Volume 86  ·   Issue 1  ·  2006

To investigate the molecular mechanism of multiple-drug and pan-drug resistance among Acinetobacter species.

Non-repetitive 90 carbapenem-resistant strains of Acinetobacter species were collected in Beijing, Guangzhou, and Fuzhou 1999-2004. The homology of the isolates was determined by both pulsed field gel electrophoresis and randomly amplified polymorphic DNA typing. Seven representative clones were selected from the 90 strains of Acinetobacter isolated from different hospitals to be used for further study. Analytical isoelectric focusing was used to measure the isoelectric point of the β-lactamase. Plasmid DNA was extracted and purified Genes of different β-lactamase, including bla_TEM--, bla_SHV-, bla_PER-, blaI_MP-, bla_VIM-, and bla_OXA. genes, in these clone strains were amplified and sequenced. PCR was used to analyze the integrons.

The P clone strain isolated during an outbreak of pan-drug-resistant Acinetobacter species in Peking Union Medical College Hospital 2004 was not susceptible to most common antimicrobial agents tested. The 7 representative clones produced multiple β-lactamases: TEM-1, high-level AmpC, SHV-type, OXA-23 carbapenemase and IMP-8 and metalloenzyme respectively. One clone produced PER-1 enzyme. These 7 clone strains were resistant to most β-lactams(including carbapenems), erythromycin, chloramphrenicol, and rifampin. Two clone strains were susceptible to cefoperazone/sulbactam and amikacin while 4 clone strains susceptible to levofloxacin. All of the 7 clones were susceptible to minocycline and colistin. Five different integrons were found, harboring the genes mediating the resistance to aminoglycosides, rifampin, chloramphenicol, and carbapenems (bla_IMP-8).

The molecular bases of multiple-drug or pan-drug resistance in Acinetobacter species include production of OXA-23 carbapenemase or IMP type metalloenzyme and integrons with different resistance gene cassettes. Pan-drug-resistant Acinetobacter species are susceptible to old antimicrobials agents, such as colistin and minocycline.

To investigate the trend of resistance to antimicrobial agents among clinical isolates of Escherichia coli 1990-2004.

Agar diffusion test was used to analyze the changes of drug susceptibility of 33495 strains of E. coli isolated from 11 hospitals in Shanghai to 21 antimicrobial agents 1990-2004.

The resistance rates of 33 495 E. coli isolates to 21 antimicrobial agents mostly increased 1990-2004. The resistance rates to ampicillin and piperacillin increased from 69% and 30% to 85% and 71.4% respectively. The resistance rates to cephalosporins, except ceftazidime and cefepime, all increased, e. g., the resistance rates to cefazolin (24.0%→48.3%), cefuroxime (18.0%→45.7%), and cefaclor (33.3%→46.8%), especially that to cefotaxime (6.0%→35.2%). The resistance rate to fluoroquinolones increased from 11.0% to 55.4%. The resistance rate to gentamicin increased from 44.0%to 54.0%. The resistance rates to tetracycline, chloramphenicol, SMZ/TMP remained at high levels. However, ceftazidime, cefepime, imipenem, amikacin, β-lactams/β-lactamase inhibitors, and nitrofurantoin remained active against the E. coli isolates. The detectable rate of extended-spectrum β-lactamase-producing strains in E. coli increased from 14.7% to 36.5%.

The trend of resistance of E. coli to commonly used antimicrobials was upward 1990-2004.

To screen a sub-clone of human breast cancer cell of the MCF-7 line with high metastasis potential.

Human breast cancer cells of the MCF-7 line were injected subcutaneously into 10 severe combined immunodeficiency (SCID) mice. Sixty-eight days after the mice were killed and their lungs were taken out. Primary cell culture was conducted. When the cells were passed on to the third generation a sub-clone was screened from the lung tissue and termed LM-MCF-7. Microscopy was performed on the lung tissues. The growth curve was drawn. Flow cytometry was used to examine the cell cycle. Chromosome analysis was done. Immunohistochemistry was used to detect the expression of breast cancer specific antigen CAI5-3. Western blotting was used to detect the protein expression of the protein associated with tumor metastasis: nm23 (a metastasis-suppressing gene), myosin light chain kinase(MLCK, a kinase related to cell movement), surviving, bcl-2 and p27 (a gene related to cell cycle). LM-MCF-7 cells were injected into other SCID mice and these mice were killed 30 days later to observe the metastasis of cancer so as to detect the tumorigenic ability of the LM-MCF-7 cells.

When the cells from the mouse lung tissues were passed on to the third generation a sub-clone with high metastasis potential was screened and termed LM-MCF-7. The morphology of the new cell line was typically epithelioid. Flow cytometry showed that the DNA relatively contents were 53.40% of the LM-MCF-7 cells were in the G₀/G₁ phase, a lower percentage than that of the MCF-7 cells, and 17.10% in the S phase and 23.20% in the G₂+ M phase, both percentages higher than those of the MCF-7 cells. The proliferating time of the LM-MCF-7 cell population was about 20 ± 14 hours, much shorter than that of the parent strain cells. The chromosomes of the LM-MGF-7 cells, numbering 16~123, showed the morphology characteristic c of human chromosomes. The marker of human breast cancer CA15-3 was detected in both MCF-7 and LM-MCF-7 cells. The protein expression of nm23 and p27 was down-regulated, but the protein expression of MLCK, bcl-2 and survivin was up-regulated in LM-MCF-7 cells in comparison with those in MCF-7 cells. The tumorigenesis rate of LM-MCF-7 cells was 100% (5/5), with a latent period of 5.0 ± 0.0d, and the tumor metastasized to lung, kidney, spleen, bone marrow, lymph node and heart.

A human breast cancer line, LM-MCF-7 cell line, with high metastasis potential has been derived from the human breast cancer cells of MCF-7 line.

To investigate the expression of α-tubulin and γ-tubulin, 2 kinds of centrosome proteins, in premalignant lesion and carcinoma of breast and the significance thereof.

Forty specimens of premalignant lesions of breast, 40 specimens of infiltrating ductal carcinoma of breast, 40 specimens of intraductal carcinoma (IDC), and 30 specimens of normal breast tissues were obtained during operation. Immunohistochemistry was used to analyze the protein expression of α-tubulin and γ-tubulin, and the percentages of ki67 positive cells. Western blotting was used to examine the mRNA expression of α-tubulin and γ-tubulin.

The protein and mRNA expression values of α-tubulin and γ-tubulin in breast carcinoma were higher than those in the premalignant lesions and normal breast tissues with significant differences between the premalignant lesions and normal breast toques and without significant differences between infiltrating ductal carcinoma of breast and IDC. The ki67 positive rates of the infiltrating ductal carcinoma of breast group, IDC group, premalignant lesion group, and normal breast tissues group were 16.0%, 37.0%, 53.6%, and 67.8% (P=0.001). A positive correlation existed between the expression of α-tubulin and the expression of γ-tubulin in the same case and the same group (all P=0.00) and there was no significant correlation between the expression of α-tubulin and the expression of γ-tubulin in the same case and the same group (all P> 0.05). Both the expression of α-tubulin and the expression of γ-tubulin were significantly associated with the prognosis.

Centrosome protein is one of the distinct phenotypes of breast cancer cells. Aberration of centrosome proteins may be used to screen high risk cases of breast carcinoma and to estimate the prognosis.

To analyze the management, prognosis and prognostic risk factors of recurrent gestational trophoblastic tumor (GTT).

One thousand one hundred and thirty GTT patients, aged 29± 6, were hospitalized and treated and 901 of them got complete remission (CR). Among these CR patients, 31 suffered relapsed. The clinical data of these 31 cases were analyzed retrospectively.

Thirty-one patients suffered 15.3 months (6~72 months) after the cessation of treatment with an overall recurrence rate of 3.4% (31/901). Four of the 31 patients suffered relapse repeatedly (totally seven times), resulting in an overall re-recurrence rate of 22.6% (7/31). Twenty-five of the 31 patients were rehospitalized and received treatment. Eighteen of them got complete remission (CR), 3 got partial remission(PR), and 4 died of progress of the disease (PD). The major adverse prognostic risk factors included: clinical stage (P <0.05), an interval of more than 12 months from the antecedent pregnancy to chemotherapy (OR=3.170, P <0.05), declination of β-hCG level back to normal titer after more than seven courses of chemotherapy (OR=4.775, P <0.05), and less than two courses of consolidation chemotherapy (OR=0.441, P<0.05).

More attention should be given to those GTT patients with adverse prognostic risk factors. Multi-drug and multiple route chemotherapy and/or combined surgical intervention can be used to improve the cure rate and lower the re-recurrence rate of the GTT recurrent patients.

To explore a new method to evaluate the dose-response relationship between the noise exposure and prevalence of noise induced high frequency hearing loss.

Physical examination and questionnaire survey were conducted among 32 mechanical workers, 12 males and 20 females, aged 35.1 ± 7.2, exposed to industrial impulse noise (impulse noise group), and 163 textile workers, 82 males and 81 females, aged 31.5 ± 8.7, exposed continuous noise (continuous noise group). SH-126 dosimeter was used to measure the A weighted equal sound level in continuous eight working hours (L_Aeq.8h) so as to evaluate the individual noise exposure. Cumulative noise exposure (CNE) was calculated with the values of L_Aeq.gh and noise working year adjusted by different exchange rates (ERs) for each worker. Hearing threshold was measured by audiometer with routine method and adjusted by age and gender according to GBZ49-2002. High frequency hearing loss was diagnosed based on the GBZ49-2002 for each worker.

According to the equal energy rule, with the ER=3 the CNE of the impulse noise group was [103.2 dB (A)·year ± 4.2dB (A)·year], significantly lower than that of the continuous noise group [110.6 ± 6.0dB (A)·year, P<0.05]. With the ER=5.5, the CNE of the impulse noise group was 110.3 ± 6.6 dB (A)·year, not significantly different from that of the continuous noise group [110.6 ± 6.0dB (A)·year]. The high frequency hearing loss prevalence of the impulse noise group was 68.8%, not significantly different that of the continuous noise group (65%, P> 0.05). Trend chi square test showed significant differences in the relationship between CNE and hearing loss prevalence among the impulse noise group with the ER=3, the impulse noise group with the ER=5.5, and continuous noise group (all P <0.01). With the ER=3, the high frequency hearing loss prevalence of the impulse noise group with the CNE of 100-104 dB (A)·year was 76.9%, significant higher than that of the continuous noise group (30.4%, P <0.05); and the high frequency hearing loss prevalence of the impulse noise group with the CNE of 105-108 dB (A)·year was 90.9%, significantly higher than that of the continuous noise group (50.0%, P <0.05). With the ER=5.5, there was no difference in the prevalence of high frequency hearing loss between the continuous noise group and impulse noise group. Logistic regression model showed that with the ER=3, the dose-response curve of the impulse noise group presented a left shift and with a slope sharper than that of the continuous noise group. With the ER increasing the dose-response curve of the impulse noise group showed a right shift and with a gradually lowering slope. When the ER equaled to 6 in the impulse noise group and equaled to 3 in the continuous noise group, the two dose-response curves were almost superposed.

Impulse noise causes more serious damage in causing high frequency hearing loss than continuous noise according to equal energy rule. Increasing the ER value in the impulse noise group adjusts the dose-response curve to be close to that of the continuous noise group.

To investigate the feasibility of hydroxyapatite (HA) middle ear prostheses in otosurgery.

In the animal experiment HA prostheses were put in the bilateral bullae of the ears of 12 healthy white guinea pigs (totally 24 ears). Three guinea pigs were killed every month 1~4 months later. Morphological examination and electron microscopy were conducted to observe grafts. In the clinical experiment HA prostheses was used in ossiculoplasty in 31 cases, reconstruction of posterior wall of external auditory canal in 5 cases, and mastoid obliteration in 11 cases.

The HA prostheses taken out from 23 ears of the guinea pigs were with normal and complete structure, and clear margin in gross anatomy and microscopically. Only one ear suffered infection. Follow-up or the patients for 1~4 years showed excellent healing of the operated parts and an improvement of hearing threshold by about 16 dBHL.

Hydroxyapatite With excellent physical and chemical properties and biological tolerance HA is a good material in ossiculoplasty, canaloplasty, mastoid obliteration, and other otological operations.

To evaluate the efficacy and feasibility of laparoscopic aid in upper urinary reconstructive operation.

Fifty-eight patients with ureteropelvic junction obstruction, 5 patients with upper ureter polypus, 2 patients with upper ureter stenosis, and 13 patients with upper ureter lithiasis underwent upper urinary reconstructive operation with laparoscopic aid described as follows: an incision 1 cm long was made, a 10 mm trocar and a 30°laparoscope were wt in, the part with lesion was isolated and resected, and then pyeloplasty or end-to-end anastomosis of ureter was performed.

The mean operative time was 33 minutes (25~45 minutes). The mean blood loss was 20 ml (15~25 ml). Complications such as urinary leakage and infection were observed. The double J stent was removed at 1 month after the operation. Follow-up for 3 to 15 months in 20 cases showed alleviation of hydronephrosis.

An effective and safe method with less wound and operative time, combination of laparoscopic aid and open surgery in upper urinary reconstructive operation helps avoid difficult laparoscopic operation, does not increase trauma of the abdominal wall, and is worth promoting clinically.

To investigate the etiology, diagnosis, and management of spontaneous perirenal hemorrhage (SPH).

The clinical data of 35 patients, 10 males and 12 females, aged 35.9 (12-77), with the diagnosis of SPH, without history of trauma, anticoagulant use, dialysis, and renal transplantation, were analyzed.

The underlying disease of SPH included angiomyolipoma (18 cases), renal cell carcinoma (7 cases), kidney cyst (2 cases), renal artery aneurysm (3 cases), rupture of renal artery aneurysm accompanied with pregnancy (2 cases), renal pheochromocytoma (3 cases 2 of which accompanied with pregnancy), congenital stricture of pelviureter junction (1 case), and liver cancer(1 case). The most common underlying diseases were nephrigenic (96%) with angiomyolipoma ranking first (54%) followed by renal cell carcinoma (21%). The underlying diseases were diagnosed correctly in 23 cases (69%). CT helped in diagnosis of 34 cases. Surgery was performed on most of the cases.

The most common causes of SPH is renal neoplasms more than 50% of which are benign. Renal artery aneurysm and pheochromocytoma tend to rupture during pregnancy. CT is the first method of choice in diagnosis.

To investigate the role of CD4⁺CD25⁺regulatory T cells (Tr cells) in the pathogenesis of asthma in children.

Peripheral blood samples were collected from 20 pediatric patients with asthma, 10 male and 10 female, aged 7 (3-12), and 20 healthy children, 10 male and 10 female, aged 6.5 (2-11). Lymphocytes were isolated. Flow cytometry was used to examine the percentages of CD4⁺CD25⁺regulatory T cells, IL-10 secreting CD4⁺CD25⁺regulatory T cells (CLD4⁺CD25+-IL-10), and transforming growth factor (TGF)-β secreting CD4⁺CD25⁺ regulatory T cells (CD4⁺CD25⁺-TGF-β). RT-PCR and real-time PCR were used to detect the mRNA expression of suppressor of cytokine signal 1(SOSC1) and Foxp3.

The percentages of CD4⁺CD25⁺ regulatory T cells of the asthma children was 6.51% ± 1.94%, significantly lower than that of the healthy children (11.96% ± 2.30%, P <0.01); the percentage of CD4⁺CD25⁺-IL-10 of the asthma children was 1.46%± 0.35%, significantly lower than that of the healthy children (5.65% ± 1.70%, P<0.01); and the percentage of CD4⁺CD25⁺-TGF-β of the asthma children was 1.24%± 0.21%, significantly lower than that t of the healthy children (4.23% ± 1.65%, P <0.01). The Foxp3 mRNA expression of the asthma children was 0.12± 0.05, significantly lower than that of the healthy children (1.71 ± 0.58, P <0.01); and the SOCS1 mRNA expression of the asthma children was 0.38± 0.19, significantly lower than that of the healthy children (1.51 ± 0.41, P < 0.01).

The decrease of CD4⁺CD25⁺ regulatory T cells may be involved in the pathogenesis of asthma. The decreased mRNA expression of Foxp3 and SOCS1 may be associated with the aberrant development of CD4⁺CD25⁺ regulatory T cells.

To explore the mechanism of relaxation mediated by nitric oxide on the human lower esophageal sphincter(LES), and compare the difference in relaxation response between clasp fibers and sling fibers.

32 LES specimens were obtained from 32 patients with high-positioned carcinoma of the mid-esophagus, 12 males and 16 females, aged 55.9 ± 9.3, during operation. The clasp fibers and sling fibers were isolated and suspended in perfusion tough. Electric field stimulation (EFS) was applied to the clasp and sling fibers in vitro. Nitric oxide synthase (NOS) inhibitor L-NNA, NOS substrate L-arginine, neurotoxin tetrodotoxin (TTX), and atropine were added respectively to observe their effects on the clasp and sling fibers under EFS. Sodium nitroprusside was added on the two kinds of smooth muscle stripes to observe its influence as well.

EFS induced frequency-dependent relaxation to clasp fibers and some of sling fibers, which was inhibited by L-NNA in a concentration-dependent manner and was reversed by L-arginine partially. Maximal relaxation in clasp fibers and sling fibers was observed at 512 Hz and 16 Hz respectively. The higher amplitude relaxation was induced in the sling fibers at lower stimulus frequencies (<32 Hz). Conversely, the same response was induced in the clasp fibers at higher stimulus frequencies (>64 Hz). Meanwhile, off-contraction was induced by EFS in some sling fibers and clasp fibers. In some sling fibers, contraction was induced by EFS which was inhibited by atropine. Maximal contraction in these fibers was observed at 128 Hz. TTX abolished the effect of EFS on both clasp and sling fibers, which was considered neurogenic. Sodium nitroprusside elicited the similar response to EFS.

Relaxation of clasp and sling fibers is related to L-NNA, TTX, and sodium nitroprusside, and can be mediated by nitric oxide. Lower stimulus frequencies induce higher amplitude relaxation to sling fibers, and conversely, higher stimulus frequencies induce higher amplitude relaxation to clasp fibers. EFS induces contraction response in some sling fibers.

to investigate the expression of non-T cell derived cytokines in acute rejection after heart transplantation.

The hearts of 26 C57BL/6 mice were transplanted into the abdominal cavities of 26 Balb/c mice (acute rejection group). The hearts of 26 Balb/c mice were transplanted into the abdominal cavities of another 26 Balb/c mice (syngeneic control group). After the operation, heart beat was felt with finger at the abdomen every day to determine the survival of the transplanted heart. One, three, five, and seven days after the operation the transplanted hearts were taken out from 5 mice in each group respectively to undergo microscopy. RT-PCR was used to detect the mRNA expression of these cytokines: interleukin (IL)-2, IL-15, tumor necrosis factor (TNF)-α, and interferon(IFN)-γ. Western blotting was used to detect the protein expression of these cytokines. Six mice in each group, totally 12, were used to observe the survival time of transplanted heart.

The mice of the control group recovered more rapidly in comparison with the nice of the acute rejection group. The survival times of the transplanted hearts of the control group were all longer than 100 days, significantly longer than those of the acute rejection group (7-9 days, P <0.01). No sign of acute rejection was found in the hearts of the control group, and significant lymphocyte infiltration was seen in the myocardium of the acute rejection group 3-7 days after the operation. In the control group, no mRNA expression of IL-2 and IFN-was seen at any time-point, and IL-15 mRNA and TNF-α mRNA were expressed at low level at any time-point. However, in the acute rejection group the mRNA expression of IL-2, TNF-α, and IL-15 could be detected since the first day post-operatively and peaked on the day 5, but the mRNA expression of IFN-γ could be detected and peaked only on the 5 th post-operative day. The mRNA expression levels of these 4 cytokines at any time point of the acute rejection group were all significantly higher than those of the control group (P < 0.01 or P <0.05). The proteins of the 4 cytokines were all expressed at low level at any time point in the control group. However, in the acute rejection group the protein expression levels of IL-15 and IL-2 began to increase since the third day after operation and the protein expression levels of TNF-α and IFN-γ began to increase since the first day after operation.

Many cytokines, including T cell derived and non-T cell derived cytokines, are involved in the pathogenesis of acute rejection after allogeneic organ transplantation.