2002年 · 第115卷第10期

Objective To characterize the immune response induced by SAG1 encoding plasmid combined with IL-2 gene adjuvant in mice and to assess the protective effect of this vaccination against toxoplasmosis. Methods Mice were co-injected intramuscularly with plasmid encoding Toxoplasma gondii SAG1 plus murine IL-2 expression vector at a dose of 100 μg. Booster immunizations were employed 2 more times at 3-week interval. As controls, mice were inoculated with PBS or empty plasmid pcDNA3. Humoral and cellular responses were assayed using ELISA for the determination of Ab, Ab isotype and IFN-γ, as well as IL-4. To detect the integration and dissemination of DNA in the injected mice, PGR and in situ hybridization were performed. All mice were then infected with highly virulent RH tachyzoites of Toxoplasma gondii intraperitoneally. Results Significant increases in specific IgG levels were observed in mice after immunization three times with SAG1 expression plasmid. With respect to the IgG isotype, co-inoculation of IL-2 expression plasmid enhanced the level of IgG2a and the production of IFN-γ. Challenging mice by vaccinating with combined plasmids with RH tachyzoites resulted in prolonged survival. Conclusion Humoral and cytokine responses elicited by SAG1 DNA immunization can be modulated by co-inoculation with IL-2 expression plasmid. The use of DNA vaccine in combination with an appropriate cytokine gene to prevent T. gondii infection warrants further investigation. Chin Med J 2002; 115(10): 1448-1452

Objective To establish a C57BL/6N mouse model infected with Giardia lamblia (G. lamblia) isolates from human origin. Method Two groups of C57BL/6N mouse were inoculated with purified cysts of two G. lamblia isolates(CD and XZ) by gavage separately. Patterns and curves of cyst excretion of the infected mice were observed and summarized. Histopathological changes of the small intestines of the infected mice were observed. Results Thirty-six mice receiving 1 x 104 cysts each were all infected. The C57BL/6N mouse showed high susceptibility to G. lamblia infection. There was no notable distinction between the two groups of the mice infected by the cysts of CD and XZ isolates. Cyst excretion occurred with intermittence. Of 36 infected mice, 32 (89%) passed cysts intermittently and 4 (11%) others persistently. The latent period of cyst excretion was 0-3 days p.i. (post-inoculation). The interruption of cyst excretion ranged from 12 to 20 days p. i. The fastigium of the cyst excretion was on day 6 p. i. The peak count of the cysts passed during a 2 h collection period was 2.3 × 107/g fecal specimen. Edema, inflammation, cell infiltration, small blood vessels congestion, mitotic figures and mucosa necrosis appeared in sections of intestines. Conclusion C57BI/6N mouse is a suitable animal model of G. lamblia. Chin Med J 2002; 115(10): 1453-1456

Objective To confirm the existence of point mutations in the SSU rDNA variable regions of 5 Leishmania donovani (L. d.) isolates from different epidemic foci in China. Methods Specific SSU rDNA fragments from nuclear DNA of 7 Leishmania species/isolates were amplified by PCR and then cloned into pGEMR-T Easy Vectors. After that, the specific fragments were sequenced by an automated DNA sequencer. Results Sequence analysis showed that the amplified DNA fragments of 7 Leishmania species/isolates were all 392 bp in length. All 5 point mutations were located in two unique sequence blocks (UQ-I and UQ-Ⅱ), and no insertions or deletions were found. The identities of comparison of Leishmania in GeneBank were more than 98%. Conclusion Five point mutations exist in the SSU rDNA variable region of 5 L. d. isolates from different epidemic foci of visceral leishmaniasis (VL) in China. Sequence differences of the SSU rDNA variable region exist among L.d. isolates from different foci. Chin Med J 2002; 115(10): 1457-1459

Objective To assess the feasibility of using saliva for Schistosomiasis japonica diagnosis. Methods Schistosoma japonicum infected animal model was established. Pairs of saliva and serum samples from rabbits and chronic schistosomiasis patients were collected. Anti-schistosoma specific antibodies in saliva and serum were detected by indirect ELISA. Results The specificities of antibody detection of rabbit saliva and serum were 93%(28/30) and 97%(29/30), respectively, and the sensitivities of antibody detection of rabbit serum and saliva were 100%(24/24) and 88%(21/24), respectively. A significant correlation (r=0.5307, P=0.0038 <0.05)existed between anti-SEA IgG levels in serum and saliva. As with those in serum, anti-SEA IgG levels in saliva could reflect the state of infection and treatment. The sensitivity of antibody detection was 91 %(29/32) for patient saliva samples and 100%(32/32) for their sera. 8 samples were positive in 140 normal saliva samples (i.e. 6%false positive rate) and 6 samples were positive in 156 normal serum samples(4%false positive rate). There was a significant correlation (r=0.4227, P=0.008 <0.05) between specific antibodies in saliva and serum. Conclusion The detection of specific antibodies in saliva can be used as a non-invasive immunodiagnosis method of Schistosomiasis japonica . Chin Med J 2002; 115(10): 1460-1464

Objective To perform cloning of the gene encoding Chinese Schistosoma japonicum tropomyosin(SjcTM) and its expression in Escherichia coli. Methods SjcTM cDNA fragment, except for 14 amino acids at the amino terminus, was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) with total RNA extracted from adult worms of S. japonicum. The RT-PCR product was cloned into T vector and sequenced. The SjcTM cDNA, derived from the constructed TA clone pGEM-SjcTM, was then subcloned into the expressing vector pBV220. After characterization by agarose gel electrophoresis, endonucleases digestion and PCR, the resultant recombinant plasmid was used for expression under the temperature-dependent condition. Results The RT-PCR product, cloned into a T vector, was sequenced and shown to be 96.5%identical at the nuclei acid level and 98.1 %identical in deduced amino acid sequence to that of S. mansoni tropomyosin. The target DNA fragment was then subcloned into a prokaryotic vector pBV220. Induced expression in E. coli DH5α cells resulted in a constant level of recombinant protein production. The results of SDS-PAGE and Western blot revealed that the molecular weight of non-fusion recombinant protein(rSjcTM) was approximately 32 kDa and could be recognized specifically by a polyclonal antiserum specific for native S. japonicum tropomyosin (SjcTM). Conclusion The engineering of the cDNA encoding S. japonicum tropomyosin and its bacterial expression was successfully made. Chin Med J 2002; 115(10): 1465-1469

Objective To investigate adenoviral vector mediated exogenous gene expression in mouse lungs and the effect of mIFN-γ transgene expression on allergen-induced pulmonary eosinophil infiltration in a murine asthmatic model. Methods LacZ marker gene was transduced into CD-1 mouse airway epithelial cells by installation of a replication-deficient adenovirus with LacZ gene (AdCMVLacZ) 5 x 109 plaque forming unit (pfu) in the intratrachea or nostril. C57 mice were sensitized intraperitoneally and challenged by aerosol with ovalbumin(OVA) to produce an asthmatic model. AdCMVmlFNγ 5 × 109 pfu was administered via nostril in asthmatic mice 48 h before OVA challenge. Sera, bronchial alveolar lavage (BAL) and lungs were recovered 48 h after OVA challenge. Results After administration with AdCMVLacZ by intratracheal installation or nose-drop, the lungs revealed a high level of widespread LacZ transduction with X-gal staining, mainly along airways. IFN-γ via adenoviral vector transduction could be overexpressed both in vitro and in vivo (1624.7±1321.5 pg/ml in BAL 96 h after AdCMVIFNy infection). In AdCMVIFNy treated asthmatic models, histological evaluation revealed marked suppression of eosinophil peribronchial and perivascular infiltration; the recoverable percentage of eosinophils in BAL was an average of 9.00% ± 4.58%, which was a statistically significant decrease versus that of the positive control group (75.13% ± 6.85%) (P <0.001). The total cell number in BAL((145 ± 55.6) x 103 cells/ml) in AdCMVmlFNγ treated mice also was tremendously reduced compared to the positive control group ((216. 6±71 . 1) x 103 cells/ml). Conclusions Adenoviral vector was able to overexpress exogenous gene in murine lungs. IFN-γ overexpression via adenoviral vector in pulmonary epithelia in vivo can abrogate allergen-induced eosinophilic infiltration in lungs in an asthmatic model, which may suggest a new preventively therapeutic method for cytokine immunogenetic transfer in allergic asthma. Chin Med J 2002; 115(10): 1470-1474

Objective To study telomerase activity (TA) and its variation in bone marrow mononuclear cells from patients with myelodysplastic syndrome (MDS) at different stages in comparison with normal bone marrow cells and leukemic cells. Methods The TA was semi-quantitatively determined in mononuclear cells from 20 normal bone marrow samples, 21 patients with MDS at different stages and 32 cases of acute leukemia by using a polymerase chain reaction-enzyme linked immuno-sorben assay (PCR-ELISA) kit. Results The TA in normal bone marrow cells was in the range of 0 to 0.3 units (U) with a mean of 0. 11 ± 0.08 U. Among them, 3 samples were considered positive in accordance with the standard recommended by the kit's pamphlet. In bone marrow cells from patients with acute leukemia, the TA was ranging from 0 to 0.96 U with a mean value of 0.42 ±0.26 U. The positive rate was 78.1 %which was significantly different from that in normal bone marrow (BM) (P <0.01) . In case of myelodysplastic syndrome, the average level of TA was 0.27 ± 0.19 U (ranging from 0 to 0.97 U) with a positive rate of 66.7%. In comparison with normal BM cells, the difference was significant (P <0.05). Particularly, the MDS high-risk subgroup exhibited a significantly higher activity of telomerase (P <0.05). In comparison with INT-1 and INT-2 subgroups in MDS patients based on international prognostic scoring system (IPPS),the difference in TA was also significant (P <0.05). The abnormality in cell karyotype was not correlated with TA. Conclusion The normal bone marrow cells demonstrate TA at a marginal level while a remarkably increasing level may be seen in acute leukemia patients. The BM cells from MDS patients display a moderate TA among which the high risk MDS subgroup with a poor prognostic IPPS score exhibited markedly higher TA. Chin Med J 2002; 115(10): 1475-1478

Objective To investigate the epidemiological status of extended-spectrum β-lactamase (ESBL) producing Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) and the drug resistance profiles of such organisms. Methods A total of 282 clinical isolates of E. coli and 180 of K. pneumoniae were collected from different districts of Zhejiang Province. Inhibitor potentiated broth dilution tests were performed for detecting extended-spectrum β-lactamases. Etests were performed to detect the drug resistance of these strains against nine commonly used antibiotics. Results The prevalence of extended-spectrum β-lactamases in E. coli and K. pneumoniae was 34.0%and 38.3%, respectively. The average prevalence of extended-spectrum β-lactamases in E. coli and K. pneumoniae was 35.7%. The resistance prevalence of extended spectrum β-lactamase producing strains to ceftazidime and cefotaxime was 40%and 26%respectively, so were those to cefepime, cefoxitin,piperacillin-tazobactam, cefoperazone-sulbactam, amikacin and ciprofloxacin. All these strains were sensitive to imipenem. Conclusion The results in this study showed that the prevalence of extended-spectrum β-lactamases was high, while extended-spectrum β-lactamase producing strains were resistant to most antimicrobial agents except imipenem. Chin Med J 2002; 115(10): 1479-1482

Objective To evaluate the immunogenicity, safety, and dosage of a new inactivated hepatitis A vaccine administered to young adults. Methods One hundred and four normal adult volunteers, seronegative for hepatitis A virus and hepatitis B surface antigen, were randomly assigned to one of three groups. The high-dose group received a primary dose of 1000 units of the new vaccine, the low-dose group received a primary dose of 500 units of the same vaccine, and the Havrix group received a primary dose of 1440 enzyme-linked immunosorbent assay units of Havrix, a licensed inactivated hepatitis A vaccine. All groups received a booster dose of the same vaccine 6 months after the primary dose. Local and systemic adverse reactions, seroconversion rates, and geometric mean titers of hepatitis A virus antibodies were measured in all three groups. Results Local and systemic reaction types and rates were similar in all three groups after primary and booster doses, although local reactions were more frequent in the Havrix group following the primary dose. No serious adverse reactions occurred. One month after the primary dose, the seroconversion rate was 87.5%in the high-dose group, 70.0%in the low-dose group, and 50.0%in the Havrix group (P=0. 001, versus the high-dose group). At month 6 (before administration of the booster dose), seroconversion rates were 96.9%in the high-dose group, 65.0%in the low-dose group (P=0.0029), and 68.8%in the Havrix group (P=0.007). All subjects in all groups seroconverted by one month after receipt of the booster dose. Geometric mean titers were similar in all three groups at month 1, but were higher in the high-dose group (264 mlU/ml) than those in the Havrix group (135 mlU/ml) at month 6 (P=0.0013). One month after the booster dose, geometric mean titers in the high-dose group (2747 mlU/ml) were higher than those in the low-dose group (1657 mlU/ml) (P=0.0223) or in the Havrix group (1316 mlU/ml) (P=0.01). Conclusions This new inactivated hepatitis A vaccine is immunogenic and safe; two doses of either 500 or 1000 units can induce hepatitis A virus antibodies well above the protection level. Chin Med J 2002; 115(10): 1483-1485

Objectives To investigate the effects of glucose and free fatty acids (FFAs) on the proliferation and cell cycle of human vascular endothelial cells in vitro, and to examine whether the combined presence of elevated FFAs and glucose may cross-amplify their individual injurious effects. Methods Cultured human vascular endothelial cells (ECV304) were incubated with various concentrations of glucose and/or FFAs (palmitate and/or oleate) for 24 - 96 h. Morphologic alterations were observed using a phase contrast microscope and an electron microscope. Inhibition of proliferation was measured by a colorimetric 3-[4, 5-dimethyl thiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. Cell viability was determined using trypan blue exclusion. Distribution of cells along phases of the cell cycle was analyzed by flow cytometry. Results Glucose 15 or 30 mmol/L, palmitate (PA) 0.25 or 0.5 mmol/L, and oleate (OA) 0.5 mmol/L inhibited proliferation and accelerated death of endothelial cells in a dose-and-time-dependent manner. After treatment with elevated glucose and/or FFAs, the G0/G1 phase cells increased, whereas S phase cells decreased, suggesting that high glucose and/or FFAs mainly arrested endothelial cells at G0/G1 phase. The inhibitive rates of proliferation and population of dead cells in endothelial cells incubated with glucose plus FFAs (glucose 30 mmol/L + PA 0.25 mmol/L, glucose 30 mmol/L + OA 0.5 mmol/L, glucose 30 mmol/L + PA 0.25 mmol/L+OA 0.5 mmol/L) increased more markedly than those treated with high glucose or FFAs (PA and/or OA) alone. Conclusion Both high ambient glucose and FFAs can inhibit proliferation and accelerate death of endothelial cells in vitro. These changes were cross-amplified in the combined presence of high levels of glucose and FFAs. Chin Med J 2002; 115(10): 1486-1490

Objective To evaluate the efficacy of a novel compound derived from Traditional Chinese Medicine (TCM)for treatment of alveolar echinococcosis in the Kuming mouse. Methods The compound was extracted from TCM by serial procedures including amalgamation of TCM drugs, boiling in a beaker, cold condensation using a reflux instrument, evaporation by mild heating and finally, formation of a deep brown powder. An animal model of alveolar echinococcosis was established concurrently. A bit of alveococcus tissue derived from alveolar echinococcosis in the mouse was transplanted into the peritoneal cavity of the musculus of a Kuming mouse. The duration of infection after inoculation was classified as either 1 week or 10 weeks; the animals were again divided into treated groups and control groups. Estimation of the alveococcus inhibitory rate and electron microscopic observation served as the main methods for assessment of efficacy. The infected mice in the treated groups were intra-gastrically administered once per day with the TCM extractive powder at doses of 20 mg • kg-1 for three months continuously. All the animals were sacrificed and examined half a month after the end of TCM treatment. Results Total wet weight of alveococcus collected from the 9 mice in the treated group after one week of infection was 37.8 g (±s, 4.196±2.090 g), which was markedly lower than the amount collected from 10 mice of the control group (121 .3 g, ±s, 12.1 ±4.3 g) . Therefore, the inhibitory rate of the alveococcus was 65.7%(P <0.01). Similarly, the total wet weight of alveococcus from 7 mice of the treated group after 10 weeks of infection was 4.3 g (±s, 0.62 ±1.21 g), which was much lower than the amount collected from 6 mice of the control group (17.85 g, ±s, 2.93±3.28 g). The inhibitory rate of alveococcus was 80.6%(P <0.01) . Furthermore, the ultrastructural appearances of alveococcus showed obvious differences between the two groups. Conclusion TCM extractive powder for treatment of murine alveococcosis is considered to be a hopeful anti-echinococcus compound it is a topic for further study. Chin Med J 2002; 115(10): 1576-1578

Objective To investigate the cross-reactivity between glutamic acid decarboxylase (GAD)-I-A97 and I-Ad tetramer in diabetes-prone non-obese diabetic (NOD) mice (I-A97) and diabetes-free Balb/c mice (I-Ad). Methods Two GAD peptide I-A97 and I-Ad tetramers were generated and compared for phenotype and function of sorted GAD peptide I-A97 and I-Ad tetramer-positive (tet+) T cells. Results The cross-reactivity is shown in either tetramer positive percentage or tetramer staining intensity. The NOD and Balb/c derived-tet+ T cells were able to be cross-stained by GAD peptide I-A97 and I-Ad tetramers, and responded to both irradiated NOD and Balb/c splenotyes under stimulation by synthetic and recombinant GAD peptides. Conclusion Although I-A97 and I-Ad are closely related in biochemical and biological aspects, their most notable difference is the presence or absence of a negatively charged residue at position β57 that links to insulin-dependent diabetes mellitus. Chin Med J 2002; 115(10): 1579-1581

Purpose To review 54 cases of human myiasis in China reported from 1995 - 2001 in order to characterize the current epidemic situation. Date sources All reference data originated from some Chinese literature in Chinese medical journals. Twenty-four short reports published from 1995-2001 were selected according to the stated purpose. Due to the varied outcomes of myiasis at different sites in the body, the present paper is divided into 7 types of infection: ocular, nasal cavity, external auricular, cutaneous, gastrointestinal, urogenital, and traumatic myiasis . Results Of these 54 selected cases of myiasis in China from 1995 - 2001, hypodermic myiasis (31 patients) and ocular myiasis (12 patients) were the most common, comprising 43 cases (80%). The cases were equally distributed amongst both genders. Furthermore, most of the infected were children and infants, totaling 39 cases, or 72%. The regional distribution of myiasis was widespread throughout the sixteen provinces and autonomous regions in question. Conclusions Although not always reported, myiasis is a common zoonotic parasitosis occurring in most of China's territories. Due to its prevalence, preventive measures for myiasis are urgently needed. Chin Med J 2002; 115(10): 1445-1447