[Role of allitridin in the blocking of murine cytomegalovirus induced regulatory T cells amplification in vitro].

OBJECTIVE: To investigate the effect of allitridin on murine cytomegalovirus (MCMV) induced regulatory T cells (Treg) amplification in vitro. METHODS: A co-culture system of T cells and MCMV infected mouse embryo fibroblasts (MEF) was established. A maximum tolerance concentration (MTC) of allitridin was added into the co-culture system. After 3 days, the change of Foxp3 mRNA was measured by real-time PCR. And the percentages of Tc1 (CD3(+)CD8(+)IFN-γ(+)), Tc2 (CD3(+)CD8(+)IL-4(+)), Th1 (CD3(+)CD8(-)IFN-γ(+)) and Th2 (CD3(+)CD8(-)IL-4(+)) were analyzed by flow cytometry. The production of IL-10 and TGF-ß in supernatants was detected with double-antibody sandwich ELISA while the viral load of culture quantified by plaque assay. All results were compared with those of the placebo group. RESULTS: The MTC of allitridin was 9.83 µg/ml in MEF. The treatment of 9.83 µg/ml allitridin could partly block the MCMV induction of Foxp3 mRNA expression [(87 ± 5) vs (114 ± 8), P < 0.01]. The percentages of Tc1, Tc2 and Th1 significantly increased to the levels of (12.42 ± 1.23)%, (4.28 ± 0.56)% and (13.25 ± 0.68)% respectively. They showed statistic differences with those of placebo controls [(6.85 ± 0.92)%, (2.34 ± 0.42)% and (9.32 ± 0.86)%; all P < 0.01]. Meanwhile, the levels of IL-10 and TGF-ß1 in supernatants also significantly decreased to (29.98 ± 3.15) pg/ml and (3.48 ± 0.23) ng/ml by allitridin treatment as compared with placebo controls [(38.21 ± 4.02) pg/ml and (5.31 ± 0.59) ng/ml, all P < 0.05]. In addition, the MCMV plaque assays showed that allitridin significantly suppressed the viral loads by one order of magnitude. CONCLUSION: Allitridin can partly retrieve MCMV-induced Treg expansion and Treg-mediated anti-MCMV immunosuppression so as to enhance the specific cellular immune responses against CMV.

Chinese clinical practice guidelines for acute infectious diarrhea in children.

BACKGROUND: The guidelines addressed the evidence-based indications for the management of children with acute infectious diarrhea in Chinese pediatric population. DATA SOURCES: The experts group of evidence development put forward clinical problems, collects evidence, forms preliminary recommendations, and then uses open-ended discussions to form recommendations. The literature review was done for developing this guideline in databases including PubMed, Cochrane, EMBASE, China Biomedical Database, and Chinese Journal Full-text Database up to June 2013. Search the topic "acute diarrhea" or "enteritis" and "adolescent" or "child" or "Pediatric patient" or "Baby" or "Infant". RESULTS: For the treatment of mild, moderate dehydration, hypotonic oral rehydration solutions (ORS) are strongly recommended. Intravenous (IV) rehydration is recommended for severe dehydration, with a mixture of alkali-containing dextrose sodium solution. Nasogastric feeding tube rehydration is used for children with severe dehydration without IV infusion conditions with ORS solution. Regular feeding should resume as soon as possible after oral rehydration or IV rehydration. The lactose-free diet can shorten the diarrhea duration. Zinc supplements are recommended in children with acute infectious diarrhea. Saccharomyces boulardii and Lactobacillus Rhamnus are recommended to be used in acute watery diarrhea. Saccharomyces boulardii is recommended in children with antibiotic-associated diarrhea as well. Montmorillonite and Racecadotril (acetorphan) can improve the symptoms of diarrhea or shorten the course of acute watery diarrhea. Antibiotics are recommended with dysenteric-like diarrhea, suspected cholera with severe dehydration, immunodeficiency, and premature delivery children with chronic underlying disease; otherwise, antibiotics are not recommended. CONCLUSION: The principles of the most controversial treatments with of acute infectious disease are reaching to a consensus in China.

Experimental study on the action of allitridin against human cytomegalovirus in vitro: Inhibitory effects on immediate-early genes.

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.

An experimental study on astrocytes promoting production of neural stem cells derived from mouse embryonic stem cells.

BACKGROUND: 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. METHODS: 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. RESULTS: 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, P < 0.01), 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, P < 0.01). 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 (P < 0.01). CONCLUSIONS: Astrocytes can not only increase the production of NSCs derived from ES cells but also promote the differentiation of NSCs toward neuronal lineage.

Experimental study on the prevention and treatment of murine cytomegalovirus hepatitis by using allitridin.

Allitridin (diallyl trisulfide), a main effective compound of Allium sativum (garlic), was previously shown to inhibit the expression of immediate-early antigens and viral proliferation of human cytomegalovirus (HCMV) in vitro. Here we have examined the prophylactic and therapeutic efficacy of allitridin in a non-lethal murine cytomegalovirus (MCMV) hepatitis in methylprednisolone-immunosuppressed BALB/c mice. Allitridin was administered at 25mg/kg per day (equal to the mean human dose) and 75 mg/kg per day in two regimens: prophylaxis plus therapy beginning at 2 days before infection and lasting for 18 days, and therapy lasting for 14 days initiated at 2 days after infection. Ganciclovir (GCV)-treated, infected, and non-infected mice served as controls. MCMV DNA load in the liver, plasma alanine aminotransferase (ALT) level and Knodell's histological activity index (HAI) score of liver section were evaluated. We found that MCMV DNA load was significantly decreased in all allitridin- and GCV-treated mice, compared with infected controls. Concomitantly, histopathological lesions in the liver and plasma ALT levels were reduced. Statistically, no significant differences were detected between the combined allitridin prophylaxis plus therapeutic and therapeutic groups regardless of dose and the GCV groups. Our results demonstrate the therapeutic efficacy of allitridin in mouse models with MCMV hepatitis.

[An experimental study on the effect of allitridin on inhibiting the expression of HCMV immediate-early antigens in vitro].

OBJECTIVE: 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. METHODS: 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. RESULT: 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%. CONCLUSION: 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.

[Inhibitory effect of murine cytomegalovirus infection on neural stem cells' differentiation and its mechanisms].

OBJECTIVE: Cytomegalovirus (CMV) is the leading infectious cause of congenital anomalies of the central nervous system caused by intrauterine infection. However, the exact pathogenesis of these brain abnormalities has not been fully elucidated. It has been reported that periependymitis, periventricular necrosis and calcification are the most frequent findings in the brains of congenital CMV infection. Because a number of multipotential neural stem cells (NSCs) have been identified from ventricular zone, it is possible that NSCs in this area are primary targets for viral infection, which seems to be primarily responsible for the generation of the brain abnormalities. Therefore, the objective of the present study was to investigate the effect and mechanism of murine cytomegalovirus (MCMV) infection on neural stem cells' differentiation in vitro and its role in the mechanisms of brain abnormalities caused by congenital cytomegalovirus infection. METHODS: NSCs were prepared from fetal BALB/c mouse and were infected with recombinant MCMV RM461 inserted with a report gene LacZ at 1 multiplicity of infection (MOI = 1). The effect of MCMV infection on neural stem cells' differentiation was observed by detecting the ratio of nestin, GFAP and NSE positive cells with immunohistochemistry and flow cytometry on day 2 postinfection. The effects of MCMV infection on gene expression of Wnt-1 and neurogenin 1 (Ngn1) related to neural differentiation were detected by RT-PCR. RESULTS: NSCs isolated from embryonic mouse brains strongly expressed nestin, a specific marker of NSCs and had the capacity to differentiate into NF-200 and NSE positive neurons or GFAP positive astrocytes. At MOI = 1, the results of flow cytometry assay showed that nestin positive cells' proportion in the infection group [(62.2 +/- 1.8)%] was higher than that in the normal group [(37.2 +/- 2.4)%] (t = 4.62, P < 0.01). At the same time, the rates of GFAP and NSE positive cells' in the infection group were significantly lower than those in the normal group (P < 0.01). The scanning densities of Wnt-1 was 0.14 +/- 0.03 in the infection group while 0.32 +/- 0.04 in the control group (t = 7.21, P < 0.01). The scanning densities of Ngn1 were 0.09 +/- 0.01 and 0.21 +/- 0.02 in the two groups (t = 10.7, P < 0.01). CONCLUSIONS: These results suggest that MCMV infection could inhibit neuronal differentiation, which may be primary causes of disorders of brain development in congenital CMV infection. The decreased expression of Wnt-1 and Ngn1 may be involved in the inhibitory effect of murine cytomegalovirus infection on neural stem cells' differentiation, which may lead to a new strategy for preventing and treating brain abnormalities caused by CMV infection through regulating these two signal pathways.

[T cell-mediated protective effect in cytomegalovirus infected murine astrocytes].

AIM: To explore T cell-mediated restriction of cytomegalovirus (CMV) in murine astrocytes. METHODS: A T cell-astrocyte coculture system was established, in which astrocytes were infected with mouse cytomegalovirus. Proliferation of T cells was observed under inverted microscope and detected after CFSE staining by flow cytometry. ELISA was used to detect the levels of IFN-gamma and TNF-gamma in coculture supernatants. Observation of astrocyte cytopathic effect (CPE) and PCR assay of cytomegalovirus DNA were also performed. RESULTS: After coculture for 3 d, T cells proliferation was observed under inverted microscope, and flow cytometry assay showed (6.68+/-0.61)% T cells had proliferated (P<0.01 compared with uninfected control). The level of IFN-gamma was (22.9+/-3.4) ng/L in coculture supernatants (P<0.05 compared with uninfected control). While levels of TNF-gamma were under 8 ng/L in both cytomegalovirus infected and uninfected groups. After coculture with T cells 31.25%-75% astrocyte CPE was diminished, and -75% cytomegalovirus DNA load was reduced. Cell-free coculture supernatants also exerted suppressive effect on CPE and viral DNA load. However, the antiviral effect of T cells diminished when added to fresh cytomegalovirus infected astrocytes, accompanied with the decreased level of T cells proliferation and IFN-gamma in coculture supernatants, suggesting the inhibition of T cells' function. CONCLUSION: Cytomegalovirus infected astrocytes could stimulate T cells proliferation and activation. Activated T cells had antiviral effect, which was partly mediated by soluble factors. The function of T cells was rapidly inhibited after activation, of which the underlying complicated mechanisms remain unclear and need further study.

[Detection of human rhinovirus genes from clinical sample by one-step RT-PCR].

OBJECTIVE: Human rhinovirus (HRV) is the most common respiratory pathogen, which causes not only acute respiratory infection and community acquired pneumonitis in children, but also asthma episode and deterioration. However, the detection of respiratory pathogen, which mainly focuses on respiratory syncytial virus, influenzaviruses A and B, parainfluenza viruses 1-3 and adenoviruses, does not include HRV yet by now in China. The absence of detection method limits the clinical understanding of HRV pathogenicity, and causes unreasonable use of antibiotics. This study aimed to establish a one-step reverse transcription (RT) PCR system for detecting specific fragment of HRV RNA, and to analyze the sequences of amplicons. METHODS: A pair of degenerate primers based on the HRV highly conserved 5'' noncoding region (NCR) were used to develop a one-step RT-PCR system for detecting HRV RNA in nasopharyngeal aspirates from 78 children with acute respiratory tract infections in the spring of 2004. All the positive PCR products were sequenced, and the sequences of the nucleotides were analyzed by using biological software and compared with those in the GeneBank. RESULTS: Eleven (14.1%) of 78 samples were positive on RT-PCR, these patients were clinically diagnozed as upper respiratory tract infection (n = 7), bronchitis (n = 3) and bronchopneumonia (n = 1), respectively. Compared with the sequences of clinical and standard HRV viruses in the GeneBank, the nucleotide sequences of these 11 amplicons shared high homology of 89%-95.5%. Within the 11 amplicons, nucleotide identity varied from 75.2% to 91.8%, and the ratio of genetic variation was from 8.8% to 31.0%, which occurred in highly conserved regions and usually showed single nucleotide mutation in some special locations. These 11 amplicons attribute to the two branches of HRV cladogram, respectively. Most of mutations in highly conservative domain occurred on single ribonucleotide, mainly as transversion (C/G, A/G) and transition (T/C, A/G), some were mutations among 3 bases (A/C/G, A/T/G, A/C/T). And a few mutations involved two nearby ribonucleotide which were also found in highly conservative domain. However, ribonucleotide deletion and insertion were usually found in highly variable domain. CONCLUSION: The findings showed that this one-step RT-PCR system was highly specific, rapid and convenient for the detection of HRV RNA in nasopharyngeal secretions of patients with acute respiratory tract infections and that the genome of HRV viruses was highly variable.

[Dynamic examination of duodenal fluid in the differential diagnosis of infantile cholestatic jaundice].

OBJECTIVE: To investigate the value of dynamic examination of duodenal fluid in the differential diagnosis of infantile hepatitis syndrome (IHS) and extrahepatic biliary atresia (EHBA). The aim of the study was to establish a simple, rapid and accurate diagnostic procedure for infantile cholestatic jaundice. METHODS: The authors developed a special duodenal drainage-tube and established a specific duodenal fluid drainage technique. The duodenal fluids were collected and the colors were documented. The bilirubin, gamma-glutamyltranspeptidase (gamma-GT) and bile acid concentrations in the duodenal fluids were measured. RESULTS: Duodenal fluid drainages were initially performed on 561 cases of infants with cholestatic jaundice. The yellow duodenal fluids were drained within 3-8 minutes after intubation in 342 cases. The yellow fluids were obtained in more patients after continuous drainage for 24 hours (21 cases) and 48-72 hours (16 cases), respectively. The duodenal fluids were light yellowish in 71 cases and white in 111 cases. The drainage techniques were subsequently performed in 182 infants with light yellowish or white duodenal fluids after conservative treatment. The duodenal fluids were yellow in 91 cases, white in 89 cases, and slightly yellowish in 2 cases. The increased levels of bilirubin (> or = 8.5 micromol/L), gamma-GT (> 20 IU/L) and bile acid (positive or 33-260 micromol/L) were observed in the yellow duodenal fluids. While the bilirubin levels were 0-2 micromol/L or 5-8 micromol/L in the white or slightly yellowish duodenal fluids, with gamma-GT levels at 0-5 IU/L and bile acid tested negative. According to the criteria set as bilirubin > or = 8.5 micromol/L, bile acid tested positive and gamma-GT > 20 IU/L in duodenal fluid, 470 infants were diagnosed as HIS; 91 cases were diagnosed as EHBA with duodenal fluid bilirubin < 8.5 micromol/L, bile acid tested negative and gamma-GT < 20 IU/L. The diagnoses of these patients were confirmed by surgical operation. CONCLUSION: Dynamic examination of duodenal fluid is a simple, rapid, safe and reliable method in the differential diagnosis of infantile cholestatic jaundice.

[Analysis of 187 children with enteroviral central nervous system infection in Shandong area].

OBJECTIVE: To evaluate the diagnostic potential of previously published enterovirus (EV) reverse transcription polymerase chain reaction (RT-PCR) assay in detection of EV in CSF samples from children with a diagnosis of aseptic meningitis and to investigate the clinical characteristics of the patients seen in Shandong. METHODS: EV RNA was detected in 187 CSF samples and serum and/or urine samples of a part of patients by RT-PCR and viral culture technique. RESULTS: RT-PCR was positive in all 62 CSF specimens which were positive by cell culture (100%). In addition, 93 of 125 (74.4%) CSF samples negative by cell culture were RT-PCR positive. In 4 of these 93 (4.3%) patients, viral culture of specimens from other sites (serum or urine) was also positive. The sensitivity of CSF RT-PCR based on clinical diagnosis in patients with meningitis of negative bacterial culture results was 82.9% (155/187), which was considerably higher than the sensitivity of CSF virus culture 33.2% (62/187). The results of RT-PCR can be reported within 4 hours, whereas the viral culture of CSF requires 4.6 days for a cytopathic effect to develop. EV meningitis occurred in a sporadic form and in some areas there were outbreaks. The clinical characteristics of 155 patients with EV meningitis were different in different age groups. CONCLUSION: EV was one of the most common causes of aseptic meningitis in Shandong area. The RT-PCR assay was rapid, sensitive and specific for the diagnosis of EV meningitis and may be a potential tests to shorten hospital stay and reduce the use of antibiotics.

[Application of gas chromatography-mass spectrometry analysis on urine filter paper in the high-risk screening and diagnosis of inherited metabolic diseases].

OBJECTIVE: To establish a specific procedure for the high-risk screening and diagnosis of organic acidurias and other inherited metabolic diseases in China. METHODS: A nation-wide network for the high-risk screening and diagnosis of genetic metabolic diseases was formed to facilitate the collaboration. Urine samples were collected using filter paper from patients with clinical symptoms suspicious of inherited metabolic diseases. The samples were eluted with distilled water and internal standards were added. Samples were treated with hydroxylamine hydrochloride to form oximes to improve the recoveries of 2-ketoacids. Urinary organic acids were extracted with ethyl acetate and diethyl ether under acidic condition. After dehydration, the combined organic phase was evaporated to dryness with nitrogen. The residues were added with BSTFA + 1%TMCS and heat incubated to form the trimethylsilyl derivatives, and then were analyzed on an Agilent 5890/5973N gas chromatography-mass spectrometer (GC-MS), with a 7683 series auto-sampler. The peaks were identified by reference to a mass spectral library. RESULTS: Totally 352 samples were collected from the network collaborating hospitals since 2001. Thirty-four (9.66%) cases of various inherited metabolic diseases were diagnosed with an age range of 2 days to 14 years. The disease profile was consisted of methylmalonic acidemias (6), alpha-keto-glutaric aciduria (5), tyrosinemia type I (4), dicarboxylic aciduria (4), multiple carboxylase deficiency (3), phenylketonuria (3), lactic acidemia (3), propionic acidemia (2), ornithine transcarbamoylase deficiency (1), ethylmalonic-adipic aciduria (1), glutaric aciduria type II (1) and 3-methylcrotyl CoA carboxylase deficiency (1). The most common clinical symptoms and signs included mental and developmental retardation, convulsion, musculotonic abnormality and jaundice. Routine laboratory tests often revealed metabolic acidosis, hypoglycemia and hyperammonemia, etc. CONCLUSION: Urine organic acids analysis by GC-MS remains to be the most important technique for the high-risk screening and diagnosis of inherited metabolic diseases. Use of urine filter paper for sample collection and analysis in advanced genetic metabolic centers is a practical approach to extend the diagnostic capacity and improve the management of such diseases in China. Collaborative network played a critical role in the success of the program.