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Background: A negative-pressure operating theater is required to limit the spread of respiratory diseases in patients with severe acute respiratory syndrome, tuberculosis, avian influenza, or similar infectious diseases. In Hong Kong, we converted a conventional operating theater into a negative-pressure operating theater that has been in service for more than a year. In this article, we introduce its ventilation design and evaluate the airflow performance in relation to different combinations of medical lamp configurations and modes of launching infectious particles into the room air., Methods: We used a computational fluid dynamics technique for the numerical analysis., Results: Our analyses showed that the airflow performance in the negative-pressure operating theater was satisfactory and comparable to the original positive-pressure design. The airflow pattern effectively controlled the dispersion of infectious particles. Our calculations demonstrated that the airflow contained the dispersion of infectious particles released from the patient sufficiently to protect the surgical team, and vice versa., Conclusions: Computational fluid dynamics can be used to assess airflow in a negative-pressure operating room and model the dispersion of infectious particles from the patient.

Aim: To develop and optimize a competitive fluorescence polarization (FP) method, and use it as a high-throughput screening (HTS) assay for drug discovery., Methods: Human lectin-like oxidized low-density lipoprotein receptor-1 (hLOX-1) and oxidized low-density lipoprotein (oxLDL) were used to establish a high-throughput fluorescence polarization assay to screen ligands of human LOX-1. A 96-well plate assay was performed with a fast plate reader. Three fluorescein isothiocyanate-labeled hLOX-1 concentrations (100, 200, and 400 nmol/L) were selected to be titrated by oxLDL (from 0.05 nmol/L to 100 micromol/L) in order to obtain optimal reactive concentrations. The concentration of Me2SO used (0%, 1%, 3%, 5%) and incubation time (15 min, 30 min, 1 h, 2 h) were optimized. The Z' factor was calculated to estimate the quality of FP-based HTS., Results: Concentrations of 200 nmol/L for human LOX-1 and 50 micromol/L for oxLDL were used in the actual assay. Concentrations of 0% to 5% Me2SO and different reaction times did not affect the FP-based HTS. The Z' value was 0.66. By using this detection and screening system, 12 700 compounds were screened and 3 ligands with an IC50 of less than 4.5 micromol/L were found., Conclusion: The established competitive FP-based assay is sensitive, stable, highly reproducible and robust, and suitable for HTS for ligands of the hLOX-1 receptor.

Endothelin-1 (ET-1) plays a key role in the pathogenesis of pulmonary hypertension. The present study was conducted to examine the effects of a novel compound p-chlorobenzyltetrahydroberberine (CPU 86017) on endothelin-1 system of hypoxia-induced pulmonary hypertension in rats. SD male rats were divided into control, untreated pulmonary hypertension, nifedipine (10 mg/kg p.o.), and CPU 86017 (80, 40, and 20 mg/kg p.o.) groups. The pulmonary hypertension was established by housing the rats in a hypoxic (10 +/- 0.5% oxygen) chamber 8 hours per day for 4 weeks. Hemodynamic and morphologic assessment exhibited a significant increase in the central vein pressure (CVP), right ventricular systolic pressure (RVSP), and pulmonary arteriole remodeling in the pulmonary hypertensive rats, which were improved by CPU 86017 80 and 40 mg/kg administration (P < 0.01). The elevated pulmonary endothelin-1 level and the over-active preproET-1 and iNOS mRNA expression were also decreased significantly (P < 0.01) in CPU 86017 groups. The maladjustment of redox enzyme system in pulmonary hypertension rats was corrected after treatment. We concluded that CPU 86017 improves pulmonary hypertension mainly to suppress the endothelin-1 pathway at the upstream and downstream via calcium antagonism and antioxidative action, then, resulting in a relief in pathogenesis of the disease.

Aim: To elucidate the involvement of the endothelin (ET) pathway in the pathogenesis of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) and the therapeutic effect of CPU 86017 (p-chlorobenzyltetrahydroberberine chloride) in rats., Methods: Rats were injected with a single dose (60 mg/kg, sc) of MCT and given CPU 86017 (20, 40, and 80 mg/kg-1/d-1, po) or saline for 28 d. The hemodynamics, mRNA expression, and vascular activity were evaluated., Results: Right ventricular systolic pressure and central venous pressures were elevated markedly in the PAH model and decreased by CPU 86017. In the PAH group, the endothelin-1 (ET-1) in serum and lungs was dramatically increased by 54% (79.9 pg/mL, P<0.01) and 93% (166.2 pg/mL, P<0.01), and mRNA levels of preproET-1, eNOS, and iNOS also increased dramatically compared with control. Compared with PAH group, CPU 86017 decreased the content of ET-1 to the normal level in lung tissue, but was less effective in serum. The level of NO was significantly increased in CPU 86017 at 80 and 40 mg/kg-1/d-1 groups in tissue, whereas the difference in serum was not significant. A significant reduction in MDA production and an increase in the SOD activity in the serum and lungs was observed in all three CPU 86017 groups. CPU 86017 80 mg/kg-1/d-1 po increased the activity of cNOS by 33% (P<0.01). The up-regulation of eNOS and iNOS mRNA levels induced by MCT was significantly reversed in 3 CPU 86017 groups, and preproET-1 mRNA abundance was also reduced notably in CPU 86017 80 mg/kg-1/d-1 group vs the PAH group. The KCl-induced vasoconstrictions in the calcium-free medium decreased markedly in PAH group but recovered partially after CPU 86017 intervention. The constrictions in the presence of Ca(2+) was not improved by CPU 86017. The phenylephrine-induced vasoconstrictions in the calcium-free medium decreased markedly in PAH group but not recovered after CPU 86017 intervention. The constrictions in the presence of Ca(2+) completely returned to the normal after CPU 86017 intervention., Conclusion: CPU 86017 suppressed MCT-induced PAH mainly through an indirect suppression of the ET-1 system, which was involved in the pathogenesis of the disease.

Aim: To develop a fluorescence polarization-based high throughput screening and identify ligands for human Lectin-like oxidized low-density lipoprotein receptor-1 (hLOX-1)., Methods: Sequential ultracentrifugation at 4 degrees C from normolipidemic fasting volunteers to obtain low density lipoprotein (LDL), which was modified by CuSO4 (5 micromol x L(-1)) at 37 degrees C for 24 h. The assay was based on the interaction between receptor and ligand, and hLOX-1 was labeled by FITC and bound to its specific ligand, oxLDL. Different reaction time and DMSO concentration were optimized to determine the stability and tolerance of fluorescence polarization (FP) assay. 3 200 compounds were screened in black 384-well microplate by FP-based competitive displacement assay, at excitation filter of 485 nm and emission filter of 530 nm. Z' was used to assess the assay quality., Results: The FP-based HTS was formatted in a 384-well microplate with a Z' factor of 0. 75, and three active compounds for hLOX-1 were identified with IC50 below 40 micromol x L(-1) from total 3 200 compounds., Conclusion: The results indicated that the fluorescence polarization assay is stable, sensitive, reproducible and well suited for high throughput screening efforts.

Preoccupied with the diagnosis of SARS (severe acute respiratory syndrome) in a SARS outbreak, doctors tend to overlook other endemic diseases, such as tuberculosis. Incorrectly labeling a patient as SARS may result in serious consequences. Single isolation being not always possible, the patient may have to be isolated with a group of SARS patients. The following article reports how a young girl suffering from pulmonary tuberculosis was erroneously diagnosed as SARS in an outbreak. Isolated with other SARS patients, she was infected with the virus. The treatment of SARS and tuberculosis resulted in liver dysfunction. Fortunately, the patient recovered uneventfully. The importance of keeping an open mind in an outbreak is highlighted.

Aim: To investigate the differences in gene expression of transient outward potassium ion channel between the free wall of right ventricle, free wall of left ventricle, and the septum in monocrotaline (MCT)-induced right ventricular hypertrophy of rat., Methods: Twenty rats were randomly divided into two groups: a single injection of monocrotaline (MCT) 60 mg/kg (model) or saline (control). Four weeks later, hemodynamic parameters were measured and the gene expression of Ito channels were detected by semi-quantitative RT-PCR., Results: After 28 d, the right ventricular systolic pressure and central venous pressure were remarkably elevated by 128% and 533% in the MCT-treated group, accompanied by an overt right ventricle (RV) remodeling. The difference of mRNA expression of Kv1.4 was not significant in free wall of RV, left ventricle (LV), and septum in MCT group compared with control group. In contrast, mRNA of Kv4.2 and Kv4.3 in the free wall of RV in MCT-induced rat was dramatically decreased by 45.2% and 51.1% vs control, however, in free wall of LV and septum, no difference was found. In addition, mRNA expression level of Kv4.2 in control rat was significantly lower in septum than that in free wall of RV and LV., Conclusion: Expression of Kv1.4, Kv4.2, and Kv4.3 differs between regions in normal rat hearts. The down-regulation of Kv4 family gene expression of Ito contributed to the pathophysiological changes in ventricular hypertrophy and pulmonary hypertension induced by MCT.

Aim: Cytotoxic T lymphocytes (CTLs) play an important role in resolving HBV infection. In the present study, we attempted to evaluate the efficiency of bone marrow-derived dendritic cells (DCs) transduced with recombinant retroviral vector bearing hepatitis B virus (HBV) core gene and the capability of generating CTLs against HBcAg by genetically modified DCs in vivo., Methods: A retroviral vector containing HBV core gene was constructed. Replicating DC progenitor of C57BL/6 mice was transduced by retroviral vector and continually cultured in the presence of recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and interleukin-4(IL-4) for 6 days. LPS was added and cultured for additional two days. The efficiency of gene transfer was determined by PCR, Western blot and FACS. Transduced DCs immunized C57BL/6 mice subcutaneously 2 times at an one-week interval. Intracellular IFN-gamma and IL-4 of immunized mice lymphocytes were analyzed. Generation of CTLs in lymphocytes stimulated with mitomycin C-treated EL4-C cell which stably expresses HBcAg was determined by LDH release assays., Results: Recombinant retroviral expression vector (pLCSN) was positively detected by PCR as well as enzyme digestion with EcoRI and BamH I. Retroviruses were generated by pLCSN transfection packing cell and the virus titer was 3 x 10(5) CFU/ml. Indirect immunofluorescence and FACS showed that HBV core gene was expressed in murine fibroblasts. Transduced bone marrow cells had capability of differentiating into DCs in vitro in the presence of rmGM-CSF and rmIL-4. The result of PCR showed that HBV core gene was integrated into the genome of transduced DCs. Western blot analysis showed that HBV core gene was expressed in DCs. The transduction rate was 28 % determined by FACS. Retroviral transduction had no influence on DCs expressions of CD80 and MHC class II. HBcAg specific CTLs and Th1 type immune responses could be generated in the mice by using transduced DCs as antigen presenting cells (APCs)., Conclusion: Retroviral transduction of myeloid DCs progenitors expresses efficiently HBcAg, and genetically modified DCs evoke a higher CTLs response than HBcAg in vivo.

Aim: To evaluate the transfection efficiency of recombinant retrovirus vector bearing hepatitis B virus(HBV) core gene to bone marrow-derived dendritic cells(DCs) and the capability of these DCs to induce cytotoxic T lymphocyte(CTL) response., Methods: C57BL/6 mice bone marrow cells were stimulated with recombinant mouse granulocyte-macrophage colony-stimulating factor(rmGM-CSF) and interleukin-4(rmIL-4) for 6 days. Expanding DC progenitors were transfected by retrovirus vector containing HBV core gene. Integration and transcription of HBV core gene were determined by PCR and RT-PCR, respectively. Expression of HBcAg was analyzed by fluorescence activated cell sorter (FACS) and Western blot. Cytokines were quantified by enzyme immunoassay. The expressions of CD80 and MHC class II molecules on DCs were measured by FACS. Generation of CTLs in mixed leukocyte reaction were determined by LDH release assays., Results: Transfected bone marrow cells were capable of differentiating into DCs in-vitro at the presence of rmGM-CSF and rmIL-4. The result of PCR and RT-PCR showed that the HBV core gene was integrated into the genome of infected DCs. Western blot analysis showed that HBV core gene was expressed in DCs. Transfection rate was 28% determined by FACS. Retroviral transfection had no influence on expressions of CD80 and MHC class II molecules, as well as IL-12 production. HBcAg-specific CTLs could be generated by using transfected DCs as antigen presenting cell (APC)., Conclusion: Retroviral transfected myeloid DC progenitors could efficiently express HBcAg, without significant change on development and function of DCs, which lays a solid foundation for immunotherapy of chronic hepatitis B.

Background: To construct recombinant retroviral vector expressing HBcAg in eukaryotic cells., Methods: The HBV core gene fragment was amplified by using PCR from pADR which contains complement nucleotide sequence of HBV subtype adr and cloned into retroviral expression plasmid pLXSN, then transfected into packing cell (PT67) with lipofec AMINE. After 2-3 weeks selection with G418, large colonies were isolated and transferred to individual plates. Virus-containing medium was collected and used to infect NIH3T3, EL4 and mouse bone marrow derived dendritic cells(DC). DNA was extracted from infected cells and tested by PCR. Indirect immunofluorescence and FACS were used to detect the expression of HBcAg. Cell mediated immunity of immunized C57BL/6 mice with transduced DC was analyzed., Results: The insertion of HBV core gene fragment in the recombinant retroviral plasmid was confirmed by PCR as well as enzyme digestion with EcoR1 and BamH2. The viral titer reached 3 x 10(5) CFU/ml. The result of PCR showed that the HBV core gene had been integrated into the genome of infected NIH3T3 cells. Indirect immunofluorescence and FACS analysis showed that HBcAg had been expressed in these cells. HBcAg specific CTL responses could be generated in mice immunized with retrovirus transduced DC., Conclusions: HBV core gene had been integrated into eukaryotic cells with retroviral vector and target gene had been expressed efficiently. These results may have some impact on gene therapy of chronic hepatitis B.