Synthesis and antifungal activities of N-1,3,4-thiadiazol-2-yl-4-oxo-thiochroman-2-yl-formamide derivatives / 药学学报

Thiochromanones and 1,3,4-thiadazoles as heterocyclic compounds have broad biological activities. In order to find novel compounds with antifungal bioactivity, substituted thiophenol and maleic anhydride were used to synthesize the intermediate 4-oxothiochromane-2-carboxylic acid. It was reacted with 2-amino-1,3,4-thiadiazol to get fourteen target compounds containing 1,3,4-thiadazole moiety. The structures of the obtained compounds were confirmed by 1H NMR, 13C NMR and HR-MS. All compounds were investigated for antifungal activity via microdilution broth method. The results showed that the target compounds 3a and 3c to Epidermophyton floccosum and Mucor racemosus exhibited better antifungal activity than the positive control fluconazole, in which the minimum inhibition concentration can reach 8 μg·mL-1 and 16 μg·mL-1. Compound 3e showed significant inhibitory activity to Helminthosporium maydis, Sclerotinia sclerotiorum and Botrytis cinerea compared with that of the positive control carbendazim. Compound 3b exhibited inhibitory activity to Helminthosporium maydis better than the positive control carbendazim.

Synthesis, biological activity and molecular docking research of N-{(4-oxo-thiochroman-3-yl)phenyl-methyl}acetamide derivatives as α-glucosidase inhibitors / 药学学报

In order to develop potent antidiabetic agents that have inhibitory effect to α-glucosidase, twelve β-acetamido ketone derivatives such as N-{[(substituted-4-oxo-thiochroman-3-yl)phenyl]-methyl}acetamide are designed and synthesized through one-pot Dakin-West reaction. Their chemical structures are confirmed by 1H NMR, 13C NMR, IR and HR-MS. In vitro α-glucosidase inhibition assays of compounds 4a-4l were carried out using glucose oxidase method. The result indicated that most of them possess inhibitory activity in vitro. Compound 4k showed the most potent inhibitory activity with 87.3% inhibition of α-glucosidase at the concentration of 5.39 mmol·L-1. The structure-activity relationship of these β-acetamido ketone derivatives was discussed preliminarily. Moreover, the molecular docking method was used to study the interaction mode of compound 4k and α-glucosidase. Our results will be helpful for designing of α-glucosidase inhibitors in the future.

Synthesis, biological activity and molecular docking research of N-{(4-oxo-thiochroman-3-yl)phenyl-methyl}acetamide derivatives as α-glucosidase inhibitors / 药学学报

In order to develop potent antidiabetic agents that have inhibitory effect to a-glucosidase, twelve β-acetamido ketone derivatives such as N-{[(substituted-4-oxo-thiochroman-3-yl)phenyl]-methyl}acetamide are designed and synthesized through one-pot Dakin-West reaction. Their chemical structures are confirmed by 1H NMR, 13C NMR, IR and HR-MS. In vitro α-glucosidase inhibition assays of compounds 4a-41 were carried out using glucose oxidase method. The result indicated that most of them possess inhibitory activity in vitro. Compound 4k showed the most potent inhibitory activity with 87.3% inhibition of α-glucosidase at the concentration of 5.39 mmol x L(-1). The structure-activity relationship of these β-acetamido ketone derivatives was discussed preliminarily. Moreover, the molecular docking method was used to study the interaction mode of compound 4k and α-glucosidase. Our results will be helpful for designing of α-glucosidase inhibitors in the future.

Expression of PSF1 in colon cancer tissues and its effect on the proliferation of colon cancer cells / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To detect the expression of PSF1 (partner of Sld five 1) in colon cancer specimens, and to explore the effect of RNA interference targeting PSF1 on the proliferation of colon cancer cells and its mechanism.</p><p><b>METHODS</b>Expression level of PSF1 protein in colon cancer specimens was detected by Western blot in 40 patients with colon cancer from May 2004 to December 2006. The short hairpin RNA (shRNA) plasmid targeting PSF1 was transfected into LOVO, HT-29 and HCT116 cells with liposome, then the expression level of PSF1 protein was measured by Western blot, the effect of PSF1 shRNA plasmid transfection on cell proliferation by MTT assay, anchorage-independent growth by soft agar colomy-formation assay, and PSF2, PSF3 and SLD5 mRNA expression by quantitative reverse transcription polymerase chain reaction.</p><p><b>RESULTS</b>The relative expression level of PSF1 protein in colon cancer tissues was 0.485±0.113, which was significantly higher than that in adjacent normal mucosa tissues (0.056±0.014, P<0.01). Western blot showed that the expression level of PSF1 protein was significantly decreased in colon cancer cells transfected with PSF1 shRNA plasmid. After PSF1 shRNA plasmid transfection, cell proliferation was significantly suppressed, the soft agar colony-forming rates of LOVO, HT-29 and HCT116 cells were significantly lower than those in control groups (P<0.05), meanwhile the expression levels of PSF2, PSF3 and SLD5 mRNA were significantly decreased (P<0.05).</p><p><b>CONCLUSIONS</b>PSF1 is significantly up-regulated in colon cancer tissues compared with adjacent normal mucosa tissues. ShRNA plasmid targeting PSF1 can inhibit the expression of PSF1 gene, suppress the proliferation of colon cancer cells, suggesting that it may be a new therapeutic target for colon cancer.</p>

Expression and clinical significance of GINS complex in colorectal cancer / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the expression and clinical significance of GINS complex in colorectal cancer (CRC).</p><p><b>METHODS</b>The expression level of GINS complex including PSF1, PSF2, PSF3 and SLD5 in CRC specimens (n=76) were detected by real-time fluorescent quantitative polymerase chain reaction. The association of GINS complex with clinicopathological parameters and prognosis of CRC patients were analyzed.</p><p><b>RESULTS</b>The relative expression level of PSF1, PSF2, PSF3, and SLD5 mRNA in CRC tissues was 0.001 853±0.000 651, 0.007 757±0.004 260, 0.000 967±0.000 481 and 0.003 248±0.001 721, which was significantly higher than that in normal colorectal mucosa tissues (0.000 352±0.000 169, 0.002 951±0.001 216, 0.000 472±0.000 271, and 0.001 675±0.001 156) (all P<0.01). PSF1 mRNA expression was associated with tumor size (P<0.01), and PSF2 mRNA expression with age (P<0.05) and lymph node metastasis (P<0.05). No correlations between PSF3 mRNA expression and clinicopathological parameters were observed. SLD5 mRNA expression was associated with lymph node metastasis (P<0.01). Patients with high expression of PSF1, PSF2 and SLD5 had worse 5-year overall survival rate (57.1%, 54.3%, and 54.3%) than those with low expression (77.1%, 80.0%, and 80.0%) (all P<0.05). Multivariable Cox regression analysis indicated that PSF1 mRNA expression (P<0.05) was an independent factor associated with prognosis of colorectal cancer.</p><p><b>CONCLUSIONS</b>Overexpression of GINS complex in CRC is associated with clinicopathological characteristics and prognosis of colorectal cancer. PSF1 expression is prognostic for CRC patients.</p>

Mullerian adenosarcoma of the uterus: A clinicopathologic analysis of 9 cases / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the clinicopathologic features, diagnosis, treatment and prognosis of uterine mullerian adenosarcoma.</p><p><b>METHODS</b>The clinicopathological data of 9 cases of uterine mullerian adenosarcoma in PUMC hospital from January 2003 to February 2009 were retrospectively analyzed.</p><p><b>RESULTS</b>There were 6 uterine endometrial adenosarcomas and 3 cervical adenosarcomas. The main clinical manifestations were abnormal vaginal bleeding and pelvic pain. Physical examination showed cervical/vaginal mass, enlarged uterus or pelvic mass. The adenosarcoma was characterized by benign or atypical-appearing neoplastic glands within a sarcomatous stroma. This stroma could appear as periglandular cuffs or intraglandular polypoid projections of increased cellular structure. The primary diagnostic rate was 66.7% and the most common clinical stage was stage I (7/9). All patients received surgical treatment and seven had postoperative chemotherapy, radiotherapy or hormone therapy. Conservation of unilateral ovary or bilateral ovaries was performed in 5 cases. Three patients underwent local excision, which resulted in the preservation of reproductive function. During the follow-up, 2 cases of uterine endometrial adenosarcoma recurred. One patient of clinical stage III containing sarcomatous overgrowth died from recurrence 13 months after surgery. The other one recurred 2 years after local excision of the tumor in the uterine cavity and she remained healthy since hysterectomy.</p><p><b>CONCLUSION</b>Uterine mullerian adenosarcoma is a rare tumor without specific clinical symptoms and signs. The diagnosis depends on pathomorphologic examination. The tumors show low malignant potential and the vast majority are at early stage. Surgical excision is the main treatment strategy with a good prognosis in the early stage disease with complete removal of tumors. The prognosis is poor in advanced adenosarcoma with sarcomatous overgrowth. Due to the relatively high rate of recurrence, long-term follow-up is recommended.</p>

Selectivity for the rate of frequency-modulated sweeps and its affecting factors in the inferior collicular neurons of mouse / 生理学报

Both animal communication sounds and human speech contain frequency-modulated (FM) sweeps. Although the selectivity for the rate of FM sweeps in neurons has been found in many kinds of animals at different levels of the central auditory structures, the underlying neural mechanism is still not clear. Using extracellular single unit recording techniques, we examined the selectivity for the rate of FM sweeps in the inferior colliculus (IC) neurons of the Kunming mouse (Mus musculus, Km) in the free-field stimulation conditions and determined its affecting factors. Totally, 102 neurons were recorded successfully, among which 42 neurons (41.2%) displayed a duration tuning pattern under pure tone (PT) stimulus. The percentages of short-pass, band-pass, and long-pass neurons were 22.6% (23/10), 8.8% (9/102), 9.8% (10/102), respectively. The other 60 neurons (58.8%) did not show any duration tuning features. Under FM stimulus, the majority of duration tuning neurons (78.6%, 33/42) showed the selectivity for the rate of FM sweeps. For these neurons, the type of rate selectivity was determined by the duration tuning features, but it was not related to the modulation range (MR) of FM. In a small fraction of duration tuning neurons (21.4%, 9/42), the rate selectivity was correlated with the MR, but uncorrelated with the duration tuning features. On the other hand, more than half of the non-duration tuning neurons (53.3%, 32/60) exhibited the rate selectivity under FM stimulus, and almost all of them (31/32) showed fast-rate selectivity. Nevertheless, there were 8 neurons (in 32) displaying the same best rate at different MR, indicating that they were real rate-selective neurons. Our results indicate that the selectivity for the rate of FM sweeps is co-determined by duration tuning features and sweep bandwidth. Only a few of inferior colliculus neurons belong to real rate-selectivity neurons in the mouse.

Gene expression profile difference between colorectal cancer tissue and pericancerous mucosa by DNA microarray / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To study the difference of gene expression profile among colorectal cancer tissue, pericancerous mucosa and normal mucosa, and to screen associated novel genes in colorectal carcinogenesis by DNA microarray.</p><p><b>METHODS</b>cDNA chip containing approximate 8000 genes was used to detect differentially expressed genes in colorectal cancer tissues, pericancerous mucosa and normal mucosa, and to screen associated novel genes in colorectal carcinogenesis by DNA microarray.</p><p><b>RESULTS</b>As compared with normal mucosa, 769 genes differentially expressed in cancerous tissue were identified, which included 363 up-regulated and 406 down-regulated genes. In pericancerous mucosa 3 cm away from cancerous tissues, 155 genes differentially expressed were identified, of whom 52 genes were up-regulated and 103 were down-regulated. In pericancerous mucosa 5 cm away from cancerous tissues, 230 genes differentially expressed were identified, of whom 46 genes were up-regulated and 184 genes were down-regulated. The genes expressed differentially were associated with several functional types. According to the primary results, the differentially expressed genes with prominent functions included tumor-related genes, genes regulating cell proliferation and apoptosis, transcriptional control genes, and construction and degradation of extracellular matrix-associated genes. The cancerous mucosa was obviously different from the normal mucosa(about 20%, 769/3944). The differences between the normal mucosa and pericancerous mucosa were relatively small (3.9%,5.8%).</p><p><b>CONCLUSIONS</b>Different tissues have their own biological property. Several genes play roles in the development of colorectal carcinogenesis. Genes in adjacent non-cancerous tissues are also expressed differentially, leading to a malignant change.</p>

Hepatitis B virus X protein suppressing adriamycin-induced apoptosis of HepG2 cells / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate the effect of hepatitis B virus X protein (HBx) on adriamycin-induced apoptosis of hepatocellular carcinoma cells.</p><p><b>METHODS</b>HBx gene fragment was amplified from subtype adr HBV plasmid by PCR, and inserted into Hind III and Kpn I sites of green fluorescent protein (GFP) eukaryotic expression vector pEGFP-C1 to construct recombinant pGFP/HBx. The pEGFP-C1 and pGFP-HBx were introduced into HepG2 cells by Lipofectamine 2000 to obtain HepG2 cells expressing GFP. GFP-HBx fusion protein was selected using G418. The expression of HBx gene was demonstrated by RT-PCR analysis. HepG2, HepG2/GFP and HepG2/GFP-HBx cells were treated with adriamycin (2.5 microg/ml), and apoptosis of the cells was determined by their morphological changes, trypan blue exclusion, and flow cytometry analysis.</p><p><b>RESULTS</b>Under a fluorescence microscope, visible expression of GFP and GFP-HBx fusion proteins were observed in HepG2/GFP and HepG2/GFP-HBx cells, even after growing over 70 generations. RT-PCR analysis showed that HBx gene was expressed in HepG2/GFP-HBx cells. Trypan blue exclusion showed adriamycin induced time-dependent cell death in HepG2 and HepG2/GFP cells while no significant cell death was observed in HepG2/GFP-HBx cells. Flow cytometry analysis showed that apoptosis rates in HepG2/GFP-HBx (3.94%) cells were significantly lower than those in HepG2 (59.03%) and HepG2/GFP cells (61.38%) at 36 hours after the adriamycin treatment (P < 0.01). No significant differences of apoptosis rates of HepG2/GFP-HBx (3.94%) and of the untreated cells (2.12%, 2.78%, 2.55%) (P > 0.05) were observed.</p><p><b>CONCLUSION</b>A HepG2 cell line expressing GFP and GFP-HBx fusion proteins was successfully established. HBV X protein blocks adriamycin-induced apoptosis of these HepG2 cells.</p>

Effect of a selective inducible nitric oxide synthase inhibitor on cell growth in human colorectal cancer Lovo cell line / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of a selective inducible nitric oxide synthase(iNOS) inhibitor, aminoguanidine (AG), on the proliferation and apoptosis of human colorectal cancer (CRC) Lovo cell line, and explore its possible mechanism.</p><p><b>METHODS</b>MTT assay was used to detect the inhibition of Lovo cell growth by aminoguanidine. Apoptosis and cell cycle of Lovo cells were examined by flow cytometry (FCM). Morphologic change of Lovo cell treated by AG was observed with AO/EB staining.</p><p><b>RESULTS</b>There were significant differences in 0.5 mmol/L and 1.0 mmol/L AG groups as compared to the control group (P<0.05). The absorbance (A) values of Lovo cells in each time point were significantly different (P<0.05). Growth of Lovo cells was inhibited by aminoguanidine in a dose- and time-dependent manner. FCM analysis showed that the cell ratio of G(0)/G(1) phase increased with the increasing of the concentration of aminoguanidine, but the cell ratio of S-and G(2)/M phase decreased correspondingly (P<0.05). S phase fraction and proliferation index (PI) decreased remarkably, and the apoptotic rate of Lovo cells increased. After AG treatment, AO/EB staining revealed some apoptotic morphological features such as cell shrinkage, nuclear condensation, DNA fragmentation, and formation of apoptosis bodies.</p><p><b>CONCLUSIONS</b>Aminoguanidine inhibits the proliferation and facilitates the apoptosis of human CRC Lovo cells. One of the mechanisms may be explained as blocking the progress of cell cycle of CRC Lovo cells by aminoguanidine.</p>

Effect of all-trans retinoic acid on drug sensitivity and expression of survivin in LoVo cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>All-trans retinoic acid (ATRA) can influence the tumor cell proliferation cycle, and some chemotherapeutic drugs are cycle specific. In this study, we hypothesize that ATRA can enhance chemotherapeutic drug sensitivity by affecting the cell cycle of tumor cells.</p><p><b>METHODS</b>The cell cycle of LoVo cells was evaluated using flow cytometry (FCM). Cell viability was analyzed using the MTT assay. The morphologic changes in the treated LoVo cells were measured with acridine orange (AO)/ethidium bromide (EB) staining. Expression of survivin in LoVo cells was analyzed by immunofluorescence assay.</p><p><b>RESULTS</b>After LoVo cells were treated with ATRA, the G0/G1 ratio of the tumor cells increased and the cell ratio of S- and G2/M-phase decreased. Viability of the cells decreased significantly after combined treatment with ATRA and 5-fluorouracil (5-FU) or mitomycin c (MMC) and was evaluated by fluorescence microscopy. Expression level of survivin in the tumor cells decreased after ATRA combination treatment.</p><p><b>CONCLUSIONS</b>ATRA enhances drug sensitivity of the LoVo cell line to cell cycle-specific agents and inhibits the expression of survivin in LoVo cells. The combination of ATRA and 5-FU or MMC promoted cell apoptosis, and the mechanism involved in apoptosis may be related to inhibition of survivin gene expression.</p>

Effect of different treatments of dentin surface on sheer bond strength between different bonding agents and dentin / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To evaluate the effect of Zinc oxide-eugenol cement and Gluma desensitizer on the sheer bond strength of three kinds of dentin bonding agents. The three dentin bonding agents were Zinc phosphate cement, Glass ionomer cement and Super-Bond C&B. To find the theory depending for the using of different protective methods and the selecting of different kinds of dentin bonding agents in prepared abutment teeth.</p><p><b>METHODS</b>The buccal surfaces of ninety freshly extracted human premolars were flattened to expose an adequate area of lower dentin. Followed by wet-grinding on a series of silicon carbide paper from number 320, 400, 600 grit to produce the dentin bonding surface. The teeth roots were embedded in self-curing resin with the crown out of the resin. The embedded ninety teeth were divided randomly into three groups. The group A was control and the dentin surfaces were not treated. The group B was covered with a paste of Zinc oxide-eugenol cement. The group C was covered with Gluma desensitizer. Calculating the sheer strength between three bonding agents and dentin after the two treatments of dentin surface. The results were statistically assessed with SPSS software. Dentin surfaces were observed with scanning electron microscope (SEM).</p><p><b>RESULTS</b>The sheer bond strengths of Zinc phosphate cement had significant decrease (P<0.05), especially the C1 group. The sheer bond strengths of Glass ionomer cement and Super-Bond C&B had no significant difference.</p><p><b>CONCLUSION</b>Zinc oxide-eugenol cement and Gluma desensitizer could reduce the sheer bond strength of Zinc phosphate cement with dentin surface. Zinc oxide-eugenol cement and the Gluma desensitizer could not effect Glass ionomer cement and the Super-Bond C&B with dentin.</p>

Analysis of the inhibitory effect of gypenoside on Na(+), K (+)-ATPase in rats' heart and brain and its kinetics / 中国结合医学杂志

<p><b>OBJECTIVE</b>To study the effects of gypenoside (Gyp) on the activity of microsomal Na(+), K(+)-ATPase in rat's heart and brain in vitro.</p><p><b>METHODS</b>The microsomal Na(+), K(+)-ATPase was prepared from rat's heart and brain by differential centrifugation. The activity of microsomal Na(+), K(+)-ATPase was assayed by colorimetric technique. Enzyme kinetic analysis method was used to analyze the effect of Gyp on the microsomal Na(+), K(+)-ATPase of rats.</p><p><b>RESULTS</b>Gyp reversibly inhibited the brain and heart's microsomal Na(+), K(+)-ATPase in a concentration-dependent manner, and showed a more potent effect on enzyme in the brain. The IC(50) of Gyp for the heart and brain were 58.79+/-8.05 mg/L and 52.07+/-6.25 mg/L, respectively. The inhibition was enhanced by lowering the Na(+), or K(+) concentrations or increasing the ATP concentration. Enzyme kinetic studies indicated that the inhibitory effect of Gyp on the enzyme is like that of competitive antagonist of Na(+), the counter-competitive inhibitor for the substrate ATP, and the mixed-type inhibitor for K(+).</p><p><b>CONCLUSION</b>Gyp displays its cardiotonic and central inhibitory effects by way of inhibiting heart and brain's microsomal Na(+), K(+)-ATPase activities in rats.</p>

Reevaluation resection margin rectal cancer by flow cytometry and pathological examination / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the appropriate distal resection margin in rectal cancer patients.</p><p><b>METHODS</b>Thirty specimens of rectal carcinoma with total mesorectal excision(TME) were studied by flow cytometry and pathological examination. The differences of DNA ploidy status, DNA index (DI), proliferative index (PI), S-phase fraction (SPF) among rectal cancer, 3 cm and 5 cm below the tumor, normal rectum, distal mesorectum 3 cm and 5 cm below the tumor, and normal colon mesentery were analysed by flow cytometry, and were compared with the data of pathological examination.</p><p><b>RESULTS</b>Pathological examination showed that there was no tumor invasion 3 cm and 5 cm below the tumor,but the metastasis rates of distal mesorectum 3 cm and 5 cm below the tumor were 26.7% and 6.7% respectively. The DI, PI and SPF of rectal cancer by flow cytometric examination were significantly higher than those of distal rectum 3 cm and 5 cm below the tumor, and normal rectum (P<0.05). The DI, PI and SPF of distal rectum 3 cm below the tumor were also significantly higher than those of distal rectum 5 cm below the tumor, and normal rectum (P<0.05), but there were no significant differences between DI, PI and SPF of distal rectum 5 cm below the tumor and those of normal rectum (P>0.05). The rate of DNA aneuploid of tumor was significantly higher than those of normal rectum and distal rectum 5 cm below the tumor,but there was no significant difference between the rate of DNA aneuploid of tumor and that of distal rectum 3 cm below the tumor. The DI and DNA aneuploid of rectal cancer and distal mesorectum 3 cm and 5 cm below the tumor were significantly higher than those of normal mesorectum,but there were no significant differences between DI and DNA aneuploid of rectal cancer and those of distal mesorectum 3 cm and 5 cm below the tumor. The PI and SPF of rectal cancer were significantly higher than those of normal mesorectum and distal mesorectum 3 cm and 5 cm below the tumor.</p><p><b>CONCLUSIONS</b>Rectal cancer is able to invade distal rectum 3 cm below the tumor and distal mesorectum 5 cm below the tumor, and radical resection of rectal cancer should beyond that range.</p>

Changes of antigen-specific cytotoxic T lymphocytes during acute flare-ups in chronic hepatitis B patients / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To detect HBV antigen specific cytotoxic T lymphocyte (CTL) changes in patients during acute flare-ups and to study their association with flare-ups and aggravations into grave hepatitis by quantitative analysis of HLA-A2* restricted HBcAg-specific CTL cells.</p><p><b>METHODS</b>The frequency of HBcAg-specific CTL cells in the peripheral blood mononuclear cells (PBMC) from 29 patients with persistent infection with HBV were quantified by flow cytometry using one HLA-A2*HBV peptide pentamers complex (Pro5TM MHC Pentamers).</p><p><b>RESULTS</b>There was a statistical difference of HBcAg specific CTLs between the patients with acute exacerbations (1.4%+/-0.8%) and the patients with immune tolerance (0.6%+/-0.4%) (t = 2.180, P = 0.01-0.05); There was no significant difference between the grave hepatitis group (1.3%+/-1.0%) and the chronic hepatitis group (1.4%+/-0.8%) regarding frequencies of antigen specific CTL (t = 0.215, P = 0.833-0.05). The level of antigen specific CTLs in PBMC in the 6 cases of chronic hepatitis B with acute exacerbations maintained a relatively high level (more than 0.7%) within the 12 week follow-up period.</p><p><b>CONCLUSION</b>HBcAg-specific CTLs may play an important role in hepatic flare-ups in patients with chronic HBV infection, but there was no direct relationship between antigen- specific CTLs and grave hepatitis.</p>