Hepatitis C virus in human B lymphocytes transformed by Epstein-Barr virus in vitro by in situ reverse transcriptase-polymerase chain reaction

AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR. RESULTS: HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion. CONCLUSION: HCV may exist and remain functional in a cultured cell line for a long period.

SEQUENCE ANALYSIS OF THE NS5 REGION OF GBVC/HGV AND DETECTION OF THE VIRUS BY REVERSE TRANSCRIPTASE PCR

GBV C/HGV is a newly identified virus associated with human hepatitis In this study, the nucleotide sequences of the partial NS5 gene of GBV C/HGV derived from sera of 8 Chinese patients were determined The nucleotide homology among the 8 isolates were 92% on average On the basis of sequence analysis, two sets of oligonucleotide primers derived from highly conserved region of GBV C/HGV NS5 gene were designed to establish both sensitive and specific nested PCR for detection of GBV C/HGV RNA 253 Chinese patients were examined for the virus RNA GBV C/HGV RNA positive rates in patients infected with HBV, HCV and patients with chronic non B,non C hepatitis were 18 4%, 19 8% and 8 9% respectively This result suggested that HBV,HCV and GBV C/HGV shared the same transmission risk factors 8 patients with GBV C/HGV and HCV coinfection were retrospectively observed for the response to interferon Coinfection with GBV/HGV did not negatively influence the responsiveness of HCV, and GBV C/HGV was sensitive to interferon to a certain degree

Detection of SYT-SSX fusion transcripts in paraffin-embedded tissues of synovial sarcoma by reverse transcription-polymerase chain reaction

OBJECTIVE: To assess the feasibility of detecting SYT-SSX fusion transcripts in paraffin-embedded tissues of synovial sarcoma by reverse transcription-polymerase chain reaction (RT-PCR). METHODS: RT-PCR was used to amplify the SYT-SSX fusion transcripts using archival formalin-fixed paraffin-embedded tumor specimens from a series of 37 synovial sarcoma cases. To investigate the specificity of the SYT-SSX fusion transcripts, a variety of non-synovial sarcoma tumors were included in the study as negative controls. The detected messages derived from fusion genes were confirmed by subsequent sequence analysis. RESULTS: SYT-SSX fusion transcripts were detected in 33 of 37 (89.2%) synovial sarcomas. None of the 34 cases of non-synovial sarcoma tumors showed amplified products of SYT-SSX fusion transcripts, although PBGD mRNA was detected in all specimens. Among 33 SYT-SSX-positive synovial sarcomas, 22 tumors had an SYT-SSX 1 fusion transcript, whereas 6 tumors had an SYT-SSX2 fusion transcript. Fusion types can not be distinguished in the remaining 5 cases. There was a significant relationship between SYT-SSX fusion type and histologic subtype. All 10 biphasic synovial sarcomas had the SYT-SSX1 fusion, whereas all tumors with SYT-SSX2 were of monophasic morphology (P

Epidemiological and Subtype Characterization of Influenza Viruses Infection in Children in Shenzhen, China during Three Consecutive Seasons (January 2016-December 2018)

Background: Children with seasonal influenza infection cause a significant burden of disease each year in the pediatric clinic. Influenza A and B viruses are the major types responsible for illness. A better understanding of the periodicity facilitates the prevention and control of influenza in children. Objective: This study aims to analyze the epidemiological patterns and subtype characterization of influenza viruses among children in Shenzhen, China. Methods: Influenza samples were collected by nasopharyngeal swabs from influenza like illness patients in Shenzhen Children’s Hospital from January 2016 to December 2018. The positive cases and influenza subtypes were determined by gold labeled antigen detection and reverse transcriptase polymerase chain reaction. The influenza periodicity and age, subtype distribution as well as the association between climate parameters and different influenza subtypes were analyzed by SPSS 22.0. Results: The influenza positive rate during 2016-2018 was 21.0%, with a highest positive rate in the year 2018. The positive rate varied by month, season, and year describing a sequence of peaks presenting primarily in all year including spring, summer and winter. The characteristics of influenza peak were different in each year, with a spring peak in 2016 and a summer plus a winter-spring peaks in 2017 and 2018. In addition, influenza B exhibited a winter-spring seasonal pattern while influenza A displayed a more variable seasonality, highlighting influenza B rather than influenza A which had a negative association with climate parameters. Influenza-positive cases were older than influenza-negative cases (P P Conclusion: Influenza activity in children from Shenzhen typically displays both winter-spring and summer peaks. Influenza A epidemic occurred separately or co-circulated with influenza B, with a winter-spring pattern for influenza B and a much more variable seasonality for influenza A. Influenza B had a negative association with climate parameters. In addition, hospitalization with influenza often occurs in younger individuals infected with influenza A.

mRNA Expression of the Cancer-testis Antigens SSX1 and SSX4 in Human Hepatocellular Carcinomas

Objective: To detect the mRNA expression of the cancer-testis antigens (CT) SSX1 and SSX4 gene in human hepatocellular carcinomas (HCCs) and to investigate the specificity of their expression in HCCs. Methods: The mRNA expression of SSX1 and SSX4 in HCC tissues and the corresponding nearby liver tissues in 35 cases was detected by using RT-PCR; Six positive RT-PCR products were randomly selected and sequenced. Results: In all 35 HCC tissues, SSX1 in 27 cases (81%) and SSX4 in 23 cases (73%) were detected, and their expression was negative in the liver tissues nearby HCC and the non-tumor liver tissues (12 cirrhotic tissues and 15 normal tissues). In all 6 cases selected randomly, the results of DNA sequencing were identical with the cDNA sequence of SSX1 and SSX4 genes. The SSX1, SSX4 mRNA expression was not significantly correlated with age, sex, the tumor size, the level of tumor differentiation, the serum AFP level and the infection rate of HBV and HCV respectively (P>0.05). Conclusion: The SSX1, SSX4 mRNA expression was greatly specific in HCCs, which would not only provide the ideal target molecular sites for HCC tumor vaccines, but also establish the potential value of the polyvalent tumor-antigen vaccines for HCC therapy and its theory bases.

HCV-RNA positivity in peripheral blood mononuclear cells of patients with chronic HCV-infection: does it really mean viral replication?

AIM: To analyze the association of HCV-RNA with peripheral blood mononuclear cells (PBMC) and to answer the question whether HCV-RNA positivity in PBMC is due to viral replication. METHODS: HCV-RNA was monitored in serum and PBMC preparations from 15 patients with chronic HCV infection before, during and after an IFN-alpha therapy using a nested RT/PCR technique. In a second approach, PBMC from healthy donors were incubated in HCV positive plasma. RESULTS: In the IFN-alpha responding patients,HCV-RNA disappeared first from total RNA preparations of PBMC and then from serum. In contrast, in relapsing patients, HCV-RNA reappeared first in serum and then in PBMC. A quantitative analysis of the HCV-RNA concentration in serum was performed before and after transition from detectable to non detectable HCV-RNA in PBMC-RNA and vice versa. When HCV-RNA was detectable in PBMC preparations, the HCV concentration in serum was significantly higher than the serum HCV-RNA concentration when HCV-RNA in PBMC was not detectable. Furthermore, at no time during the observation period was HCV specific RNA observed in PBMC, if HCV-RNA in serum was under the detection limit. Incubation of PBMC from healthy donors with several dilutions of HCV positive plasma for two hours showed a concentration dependent PCR positivity for HCV-RNA in reisolated PBMC. CONCLUSION: The detectability of HCV-RNA in total RNA from PBMC seems to depend on the HCV concentration in serum. Contamination or passive adsorption by circulating virus could be the reason for detection of HCV-RNA in PBMC preparations of chronically infected patients.

Interleukin-1 beta up-regulates tissue inhibitor of matrix metalloproteinase-1 mRNA and phosphorylation of c-jun N-terminal kinase and p38 in hepatic stellate cells

AIM： To study the relationship between interleukin-lbeta （IL-1β） up-regulating tissue inhibitor of matrix metalloproteinase-1 （TIMMP-1） mRNA expression and phosphorylation of both c-jun N-terminal kinase （INK） and p38 in rat heffatic stellate cells （HSC）. METHODS： RT-PCR was performed to measure the expression of TIMMP-1 mRNA in rat HSC. Western blot was performed to measure IL-1β-induced JNK and p38 activities in rat HSC. RESULTS： TIMMP-1 mRNA expression （1.191± 0.079） was much higher after treatment with IL-1β （10 ng/mL） for 24 h than in control group （0.545±0.091） （P〈0.01）. IL-1β activated INK and p38 in a time-dependent manner. After stimulation with IL-1β for 0, 5, 15, 30, 60 and 120 min, the INK activity was 0.982±0.299, 1.501±0.720, 2.133±0.882, 3.360±0.452, 2.181±0.789, and 1.385 ± 0.368, respectively. There was a significant difference in JNK activity at 15 min （P〈 0.01）, 30 min （P〈 0.01） and 60 min （P〈0.01） in comparison to that at 0 min. The p38 activity was 1.061±0.310, 2.050±0.863, 2.380±0.573, 2.973±0.953, 2.421±0.793, and 1.755 ± 0.433 at the 6 time points （0, 5, 15, 30, 60 and 120 min） respectively. There was a significant difference in p38 activity at 5 min （P〈0.05）, 15 min （P〈0.01）, 30 min （P〈0.01） and 60 min （P〈0.01） compared to that at 0 min. TIMMP-1 mRNA expression trended to decrease in 3 groups pretreated with different concentrations of SP600125 （10 μmol/L, 1.022±0.113; 20 μmol/L, 0.869±0.070; 40 μmol/L, 0.666±0.123）. Their decreases were all significant （P〈0.05, P〈0.01, P〈0.01） in comparison to control group （without SP600125 treatment, 1.163±0.107）. In the other 3 groups pretreated with different concentrations of SB203580 （10 μmol/L, 1.507±0.099; 20 μmol/L, 1.698±0.107; 40 μmol/L, 1.857±0.054）, the expression of TIMMP-1 mRNA increased. Their levels were higher than those in the control group （without SB203580 treatment, 1.027 ± 0.061） with a significant statistical significance （P〈 0.01）. CONCLUSION： IL-1β has a direct action on hepatic fibrosis by up-regulating TIMMP-1 mRNA expression in ratessionin in rate HSC.JNK and p38 mitogen-activated protein kinases （MAPKs） are involved in IL-1β-induced TIMMP-1 gene expression, and play a distinct role in this process, indicating that p38 and .INK pathways cooperatively mediate TIMP-1 mRNA expression in rat HSC.

Expressions of ICAM-1 and its mRNA in sera and tissues of patients with hepatocellular carcinoma

INTRODUCTIONThe increased expression of ICAM-1 on a widerange of cells and in the sera of patients withmalignancies, chronic liver diseases andinflammation diseases has been described since thelate 1980s[1-22]. Recently rapid progress in studieson expression of ICAM-1 in patients withhepatocellular carcinoma ( HCC ) have beenachieved, including clinical and experimentalresearches[23-31].

Expression of mucosal addressin cell adhesion molecule 1 on vascular endothelium of gastric mucosa in patients with nodular gastritis

AIM:The interaction of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) with integrin α4β7 mediates lymphocyte recruitment into mucosa-associated lymphoid tissue (MALT).Nodular gastritis is characterized by a unique military pattern on endoscopy representing increased numbers of lymphoid follicles with germinal center,strongly associated with H pylori infection.The purpose of this study was to address the implication of the MAdCAM-1/integrin β7 pathway in NG. METHODS:We studied 17 patients with NG and H pylori infection and 19 H pylori-positive and 14 H pylori-negative controls.A biopsy sample was taken from the antrum and snap-frozen for immunohistochemical analysis of MAdCAM- 1 and integrin β7.In simultaneous viewing of serial sections, the percentage of MAdCAM-1-positive to von Willebrand factor-positive vessels was calculated.We also performed immunostaining with anti-CD20,CD4,CD8 and CD68 antibodies to determine the lymphocyte subsets co- expressing integrin β7. RESULTS:Vascular endothelial MAdCAM-1 expression was more enhanced in gastric mucosa with than without H pylori infection.Of note,the percentages of MAdCAM-1-positive vessels were significantly higher in the lamina propria of NG patients than in H pylori-positive controls.Strong expression of MAdCAM-1 was identified adjacent to lymphoid follicles and dense lymphoid aggregates.Integrin β7-expressing mononuclear cells,mainly composed of CD20 and CD4 lymphocytes,were associated with vessels lined with MAdCAM-1-expressing endothelium.CONCLUSION: Our results suggest that the MAdCAM一1/ integrin a4p7 homing system may participate in gastric inflammation in response to H py/o}i-infection and contributes to MALT formation, typically leading to the development of NG.

Cytopathological evaluations combined RNA and protein analyses on defined cell regions using single frozen tissue block

The co-existence of multiple cell components in tissue samples is the main obstacle for precise molecular evaluation on defined cell types. Based on morphological examination, we developed an efficient approach for paralleled RNA and protein isolations from an identical histological region in frozen tissue section.The RNA and protein samples prepared were sufficient for RT-PCR and Western blot analyses, and the results obtained were well coincident each other as well as with the corresponding parameters revealed from immunohistochemical examinations. By this way, the sampling problem caused by cell-cross contamination can be largely avoided, committing the experimental data more specific to a defined cell type. These novel methods thus allow us to use single tissue block for a comprehensive study by integration of conventional cytological evaluations with nucleic acid and protein analyses.

Molecular diagnosis and therapy for occult peritoneal metastasis in gastric cancer patients

To apply an individualized oncological approach to gastric cancer patients,the accurate diagnosis of disease entities is required.Peritoneal metastasis is the most frequent mode of metastasis in gastric cancer,and the tumor-node-metastasis classification includes cytological detection of intraperitoneal cancer cells as part of the staging process,denoting metastatic disease.The accuracy of cytological diagnosis leaves room for improvement;therefore,highly sensitive molecular diagnostics,such as an enzyme immunoassay,reverse transcription polymerase chain reaction,and virusguided imaging,have been developed to detect minute cancer cells in the peritoneal cavity.Molecular targeting therapy has also been spun off from basic research in the past decade.Although conventional cytologyis still the mainstay,novel approaches could serve as practical complementary diagnostics to cytology in near future.

Impact of endoscopically minimal involvement on IL-8 mRNA expression in esophageal mucosa of patients with non-erosive reflux disease

AIM:Little has been known about the pathogenesis of non- erosive reflux disease(NERD).Recent studies have implicated interleukin 8(IL-8)in the development and progression of gastroesophgeal reflux disease(GERD).The purpose of this study was to determine IL-8 RNA expression levels in NERD patients with or without subtle mucosal changes. METHODS:We studied 26 patients with NERD and 13 asymptomatic controls.Biopsy sample was taken from the esophagus 3 cm above the gastroesophageal junction and snap frozen for measurement of IL-8 mRNA levels by real-time quantitative polymerase chain reaction(PCR).We also examined mRNA expression of IL-8 receptors,CXCR-1 and -2 by reverse transcriptase PCR.The patients were endoscopically classified into grade M(mucosal color changes without visible mucosal break)and N(neither minimal involvement nor mucosal break)of the modified Los Angeles classification. RESULTS:The relative IL-8 mRNA expression levels were significantly higher in esophageal mucosa of NERD patients than those in esophageal mucosa of the controls.There was a significant difference in IL-8 mRNA levels between grades M and N.The CXCR-1 and -2 mRNAs were constitutively expressed in esophageal mucosa.CONCLUSION: Our results suggest that high IL-8 levels in esophageal mucosa may be involved in the pathogenesis of NERD through interaction with its receptors. NERD seems to be composed of a heterogeneous population in terms of not only endoscopically minimal involvement but also immune and inflammatory processes.

TT virus and hepatitis G virus infections in Korean blood donors and patients with chronic liver disease

AIM:To determine the prevalences of TTV and HGV infections among blood donors and patients with chronic liver disease in Korea,to investigate the association of TTV and HGV infections with blood transfusion,and to assess the correlation between TTV and HGV viremia and hepatic damage. METHODS:A total of 391 serum samples were examined in this study.Samples were obtained from healthy blood donors(n=110),hepatitis B surface antigen(HBsAg)-positive donors(n=112),anti-hepatitis C virus(anti-HCV)-positive donors(n=69),patients with type B chronic liver disease (n=81),and patients with type C chronic liver disease(n=19). Trv DNA was detected using the hemi-nested PCR.HGV RNA was tested using RT-PCR.A history of blood transfusion and serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were also determined. RESULTS:TTV DNA was detected in 8.2%of healthy blood donors,16.1%of HBsAg-positive donors,20.3%of anti- HCV-positive donors,21.0%of patients with type B chronic liver disease,and 21.1%of patients with type C chronic liver disease.HGV RNA was detected in 1.8%of healthy blood donors,1.8%of HBsAg-positive donors,17.4%of anti-HCV-positive donors,13.6%of patients with type B chronic liver disease,and 10.5%of patients with type C chronic liver disease.The prevalence of TTV and HGV infections in HBV- or HCV-positive donors and patients was significantly higher than in healthy blood donors(P<0.05), except for the detection rate of HGV in HBsAg-positive donors which was the same as for healthy donors.There was a history of transfusion in 66.7%of TTV DNA-positive patients and 76.9%of HGV RNA-positive patients(P<0.05).No significant increase in serum ALT and AST was detected in the TTV or HGV-positive donors and patients. CONCLUSION:TTV and HGV infections are more frequently found in donors and patients infected with HBV or HCV than in healthy blood donors.However,there is no significant association between TTV or HGV infections and liver injury.

Comparison of gene expression profiles between primary tumor and metastatic lesions in gastric cancer patients using laser microdissection and cDNA microarray

AIM. To study the differential gene expression profiles of target cells in primary gastric cancer and its metastatic lymph nodes using laser microdissection （LMD） in combination with cDNA microarray. METHODS： Normal gastric tissue samples from 30 healthy individuals, 36 cancer tissue samples from primary gastric carcinoma and lymph node metastasis tissue samples from 58 patients during gastric cancer resection were obtained using LMD in combination with cDNA microarray independently. After P27-based amplification, aRNA from 36 of 58 patients （group 1） with lymph node metastasis and metastatic tissue specimens from the remaining 22 patients （group 2） were applied to cDNA microarray. Semiquantitative reverse transcriptase polymerase chain reaction （RT-PCR） and imrnunohistochemical assay verified the results of microarray in group 2 and further identified genes differentially expressed in the progression of gastric cancer. RESULTS： The expression of 10 genes was up-regulated while the expression of 15 genes was down-regulated in 22 gastric carcinoma samples compared with that of genes in the normal controls. The results were confirmed at the level of mRNA and protein, and suggested that four genes （OPCML, RNASE1, YES1 and ACK1） could play a key role in the tumorigenesis and metastasis of gastric cancer. The expression pattern of 3 genes （OPCML, RNASE1 and YES1） was similar to tumor suppressor genes. For example, the expression level of these genes was the highest in normal gastric epithelium, which was decreased in primary carcinoma, and further decreased in metastatic lymph nodes. On the contrary, the expression pattern of gene ACK1 was similar to that of oncogene. Four genes were further identified as differentially expressed genes in the majority of the cases in the progression of gastric cancer. CONCLUSION： LMD in combination with cDNA microaro ray provides a unique support foe the identification of early expression profiles of differential genes and the expression pattern of 3 genes （OPCML, RNASE1 and YES1） associated with the progression of gastric cancer. Further study is needed to reveal the molecular mechanism of lymph node metastasis in patients with gastric cancer.

Three new alternative splicing variants of human cytochrome P450 2D6 mRNA in human extratumoral liver tissue

AIM: To identify the new alternative splicing variants of human CYP2D6 in human extratumoral liver tissue with RT-PCR and sequencing. METHODS: Full length of human CYP2D6 cDNAs was amplificated by reverse transcription-polymerase chain reaction (RT-PCR) from a human extratumoral liver tissue and cloned into pGEM-T vector. The cDNA was sequenced. Exons from 1 to 4 of human CYP2D6 cDNAs were also amplificated by RT-PCR from extratumoral liver tissues of 17 human hepatocellular carcinomas. Some RT-PCR products were sequenced. Exons 1 to 4 of CYP2D6 gene were amplified by PCR from extratumoral liver tissue DNA. Two PCR products from extratumoral liver tissues expressing skipped mRNA were partially sequenced. RESULTS: One of the CYP2D6 cDNAs had 470 nucleotides from 79 to 548 (3' portion of exons 1 to 5' portion of exon 4), and was skipped. Exons 1 to 4 of CYP2D6 cDNA were assayed with RT-PCR in 17 extratumoral liver tissues. Both wild type and skipped mRNAs were expressed in 4 samples, only wild type mRNA was expressed in 5 samples, and only skipped mRNA was expressed in 8 samples. Two more variants were identified by sequencing the RT-PCR products of exons 1 to 4 of CYP2D6 cDNA. The second variant skipped 411 nucleotides from 175 to 585. This variant was identified in 4 different liver tissues by sequencing the RT-PCR products. We sequenced partially 2 of the PCR products amplified of CYP2D6 exon 1 to exon 4 from extratumoral liver tissue genomic DNA that only expressed skipped mRNA by RT-PCR. No point mutations around exon 1, intron 1, and exon 4, and no deletion in CYP2D6 gene were detected. The third variant was the skipped exon 3, and 153 bp was lost. CONCLUSION: Three new alternative splicing variants of CYP2D6 mRNA have been identified. They may not be caused by gene mutation and may lose CYP2D6 activity and act as a down-regulator of CYP2D6.

Current status quo on COVID-19 including chest imaging

With each day the number coronavirus disease 2019(COVID-19)cases continue to rise rapidly and our imaging knowledge of this disease is expeditiously evolving.The role of chest computed tomography(CT)in the screening or diagnosis of COVID-19 remains the subject of much debate.Despite several months having passed since identifying the disease,and numerous studies related to it,controversy and concern still exists regarding the widespread use of chest CT in the evaluation and management of COVID-19 suspect patients.Several institutes and organizations around the world have released guidelines,recommendations and statements against the use of CT for diagnosing or screening COVID-19 infection and advocating its use only for those cases with a strong clinical suspicion of complication or an alternate diagnosis.However,these guidelines and recommendations are in disagreement with majority of the widely available literature,which strongly favour CT as a pivotal tool in the early diagnosis,management and even follow-up of COVID-19 infection.This article besides comprehensively reviewing the current status quo on COVID-19 disease in general,also writes upon the current consensus statements/recommendations on the use of diagnostic imaging in COVID-19 as well as highlighting the precautions and various disinfection procedures being employed world-wide at the workplace to prevent the spread of infection.

Novel flutamide regulated genes in the rat veritral prostate： differential modulation of their expression by castration and flutamide treatments

Aim： To identify flutamide regulated genes in the rat ventral prostate. Methods： Total RNA from ventral prostates of control and flutamide treated rats were isolated. Differentially expressed transcripts were identified using differential display reverse transcriptase polymerase chain reaction. The effect of castration on the expression of flutamideregulated transcripts was studied. Results： We have identified β2-microglobulin, cytoplasmic FMR1 interacting protein 2 and pumilio 1 as flutamide induced and spermine binding protein and ribophorin Ⅱ as flutamide repressed targets in the rat ventral prostate. Although flutamide treatment caused an induction of pumilio 1 rnRNA, castration had no effect. Conclusion： Castration and flutamide treatments exert differential effects on gene expression. Flutamide might also have direct AR independent effects, which might have implications in the emergence of androgen independent prostate cancer and the failure of flutamide therapy.

Detection of CEA mRNA in patients with non-small cell lung cancer and it’s significance

Objective: To detect the expression of CEA mRNA in patients with non-small cell lung cancer (NSCLC) and to investigate it's significance. Methods: The blood samples were taken from peripheral veins of 70 patients with NSCLC and 18 patients with benign diseases at 3 intervals during the surgery. The transcription of carcinoembryonic antigen messenger ribonucleic acid (CEA mRNA) was assayed by means of nested reverse transcriptase polymerase chain reaction (RT-PCR) and micro-fluid chip. Results: The CEA mRNA positive rates at each of the 3 time spots were as follows: 50% at beginning of the surgery (group 1), 62.8% in the samples collected when ligating the pulmonary vein (group 2) and 57.1% in samples collected 1 h after ligation (group 3). A significant difference was found between groups 1 and 2 (χ2 = 7.114, P < 0.05). Con-clusion: Cancer cell dissemination during surgery is demonstrated indirectly in our study, when to ligate the pulmonary vein (earlier or later) may affect the quantity of tumor cells spread into the circulation.

The effect of FasL expression on pancreatic islet allografts

OBJECTIVE: To investigate the immune privilege induced by the Fas ligand (FasL) expressed by cotransplanted testicular Sertoli cells in islet allografts, and the effect of FasL gene transfection on islet cells in pancreatic islet allografts. METHODS: Allogeneic islets and testicular cells were cotransplanted into diabetic recipients.Pancreatic islets were infected with the recombinant adenovirus, AdV-FasL, and transplanted into diabetic recipients. Allograft survival, islet function, apoptosis of infiltrative lymphocytes in allografts and gene transfected islet allografts were analyzed. RESULTS: All animals receiving islet allograft alone returned to a diabetic state in a few days (mean survival time 6.3 +/- 0.6 days). When the quantity of testicular cells cotransplanted with islets increased to 1 x 10(7), all animals remained normoglycemic throughout the follow-up period (60 days). FasL expression by cotransplanted Sertoli cells induced apoptosis of activated lymphocytes. Rejection of allografts in the FasL gene transfer group was accelerated and allograft survival was shortened to 3.4 +/- 0.2 days (P

Stable expression of antisense hTR inhibits in vitro pancreatic cancer cell growth

OBJECTIVE: To clarify growth inhibition in pancreatic cancer cells by interference with the hTR component of the telomerase reverse transcriptase enzymatic complex. METHODS: A 593 bp full length hTR cDNA was subcloned into a mammalian expression vector pcDNA3.1(-) in the antisense orientation to construct an antisense hTR expression plasmid. These were introduced into panc1 cells, a human pancreatic carcinoma cell line, by lipofectin and G418-resistant stable transformants were expanded. Resulting stable clones were screened for the presence of the hTR insert by PCR with T7 and BGH reverse primers located on the flanks of the multiclonal site of the pcDNA3.1 vector. Cell growth rate, hTR expression, telomerase activity and anchorage-independent growth properties were analyzed. RESULTS: Significant downregulation of endogenous hTR was evident in the antisense-hTR transformed cells and telomerase activity was markedly decreased compared to control cells in standard TRAP assays. Furthermore, cell proliferation and the anchorage-independent growth ability in antisense-hTR expressing cells were significantly decreased compared with control parental cells. However, no crisis or senescence phenomena were observed. CONCLUSIONS: These data indicate that hTR may be a critical component of human telomerase activity and suggest that downregulation of the RNA component of human telomerase is a possible target for anticancer strategies.