Volume 114 · Issue 01 · 2001

Objective To study whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH-1) and multidrug resistance gene (MDR1) have increases resistance to 4-Hydroperoxycyclo-phosphamide (4-HC) and P-glycoprotein effluxed drugs. Methods A bicistronic retroviral vector G1 Na-ALDH1 -IRES-MDR1 was constructed and used to transfect the packaging cell lines GP+E86 and PA317 by LipofectAMINE method, using the medium containing VCR and 4-HC agents for cloning selection and ping-ponging supernatant infection between the ecotropic producer clone and the amphotropic producer clone, we obtained high titer amphotropic PA317 producing cells with high titers up to 5.6 x 105 CFU/ml. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human ALDH-1 and MDR1 cDNA under the stimulation of hemopoietic growth factors. Results Bicistronic retroviral vector construction was verified by restriction endonuclease analysis. Polymerase chain reaction (PGR), reverse transcription (RT)-PCR, Southern blot, Northern blot,fluorescenceactivated cell sorting (FACS) method and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) analyses showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgenes recipient cells confered 4-fold stronger resistance to 4-HC and 5.5 to 7.2-fold P-glycoprotein effluxed drug than untransduced cells. Conclusion The bicistronic retroviral vector-mediated transfer of two different types of drug resistance genes into human cord blood CD34+ cells and co-expression provided an experimental foundation for improving combination chemotherapy tolerance in tumor clinical trial. Chin Med J 2001; 774(7): 25-29

Objective To study the effect of all-trans retinoic acid (ATRA) and arsenic trioxide (AS2O3) on tissue factor (TF) expression and procoagulant activity (PCA) of acute promyelocytic leukemia (APL) cells in vivo and in vitro. Methods PCA from freshly isolated APL blasts from APL patients treated with ATRA or AS2O3 was detected using a one-stage clotting assay. TF antigen was detected by ELISA and TF mRNA by RT-PCR. The maturation sensitive (NB4) or resistant subclones (NB4-R1) of the promyelocytic NB4 cell line, as well as U937 cells infected with pMSCV-PML-RARa treated with or without ATRA or As2O3, were also examined. Results Both ATRA and AS2O3 can down-regulate the TF antigen, its mRNA transcription and membrane PCA of APL cells in vivo and in vitro, in a time-dependent manner. The TF antigen level in PML-RARa+U937 cells was significantly higher than that in U937 cells infected with retrovirus vector. Both ATRA and AS2O3 can also down-regulate the TF antigen in U937 cells transfected with or without PML-RARa. Conclusion Tissue factor expression and PCA in APL cells may be down-regulated by ATRA and AS2O3. By down-regulating TF expression, AS2O3 might also be used to improve the DIG-related hemorrhage in APL. Our data indicate that elevated TF antigen in PML-RARa+ U937 may be related to the fusion protein PML-RARa. The down-regulating effect of ATRA and AS2O3 on TF expression in U937 cells might not involve this fusion protein. Chin Med J 2000; 114(1): 30-34

Objective To investigate genes involved in cancer metastasis, mRNA differential display was used to compare the levels of gene expression in two cell sublines derived from human giant cell carcinoma of lung(PG) which had different metastatic potentials. Methods Using in vivo tumorigenicity and a spontaneous metastasis assay in nude mice, two sublines(BE1, LH7) from human giant cell carcinoma of lung (PG) with different metastatic potentials were isolated and characterized. mRNA differential display was used to compare the levels of gene expression between them and the obtained results were confirmed by Northern hybridization. Results One differentially expressed band was nearly identical (99%homology) to Ras-GTPase-Activating protein SH3 domain binding protein (G3BP). G3BP displayed a strong expression in LH7 (non-metastatic in recipient nude mice) and a very weak expression in BE1 (100%metastatic frequency). The same different expression level of G3BP was detected in Northern hybridization with another panel of cell sublines with different metastatic potentials (established in our lab) derived from human prostate carcinoma cell line PC-3M. Conclusion Our results indicate that G3BP was implicated in cancer metastasis because of its differential expressions in the two panels of cell sublines with different metastatic potentials. Chin Med J 2001; 114 (1): 35-38

Objective To investigate the relationship between p53 gene and tumors of adipose tissue at the level of protein and gene. Methods Immunohistochemical LSAB, PCR-SSCP and DNA sequencing were used in 82 cases. Results p53 protein is expressed only in liposarcomas, in which the positive staining rate was 48.08%(25/52). In different subtypes of liposarcomas, the positive staining rate in well differentiated liposarcomas was 30.00%(9/30), which is much lower than that of the poorly differentiated liposarcomas (P<0.005). Abnormality in the single-stranded DNA pattern was determined in 2 samples (pleomophic liposarcomas) by PCR-SSCP analysis. Missense mutations in exon 8 codon 268 of p53 gene (AAC→ATC) were detected by DNA sequencing. Another heterozygotic cosense mutation may exist at exon 6 codon 221 of p53 gene(GAG→GAA). Conclusions The data suggest that the p53 protein has a relationship with development, differentiation and malignancy of liposarcoma. Detecting the level of p53 protein expression may be valuable in evaluating the level of differentiation and malignancy of liposarcoma. There appear point mutation on exon 8, 6 of p53gene. Chin Med J 2001; 114(1): 39-43

Objective To study the histopathologic features and pathogenesis of mucosa-associated lymphoid tissue lymphoma (MALT-oma) of salivary glands. Methods Clinical data, paraffin-embedded sections, immunohistochemical slides (SP method) and electron microscopic features of surgical specimens of 32 cases of salivary gland MALT-oma were studied. Results The patients were 27 males and 5 females, with a mean age of 54.76 years. The lesions were located in the parotid area in 17 cases, and in the submandibular gland in the remaining 15 cases. Much of the MALT-oma was replaced by infiltration of a great amount of centrocyte-like cells (CCL) as background and occasional large cells (centroblast- or immunoblast-like). In MALT-omas "lymphoepithelial lesions" were present. Immunohistochemically, CD20 expression was found to be positive and CD45RO expression was negative in all MALT-omas. Conclusion Most of the MALT-omas are low grade malignant tumors and have a "homing back" phenomenon. The cases were managed by surgery and chemotherapy. In a few MALT- omas which turned into high grade malignant tumors, the prognosis was poor. Acquired MALT may develop as a reaction to autoimmune disease and infection. Hyper-immune reaction and MALT hyperplasia under stimulation may result in myoepithelial sialadenitis and lead to MALT-oma of the salivary gland. Chin Med J 2001; 774(7): 44-47

Objective To get insight on the regulatory mechanism of Ki-67 gene expression in malignant cell cycle. Methods Non-radioactive in situ hybridization (ISH) was undertaken, combined with immunohistochemistry to study the Ki-67 gene transcription and translation in various human cells and tissues. HeLa cells and fresh colon cancer cells, tonsil, normal pancreas and pancreatic cancer tissues were used in this study. A 435 bp cDNA fragment located in exon 13 of the Ki-67 antigen gene was amplified by polymerase chain reaction (PCR). Digoxigenin-labelled antisense and sense RNA probes were prepared for detecting Ki-67mRNA, combined with MIB-1 immunohistochemistry. Results Successful localization of Ki-67 mRNA in human HeLa cells, colon cancer cells, tissues specimen of the tonsil and pancreatic cancer tissue sections was accomplished by digoxigenin-labelling in situ hybridization technique. ISH to colon cancer cells and pancreatic cancer tissue slides showed that much stronger cytoplasm and perinuclear mRNA signals of the Ki-67 gene were present in malignant cells than in normal cells, which was in accordance with MIB-1 nuclear protein signals. Conclusions A sensitive and practical in situ hybridization method for the analysis of Ki-67 antigen mRNA in human cell and tissue was developed. Abnormal transcription of exon 13 of Ki-67 gene might be responsible for malignant cell proliferation in colon and pancreatic cancer. Chin Med J 2001; 114(1): 48-53

Objective To evaluate the clinical application of helical CT multiplanar reformation (MPR), three-dimensional reconstruction (3D) and virtual laryngoscopy (CTVL) in laryngeal and hypopharyngeal carcinoma. Methods Axial helical CT scans were performed in 22 patients with laryngeal or hypopharyngeal carcinoma, along with MPR, 3D and CTVL. These results were compared with the findings of fiber optic laryngoscopy and surgery. Results Combining axial and MPR images, both the accuracy in preoperative tumor staging and the diagnosis of metastatic lymph nodes were 95%. MPR demonstrated more information about the extent of tumor than axial images in 23%cases; 3D image displayed clearly the extension of tumor, the vessels and airway from multiple views. The location, size and extent of tumors found in cranio-caudal CTVL corresponded well with that of laryngoscopy, and CTVL demonstrated the relationship between the tumor and vocal cords and anterior commissure by caudo-cranial approach, which was inaccessible to fiber optic laryngoscopy in 3 cases. Conclusions Axial images of helical CT clearly demonstrate the location, size and extent of laryngeal and hypopharyngeal carcinoma, while MPR and 3D images are useful in displaying the three-dimensional images and anatomical relation of the tumor. CTVL can clearly display the mucosal surface structures of the larynx and hypopharynx and is a good complementary method of laryngoscopy. Chin Med J 2001; 114(1): 54-58

Objective To assess the efficacy and safety of intracoronary stenting in the acute phase of unstable angina pectoris (UAP). Methods Fifty-five patients with UAP were randomized to early (Group Ⅰ, n=29) and delayed interventional treatment (Group Ⅱ, n=26). Coronary angiography and stenting were performed within 48hours in Group I and 7-10 days later in Group Ⅱ . Procedural success rate, time interval from admission to angina relief and duration of hospitalization were recorded. Cardiac events within 30 days were observed as well. Results Clinical characteristics and angiographic features were similar between the two groups. There was no significant difference in the procedural success rate (93%versus 96%), but the cardiac event rate within 30 days was significantly lower in Group I than in Group Ⅱ (0%versus 9.2%, P <0.05). The time interval from admission to angina relief (4.4 ± 3.1 days versus 5.7 ± 2.9 days) and the duration of hospitalization (8.8 ± 3.2 days versus 13.5 ± 3.1 days) were significantly reduced in Group Ⅰ(both P<0.05). Conclusions Intracoronary stent implantation is effective and safe in the acute phase of UAP. Early percutaneous coronary intervention results in rapid improvement in symptomatology and a shorter hospitalization. Its long-term effect has to be confirmed in a future randomized study. Chin Med J 2001; 114(1): 59-61

Objective To examine the effect of endovascular irradiation on restenosis after balloon angioplasty in rabbit. Methods After the establishment of rabbit iliac atherosclerosis model, balloon angioplasty was performed at the lesion segment of the iliac artery. Rabbits were randomly divided into three groups: control group, 10Gy irradiated group and 18 Gy irradiated group. Endovascular irradiation was performed for irradiated groups at the dilated sites by introducing the 192lr radioactive guidewire through a catheter. After four weeks, the animals were killed and the target segments were cut down. Histopathologic and morphometric analyses were carried out. Results The mean final lumen area in the 18 Gy group was larger than that in the control or 10 Gy group(P<0.05) . The intimal area in the 18 Gy group was smaller (P<0.05). Conclusions 192lr endovascular irradiation may prevent restenosis after balloon angioplasty. The effect is related to the delivered dose. The mechanism is involved in inhibition of neointimal proliferation. Chin Med J 2001; 114(1): 62-63

Objective To identify aldosterone synthase gene-CYP11B2 mRNA expression in normal and fibrotic liver in rats and evaluate the curative effect of antisterone. Methods 160 Wistar rats weighing about 250 g were divided into 4 groups. In the model group (n=40),the rats were injected with 40%CCI4 (0.25 ml/100 g) subcutaneously three times a week. In the antisterone group (n=40), the rats were injected with 40%CCI4 (0.25 ml/100 g) subcutaneously three times a week. Antisterone equivalent to 20 mg•kg-1 • d-1 was given intragastrically (ig) . In the malotilate group (n=40), the rats were injected with 40%CCI4(0.25 ml/100 g) subcutaneously three times a week. Malotilate equivalent to 50 mg•kg-1 • d-1 was given ig. In the control group (n=40), the rats were injected with olive oil only. After 2, 4, 6, 8 and 10 weeks, animals were sacrificed, and morphological examination was carried out. The area of collagen was examined with an Image Analyse System. Expression of the aldosterone synthase gene, CYP11B2 mRNA, in fibrotic and normal liver was detected by means of reverse transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization. Results In situ hybridization and RT-PCR showed that the expression of CYP11B2 mRNA, which localized in the endoplasm of hepatic stellate cells (HSCs), was up regulated when fibrogenesis occurred. Histological observation indicated that the grade of fibrosis and the area of collagen in the antisterone group were less than those in model group before 6 weeks (P <0.05). There was no significant difference between the antisterone and malotilate groups (P> 0.05) . After that, however, the grade of fibrosis and the area of collagen in the antisterone group were higher than those in the malotilate group (P <0.05). There was no significant difference between the antisterone and model groups (P> 0.05). Conclusions The expression of CYP11B2 mRNA is up regulated in fibrotic liver. Antisterone can have a partial fibrogenesis-inhibiting effect in the early stages. Chin Med J 2001; 114(1): 64-68

Purpose To investigate the role of cationic antimicrobial protein of Mr 37 kDa (CAP37) a neutrophil-derived inflammatory mediator on endothelial cell function. Data sources Endothelial cells used in this study were obtained from human lung microvessels and rat aorta. The latter was a kind gift of Dr. Paula Grammas. The mono-mac 6 cell line used in this study was the generous gift of Dr. H.W. Loms Ziegler-Heitbrock. Study selection and data extraction Endothelial cell proteins kinase C activity was determined by measuring calcium- and phospholipid-dependent phosphorylation of histone. Endothelial cell migration was determined using Costar™ Transwell apparatus. Cell surface expression of adhesion molecules, ICAM-1and PECAM-1 was determined using flow cytometry. RT-PCR was used to amplify the CAP37 from endothelial cells treated with LPS . Results We demonstrated that CAP37 which was originally identified as having potent antimicrobial activity and chemotactic activity for monocytes was capable of modulating endothelial cell functions. CAP37activated endothelial cell protein kinase C in a dose- and time-dependent fashion. Importantly CAP37increased the adhesive properties of the endothelium for monocytes. CAP37 upregulated the well known adhesion molecules, ICAM-1 and PECAM-1 in a dose- and time- dependent manner. In addition, CAP37promoted endothelial cell migration. Further investigations indicated that CAP37 was induced in endothelial cells in response to pro-inflammatory cytokines such as tumor necrosis factor-α and interleukin-1α as well as inflammatory mediators such as lipopolysaccharide. Unstimulated endothelial cells did not constitutively express CAP37. The cDNA sequence of endothelial CAP37 was determined and found to be highly homologous to the sequence obtained for neutrophil-derived CAP37. Conclusions Our studies strongly suggest that CAP37 plays a pivotal role in monocyte-endothelial interactions and the transmigration of monocytes from the vasculature into extravascular tissues. Chin Med J 2000; 114(1): 9-13

Objective To study the pathologic features of occult lymphatic metastasis in supraglottic carcinoma. Methods Serial sections of 153 neck dissection specimens in 100 patients with supraglottic carcinoma were evaluated under the microspcope. Results In 100 patients, 38 had occult metastatic lymph nodes. 51 metastatic lymph nodes were found in pathology, and their sizes ranged from 0.5 cm to 2.6 cm (average 1.1 cm). The distribution of 51 lymph nodes was 1 in level I (2%), 37 in level Ⅱ (73%), 12 in level Ⅲ (24%), and 1 in level N (2%). Among the 51 nodes, 21 (41 %) were early stage, 18 (35%) were growth stage, 7 (14%) were tull stage, and 5 (10%) were extracapsular stage. The differentiation degree and appearance of supraglottic carcinoma was not directly related with occult metastasis. Conclusion The occult metastatic rate of supraglottic carcinoma is high, and selective neck dissection may be necessary. Chin Med J 2001; 114(1): 88-89

Objective To investigate the prevalence of significant enterococcal isolates from urine and determine what factors are associated with the increased prevalence, with particular reference to antibiotic susceptibilities. Methods Retrospective analysis over an 8-year period of hospital laboratory records of urinary isolates of enterococci was done. Species were identified via colony morphology, growth in 6.5%sodium chloride and their ability to hydrolyze esculin in the presence of 40%bile salts. Susceptibility testing via the disc diffusion technique with 9 commonly used antibiotics was also done as defined by the National Committee for Clinical Laboratory Standards. Results From 39 881 urine specimens, 9116 (22.9%) were culture positive. Of this 9116, 1001(11.0%) were enterococci, the 4th most common urinary isolate. E. coli was the most common(36.2%). Most enterococci were from pediatric patients (28.4%) and the urology unit (24.5%). All enterococci were fully sensitive to ampicillin and augmentin (amoxicillin-clavulanic acid) . Sensitivity to gentamicin decreased significantly from 79%in 1990 to 58%in 1997 (P <0.005) . Sensitivity to the cephalosporins and nitrofuratoin were relatively stable, but sensitivity to nalidixic acid varied. No resistance to vancomycin was detected during the study, and no cases of bacteremia complicated bacteriuria were seen. Conclusion Isolation of enterococci was relatively stable during the 8-year period, and all isolates were fully sensitive to the older β-lactams^ ampicillin, cefaclor and augmentin, but displayed varying degrees of multi-resistance to other commonly used urinary agents such as nalidixic acid and co-trimoxazole(trimethoprim-sulfamethoxazole). Because of the emergence of multi-resistant enterococci in many countries and the high cost of drugs in our society, it is imperative that vigilance be maintained in monitoring enterococcal infections in hospitals. Chin Med J 2001; 114(1): 90-92

Objective To compare the endocytic routes of exogenous antigen between murine dendritic cells (DCs)and macrophages (Mϕs). Methods Murine bone marrow-derived DCs and peritoneal Mϕs were pulsed with horseradish peroxidase(HRP)-5 nm colloidal gold for 10 minutes, then grouped and chased for 0-120 minutes in culture medium. Intracellular distribution of 5 nm colloidal gold was explored by means of the cellular enzymatic-chemistry of acid phosphatase and MHC Ⅱ immuno-cytochemistry under electron microscope. Results After 10 minutes of pulse with HRP-5 nm colloidal gold and 30 minutes of chase, most HRP-5 nm gold particles internalized by DCs entered into MHC class Ⅱ compartments (MⅡCs), and a small portion entered into acid phosphatase-positive lysosomes. In contrast to DCs, most Mϕs lysosomes were accessed by HRP-5 nm gold particles, and a small portion of HRP-5 nm gold particles entered into M Ⅱ Cs. After 60minutes of chase, 5 nm gold particles could hardly be seen within Mϕs, whereas most 5 nm gold particles were still retained in DCs. Conclusions The endocytic route of exogenous antigen in DCs seems to be different from that in Mϕs. Antigens taken by Mϕs mainly enter into lysosomes within 30 minutes. In the case of DCs, most internalized antigens enter into MⅡCs, which may be related to their unique antigen-presenting function. In addition, Mϕs seem to have more powerful capacity to scavenge exogenous antigen than DCs. Chin Med J 2001; 114(1): 93-96

Purpose and Methods Open-heart surgery with the use of cardiopulmonary bypass (CPB) is associated with an inflammatory cascade which contributes to the development of postoperative complications including multiple organ failure. To provide an update on the subject, we briefly review the recent English-language literature. Results During CPB, various factors have been recognized to induce a complex inflammatory response. Based on an enhanced understanding of the underlying mechanisms, therapeutic strategies have been developed to reduce this inflammatory reaction and its subsequent damaging effects. Off-pump coronary artery bypass grafting may result in less inflammatory injury as compared with the conventional maneuver,which can in turn, diminish the incidence of cardiac, renal, or neurological dysfunction. It is also clear that improving the biocompatibility of CPB materials can lead to a better patient recovery. Inasmuch as the pathophysiology involved appears to be multifactorial, it is unlikely that a single intervention could achieve the desired goal. Both pharmacologic strategies, such as steroid pretreatment, and modification of mechanical devices, such as the use of heparin-coated CPB circuits, could have important clinical implications. The balance between pro- and anti-inflammatory responses may be crucial in limiting the extent of inflammatory injury. Conclusions To date, the concept of organ protection should no longer be limited to the individual organ. Instead, investigations must be extended to focus on a systemic level. Chin Med J 2001; 114(1): 3-8