Evaluation of Etest MBL for detection of blaIMP-1 and blaVIM-2 allele-positive clinical isolates of Pseudomonas spp. and Acinetobacter spp

The Etest MBL (AB BIODISK, Solna, Sweden) correctly differentiated all 57 isolates of Acinetobacter spp. and Pseudomonas aeruginosa with the bla(IMP-1) allele and 135 of 137 (98.5%) Acinetobacter spp. and Pseudomonas spp. isolates with the bla(VIM-2) allele. The Etest MBL was reliable for detecting the IMP-1- and VIM-2-producing Pseudomonas and Acinetobacter isolates.     

Fibroblast growth factor 2 induces differentiation and apoptosis of Askin tumour cells

Peripheral primitive neuroectodermal tumour (PNET)/Ewing's sarcoma (ES) and neuroblastoma (NB) are related tumours of neural crest origin with primitive neural characteristics. Fibroblast growth factor 2 (FGF2) is a critical signalling molecule for primitive neural crest cells. The treatment of NB cells with FGF2 variably affects biological characteristics such as growth and differentiation, while in PNET/ES, FGF2 predominantly induces apoptosis. The JK-GMS Askin tumour cell line can be induced to differentiate upon treatment with nerve growth factor (NGF), indicating the integrity of the cellular machinery necessary for differentiation. The present study assesses whether FGF2 can induce differentiation in JK-GMS cells. JK-GMS cells expressed high-affinity FGF receptors (FGFRs), and treatment with FGF2 induced phosphorylation of FGFR1 together with activation of extracellular signal-regulated kinases (ERK1/ERK2) and c-Jun N-terminal kinase (JNK). Subsequent biological effects were growth inhibition, neuronal differentiation, and apoptosis, and these changes were associated with increased expression of neurofilaments, reduction of c-myc and bcl-2 expression, and activation of caspase 3. Treatment of the cells with a specific inhibitor of the MAPK/extracellular signal-regulated kinase (MEK)-1, PD98059, predominantly inhibited the effects of FGF2 on growth, differentiation, and apoptosis, while an inhibitor of JNK reduced apoptosis, indicating that the ERK1/2 and JNK pathways are critical components of FGF2-mediated effects in JK-GMS cells. Additional comparative analyses of FGF2-mediated effects in two ES cell lines (CADO-ES, RD-ES) and a PNET cell line (SK-N-MC) showed pronounced differentiation in SK-N-MC, but not in CADO-ES or RD-ES cells. This study demonstrates that FGF2 can induce neuronal differentiation of PNET including Askin tumour. These findings clearly indicate that the FGF2-mediated signalling pathway plays a critical role in controlling the major properties of PNET cells and may provide a potential therapeutic target for PNET.     

Effects of interleukin 4 on CD25+CD4+ regulatory T cell function

CD25+CD4+ regulatory T cells (Tregs) contribute to the maintenance of peripheral tolerance against self and non-self. The modulatory effects of cytokines, such as interleukin 4 (IL-4) on the function of Tregs have not been explored in detail. We here report that IL-4 prevents spontaneous apoptosis and the decline of foxp3 mRNA which were found to occur during culture of isolated Tregs. Tregs exposed to IL-4 were more potent in suppressing the proliferation of na?ve CD4+ T cells and they better inhibited IFN-gamma production by CD4+ T cells as compared to Tregs cultured in medium. IL-4 also enhanced membrane IL-2Ralpha (CD25) expression on Tregs above the levels observed on freshly isolated cells. IL-4-mediated effects on Treg function persisted in Tregs from Stat6-/- mice, pointing to a Stat6-independent intracellular transduction pathway. In conclusion, our data suggest that the anti-inflammatory function of IL-4 could partly be mediated by effects on Tregs function.     

No association between schizophrenia and homozygosity at the D3 dopamine receptor gene

The D₃ dopamine receptor gene is an important candidate gene for schizophrenia, since (because of its almost exclusive expression in the limbic system) it combines the dopamine receptor hypothesis with the limbic system hypothesis of schizophrenia. A BalI restriction fragment length polymorphism of the D₃ dopamine receptor gene has been typed in 107 schizophrenic patients and 98 normal controls from Sichuan (China). With regard to alleles or genotypes, no significant differences were obtained between controls from Europe and China, between patients and controls, and between patient subgroups and controls. These results indicate a lack of association between schizophrenia and the D₃ dopamine receptor gene in our sample. Our findings are at variance with reports of a significant excess of homozygosity at the D₃ dopamine receptor gene in schizophrenic patients from Wales (United Kingdom) and Alsace (France). In conclusion, further studies will be needed with larger samples of patients from Wales and Alsace as well as with samples of different racial groups to prove or disprove the initial positive association between schizophrenia and genotypes of the D₃ dopamine receptor gene. © 1993 Wiley-Liss, Inc.     

Development of a Western Blot Assay for Detection of Antibodies against Coronavirus Causing Severe Acute Respiratory Syndrome

To identify a major antigenic determinant for use in the development of a rapid serological diagnostic test for severe acute respiratory syndrome (SARS) coronavirus infection and to study the immune response during SARS coronavirus infection in humans, we cloned the full length and six truncated fragments of the nucleocapsid gene, expressed them, and purified them as glutathione S-transferase-tagged recombinant proteins. The reactivities of the recombinant proteins to a panel of antibodies containing 33 SARS coronavirus-positive sera and 66 negative sera and to antibodies against other animal coronaviruses were screened. A truncated 195-amino-acid fragment from the C terminus of the nucleocapsid protein (N195) was identified that had a strong ability to detect antibodies against SARS coronavirus. No cross-reaction was found between the N195 protein and antibodies against chicken, pig, and canine coronaviruses. The N195 protein was used to develop a Western blot assay to detect antibodies against SARS coronavirus in 274 clinically blinded samples. The specificity and sensitivity of this test were 98.3 and 90.9%, respectively. The correlation between our Western blotting assay and an immunofluorescence assay (IFA) was also analyzed. The results of our Western blot assay and IFA for the detection of SARS coronavirus-positive sera were the same. Thus, the N195 protein was identified as a suitable protein to be used as an antigen in Western blot and other possible assays for the detection of SARS coronavirus infection.     

Model of Differential Susceptibility to Mucosal Burkholderia pseudomallei Infection

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease with protean clinical manifestations. The major route of infection is thought to be through subcutaneous inoculation of contaminated soil and water, although ingestion and inhalation of contaminated aerosols are also possible. This study examines infection through the intranasal route in a murine model to mimic infection through inhalation. Two strains of mice, C57BL/6 and BALB/c, exhibit differential susceptibilities to the infection, with the C57BL/6 mice being considerably more resistant. To examine host factors that could contribute to this difference, bacterial loads and cytokine profiles in the two strains of mice were compared. We found that infected BALB/c mice exhibited higher bacterial loads in the lung and spleen and that they produced significantly higher levels of gamma interferon (IFN-gamma) in the serum than C57BL/6 mice. Although tumor necrosis factor alpha and interleukin-1 could be detected in the nasal washes and sera of both strains of mice, the production in serum was transient and much lower than that of IFN-gamma. C57BL/6 mice also exhibited memory responses to bacteria upon reinfection, with the production of serum immunoglobulin G (IgG) and mucosal IgA antibodies. Thus, it is possible that the production of systemic and mucosal antibodies is important for protection against disease in C57BL/6 mice.     

小鼠趋化因子COL19Ig融合蛋白表达载体的构建、表达及鉴定

通过特异引物扩增出去掉终止密码的mCCL19编码序列，经酶切、亚克隆、拼接构建了真核表达载体pcDNA3．1-CCL19Ig，酶切和测序鉴定插入序列；将重组质粒转染CHO细胞进行体外表达，通过RT—PCR、Western blot鉴定目的基因的表达，利用趋化小室法测趋化活性。结果测序证实重组表达载体含mCCL19编码序列和人IgGI Fc段序列，其序列分别与GenBank中公布序列比对一致，IgG1-Fc段读码框未发生改变；Western blot结果证实转染了pcDNA3．1-mCEL19Ig的CHO细胞培养上清中有相对分子质量为38000的融合蛋白mCCL19Ig表达；体外趋化实验表明融合蛋白mCCL19Ig对小鼠脾细胞有趋化活性，且呈剂量依耐关系。     

Invasive pulmonary aspergillosis: high incidence of disseminated intravascular coagulation in fatal cases.

BACKGROUND AND PURPOSE: Disseminated intravascular coagulation (DIC) is a rarely described finding in invasive pulmonary aspergillosis (IPA) with unclear impact on mortality. METHODS: This study included patients with positive cultures of Aspergillus spp. from respiratory specimens, serological evidence of aspergillosis, or lung biopsy findings supporting aspergillosis treated at National Taiwan University Hospital from January 1999 to June 2005. IPA was defined based on the consensus of the European Organization for Research and Treatment of Cancer, and the Mycosis Study Group of the National Institute of Allergy and Infectious Diseases. Univariate logistic regression analysis was used to evaluate the factors associated with mortality. RESULTS: Proven or probable IPA was diagnosed in 26 patients. Hematological malignancy was found in 11 patients (42%) and immunosuppressive agents had been administered to 17 patients (65%). Among 20 culture-proven infections (77%), the most frequently encountered fungi were Aspergillus fumigatus (46%) and Aspergillus flavus (23%). The overall mortality rate was 62%. Univariate and multivariate analyses revealed that DIC was the only factor that was significantly associated with death attributable to IPA (p<0.01). CONCLUSIONS: IPA is associated with a high mortality rate, particularly for patients with DIC.