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Background Wilson disease (WD) is an autosomal-recessive metabolic disorder characterized by excess copper accumulation predominantly in the liver, brain, and cornea. Clinical diagnosis of WD remains a challenge because of its phenotypic heterogeneity. Here we describe the novel mutation (p. K838N) in the ATP7B gene of a child with WD. The mutation affects a conserved ATP-binding domain that is involved in the catalytic cycle. We also describe the clinical outcome of this patient. Case presentation: We reported a successful early diagnosis and treatment of WD in a 5-year-old boy who presented with unexplained liver dysfunction and hepatitis. Using whole-exome sequencing (WES), we identified a novel ATP7B mutation, K838N, which is valuable for early diagnosis of WD. After combination therapy with penicillamine, zinc supplement, low-copper diet, and supportive treatments for infections, liver problems, and jaundice, the patient’s medical condition gradually improved and stabilized in a clinical follow-up. We suggested that the novel K838N mutation in the case of WD might impair protein function and contribute to WD progression. Conclusions This case emphasizes the importance of WD diagnostic tests during clinical evaluation for patients presenting with an unexplained liver disorder in childhood for better outcomes and genetic counseling.

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.

Monkeypox is a zoonotic disease. Since the first human monkeypox case was detected in 1970, it has been prevalent in some countries in central and western Africa. Since May 2022, monkeypox cases have been reported in more than 96 non-endemic countries and regions worldwide. As of September 14, 2022, there have been more than 58,200 human monkeypox cases, and there is community transmission. The cessation of smallpox vaccination in 1980, which had some cross-protection with monkeypox, resulted in a general lack of immunity to monkeypox, which caused global concern and vigilance. As of September 14, 2022, there are four monkeypox cases in China, including three in Taiwan province and one in Hong Kong city. Previous foreign studies have shown that children are vulnerable to monkeypox and are also at high risk for severe disease or complications. In order to improve pediatricians' understanding of monkeypox and achieve early detection, early diagnosis, early treatment, and early disposal, we have organized national authoritative experts in pediatric infection, respiratory, dermatology, critical care medicine, infectious diseases, and public health and others to formulate this expert consensus, on the basis of the latest "Clinical management and infection prevention and control for monkeypox" released by The World Health Organization, the "guidelines for diagnosis and treatment of monkeypox (version 2022)" issued by National Health Commission of the People's Republic of China and other relevant documents. During the development of this consensus, multidisciplinary experts have repeatedly demonstrated the etiology, epidemiology, transmission, clinical manifestations, laboratory examinations, diagnosis, differential diagnosis, treatment, discharge criteria, prevention, disposal process, and key points of prevention and control of suspected and confirmed cases.

Summary In the period of regular epidemic prevention and control of Coronavirus disease 2019 (COVID-19) in our country, work resumption has been fully advanced. But there are still new sporadic local cases and imported cases across the country. In this situation, whether kindergartens reopening will increase the risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread still remains uncertain. We reviewed two pediatric patients with moderate COVID-19, collected the epidemiologic information and monitored the cycle threshold value of rectal specimen and the viral loads, and discussed the transmission of SARS-CoV-2 in pediatric patients and the virulence of feces in children with moderate COVID-19, in order to analyze the risk of kindergartens reopening.

In the early February, 2020, we called up an experts' committee with more than 30 Chinese experts from 11 national medical academic organizations to formulate the first edition of consensus statement on diagnosis, treatment and prevention of coronavirus disease 2019 (COVID-19) in children, which has been published in this journal. With accumulated experiences in the diagnosis and treatment of COVID-19 in children, we have updated the consensus statement and released the second edition recently. The current version in English is a condensed version of the second edition of consensus statement on diagnosis, treatment and prevention of COVID-19 in children. In the current version, diagnosis and treatement criteria have been optimized, and early identification of severe and critical cases is highlighted. The early warning indicators for severe pediatric cases have been summarized which is utmost important for clinical practice. This version of experts consensus will be valuable for better prevention, diagnosis and treatment of COVID-19 in children worldwide.

Background 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. Methods 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(CQ) or rapamycin to block or stimulate autophagy. Results In cultured MEFs, the ratio of LC3I/II was reduced at 24 hours post infection (h.p.i.), but was increased at 48h.p.i. In the eyecup culture, LC3I/II ratio was also decreased at 4 and 7days post infection (d.p.i.). In addition, caspase-3 cleavage was increased at 48h.p.i. in MEFs and also elevated in MCMV infected eyecups at 4, 7, 10 and 14d.p.i. 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. Conclusions 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, suppressed autophagy process directly reduced virus release, but later caspase-3 dependent apoptosis dominated and resulted in decreased virus replication. This article is protected by copyright. All rights reserved.

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 IFNβ 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.

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-IL1β and mature IL1β 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 IL1β 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 IL1β merely increased 7 days fter infection. Thereafter, expression levels of AIM2, caspase-1 p20, mature IL1β 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.

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.

Background Cytomegalovirus (CMV) infection occurs frequently and is widespread globally. Numerous studies have shown that various types of immune cells play roles in mediating the response to CMV infection. CD11c, a commonly used dendritic cell (DC) marker, is expressed by other immune cells as well, such as T cells. This study analyzed the immune cells that express CD11c and monitored the expression level of their specific cell surface markers in the lung following a disseminated murine (M)CMV infection. Methods Mouse models of disseminated MCMV infection were used; uninfected and lipopolysaccharide (LPS)-treated mice were used as controls. At 1, 3 and 7 days following infection, single cell suspensions prepared from freshly digested lung tissue were stained for CD11c, CD86 and MHC II. Stained cells were analyzed using flow cytometry. Peripheral blood and single cell suspensions from spleen were sorted as well. Then these cells were subjected to analyze the CD11c expression pattern on natural killer (NK) cells and T cells. Results This assay showed that after MCMV infection, the expression of CD86 on pulmonary CD11chiMHC-IIhi cells (encompassing conventional DCs) was higher at 3 days post-infection than at 1 or 7 days post-infection, accompanied by a downregulation of MHC II. In addition, expression of CD11c was greatly increased in the MCMV infection group at 7 days post infection. This study also detected a large population of cells displaying an intermediate level of expression of CD11c (CD11cint); these cells were in the MCMV groups exclusively, and were subsequently identified as CD8+ T cells. In lung, spleen and blood, different proportions of CD11cint cells among the NK cell and T cell populations were observed between the BALB/c and C57BL/6 mice with or without MCMV infection. The expression level of NKp46 in NK cells dropped to a lower level after MCMV infection. Conclusions The findings collectively indicate that CD11cintCD8+ T cells might play a key role in anti-MCMV adaptive immune response in lungs, as well as in spleen and blood. B220+CD11cint NK cells might be a more effective type of NK cell, participating in anti-MCMV infection. The downregulation of NKp46, in particular, might be linked with the immune evasion of MCMV. Electronic supplementary material The online version of this article (doi:10.1186/s12985-017-0801-x) contains supplementary material, which is available to authorized users.

Aim: This study aimed to explore the potential role of IL-17 in the pathological damage to the salivary gland during CMV infection. Materials & methods: For all studies, 4-week-old female BALB/c mice were used. Mouse embryo fibroblast cells were isolated from embryos of pregnant mice. The Smith murine CMV (MCMV) strain was propagated and obtained from homogenates of the salivary glands. BALB/c mice were randomly divided into two groups. Mice in one group were infected with Smith MCMV to establish disseminated infection; mice in the other group were controls. Four mice of each group were randomly chosen to be harvested on days 3, 7, 14 and 28. Viral titers in salivary gland tissues were determined using a standard plaque assay; IL-17+ T cells in the spleen were analyzed by flow cytometry; the expressions of IL-17 and IL-17 receptor (IL-17R) mRNA was measured by reverse transcription PCR; and the pathology of the salivary glands in infected mice were assessed by hematoxylin and eosin staining. Results: The viral titers in salivary gland tissues, which were much higher than those of other organs, elevated on day 7 and peaked on day 14, then gradually declined on day 28. The percentages of CD4+IL-17+ T cells were increased in MCMV-infected mice compared with controls, with the main increase of CD4+IL-17+ T cells being Th17 cells (p < 0.05). In salivary gland tissues, the expression of IL-17 and IL-17R mRNA increased significantly and achieved the highest level on day 14, which was significantly higher than that of the controls (p < 0.05). Compared with controls, serious inflammatory cellular infiltrates in the salivary glands were present on day 7 after MCMV infection, and on day 14, many of the cellular infiltrate became larger and coalesced with neighboring foci. We found that IL-17 and IL-17R were enriched in the salivary glands when the tissues were mainly injured. Conclusion: The results illustrated that the inflammatory factors, IL-17 and IL-17R, possibly contributed to the process of viral infection and played a part in inducing pathological damage of the salivary gland.

Human cytomegalovirus (HCMV) has been associated with a wide spectrum of diseases. There is currently no effective treatment for eliminating the virus. Garlic bulb extract has been reported to possess anti-viral efficacy. This study aimed to investigate the expression of the immediate‑early (IE; ul122 and ul123), early (E; ul54) and late (L; ul83) genes of HCMV as well as the inhibitory effect of allitridin on the transcription levels of these genes. The results indicated that a HCMV gene expression cascade occurred, and that the deletion of IE72 had no influence on the transcription of the ul122 gene, while it led to significant reductions of ul54 and ul83 mRNA expression levels. Additionally, allitridin effectively suppressed the transcription of the HCMV IE, E and L genes; the inhibition rates of the transcription of the ul122 and ul123 genes were higher compared with those of ul54 and ul83 mRNA expression, while the expression of the IE genes was not significantly reduced by ganciclovir (GCV). Our results indicate that the HCMV IE72 deletion mutant strain affects the transcription of the virus downstream gene, allitridin inhibits HCMV infection in vitro, and that the IE genes may be the key target of allitridin in its action against HCMV.

This study investigated the effects of allitridin compound on murine cytomegalovirus (MCMV)-induced regulatory T cell (Treg; CD4(+) CD25(+) Foxp3(+) ) amplification in vivo and in vitro. One hundred twenty MCMV-infected mice were allocated at random into two groups for treatment with allitridin or placebo. Another 120 mock-infected mice were randomly allocated as controls for the allitridin treatment and placebo treatment groups. The mice were euthanized at various time points after infection (out to 120 days) to evaluate the effects of treatment on Treg presence and function, as well as MCMV infective load. Co-culture with mouse embryo fibroblasts (MEF) and MCMV was performed to evaluate allitridin-mediated Treg and anti-CMV effects. The maximum tolerance concentration (MTC) of allitridin was used to treat cells for 3 days. Changes in Foxp3 mRNA and protein levels, percentages of T cell subsets, and Treg-related cytokines (IL-10 and TGF-β) were measured. Allitridin treatment did not influence Foxp3 expression and Treg proportion in uninfected mice, but did down-regulate each in infected mice during the chronic infection period. Additionally, allitridin treatment reduced the MCMV load in salivary glands. MTC allitridin treatment of co-cultures partially blocked MCMV induction of Foxp3 mRNA and protein expression. In vitro treatment with allitridin also increased significantly the percentages of Tc1, Tc2, and Th1, reduced the secreted levels of IL-10 and TGF-β1, and significantly suppressed viral loads. In conclusion, allitridin can promote MCMV-induced Treg expansion and Treg-mediated anti-MCMV immunosuppression. Therefore, allitridin may be useful as a therapeutic agent to enhance the specific cellular immune responses against CMV.

This study investigated the effects of allitridin on acute and chronic mouse cytomegalovirus (MCMV) infections in vivo. The results demonstrated that allitridin reduced the titers of MCMV in salivary glands, and reductions in viral loads were confirmed by determining viral DNA and RNA levels in susceptible organs during the acute infection phase. Although allitridin did not eliminate MCMV, treatment reduced viral levels and facilitated healing of pathologic lesions in organs, particularly during the chronic infection phase. The results presented in this report suggest that allitridin could act as an effective agent against MCMV infections in vivo.

To investigate the genetic polymorphism of human cytomegalovirus (HCMV) glycoprotein H (gH) from infantile clinical isolates, to analyze the genotypic distribution of gH in different diseases of HCMV infection and try to find the correlations between the diseases and genotypes.Fresh urine specimens were collected from the hospitalized children with different diseases whose blood HCMV-IgM and HCMV-IgG were positive. Virus was isolated from these specimens. Glycoprotein H of harvest clinical isolates was genotyped by nested-PCR combined with restriction fragment length polymorphism (RFLP), the purified PCR products were digested by restriction endonuclease HhaI. The digested products were genotyped by polyacrylamide gel electrophoresis and silver staining. Classification and results of sequencing were compared.Totally 102 HCMV clinical isolates were obtained. Glycoprotein H gene of these clinical isolates (43 cases had infantile hepatitis syndrome, 38 cases had anicteric hepatitis, 13 pneumonia, 7 thrombocytopenic purpura, and 1 congenital CMV infection) were positive by nested-PCR, whose positive rate was 100%. The results showed that 62 strains were gH1 genotypes (60.8%), while 40 strains were gH2 (39.2%), mixed type or new genotype was not observed. In infantile hepatitis syndrome (26 clinical isolates were gH1 genotypes, 17 clinical isolates were gH2 genotypes), anicteric hepatitis (25 were gH1, 13 were gH2) and pneumonia (9 were gH1, 4 were gH2), the distribution of HCMV gH genotypes of infantile clinical isolates was consistent with the overall trend (χ(2) = 0.357, P0.05). However , the gH2 was more common than gH1 in the clinical isolates of patients with thrombocytopenic purpura (6 were gH2, 1 were gH2, χ(2) = 6.083, P0.05).Genotype 1 was the dominant genotype of glycoprotein H in HCMV clinical isolates from our hospital infants. There was no significant difference between the distribution of gH genotypes in infantile hepatitis syndrome, anicteric hepatitis and pneumonia. However, gH2 was the dominant genotype in thrombocytopenic purpura. These findings suggested that there may be a certain relevance between gH genotype and different clinical manifestations.

Murine cytomegalovirus (MCMV) early protein M112-113 is involved in viral DNA replication and believed to play a crucial role in the viral pathogenesis. To investigate the biological function of M112-113 protein in the pathogenesis of the brain disorders caused by cytomegalovirus (CMV), a screening for proteins interacting with M112-113 was performed by a yeast two-hybrid system.Bait plasmid pGBKT7-M112-113 was constructed and transformed into AH109 yeast. After confirmation of the expression of MCMV M112-113 in yeast, the bait yeast was mated with a prey yeast containing mouse brain cDNA library plasmid to screen the proteins interacting with M112-113. Interactions between M112-113 and the obtained proteins were verified by yeast two-hybrid assay and chemiluminescent co-immunoprecipitaion.Two proteins interacting with M112-113 were identified, including metastasis-associated 1 (MTA1) and zinc finger, CCHC domain containing 18 (ZCCHC18). M112-113 protein could interact with MTA1 or ZCCHC18 in yeast and mammalian cells.The interactions of M112-113 with MTA1 or ZCCHC18 may be related to the pathogenesis of MCMV-associated disease in central nervous system.

To analyze the clinical features of acute invasive pulmonary aspergillosis in younger children, in order to improve the levels of early recognition, diagnosis and management of this disease.Clinical data of 8 patients aged below 15 months who were diagnosed as acute invasive pulmonary aspergillosis from August 2010 to February 2011 in general pediatric wards in our hospital were retrospectively analyzed for the high-risk factors of the hosts, clinical manifestations, laboratory findings and lung CT imaging, the processes of diagnosis and treatment, and the outcomes.Five cases were tested for serum GM test absorbent index (GMI) ranged from 1.92 to 3.27; in 2 cases sputum culture was positive for Aspergillus fumigatus for twice, and 1 infant was serum GMI 2.85 and a sputum culture was positive for Aspergillus fumigatus positive, all these findings were accordant with the clinical diagnosis. Seven cases had a history of receiving intravenously broad-spectrum antibiotics or plus corticosteroids (6 hospitalized, 1 out-patient), and one was only 1 month old, whose parents had severe tinea pedis. 4 patients of high-fever type had sustained high temperature, severe changes of lungs without obvious respiratory symptoms and signs in early phase, and significant increase of the rod granulocyte rate (0.25 - 0.68), which was apparently discordant with the normal WBC count and high sensitivity C-reactive protein (hs-CRP) value. Another 4 cases of non-high-fever type were present with normal WBC count, hs-CRP value and the percentage of rod granulocyte. Among them, 3 infants had low-grade fever, with serious respiratory symptoms and signs and changes of lungs CT. Another 1-month-old case only showed lower vigor and response. Lung CT imaging often showed multiple irregular large nodules, patches and streaks of density (6 cases) and unilateral lobar consolidation (1 case), with some involving the pleura; one appeared severe peri-main bronchus lesions with stenoses of bilateral main bronchi. The first case died of multiple organ failure because of severe sepsis complication. Another 7 cases were treated with voriconazole promptly after clinical or suspected diagnosis, and the state of patients relieved rapidly within 1 - 3 d.The abuse of broad-spectrum antibiotics and corticosteroids may increase the risk of invasive pulmonary aspergillosis in younger children. There may be the risk of nosocomial infection and spread of aspergillus in general pediatric wards. Cases of high-fever type in early period of disease had two inconsistency: few symptoms and signs, while severe changes of lungs CT; apparent increase of peripheral rod granulocyte, while normal WBC count and hs-CRP value. Preemptive voriconazole therapy could obtain significant effect and reduce the mortality rate.

To explore the effects of acute and chronic murine cytomegalovirus (MCMV) infections on the regulatory T cells (Treg) ratio and protein expression of the Th1/Th2 transcription factors T-bet/GATA-3.120 BALB/c mice were randomly divided into 2 equal groups: MCMV-infected group undergoing infra-peritoneal injection of homogenate of salivary gland containing MCMV, and mock infection group undergoing infra-peritoneal injection of normal homogenate of salivary gland 1, 3, 7, 14, 28, 45, 60, 75, 90, and 120 days after infection 6 mice from each group were killed to examine the viral load of the heart, lung, liver, and kidney by plaque assay to access the status of MCMV infection. Suspension of splenocytes was prepared. The proportion of CD4+CD25+Foxp3+Treg in the splenocytes was measured by flow cytometry. Western blotting was used to detect the protein expression of T-bet/GATA-3.The cutoff point between acute and chronic points was the 28th day. The CD4+CD25+Foxp3+Treg proportion in splenocytes significantly decreased during the acute infection stage and to the lowest level of (1.46+/-0.27)% at day 28, significantly lower than that of the mock infection group [(2.78+/-0.29)%, P0.05]; then obviously increased in the chronic infection stage, increased to (4.51+/-0.24)% at day 60, significantly higher than that of the mock infection group [(2.69+/-0.12)%, P0.05], and continued to increase still. The protein level (K value) of T-bet of the MCMV infection group peaked to the level of (0.618+/-0.053) on day 3, obviously higher than that of the mock infected group [(0.205+/-0.026)], then decreased to the level similar to that of the mock infection group on day 28, and was obviously lower than that of the mock infection group on day 75. Whereas the protein level of GATA-3 of the MCMV group increased to (0.836+/-0.061) on day 3, markedly higher than that of the mock infection group (0.398+/-0.022), peaked on day 7, then gradually decreased, and remained at the levels similar to those of the mock infection group from day 75 to day 120.In the acute infection stage, MCMV up-regulates the T-bet and GATA-3 protein expression. But during the chronic infection stage, MCMV induces a marked proliferation and activation of Treg cells which further inhibit the Th1 and Th2 reactions, especially Th1 response. Treg proliferation may be an important mechanism of chronic and persistent CMV infection in the host.

Garlic (Allium sativum) extraction has been reported having anti-HCMV efficacy. This study was aimed to investigate the effect of allitridin (diallyl trisulfide, a compound from A. sativum extraction) on the replication of HCMV and the expression of viral immediate-early genes. In HCMV plaque-reduction assay, allitridin appeared a dose-dependent inhibitory ability with EC(50) value of 4.2 microg/ml (selective index, SI=16.7). Time-of-addition and time-of-removal studies showed that allitridin inhibited HCMV replication in earlier period of viral cycle before viral DNA synthesis. Both immediate early gene (ie1) transcription and IEA (IE(1)72 and IE(2)86) expression was suppressed by allitridin, but not by GCV in a single HCMV cycle format. In addition, allitridin appeared stronger inhibition on IE(2)86 than on IE(1)72. Decrease of viral DNA load in infected cells was also detected under allitridin treatment, probably due to an indirect consequence of the reduction in ie gene transcription. In summary, this study indicated that allitridin has anti-HCMV activity and the mechanism is associated with suppression of ie gene transcription.

The production of neural stem cells (NSCs) derived from embryonic stem (ES) cells was usually very low according to previous studies, which was a major obstacle for meeting the needs of clinical application. This study aimed at investigating whether astrocytes could promote production of NSCs derived from ES cells in vitro.Mouse ES cells line-D3 was used to differentiate into NSCs with astrocytes as inducing stromal cells by means of three-stage differentiation procedure. Another group without astrocytes served as control. The totipotency of ES cells was identified by observation of cells' morphology and formation of teratoma in severe combined immunodeficiency disease (SCID) mice. The quantity and purity of NSCs derived from ES cells were analyzed using clonogenic assay, immunohistochemical staining and flow cytometry assay. The plasticity of NSCs was detected by differentiating test. Octamer-binding transcription factor 4 (Oct-4) and nestin, the specific marker genes of ES cells and NSCs respectively, were detected continuously using reverse transcription-polymerase chain reaction (RT-PCR) method to monitor the process of cell differentiation.The ES cells of D3 line could maintain the ability of differentiating into cellular derivations of all three primary germ layers after continuous passage culture. At the end of two-stage of inducing process, 23.2 +/- 3.5 neurospheres per plate formed in astrocyte-induced group and only 0.8 +/- 0.3 per plate in the control group (clonogenic assay, P0.01), and the ratio of nestin positive cells was (50.2 +/- 2.8)% in astrocyte-induced group and only (1.4 +/- 0.5)% in the control group (flow cytometry, P0.01). With the induction undergoing, the expression of Oct-4 gradually decreased and then disappeared, while the expression of nestin was increased step by step, and the ratio of nestin positive cells was up to 91.4% by the three-stage differentiation. The nestin positive cells could be further induced into neurons, astrocytes, and oligodendrocytes in differentiating medium supplemented with fetal calf serum. The results of differentiating test showed that the ratio of NF-200 and NSE positive cells was (42.7 +/- 2.6)% in astrocyte-induced group and only (11.2 +/- 1.8)% in the control group (P0.01).Astrocytes can not only increase the production of NSCs derived from ES cells but also promote the differentiation of NSCs toward neuronal lineage.

To investigate the prophylactic, blocking and therapeutic effects of Allitridin on inhibiting HCMV proliferation by measuring the expression level of HCMV IEA in vitro and explore the mechanism of Allitridin anti-HCMVactivity.The cytotocity of Allitridin was evaluated through MTT colorimetry and cell morphology. HCMV IEA levels were quantitatively detected by Flow Cytometry respectively under the following conditions: Allitridin was given before (pretreated for 24 h), during, or after viral inoculation in which serial doses (maximum tolerant concentration, MTC for human embryo lung cells, HEL) of Allitridin was used to treat HCMV infected HLE cells for different durations (24, 48, 72, 96 h) after viral infection.The MTC of Allitridin was 9.60 mg x L(-1). Allitridin remarkably inhibited the expression of HCMV IEA in vitro. Within MTC, the inhibitory rate had a significant correlation with its dosage (r = 0.96). At the time of IEA highest expression (72 h after infection), inhibitory effect was the greatest (inhibitory rate: 89.3%). With pretreatment of Allitridin, the inhibitory rate was 28.6%. When Allitridin was used together with HCMV inoculation, IEA inhibitory rate was only 10.3%.Allitridin can inhibit HCMV, IEA expression in vitro remarkably which is probably one of the major mechanisms of Allitridin anti-HCMV activity because IEAs are the very important regulatory factors for the expression of all HCMV genes. Its therapeutic effect is the best at the peak stage of IE1 gene expression (72 h after infection) but it has low prophylactic and little blocking effect.

AIM: To investigate the different effects of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) on hepatic differentiation. METHODS: MSCs from rat bone marrow were isolated and cultured by standard methods. HSCs from rat bone marrow were isolated and purified by magnetic activated cell sorting. Both cell subsets were induced. Morphology, RT-PCR and immunocytochemistry were used to identify the hepatic differentiation grade. RESULTS: MSCs exhibited round in shape after differentiation, instead of fibroblast-like morphology before differentiation. Albumin mRNA and protein were expressed positively in MSCs, without detection of alpha-fetoprotein (AFP). HSCs were polygonal in shape after differentiation. The expression of albumin signal decreased and AFP signal increased. The expression of CK18 was continuous in MSCs and HSCs both before and after induction. CONCLUSION: Both MSCs and HSCs have hepatic differentiation capabilities. However, their capabilities are not the same. MSCs can differentiate into mature hepatocyte-like cells, never expressing early hepatic specific genes, while Thy-1.1+ cells are inclined to differentiate into hepatic stem cell-like cells, with an increasing AFP expression and a decreasing albumin signal. CK18 mRNA is positive in Thy-1.1+ cells and MSCs, negative in Thy-1.1- cells. It seems that CK18 has some relationship with Thy-1.1 antigen, and CK18 may be a predictive marker of hepatic differentiation capability.