Experimental infection of tick cells with Nipah virus

@#Nipah virus (NiV), a highly pathogenic henipavirus of the family Paramyxoviridae, which causes fatal encephalitis in 40-70% of affected patients, was first reported in Malaysia over 20 years ago. Pteropid bats are the natural hosts of henipaviruses, and ticks have been proposed as a possible link between bats and mammalian hosts. To investigate this hypothesis, infection of the tick cell line IDE8 with NiV was examined. Presence of viral RNA and antigen in the NiV-infected tick cells was confirmed. Infectious virions were recovered from NiV-infected tick cells and ultrastructural features of NiV were observed by electron microscopy. These results suggest that ticks could support NiV infection, potentially playing a role in transmission.

A conventional multiplex PCR for the detection of four common soil-transmitted nematodes in human feces: development and validation

@#Soil-transmitted helminth (STH) infections, mainly caused by Ascaris lumbricoides, Trichuris trichiura, and hookworms, are among the most common intestinal parasites that infect humans. The infections are widely distributed throughout tropical and subtropical countries, including Malaysia, particularly in underprivileged communities. Microscopic and culture techniques have been used as a gold standard for diagnostic techniques. However, these methods yield low sensitivity and specificity, laborious and time-consuming. Therefore, simple, rapid, and accurate alternative methods are needed for the simultaneous detection of STH infections. Although advanced technologies such as real-time multiplex PCR have been established, the use of this technique as a routine diagnostic is limited due to the high cost of the instrument. Therefore, a single-round multiplex conventional PCR assay for rapid detection of four STH species in the fecal sample was developed in this study. To perform the single-round multiplex PCR, each pair of species-specific primers was selected from target genes, including Ancylostoma duodenale (Internal Transcribed Spacer 2; accession No. AJ001594; 156 base pair), Necator americanus (ITS 2; accession No. AJ001599; 225 base pair), Ascaris lumbricoides (Internal Transcribed Spacer 1; accession No. AJ000895; 334 base pair) and Trichuris triciura (partial ITS 1, 5.8s rRNA and partial ITS 2; accession No. AM992981; 518 base pair). The results showed that the newly designed primers could detect the DNA of STH at low concentrations (0.001 ng/μl) with no cross-amplification with other species. This assay enables the differentiation of single infections as well as mixed infections. It could be used as an alternative and is a convenient method for the detection of STHs, especially for the differentiation of N. americanus and A. duodenale.

Toxoplasma gondii infection among selected indigenous community in Sarawak, East Malaysia

@#Toxoplasma gondii is an obligate intracellular protozoan parasite that causes toxoplasmosis in humans. To date, little is known about T. gondii infection among the indigenous community, particularly in East Malaysia. This study was conducted to determine the status of T. gondii infection and to investigate associated risk factors among the indigenous community of Sarawak, East Malaysia. The sociodemographic data was obtained using a pretested questionnaire. A serological test was done to detect the presence of specific IgM and IgG antibodies against T. gondii in serum samples. A nested polymerase chain reaction (PCR) was used to determine acute infection among seropositive individuals. The overall seroprevalence of T. gondii infection was 50% (95% CI = 43.3 – 56.7). From this subset, 40.1%, 5.7%, and 4.2% were positive for anti-T. Gondii IgG antibodies, IgM, and both IgG and IgM, respectively. Four seropositive samples were amplified through PCR. None of the pregnant women tested positive for T. gondii infection based on the serological and PCR assays. A significant association was found between age, low monthly household income, unemployment, usage of untreated water and close contact with T. gondii seropositive cats. These results provide basic information on T. gondii infection and may be useful for policymakers to initiate prevention and control programs, especially amongst pregnant women and women of childbearing age in the indigenous community.

Effects of high glucose on osteogenic differentiation of hBMSC / 口腔疾病防治

Objective@# To investigate the effects of different glucose concentration on the proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSC) in vivo@*Methods@#Cultured with basal medium containing different glucose concentrations, CCK-8 cell proliferation was detected at 1, 4, 7, 10 days. The osteogenic differentiation of human bone marrow mesenchymal stem cells was observed at 7 d, which was induced by osteogenic differentiation medium with different concentration of glucose. The expressions of alkaline phosphatase (ALP), osteocalcin (OC) and collagen type I (Col-1) gene were detected by real-time fluorescence quantitative PCR. Mineralized nodule formation was displayed by calciumalizarin red staining on the seventh day.@*Results @#10 mM glucose stimulated proliferation of hBMSC, while the higher (>30 mM) inhibited the proliferation (P < 0.05); Osteogenic induction can induce osteogenic differentiation of hBMSC, but the increase of glucose concentration will decrease the formation of mineralized nodules of hBMSC, inhibit the expression of osteogenic marker genes ALP, OC and Col-1 (P < 0.05).@*Conclusion@#The expression of Col-1, ALP and OC in osteoblast was down-regulated by high glucose, and the hBMSC proliferation was inhibited. At the same time, high glucose can reduce the osteogenic mineralization ability of stem cells and indirectly affect bone formation and metabolism.

Stem cell factor protects against neuronal apoptosis by activating AKT/ERK in diabetic mice

Neuronal apoptosis occurs in the diabetic brain due to insulin deficiency or insulin resistance, both of which reduce the expression of stem cell factor (SCF). We investigated the possible involvement of the activation of the MAPK/ERK and/or AKT pathways in neuroprotection by SCF in diabetes. Male C57/B6 mice (20-25 g) were randomly divided into four groups of 10 animals each. The morphology of the diabetic brain in mice treated or not with insulin or SCF was evaluated by H&E staining and TUNEL. SCF, ERK1/2 and AKT were measured by Western blotting. In diabetic mice treated with insulin or SCF, there was fewer structural change and apoptosis in the cortex compared to untreated mice. The apoptosis rate of the normal group, the diabetic group receiving vehicle, the diabetic group treated with insulin, and the diabetic group treated with SCF was 0.54 ± 0.077 percent, 2.83 ± 0.156 percent, 1.86 ± 0.094 percent, and 1.78 ± 0.095 percent (mean ± SEM), respectively. SCF expression was lower in the diabetic cortex than in the normal cortex; however, insulin increased the expression of SCF in the diabetic cortex. Furthermore, expression of phosphorylated ERK1/2 and AKT was decreased in the diabetic cortex compared to the normal cortex. However, insulin or SCF could activate the phosphorylation of ERK1/2 and AKT in the diabetic cortex. The results suggest that SCF may protect the brain from apoptosis in diabetes and that the mechanism of this protection may, at least in part, involve activation of the ERK1/2 and AKT pathways. These results provide insight into the mechanisms by which SCF and insulin exert their neuroprotective effects in the diabetic brain.

High expression of the circadian gene mPer2 diminishes the radiosensitivity f NIH 3T3 cells

Period2 is a core circadian gene, which not only maintains the circadian rhythm of cells but also regulates some organic functions. We investigated the effects of mPeriod2 (mPer2) expression on radiosensitivity in normal mouse cells exposed to 60Co-γ-rays. NIH 3T3 cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce endogenous mPer2 expression or transfected with pcDNA3.1(+)-mPer2 and irradiated with 60Co-γ-rays, and then analyzed by several methods such as flow cytometry, colony formation assay, RT-PCR, and immunohistochemistry. Flow cytometry and colony formation assay revealed that irradiated NIH 3T3 cells expressing high levels of mPer2 showed a lower death rate (TPA: 24 h 4.3 percent vs 12 h 6.8 percent and control 9.4 percent; transfection: pcDNA3.1-mPer2 3.7 percent vs pcDNA3.1 11.3 percent and control 8.2 percent), more proliferation and clonogenic survival (TPA: 121.7 ± 6.51 vs 66.0 ± 3.51 and 67.7 ± 7.37; transfection: 121.7 ± 6.50 vs 65.3 ± 3.51 and 69.0 ± 4.58) both when treated with TPA and transfected with mPer2. RT-PCR analysis showed an increased expression of bax, bcl-2, p53, c-myc, mre11, and nbs1, and an increased proportionality of bcl-2/bax in the irradiated cells at peak mPer2 expression compared with cells at trough mPer2 expression and control cells. However, no significant difference in rad50 expression was observed among the three groups of cells. Immunohistochemistry also showed increased protein levels of P53, BAX and proliferating cell nuclear antigen in irradiated cells with peak mPer2 levels. Thus, high expression of the circadian gene mPer2 may reduce the radiosensitivity of NIH 3T3 cells. For this effect, mPer2 may directly or indirectly regulate the expressions of cell proliferation- and apoptosis-related genes and DNA repair-related genes.

Scoring system for empyema thoracis and help in management.

OBJECTIVE: To evaluate the implications of a newly defined severity scoring of empyema in children for the prediction of surgical management and to compare the length of hospitalization as an outcome measure of patients treated using medical therapy, salvage video-assisted thoracoscopic surgery (VATS) vs early elective VATS. METHODS: A retrospective chart review of parapneumonic empyema of patients below 18 years of age admitted to a tertiary children's hospital in northern Taiwan from April 1993 to December 2002 was performed. Patients were categorized into a medical group who received antibiotic therapy, needle aspirations with/without tube thoracostomy; a salvage VATS group when the patients required surgery for the relief of persistent fever > 38 degrees C, chest pains or dyspneic respirations despite initial medical therapy; an early VATS group when the patients received elective surgery early after admission. The demographic data, clinical features, laboratory findings, and duration of hospitalization were compared using a severity score of empyema (SSE). RESULTS: Streptococcus pneumoniae was the most common infecting organism, followed by Staphylococcus aureus, Pseudomonas aeruginosa. No organisms were recovered in 39% of patients. A pleural pH < 7.1 increases the odds of requiring surgical intervention by 6 times among this cohort. Children who required decortication of empyema had a higher severity score (mean 4.8 vs 3.0, p < 0.005). The duration of hospitalization for patients having early VATS showed a shortening stay (mean 18 vs 28 days) as compared to salvage VATS. CONCLUSION: A pleural pH < 7.1 and a newly designed clinical severity score of empyema 4 are two predictors of surgical intervention for fibrinopurulent empyema in the present study. Early elective VATS may be adopted not later than 7 days after failure of appropriate antibiotic therapy and adequate drainage of empyema to decrease the length of stay and minimize morbidity.

Hepatic iron status in Malaysians and Singaporeans.

Non-haemoglobin liver iron was estimated in 275 presumably normal individuals from Kuala Lumpur and Singapore at necropsy. Liver Iron concentrations were highest during the first two years after birth but declined sharply during childhood. They then rose gradually and reached a value of 20 mg/100gm in adult males. Liver iron concentrations of childbearing women remained low and it was only after menopause that values in women rose to those of males. Liver iron stores increased with age to a plateau of about 300 mg in adults, suggesting that this value may represent the adult size for liver iron store. Among the three major ethnic groups in Malaysia and Singapore, Chinese, being in a better socio-eonomic class, had larger liver iron stores. The median liver iron concentrations of Malaysians and Singaporeans, on the whole, were lower than those reported from western populations and as many as 35 per cent of the women were in a subclinical state of iron deficiency.