Quercetin induces autophagy-associated death in HL-60 cells through CaMKKß/AMPK/mTOR signal pathway.

Acute myeloid leukemia (AML) is one of the most common malignancies of the hematopoietic progenitor cell in adults. Quercetin has gained recognition over the years because of its anti-cancer effect with minimal toxicity. Herein, we aim to investigate the anti-leukemia mechanism of quercetin and to decipher the signaling pathway of quercetin in HL-60 leukemic cells. We observed that quercetin induces apoptosis and autophagic cell death, in which both pathways play an important role in suppressing the viability of leukemia cells. Phosphorylated AMPK (p-AMPK) protein expressions are lower in primary AML cells, HL-60 cells, KG-1 and THP-1 cells than in peripheral blood monocular cells. After quercetin treatment, the expression of p-AMPK is increased while the expression of p-mTOR is decreased in a dose-dependent manner. Mechanistically, compound C, an AMPK phosphorylation inhibitor, upregulates the phosphorylation of mTOR and inhibits autophagy and apoptosis in quercetin-induced HL-60 cells, while silencing of CaMKKß inhibits the quercetin-induced phosphorylation of AMPK, resulting in increased mTOR phosphorylation. Furthermore, silencing of CaMKKß inhibits the autophagy in HL-60 cells. Taken together, our data delineate that quercetin plays its anti-leukemia role by inhibiting cell viability and inducing apoptosis and autophagy in leukemia cells. Quercetin inhibits the phosphorylation of mTOR by regulating the activity of AMPK, thus playing a role in the regulation of autophagy and apoptosis. CaMKKß is a potential upstream molecule for AMPK/mTOR signaling pathway, through which quercetin induces autophagy in HL-60 cells.

The role of AMP-activated protein kinase in quercetin-induced apoptosis of HL-60 cells.

Our previous studies have shown that quercetin inhibits Cox-2 and Bcl-2 expressions, and induces human leukemia HL-60 cell apoptosis. In order to investigate the role of AMP-activated protein kinase (AMPK) on quercetin-induced apoptosis of HL-60 cells, we used flow cytometry to detect cell apoptosis. The expressions of LKB1, phosphorylated AMPK (p-AMPK), and Cox-2 protein were detected in HL-60 cells and normal peripheral blood mononuclear cells (PBMCs) by western blot. The expressions of LKB1, p-AMPK, and Cox-2 were detected in HL-60 cells after culture with quercetin. The expressions of p-AMPK were detected in HL-60 cells after culture with AMPK inhibitor Compound C. Then, the expressions of LKB1, p-AMPK, and Cox-2 were detected in HL-60 cells after culture with quercetin alone or quercetin + Compound C. It was found that there was no significant difference in LKB1 between PBMCs and HL-60. p-AMPK in PBMCs was higher than that in HL-60, while Cox-2 was lower. After culture of HL-60 with quercetin, p-AMPK was increased, Cox-2 was decreased, but LKB1 remained unchanged. After culture of HL-60 with Compound C, p-AMPK was decreased. There was no significant difference in LKB1 between the quercetin-alone and the quercetin + Compound C groups. p-AMPK decreased more significantly, while Cox-2 increased more significantly in the quercetin + Compound C groups than those in the quercetin-alone groups. Taken together, these findings suggested that quercetin activates AMPK expression in HL-60 cells independent of LKB1 activation, inhibits Cox-2 expression by activating AMPK, and further regulates the Bcl-2-dependent pathways of apoptosis to exert its anti-leukemia effect.

Quercetin induces apoptosis by activating caspase-3 and regulating Bcl-2 and cyclooxygenase-2 pathways in human HL-60 cells.

Quercetin is one of the naturally occurring dietary flavonol compounds. It is present abundantly in plants and has chemopreventive and anticancer effects. To investigate its anticancer mechanism, we examined the activity of quercetin against acute leukemia cell line, HL-60. Our results showed that quercetin inhibited cell proliferation and induced apoptosis in a time- and dose-dependent manner. Furthermore, quercetin down-regulated the expression of anti-apoptosis protein Bcl-2 and up-regulated the expression of pro-apoptosis protein Bax. Caspase-3 was also activated by quercetin, which started a caspase-3-depended mitochodrial pathway to induce apoptosis. It was also found that quercetin inhibited the expression of the cycloocygenase-2 (Cox-2) mRNA and Cox-2 protein. Taken together, these findings suggested that quercetin induces apoptosis in a caspase-3-dependent pathway by inhibiting Cox-2 expression and regulates the expression of downstream apoptotic components, including Bcl-2 and Bax. Quercetin can be a potent and promising medicine which might be safely used in leukemia therapy.

[Immuno-regulatory effect of 3'-meisoindigo in mice of various germlines].

OBJECTIVE: To investigate the effect of 3'-meisoindigo on the proliferation and the biological function of the splenocyte and thymocyte of mouse, which were 8 weeks old masculinity BALB/c, C57BL/6 and F1 hybridization mouse. METHODS: Cells of thymus and spleen were harvested and prepared as the unicell suspension, then treated with 5, 10, 15, 20, 25 micromol/L 3'-meisoindigo. The cell proliferation was by MTT method, concentration of IL-12 was dectected by ELISA method, the mRNA levels of Bcl-2 and CDK2 were decected by RT-PCR. The cell cycle, apoptosis ratio, death ratio and intracellular ROS concentration were detected by FCM method. The protein level of Bcl-2, CDK2 and Bax were detected by immumofluorescence method. RESULTS: 15, 20, 25 micromol/L 3'-meisoindigo can inhibit the proliferation of thymocyte and splenocyte (P < 0.05). It had dose-dependent and time-dependent manner. 3'-meisoindigo inhibit the secretion of IL-12, even at 5 micromol/L concentration. 15 micromol/L 3'-meisoindigo decrease the mRNA level of Bcl-2 and CDK2, induced apoptosis and G2 arrestting of the thymocyte and splenocyte. (P < 0.05). The intracellular ROS level increased after treated by 3'-meisoindigo at 15 micromol/L for 24 h (P < 0.05). There were no difference among three germ line mouse. CONCLUSION: Above 15 micromol/L, 3'-meisoindigo can inhibit the proliferation and externalization function of thymocyte and splenocyte from different germ line mouse, meanwhile the mRNA and protein level of Bcl-2 and CDK2 decrease, the Bax protein expressed increased, the intracellular ROS level increase too.

SKP2 siRNA inhibits the degradation of P27kip1 and down-regulates the expression of MRP in HL-60/A cells.

S-phase kinase-associated protein 2 (SKP2) gene is a tumor suppressor gene, and is involved in the ubiquitin-mediated degradation of P27kip1. SKP2 and P27kip1 affect the proceeding and prognosis of leukemia through regulating the proliferation, apoptosis and differentiation of leukemia cells. In this study, we explored the mechanism of reversing of HL-60/A drug resistance through SKP2 down-regulation. HL-60/A cells were nucleofected by Amaxa Nucleofector System with SKP2 siRNA. The gene and protein expression levels of Skp2, P27kip1, and multi-drug resistance associated protein (MRP) were determined by reverse transcription-polymerase chain reaction and western blot analysis, respectively. The cell cycle was analyzed by flow cytometry. The 50% inhibitory concentration value was calculated using cytotoxic analysis according to the death rate of these two kinds of cells under different concentrations of chemotherapeutics to compare the sensitivity of the cells. HL-60/A cells showed multi-drug resistance phenotype characteristic by cross-resistance to adriamycin, daunorubicin, and arabinosylcytosine, due to the expression of MRP. We found that the expression of SKP2 was higher in HL-60/A cells than in HL-60 cells, but the expression of P27kip1 was lower. The expression of SKP2 in HL-60/A cells nucleofected by SKP2 siRNA was down-regulated whereas the protein level of P27kip1 was up-regulated. Compared with the MRP expression level in the control group (nucleofected by control siRNA), the mRNA and protein expression levels of MRP in HL-60/A cells nucleofected by SKP2 siRNA were lower, and the latter cells were more sensitive to adriamycin, daunorubicin, and arabinosylcytosine. Down-regulating the SKP2 expression and arresting cells in the G0/G1 phase improve drug sensitivity of leukemia cells with down-regulated MRP expression.

KPT-330 inhibition of chromosome region maintenance 1 is cytotoxic and sensitizes chronic myeloid leukemia to Imatinib.

As tyrosine kinase inhibitors (e.g., Imatinib, IM) fail to induce long-term response in some chronic myeloid leukemia (CML), novel therapies targeting leukemia-dysregulated pathways are necessary. Nuclear-cytoplasmic trafficking of proteins play a key role in the development of leukemia and drug resistance. KPT-330 (Selinexor), an inhibitor of chromosome region maintenance 1 (CRM1, nuclear receptor exportin 1, XPO1), demonstrated activities against a few hematological malignancies. We examined the anti-leukemic efficacy of KPT-330 in IM-resistant CML. Cell viability was examined by MTS assay. Apoptosis and cell cycle were assessed by flow cytometry. CRM1 mRNA was detected by PCR. Expression of CRM1 protein and its cargo proteins were determined by western blot or immunofluorescent staining. Furthermore, we engrafted nude mice subcutaneously with IM-resistant CML K562G. Mice were treated with IM, KPT-330 alone or in combination. Expression of CRM1 in CML were markedly higher than control. KPT-330 inhibited proliferation, induced cell cycle arrest and apoptosis of K562 and K562G. IC50 of IM on K562G was reduced by KPT-330. Mechanistically, KPT-330 inhibited CRM1 and increased the nuclear/cytoplasm ratio of BCR-ABL and P27. p-AKT was downregulated while p-STAT1 and caspase-3 were upregulated. Furthermore, KPT-330 showed anti-leukemic effect in primary IM-resistant CML with T315I mutation in CRM1-dependent manner. In K562G xenograft mice model, KPT-330 inhibited tumor growth and sensitized K562G to IM in vivo. To conclude, KPT-330 showed anti-leukemic activity and sensitized CML to IM in CRM1-dependent manner in vitro and in vivo. KPT-330 represents an alternative therapy for IM-refractory CML, warranting further investigation of CRM1 as therapeutic target.

Regulation of mPGES-1 composition and cell growth via the MAPK signaling pathway in jurkat cells.

Previous studies have suggested that microsomal prostaglandin E synthase-1 (mPGES-1) is highly expressed and closely associated with mitogen-activated protein kinase (MAPK) signaling pathways in various types of malignant cells. However, their expression patterns and function with respect to T-cell acute lymphoblastic leukemia (T-ALL) remain largely unknown. The present study investigated whether mPGES-1 served a crucial role in T-ALL and aimed to identify interactions between mPGES-1 and the MAPK signaling pathway in T-ALL. The results indicated that mPGES-1 overexpression in T-ALL jurkat cells was significantly decreased by RNA silencing. Decreasing mPGES-1 on a consistent basis may inhibit cell proliferation, induce apoptosis and arrest the cell cycle in T-ALL jurkat cells. Microarray and western blot analyses revealed that c-Jun N-terminal kinase served a role in the mPGES-1/prostaglandin E2/EP4/MAPK positive feedback loops. In addition, P38 and extracellular signal-regulated kinase 1/2 exhibited negative feedback effects on mPGES-1. In conclusion, the results suggested that cross-talk between mPGES-1 and the MAPK signaling pathway was very complex. Therefore, the combined regulation of mPGES-1 and the MAPK signaling pathway may be developed into a new candidate therapy for T-ALL in the future.

[Effects of shRNA Targeting mPGES-1 on Tumorigenicity of K562 Cells in Nude Mice In Vivo].

OBJECTIVE: To investigate the effects of shRNA targeting mPGES-1 on tumorigenicity of human acute leukemia K562 cells in nude mice in vivo and its mechanisms. METHODS: For experiment 3 groups including KD group(expression of mPGES-1 in K562 cells was down-regulated by shRNA), CON (cells without any treatment) and NC group (cells treated with nonspecific-sequence shRNA) were set-up. Western blot was used to test the expression of ß-catenin and cyclinD1 in cells. Then the cells of 3 groups were implanted into BALB/c nude mice subcutaneously to establish murine xenograft model. The growth state of the mice and the size of the xenograft tumor were recorded. HE staining was used to observe the morphology of xenograft tumor. Expressions of ß-catenin and cyclinD1 in xenograft tumor were detected by immunohistochemical staining. RESULTS: In vitro the expression of ß-catenin and cyclinD1 in KD group were lower than the CON group and NC group (P<0.05). In vivo the tumor volume and weight of KD group were significant smaller than the other two groups (P<0.01). HE staining showed that tissues in the KD group were relatively looser in arrangement with smaller cell nucleus and less cytoplasm. The expression of ß-catenin and cyclinD1 in the KD group were remarkable weak as compared with that in CON group and NC group (P<0.05). CONCLUSION: Down-regulating the expression of mPGES-1 by shRNA may significantly inhibit the tumorigenicity of K562 cells in nude mice in vivo and its mechanism may be related with the inhibition of expression of ß-catenin and cyclinD1.

[Experimental study on anti-platelet effects of ginsenoside -2A in vitro].

OBJECTIVE: To explore the in vitro anti-platelet effects of Ginsenoside -2A,a purified extract from Panax notoginseng. METHODS: Platelet rich plasma (PRP) was prepared routinely from venous blood samples of patients with essential hypertension and normal persons. PRP was incubated with different concentrations of Nifedipine, Ginsenoside-2A ,and SK&F96365. Maximal platelet aggregation rate[ PAG (M) ] induced by 2 micromol/L ADP was taken as the observed index. Five-minute PAG( M) was determined for 5 consecutive times. RESULTS: (1) PAG (M) in essential hypertension group was 0. 89 +/- 0. 06, which was higher than that in the normal group (0. 68 +/-0. 07 ) with significant difference (P <0.01). (2)Nifedipine of two concentrations (10 p.mol/L,20 pVmol/L) had no effect on PAG(M) in either essential hypertension group or normal group(P >0. 05). (3)Different concentrations of SK&F96365 (2.5 micromol/L,5 micromol/L,10 micromol/L and 20 micromol/L) could inhibit the PAG(M) in essential hypertension group; (4) Differen concentrations of Ginsenoside -2A (2. 5 micromol/L, 5 micromol/L, 10 micromol/L and 20 micromol/L) could inhibit PAG ( M) in essential hypertension group; three concentrations of Ginsenoside -2A (5 micromol/L, 10 micromol/L, 20 micromol/L) could inhibit the PAG(M) in the normal group (all P <0.05). CONCLUSION: Platelet aggregating function in essential hypertension patients was obviously higher than that in the normal persons and platelets was in the high reactive status. Nifedipine had no inhibitive effect on platelet aggregation. SK&F96365 could inhibit the platelet aggregation. Ginsenoside-2A could inhibit platelet aggregation, and had the definite anti-platelet action.

[Study on the variation of platelet function in pregnancy induced hypertension and gestational diabetes mellitus].

OBJECTIVE: To investigate the platelet activity and function in pregnancy induced hypertension (PIH) and gestational diabetes mellitus (GDM). METHODS: Twenty-one patients with GDM and 23 patients with PIH in third-trimester were included. Twenty normal pregnant women in third-trimester served as controls. Platelet count (PC), mean platelet volume (MPV) were determined on Cell-DYN 1600 and the expression of CD62P was analyzed on FACSC alibur. RESULTS: (1) PC was (181 +/- 56) x 10(9)/L in PIH, (206 +/- 60) x 10(9)/L in GDM and (229 +/- 56) x 10(9)/L in controls, respectively. PC in PIH was lower than that of controls (P < 0.01), but there was no significant difference between GDM and controls. (2) MPV was (11.2 +/- 2.0) fl in PIH, significantly higher than that of controls (8.7 +/- 1.6) fl (P < 0.001). In GDM, MPV was (9.5 +/- 1.6) fl, without significant difference compared with that of controls. (3) The expression of CD62P increased significantly in PIH compared with controls [CD62P: (42 +/- 13)% vs (26 +/- 7)%, P < 0.001; CD62P(I): 109 +/- 39 vs 75 +/- 13, P < 0.01]. In GDM, the expression of CD62P also increased significantly compared with the normal pregnancy [CD62P(%): (42 +/- 14)% vs (26 +/- 7), P < 0.001; CD62P(I): 100 +/- 42 vs 75 +/- 13, P < 0.05]. (4) All parameters had no significant difference between PIH and GDM. CONCLUSION: Platelet activity is enhanced in PIH and GDM. It may play an important role in the pathogenesis and development of the two diseases.

Effects of hormone replacement therapy on platelet activation in postmenopausal women.

OBJECTIVE: To assess the effects of hormone replacement therapy (HRT) on platelet activation in postmenopausal women compared with premenopausal women. METHODS: The expressions of CD41 and CD62P in fifteen postmenopausal women before and after HRT were detected using flow cytometry (FCM), with fifteen premenopausal women with a mean age of 47 years as controls. RESULTS: The expressions of CD41 and CD62P in postmenopausal women were higher than those in the control group. CD62P(%), CD62P(I) and CD41 were reduced from 36.40 +/- 5.9, 37.75 +/- 5.8, and 470.11 +/- 74.0 to 27.97 +/- 5.6, 26.64 +/- 4.9, and 303.23 +/- 72.8 after six months of HRT (P < 0.05). CONCLUSIONS: Platelet activation in postmenopausal women was higher than in premenopausal women and was reduced significantly after six months of HRT. HRT may have a favorable effect on reduction of platelet activity.

Ventilation of wards and nosocomial outbreak of severe acute respiratory syndrome among healthcare workers.

OBJECTIVE: To identify valid measures for preventing outbreaks of severe acute respiratory syndrome (SARS) among protected healthcare workers in isolation units. METHODS: Architectural factors, admitted SARS cases and infection of healthcare workers in different isolation wards between January 30 and March 30, 2003 were analyzed. RESULTS: Four types of isolation wards were analyzed, including the ward where the thirty-first bed was located on the twelfth floor, the laminar flow ward in the Intensive Care Unit where the tenth bed was located on the fifteenth floor, the ward where the twenty-seventh bed was located on the thirteenth floor of the Lingnan Building, and thirty wards on the fourteenth to eighteenth floors of the Zhongshan Building. The ratios (m(2)/m(3)) of the area of the ventilation windows to the volume of the rooms were 0, 0, 1:95 and 1:40, respectively. Numbers of SARS cases in the wards mentioned above were 1, 1, 1 and 96, respectively. Total times of hospitalization were 43, 168, 110 and 1272 hours, respectively. The infection rates of the healthcare workers in the areas mentioned above were 73.2%, 32.1%, 27.5% and 1.7%, respectively. The difference in the infection rates was of statistical significance. CONCLUSIONS: Isolating SARS cases in wards with good ventilation could reduce the viral load of the ward and might be the key to preventing outbreaks of SARS among healthcare workers along with strict personal protection measures in isolation units.

A hospital outbreak of severe acute respiratory syndrome in Guangzhou, China.

OBJECTIVE: To describe a hospital outbreak of severe acute respiratory syndrome (SARS) and summarize its clinical features and therapeutic approaches. METHODS: The outbreak started with a SARS patient from the community, and a total of 96 people (76 women and 20 men, mean age (29.5 +/- 10.3) years, 93.8% of whom were health care workers) who had exposure to this source patient became infected in a short time. Clinical data in this cohort were collected prospectively as they were identified. RESULTS: (1) The incubation period ranged from 1 to 20 (mean: 5.9 +/- 3.5) days. The duration of hospitalization was (17.2 +/- 8.0) days. (2) The initial temperature was (38.3 +/- 0.6) degrees C, while the highest was (39.2 +/- 0.6) degrees C (P < 0.001), with fever duration of (9.0 +/- 4.2) days. (3) Other most common symptoms included fatigue (93.8%), cough (85.4%), mild sputum production (66.7%), chills (55.2%), headache (39.6%), general malaise (35.4%) and myalgia (21.9%). (4) The radiographic changes were predominantly bilateral in the middle or lower lung zones. The number of affected lung fields was 1.2 +/- 0.8 on presentation, which increased to 2.9 +/- 1.4 after admission (P < 0.001). The interval from the beginning of fever to the onset of abnormal chest radiographs was (3.5 +/- 2.3) days, which increased in size, extent, and severity to the maximum (6.7 +/- 3.5) days later. The time before the lung opacities were basically absorbed was (14.9 +/- 7.8) days. (5) Leukopenia was observed in 67.7% of this cohort. The time between the onset of fever and leukopenia was (4.4 +/- 2.3) days, with the lowest white blood cell count of (2.80 +/- 0.72) x 10(9)/L. (6) The lowest arterial oxygen saturation was (94.8 +/- 3.1)% with supplementary oxygen. (7) Antibiotical therapies included tetracyclines (91.0%), aminoglycosides (83.3%), quinolones (79.2%); 18.8% of the patients received a combination of tetracyclines and aminoglycosides, while 11.5% received a combination of tetracyclines and quinolones, and 63.5% received a combination of tetracyclines, aminoglycosides and quinolones. Vancomycin was used in 13.5% of the patients. (8) 68.8% of the patients were treated with methylprednisolones for a mean interval of (4.9 +/- 2.4) days. The initial dose was (67.3 +/- 28.2) mg/d and the maximal dose was (82.4 +/- 30.5) mg/d. (9) Human gamma-globulin, interferon-alpha, antiviral drugs (oral ribavirin or oseltamivir) were used respectively in 68.6%, 46.9% and 92.7% of the patients. (10) Ninety-five patients (99.0%) had a complete clinical recovery, and only 1 patient (1.0%) died. CONCLUSIONS: SARS appears to be quickly infectious and potentially lethal among health care workers, characterized by acute onset and rapid progression, and mostly bilateral lung involvement on chest radiographs. Proper administration of glucocorticosteroids seems to be of some benefits. Antibiotics, human gamma-globulin, interferon-alpha, and antiviral drugs, although empirically, might be useful to shorten the clinical course.

Comparison of clinical course of patients with severe acute respiratory syndrome among the multiple generations of nosocomial transmission.

BACKGROUND: Severe acute respiratory syndrome (SARS) is characterized by both an atypical pneumonia and efficient nosocomial transmission. However, it remains unknown whether the infectivity and the virulence of the pathogen will change throughout the successive transmission. This study was conducted to compare the clinical features and management regimens of patients with SARS among the multiple generations from nosocomial transmission initiated by a super-spreader. METHODS: The clinical data of 84 epidemiologically-linked SARS patients from a hospital outbreak were retrospectively studied. All patients, in whom a clear-cut transmission generation could be noted, had a direct or indirect exposure to the index patient and the epidemic successively propagated through the multiple generations of cases within a short period of time. RESULTS: There were 66 women and 18 men with mean age of (29.2 +/- 10.3) years in this cluster; and 96.4% of whom were health care workers. Detailed contact tracing identified 35 (41.7%) first-generation cases, 34 (40.5%) second-generation cases, and 15 (17.8%) third-generation cases. No statistical differences among the multiple generations of transmission were found in terms of age, gender, incubation period and length of hospital stay. With the advanced transmission generations, the initial temperature lowered, the number of cases with dry cough decreased. There were no statistical differences in the peak temperature and duration of fever, other accompanying symptoms, leucopenia; however, the time from initial pulmonary infiltrates to radiographic recovery shortened (P < 0.05). No differences were found in maximum number of lung fields involved, duration from the onset of fever to the occurrence of pulmonary infiltrates and time from the initial pulmonary infiltrate to its peak among the multiple transmission generations (P > 0.05). No statistical differences were found in modes of oxygen therapy and sorts of antibiotics prescribed among the various transmission generations (P > 0.05); however, as with the advanced transmission generations, the number of cases prescribed with methylprednisolone, human gamma-globulin, interferon-alpha, antiviral drugs (oral ribavirin or oseltamivir) increased (P < 0.05) and time from admission to starting these medication shortened (P < 0.05). CONCLUSIONS: There is no evidence that SARS infection will evolve or transmit within a fashion that permits it to become less powerful throughout the successive transmission within a short time.

[Comparison of clinical features of severe acute respiratory syndrome among different transmission generations].

OBJECTIVE: To investigate the differences among various transmission generation of severe acute respiratory syndrome (SARS) by comparing the corresponding clinical data. METHODS: The clinical data of 84 patients with SARS were retrospectively studied, 66 women and 18 men, mean age (29.2 +/- 10.3) years old, 96.4% of whom were health care workers. All subjects had exposure to a source patient and the epidemic progressively propagated in a short time. For the infectious chain, we defined these patients who had exposure to a source patient as the primary cases, which included 35 patients (41.7%). Patients who got the disease by exposure to the primary cases were defined as secondary cases, which included 34 patients (40.5%). Similarly, the tertiary cases included 15 patients (17.9%). RESULTS: (1) No statistical differences in age, sex ratio, incubation period and hospitalization duration among various infectious generations were found (P > 0.05). (2) With descending in infectious generations, the initial temperature lowered, and cases with cough decreased (P < 0.05) with no statistical differences in the peak temperature, other accompanying symptoms and leukopenia (P > 0.05). (3) With descending in infectious generations, the course from the appearance of pulmonary lesions to their resolution shortened (P < 0.05). No differences were found in the maximal involved pulmonary fields, duration from initial fever to appearance of pulmonary lesions and course from the initial pulmonary lesions to their peak among the above three generations (P > 0.05). (4) No statistical differences were found in ways of oxygen therapy and classes of antibiotics prescribed (P > 0.05). With descending in infectious generation, cases treated with methylprednisolone, human gamma-immunoglobulin, interferon-alpha, and antiviral drugs (oral ribavirin or oseltamivir) increased (P < 0.05) and the duration of their use also increased (P < 0.05). CONCLUSIONS: With descending in infectious generations, the clinical features of SARS may become ameliorated.

[Clinical features of 96 patients with severe acute respiratory syndrome from a hospital outbreak].

OBJECTIVE: To describe a hospital outbreak of severe acute respiratory syndrome (SARS) and summarize the clinical features and therapeutic approaches. METHODS: Clinical data in this cohort were collected prospectively as they were identified. RESULTS: The outbreak started with a SARS patient from the community on 30 January 2003, followed by a total of 96 people [76 women and 20 men; mean age (29.5 +/- 10.3) years; 93.8% of whom were health care workers] infected in a short period of time after their exposure to this source patient. The incubation period ranged from 1 to 20 days, with a mean of (5.9 +/- 3.5) days. The initial temperature was (38.3 +/- 0.6) degrees C, while the highest was (39.2 +/- 0.6) degrees C (P < 0.001), with a mean fever duration of (9.0 +/- 4.2) days. Other common symptoms included fatigue, cough, mild sputum production, chills, headache, general malaise and myalgia. The radiographic changes were predominantly bilateral and in the middle or lower lung zones. Leukopenia was observed in 67.7% of this cohort. The mean lowest oxygen saturation was (94.8 +/- 3.1)% with supplementary oxygen through a nasal cannula. 68.8% of the patients were treated with methylprednisolone for a mean period l of (4.9 +/- 2.4) days. The initial dose was (67.3 +/- 28.2) mg/d and the maximal dose was (82.4 +/- 30.5) mg/d. Ninety-five patients (99.0%) had a complete clinical recovery, and 1 patient died of progressive acute respiratory distress syndrome. The mean hospitalized duration was (17.2 +/- 8.0) days. CONCLUSION: SARS appears to be highly contagious and potentially lethal among health care workers, characterized by acute onset and rapid progression. Corticosteroids, antibiotics, human gamma-globulin, interferon-alpha, and antiviral drugs, although used empirically, might be of some benefits in shortening the clinical course.

[A study of the architectural factors and the infection rates of healthcare workers in isolation units for severe acute respiratory syndrome].

OBJECTIVE: To investigate measures to prevent the outbreak of severe acute respiratory syndrome (SARS) in healthcare workers in isolation units. METHODS: The architectural factors and the infection of healthcare workers in different wards in our hospital between 30 January 2003 and 30 March 2003 were analyzed. RESULTS: Four kinds of isolation wards were evaluated, including the ward where the thirty-first bed lied in on the twelfth floor, the laminar flow ward in the intensive care unit (ICU) where the tenth bed lied in on the fifteenth floor, the ward where the twenty-seventh bed lied in on the thirteenth floor of Building A, and thirty wards on the fourteenth to eighteenth floors of Building B. The ratios (m2/m3) of the area of the ventilation windows to the volume of the room were 0, 0, 1:95 and 1:40, respectively. Numbers of SARS cases in the wards mentioned above were 1, 1, 1 and 96, respectively. The total lengths (hour) of hospitalization were 43, 168, 110 and 1,272, respectively. The infection rates of the healthcare workers in the areas mentioned above were 73%, 32%, 28% and 2%, respectively. The difference of the infection rates was of statistical significance. CONCLUSION: In addition to strict personal protective measures, isolation of SARS cases in wards with high ratio of the area of ventilation windows to the volume of the room and good ventilation may be the key to preventing the outbreak of SARS in healthcare workers in isolation units.

Involvement of levels of Toll like receptor-4 in monocytes, CD4+ T-lymphocyte subsets, and cytokines in patients with immune thrombocytopenic purpura.

INTRODUCTION: Toll-like receptors have been found to be associated with immune-mediated diseases but it is still not clear whether they play a role in immune thrombocytopenic purpura (ITP), especially TLR4. CD4+ T-lymphocyte abnormalities, including Th17, Th1, Th2, and regulator T cell (Treg), are considered important in ITP. There have been few studies regarding the expression of TLR4 and the relationships between TLR4 and Th17 levels in ITP. MATERIALS AND METHODS: In this study, we evaluated the expression of TLR4 in monocytes, the plasma concentrations of IL-23, IL-17 and the profiles of Th17, Th1, Th2 cells in 70 patients with ITP and 31 healthy controls. In addition, we evaluated IL-2 and Treg cells in 46 cases of 70 patients with ITP and the same 31 controls. RESULTS: Higher levels of TLR4 expression, higher relative numbers of Th17 and Th1 cells and lower levels of Treg cells were observed in patients when compared with controls (p=0.001 for TLR4; p<0.001 for Th17; p=0.014 for Th1; p=0.001 for Treg). The levels of IL-23 and IL-2 were increased (p=0.022 for IL-23; p=0.025 for IL-2), the relative levels of Th2 and concentrations of IL-17 were similar across both groups (p=0.446 for Th2; p=0.316 for IL-17). A significant negative correlation was observed between levels of TLR4 and Treg(r=-0.544, p<0.001), but a significantly positive correlation was observed between IL-2 and IL-23 concentration in patients (r=0.441, p=0.004). Neither the correlation between TLR4 and the other CD4(+) T cells and cytokines nor the correlation between the three cytokines and CD4+ T cells was found to be statistically significant. CONCLUSIONS: Our data showed that TLR4, CD4+ T cells (Th1, Th17 and Treg cells) and related cytokines (IL-23, IL-2) may take part in the pathogenesis of ITP. TLR4 may play a role through the TLR4-cytokine-CD4+ T lymphocyte cell pathway.

[Effect of mPGES-1 inhibitor MK886 on cell cycle of leukemia HL-60 cells].

To investigate the effect of a microsomal prostaglandin E synthase-1 (mPGES-1) inhibitor MK886 on cell cycle of the human acute myeloid leukemia HL-60 cells. HL-60 cells were treated with different concentration of MK886 (10, 25, 50 µmol/L) for 24 h. Flow cytometry, Western blot and ELISA were used to measure cell cycle, cyclin D1, mPGES-1, PGE(2), Akt, P-Akt and C-MYC. The results indicated that after treated with MK886, the percentage of HL-60 cells decreased in G(0)/G(1) phase and increased in S phase, and expressions of mPGES-1, cyclin D1, P-Akt and C-MYC and synthesis of PGE(2) decreased significantly. It is concluded that MK886 can arrest HL-60 cells in G(0)/G(1) phase, the mechanism of which is possibly associated to inhibition of mPGES-1 expression, reduction of PGE(2) synthesis, suppression of Akt phosphorylation and C-MYC expression, down-regulation of cyclin D1 expression.

[Effect of mPGES-1 inhibitor MK886 on apoptosis and drug resistance of HL-60/A cells].

This study was aimed to investigate the effect of MK886, a mPGES-1 inhibitor, on apoptosis and drug resistance of leukemia HL-60/A cell line. Expression of mPGES-1 was assayed by QT-PCR and Western blot. The effect of MK886 on HL-60/A cell proliferation was assayed by CCK-8 method, and flow cytometry was used to detect cell apoptosis. The expression of Akt and P-Akt was detected by Western blot. PGE2 was measured by ELISA. Effect of MK886 (10 µmol/L) on the chemotherapeutic sensitivity of HL-60/A cells and expression of mdr-1 mRNA and P170 protein were investigated too. The results indicated the expression of mPGES-1 was higher in HL-60/A cells. MK886 inhibited HL-60/A cell proliferation and induced apoptosis in a time- and concentration-dependent manner. Expression of mPGES-1 and P-Akt and synthesis of PGE2 decreased significantly. MK886 reduced expression of mdr-1 and P170 protein and enhanced the sensitivity of HL-60/A cells to chemotherapeutic drugs. It is concluded that MK886 can inhibit HL-60/A cell proliferation, induce apoptosis and enhance sensitivity to chemotherapeutic drugs, the mechanism of which possibly associates to down-regulation of mPGES-1/PGE2 synthesis, reduction P-Akt expression and decreasing mdr-1 and P170 protein expression.