reported. In this report, we describe a case of A. baumannii septic arthritis combined with incomplete Kawasaki disease in an infant. The child's chief complaint was a 2-week intermittent fever with poor response to antibiotics. Initial physical examination revealed swollen lymph nodes in the neck, pharynx congestion, and the appearance of rashes. Combined with laboratory tests, the diagnosis of incomplete Kawasaki disease was considered. After administration of high-dose intravenous immunoglobulin and corticosteroids, the child's fever improved and periungual desquamation appeared simultaneously. Swelling of the right knee occurred 5 days after the fever improved and imaging tests of MRI and ultrasound suggested the existence of infection. A diagnosis of septic arthritis was established subsequently, and arthroscopy was carried out. A. baumannii was finally identified by metagenomics next-generation sequencing of joint draining fluid for pathogenic microorganisms. Treatment with meropenem was then started. The patient eventually recovered and was discharged from the hospital after 23 days of treatment with meropenem. Although A. baumannii is not a common bacterium of septic arthritis, this rare infection can still occur in infants. Early diagnosis, pathogenic identification, and target antibiotic treatment are important to reduce the occurrence of joint sequelae.

Successful treatment with tigecycline of severe pandrug-resistant Acinetobacter baumannii pneumonia complicated with a diaphragmatic hernia: a case report.

BACKGROUND: Acinetobacter baumannii (A. baumannii) is one of the most common pathogens that cause hospital-acquired infections. In recent years, drug-resistant A. baumannii has become prevalent worldwide, and pandrug-resistant A. baumannii is increasingly being observed. However, treating pandrug-resistant A. baumannii is very difficult. CASE DESCRIPTION: We report a case of an 8-year-old girl with severe pandrug-resistant A. baumannii pneumonia complicated with a diaphragmatic hernia. The patient arrived at Tongji Hospital with a fever and cough, and she was admitted to the pediatric intensive care unit with pneumonia. The day after admission, her condition worsened. She had breathing difficulties and loss of consciousness, and a ventilator was used immediately. On the fourth day after being on the ventilator, the sputum culture was positive for A. baumannii. Initially, multiple antibiotic sensitivity tests showed resistance to all antibiotics. This suggested that using antibiotics would be ineffective. After the use of all antibiotics was discontinued for 8 days, susceptibility testing of tigecycline indicated intermediate susceptibility. Following the initiation of tigecycline treatments for 5 weeks, the bacterial infection was progressively controlled, the diaphragmatic hernia disappeared, and the patient gradually recovered. CONCLUSIONS: Antibiotic sensitivity to pandrug-resistant A. baumannii can be restored by discontinuation of antibiotics. Antibiotics should be administrated even if the antibiotic sensitivity test shows an intermediary result. For pandrug-resistant A. baumannii, a prolonged monotherapy with a large dose of intermediary tigecycline can achieve good efficacy. In addition, the complication of a diaphragmatic hernia can return to normal after treatment with effective antibiotics, and patients may not necessarily need surgery.

Case Report: Refractory Listeria innocua Meningoencephalitis in a Three-Year-Old Boy.

Listeria innocua is widely distributed in the environment and food and is considered a non-pathogenic bacterium for both humans and animals. To our knowledge, only a few cases of L. innocua infection in humans and ruminants have been reported. Moreover, there has been no report on human L. innocua meningoencephalitis. Here, we report a case of severe refractory meningoencephalitis in a three-year-old boy after infection with L. innocua. The child's first symptoms were a runny nose, high fever, and rashes, which quickly progressed to unconsciousness and convulsions. The initial analysis of cerebral spinal fluid revealed remarkably elevated protein levels and increased white blood cells count. The blood culture of the patient in the early stage was positive for L. innocua. In addition, his brain imaging tests were observed dynamically, and the result showed a speedy progression from multiple intracranial abnormal signals to hydrocephalus and interstitial cerebral edema. After receiving antibiotics and symptomatic treatment for nearly 3 months, the patient's condition improved markedly. However, he still had residual complications such as hydrocephalus. Although L. innocua is considered harmless, it can still cause disease in humans, even severe meningoencephalitis, with rapid progression and poor prognosis. Early discovery, diagnosis, and treatment are necessary to elevate the survival rate and life quality of those patients. Antibiotics should be used with sufficient duration and dosage. Cephalosporins are not suitable for the treatment of L. innocua meningoencephalitis and penicillin antibiotics are preferred for children. The presentation of this case will help to expand our knowledge of Listeria infections and provide a potential candidate for pathogens causing severe childhood central nervous system infection.

A novel variant site of Alstrom syndrome in a Chinese child: a case report.

Background: Alstrom syndrome (ALMS) is an ultra-rare multisystem genetic disorder caused by autosomal recessive inheritance of the ALMS1 gene. It manifests as multisystem dysfunction, displaying unique clinical signs and symptoms and various severity, which may lead to delayed prognosis or misdiagnosis in medical practice. Although almost 300 pathogenic variants have been reported, there are some variant sites that have not been recognized yet. Case Description: We report a case of a 14-year-old boy with manifestations, including binocular vision loss, acanthosis nigricans, type 2 diabetes, insulin resistance, elevated transaminase, hepatic fibrosis, and proteinuria. Compound heterozygous variants in the ALMS1 gene have been discovered by whole exon sequencing. One of his variant sites was C. 8158C>T, which was from his father. And the other variant site was C. 3575C>A, which was from his mother. To the great of our knowledge, this site has not been reported before. Both of the variants make the synthesis of the peptide chain terminated in advance and an incomplete polypeptide chain is formed. Conclusions: The clinical presentations of ALMS are complicated and varied. Although early diagnosis can be made according to typical clinical symptoms, whole exon sequencing is necessary for the diagnosis of ALMS, as indicated by our study.

Identification of the clinical and neuroimaging characteristics in children with neuromyelitis optica spectrum disorders: a case series.

BACKGROUND: Childhood neuromyelitis optica spectrum disorders (NMOSDs) may cause visual impairment and brain or spinal cord damage, and the effects may be permanent if left untreated. Since the incidence of NMOSD cases in children is relatively low, the understanding of NMOSD among children is inadequate. METHODS: This investigation examined the clinical and neuroimaging characteristics of childhood NMOSD. We retrospectively analyzed the clinical information of 11 NMOSD patients admitted to our centre from 2012 to 2021. The disease status was assessed by the Expanded Disability Status Scale (EDSS) score. RESULTS: The two major symptoms observed in the study cohort were optic neuritis (ON) (9/11) and encephalopathy (7/11). Antibody tests were performed on 8 children, 2 of whom showed serum aquaporin 4 (AQP4) antibody positivity, and another 2 presented with serum myelin oligodendrocyte glycoprotein (MOG) antibody positivity. All patients showed white matter hyperintensity on magnetic resonance imaging (MRI) scans. Interestingly, a rare radiological sign, enlarged perivascular space (PVS), which is more commonly observed in the elderly or adults, was found in 4 participants with more severe clinical manifestations. CONCLUSIONS: While NMOSD in children is less commonly diagnosed through clinical evaluations, the symptoms of ON and encephalopathy should raise the possibility of the disease. As the diagnosis of NMOSD in children is relatively difficult, enlarged PVS may represent a promising biomarker for the diagnosis and evaluation of NMOSD.

Retinal and Choroidal Pathologies in Aged BALB/c Mice Following Systemic Neonatal Murine Cytomegalovirus Infection.

Although pathologies associated with acute virus infections have been extensively studied, the effects of long-term latent virus infections are less well understood. Human cytomegalovirus, which infects 50% to 80% of humans, is usually acquired during early life and persists in a latent state for the lifetime. The purpose of this study was to determine whether systemic murine cytomegalovirus (MCMV) infection acquired early in life disseminates to and becomes latent in the eye and if ocular MCMV can trigger in situ inflammation and occurrence of ocular pathology. This study found that neonatal infection of BALB/c mice with MCMV resulted in dissemination of virus to the eye, where it localized principally to choroidal endothelia and pericytes and less frequently to the retinal pigment epithelium (RPE) cells. MCMV underwent ocular latency, which was associated with expression of multiple virus genes and from which MCMV could be reactivated by immunosuppression. Latent ocular infection was associated with significant up-regulation of several inflammatory/angiogenic factors. Retinal and choroidal pathologies developed in a progressive manner, with deposits appearing at both basal and apical aspects of the RPE, RPE/choroidal atrophy, photoreceptor degeneration, and neovascularization. The pathologies induced by long-term ocular MCMV latency share features of previously described human ocular diseases, such as age-related macular degeneration.

The Role of Autophagy in Murine Cytomegalovirus Hepatitis.

Autophagy is involved in the pathogenesis of multiple pathogen infection. Previous studies have reported that human cytomegalovirus (CMV) activates autophagy in the early stage of infection and then inhibits autophagy. Little is known about the role of autophagy in murine CMV (MCMV) infection, especially in MCMV-induced hepatitis. The purpose of this study is to investigate the role of autophagy in MCMV hepatitis. BALB/c mice were infected with MCMV and a series of experiments involving western blot, immunofluorescence, immunohistochemistry, H&E (Hematoxylin and Eosin) staining and quantitative real-time polymerase chain reaction were performed in this study. The expression of SQSTM1/p62, PI3K, the ratio of phosphorylated Akt to total Akt, and the ratio of phosphorylated mammalian target of rapamycin (mTOR) to total mTOR were increased, and the expression of light-chain 3 (LC3)-II were decreased in the livers of infected mice on days 3 and 7 postinfection (p.i.). Compared with the untreated infected group, increased transcription level of MCMV glycoprotein B (gB), increased expression levels of interleukin1-ß (IL-1ß), aspartate aminotransferase (AST) and alanine aminotransferase (ALT), decreased expression level of type I interferon α (IFN-α), as well as aggravated liver pathological injury were detected in starvation-treated infected group on days 3 and 7 p.i.; whereas decreased transcription level of MCMV gB, decreased expression levels of IL-1ß, AST and ALT, increased expression level of type I IFN-α, as well as alleviated liver pathological injury were detected in chloroquine (CQ)-treated infected group on day 3 p.i. In conclusion, autophagy is inhibited through activating the PI3K/Akt/mTOR pathway in the liver of BALB/c mice during MCMV infection, and autophagy may promote MCMV replication and aggravate liver pathological damage and inflammation. Further understanding of the interactions between autophagy and MCMV infection and its potential mechanism may bring new important cues to the control of MCMV infection and antiviral therapy.

Inhibition of autophagy by 3-methyladenine restricts murine cytomegalovirus replication.

Cytomegalovirus (CMV) induced autophagy affects virus replication and survival of the infected cells. The purpose of this study was to investigate the role of autophagy inhibition by 3-methyladenine (3-MA) on murine cytomegalovirus (MCMV) replication and whether it is associated with caspase-3 dependent apoptosis. The eyecup isolated from adult C57BL/6J mice (6-8 weeks old) and mouse embryo fibroblast cells (MEFs) were infected with MCMV K181 strain, followed by the treatment of 3-methyladenine (3-MA), chloroquine, or rapamycin to block or stimulate autophagy. In cultured MEFs, the ratio of LC3I/II was reduced at 24 hours post infection (hpi), but was increased at 48 hpi In the eyecup culture, LC3I/II ratio was also decreased at 4 and 7 days post infection (dpi). In addition, caspase-3 cleavage was increased at 48 hpi in MEFs and also elevated in MCMV infected eyecups at 4, 7, 10, and 14 dpi. 3-MA treatment significantly inhibited the virus replication in MEFs and eyecups. The expression of early antigen (EA) of MCMV was also decreased in MEFs and eyecups. Meanwhile, cleaved caspase-3 dependent cell death was promoted with the presence of 3-MA in MCMV infected MEFs and eyecups, while RIPK1/RIPK3/MLKL pathway was inhibited by 3-MA in eyecups. Inhibition of autophagy by 3-MA restricts virus replication and promotes caspase-3 dependent apoptosis in the eyecup and MEFs with MCMV infection. It can be explained that during the early period of MCMV infection, the suppressed autophagy process directly reduced virus release, but later caspase-3 dependent apoptosis dominated and resulted in decreased virus replication.

The role of autophagy in human cytomegalovirus IE2 expression.

The purpose of this study was to determine whether autophagy regulates the expression of human cytomegalovirus (HCMV) immediately early two viral protein (IE2). Rapamycin and 3-methyladenine (3-MA) were used to stimulate or suppress autophagy during HCMV infection. UL122 recombinant plasmid was transfected to overexpress IE2 and small interference RNA against autophagy-related protein 3 (ATG3) was used to knockdown ATG3. Western blot was performed to measure the expression of viral proteins and autophagy levels. Immunofluorescence was used to detect the immediately early 1 viral protein (IE1) expression. In human embryonic lung fibroblasts, infection of HCMV promotes the lipidation of light chain 3 (LC3) at 6 and 24 hours post infection (hpi), which was accompanied by the increased expression of viral protein IE2. When only IE2 was overexpressed via UL122 recombinant plasmid transfection without HCMV infection, the autophagy hallmarks LC3II and ATG3 were upregulated. Furthermore, viral protein IE2 expression was reduced at 24 and 48 hpi either by the treatment of autophagy inducer rapamycin or by the inhibitor 3-MA before HCMV infection. At the same time, small interference ATG3 transient transfection, used to suppress autophagy, significantly inhibited IE2 expression. However, when 3-MA was used to regulate autophagy levels after HCMV infection, expression of IE2 and IE1 were both decreased, while autophagy inducer rapamycin treatment after HCMV infection increased IE2 expression slightly. IE2 was involved in autophagy induced by HCMV infection and blocking autophagy could inhibit the expression of HCMV viral protein IE2, which might be one way for autophagy to restrict HCMV replication.

Ocular cytomegalovirus latency exacerbates the development of choroidal neovascularization.

Age-related macular degeneration (AMD) is a complex, multifactorial, progressive disease which represents a leading cause of irreversible visual impairment and blindness in older individuals. Human cytomegalovirus (HCMV), which infects 50-80% of humans, is usually acquired during early life and persists in a latent state for the life of the individual. In view of its previously described pro-angiogenic properties, we hypothesized that cytomegalovirus might be a novel risk factor for progression to an advanced form, neovascular AMD, which is characterized by choroidal neovascularization (CNV). The purpose of this study was to investigate if latent ocular murine cytomegalovirus (MCMV) infection exacerbated the development of CNV in vascular endothelial growth factor (VEGF)-overexpressing VEGF-Ahyper mice. Here we show that neonatal infection with MCMV resulted in dissemination of virus to various organs throughout the body including the eye, where it localized principally to the choroid in both VEGF-overexpressingVEGF-Ahyper and wild-type(WT) 129 mice. By 6 months post-infection, no replicating virus was detected in eyes and extraocular tissues, although virus DNA was still present in all eyes and extraocular tissues of both VEGF-Ahyper and WT mice. Expression of MCMV immediate early (IE) 1 mRNA was detected only in latently infected eyes of VEGF-Ahyper mice, but not in eyes of WT mice. Significantly increased CNV was observed in eyes of MCMV-infected VEGF-Ahyper mice compared to eyes of uninfected VEGF-Ahyper mice, while no CNV lesions were observed in eyes of either infected or uninfected WT mice. Protein levels of several inflammatory/angiogenic factors, particularly VEGF and IL-6, were significantly higher in eyes of MCMV-infected VEGF-Ahyper mice, compared to uninfected controls. Initial studies of ocular tissue from human cadavers revealed that HCMV DNA was present in four choroid/retinal pigment epithelium samples from 24 cadavers. Taken together, our data suggest that ocular HCMV latency could be a significant risk factor for the development of AMD. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Short-lived AIM2 Inflammasome Activation Relates to Chronic MCMV Infection in BALB/c Mice.

Absent in melanoma 2 (AIM2) inflammasome is a crucial link bridging the innate host defense and the subsequent adaptive immunity when activated by exogenous double stranded DNA (dsDNA). Through establishing models of disseminated murine cytomegalovirus (MCMV) infection in BALB/c and C57BL/6 mice, we evaluated dynamic expression of AIM2 inflammasome components and its relationship with pathological damage and viral replication, trying to figure out whether AIM2 inflammasome is related to the chronic mechanism of MCMV. BALB/c and C57BL/6 mice were sacrificed on day 0, 1, 3, 7, 14 and 28 post infection. Expression levels of AIM2, pro-caspase-1, caspase-1 p20, pro-IL1ß and mature IL1ß in primary peritoneal macrophages (PMs) and spleens were detected by Western blotting. Contents of IL18 in the serum were detected by ELISA. Pathological examinations of livers were performed, and mRNA levels of MCMV glycoprotein B (gB) in salivary glands also assessed. Results showed that expression levels of AIM2 in PMs and spleens of C57BL/6 mice increased on day 3, even continued to day 28; caspase-1 p20 and mature IL1ß increased on day 7, 14 and 28; the persistently high expression of IL18 in the serum started on day 1, showing a double peak curve. As for BALB/c mice, expression of AIM2 in PMs increased on day 1 and day 7, while contents of AIM2 in spleens increased on day 1 and day 3; caspase-1 p20 and mature IL1ß merely increased 7 days fter infection. Thereafter, expression levels of AIM2, caspase-1 p20, mature IL1ß and IL18 were limited; the duration of AIM2 inflammasome activation in BALB/c mice was much shorter than that in C57BL/6 mice. The severer pathological damage and more viral replications in BALB/c mice further proved the deficient antiviral immunity to MCMV. In conclusion, the activation of AIM2 inflammasome in BALB/c mice was short-lived, which is quite possibly related to the chronicity of MCMV infection.

The Effects of IL10 and NK Cells on the Susceptibility to Mouse Cytomegalovirus in BALB/c Mice despite the Compensation of IFNγ.

OBJECTIVE: The aim of this study was to determine which factors lead to the susceptibility to mouse cytomegalovirus (MCMV) in the spleens of BALB/c mice. METHODS: BALB/c and C57BL/6 mice were randomly divided into a control group and an infection group and sacrificed on day 0, 1, 3, 7, 14, and 28 postinfection. The cytotoxicity of NK cells was determined by evaluating lactate dehydrogenase contents. Flow cytometry was used to analyze activated NK cells, IFNγ+ NK cells, and total NK cells in the spleen. The pathological changes of spleens in each group were analyzed by HE staining. The expression of IL10, IL18, IFNγ, Thpok, and IFNß of spleens was determined by quantitative reverse transcriptase PCR. The viral loads of MCMV in spleens and salivary glands were also detected. RESULTS: We found that spleen NK cells and IL10 in C57BL/6 mice possessed more powerful immunity to MCMV than BALB/c mice. In BALB/c mice, combined effects of the cytotoxicity of NK cells and IFNγ in spleens still ended up with deficient control of infection. CONCLUSION: The functional shortage of NK cells and inappropriate expression of IL10 result in the susceptibility to MCMV in BALB/c mice.

Maternal Murine Cytomegalovirus Infection during Pregnancy Up-regulates the Gene Expression of Toll-like Receptor 2 and 4 in Placenta.

Increasing evidence has revealed that maternal cytomegalovirus (CMV) infection may be associated with neurodevelopmental disorders in offspring. Potential relevance between the placental inflammation and CMV-related autism has been reported by clinical observation. Meanwhile, abnormal expression of Toll-like receptor 2 (TLR2) and TLR4 in placenta of patients with chorioamnionitis was observed in multiple studies. IL-6 and IL-10 are two important maternal inflammatory mediators involved in neurodevelopmental disorders. To investigate whether murine CMV (MCMV) infection causes alterations in placental IL-6/10 and TLR2/4 levels, we analyzed the dynamic changes in gene expression of TLR2/4 and IL-6/10 in placentas following acute MCMV infection. Mouse model of acute MCMV infection during pregnancy was created, and pre-pregnant MCMV infected, lipopolysaccharide (LPS)-treated and uninfected mice were used as controls. At E13.5, E14.5 and E18.5, placentas and fetal brains were harvested and mRNA expression levels of placental TLR2/4 and IL-6/10 were analyzed. The results showed that after acute MCMV infection, the expression levels of placental TLR2/4 and IL-6 were elevated at E13.5, accompanied by obvious placental inflammation and reduction of placenta and fetal brain weights. However, LPS 50 µg/kg could decrease the EL-6 expression at E13.5 and E14.5. This suggests that acute MCMV infection during pregnancy could up-regulate the gene expression of TLR2/4 in placental trophoblasts and activate them to produce more proinflammatory cytokine IL-6. High dose of LPS stimulation (50 µg/kg) during pregnancy can lead to down-regulation of IL-6 levels in the late stage. Imbalance of IL-6 expression in placenta might be associated with the neurodevelopmental disorders in progeny.

Inflammation and outer blood-retina barrier (BRB) compromise following choroidal murine cytomegalovirus (MCMV) infections.

Purpose: The purpose of this study was to determine whether the blood-retina barrier is compromised by choroidal murine cytomegalovirus (MCMV) infection, using electron microscopy. Methods: BALB/c mice were immunosuppressed with methylprednisolone and monoclonal antibodies to CD4 and CD8. At several time points post-MCMV intraperitoneal inoculation, the eyes were removed and analyzed with western blotting and immunoelectron microscopy for the presence of MCMV early antigen (EA) and the host protein RIP3. Posterior eyecups from RIP3-/- and RIP3+/+ mice were cultured and inoculated with MCMV. At days 4, 7, and 11 post-infection, cultures were collected and analyzed with plaque assay, immunohistochemical staining, and real-time PCR (RT-PCR). Results: MCMV EA was observed in the nuclei of vascular endothelial cells and pericytes in the choriocapillaris. Disruption of Bruch's membrane was observed, especially at sites adjacent to activated platelets, and a few RPE cells containing some enlarged vesicles were found directly beneath disrupted Bruch's membrane. Some virus particles were also observed in the enlarged vesicles of RPE cells. Levels of the RIP3 protein, which was observed mainly in the RPE cells and the basement membrane of the choriocapillaris, were greatly increased following MCMV infection, while depletion of RIP3 resulted in greatly decreased inflammasome formation, as well as expression of downstream inflammation factors. Conclusions: The results suggest that systemic MCMV spreads to the choroid and replicates in vascular endothelia and pericytes of the choriocapillaris during immunosuppression. Choroidal MCMV infection is associated with in situ inflammation and subsequent disruption of Bruch's membrane and the outer blood-retina barrier.