Anxiety Severity and Influencing Factors in Day Surgery Patients or Relatives in Covid-19 Normalized Prevention Phase.

BACKGROUND: As the 2019 Coronavirus Disease (COVID-19) repeated, the prevention and treatment will be normalized in a period. "Large number of patients" and " Turnover quickly" of the day surgery ward greatly increased the difficulty of policy formulation and implementation. The normalization also had a huge negative psychological impact on patients/family members. This study aims to introduce effective epidemic prevention and control measures in day surgery wards, and to clarify the influencing factors of anxiety and subjective discomfort of patients and their families during the normalization of COVID-19. METHODS: To prepare for normalization of epidemic, research discuss improvements in the management of staff, environment, process. A total of 148 patients admitted to West China Hospital from December 2021 to March 2022 and their relatives were asked to complete a questionnaire effectively. Using the Self-rating Anxiety Scale, Social Support Rating Scale and Subjective Units of Distress scales to analyze anxiety and its risk factors. RESULTS: Under normalized control measures, no staff was infected. The subjective discomfort score was higher in people with lower body mass index (BMI). Young and high social support score were risk factors for anxiety (P < .05), and social support was positively correlated with anxiety. CONCLUSION: The normalization of epidemic is an inevitable trend in a period. A stable and safe medical environment needs to fully eliminate the policy defects, to fit the people and focus on mental health of the people. For patients/family members, who are younger,a lower BMI and higher social support should be attention more.

Coupling freedom from disease principles and early warning from wastewater surveillance to improve health security

Infectious disease surveillance is vitally important to maintaining health security, but these efforts are challenged by the pace at which new pathogens emerge. Wastewater surveillance can rapidly obtain population-level estimates of disease transmission, and we leverage freedom from disease principles to make use of non-detection of SARS-CoV-2 in wastewater to estimate the probability that a community is free from SARS-CoV-2 transmission. From wastewater surveillance of 24 treatment plants across upstate New York beginning in May 2020, we observed a reliable limit of detection of 0.3--0.5 cases per 10,000 population. No COVID-19 cases were reported 40% of the time following a non-detection of SARS-CoV-2 in wastewater, and cases were less than 1 daily case per 10,000 population 97% of the time following non-detection. Trends in the intensity of SARS-CoV-2 in wastewater correlate with trends in COVID-19 incidence and test positivity ({rho}>0.5), with the greatest correlation observed for active cases and a three-day lead time between wastewater sample date and clinical test date. Wastewater surveillance can cost-effectively demonstrate the geographic extent of the transmission of emerging pathogens, confirming that transmission is absent or under control and alerting of an increase in transmission. If a statewide wastewater surveillance platform had been in place prior to the onset of the COVID-19 pandemic, policymakers would have been able to complement the representative nature of wastewater samples to individual testing, likely resulting in more precise public health interventions and policies.

Quantification of SARS-CoV-2 and cross-assembly phage (crAssphage) from wastewater to monitor coronavirus transmission within communities

Wastewater surveillance of SARS-CoV-2 has become an attractive tool for combating the spread of COVID-19 by assessing the presence or levels of the virus shed in a population. However, the methods to quantify viral RNA and to link those quantities to the level of infection within the community vary. In this study, we sought to identify and optimize scalable methods for recovery of viral nucleic acids from wastewater and attempted to use a constitutive member of the gut virome, human-specific crAssphage, to help account for unknown levels of SARS-CoV-2 decay and dilution in the wastewater infrastructure. Results suggest that ultracentrifugation of a small volume of wastewater through a 50% sucrose cushion followed by total nucleic acid extraction yielded quantifiable virus in an area with a modest number of COVID-19 cases. Further, the ratio of log10(SARS-CoV-2):log10(crAssphage) appears to be associated with the cumulative incidence of COVID-19 in the Syracuse, NY area. In areas where ultracentrifuges are available, these methods may be used to link SARS-CoV-2 quantities in wastewater to levels of transmission within communities with sewer service.

Charge Reversible and Mitochondria/Nucleus Dual Target Lipid Hybrid Nanoparticles To Enhance Antitumor Activity of Doxorubicin.

The experiment aims to increase antitumor activity while decreasing the systemic toxicity of doxorubicin (DOX). Charge reversible and mitochondria/nucleus dual target lipid hybrid nanoparticles (LNPs) was prepared. The in vitro experimental results indicated that LNPs released more amount of DOX in acidic environment and delivered more amount of DOX to the mitochondria and nucleus of tumor cells than did free DOX, which resulted in the reduction of mitochondrial membrane potential and the enhancement of cytotoxicity of LNPs on tumor cells. Furthermore, the in vivo experimental results indicated that LNPs delivered more DOX to tumor tissue and significantly prolonged the retention time of DOX in tumor tissue as compared with free DOX, which consequently resulted in the high antitumor activity and low systemic toxicity of LNPs on tumor-bearing nude mice. The above results indicated that charge reversible mitochondria/nucleus dual targeted lipid hybrid nanoparticles greatly enhanced therapeutic efficacy of DOX for treating lung cancer.

Mitochondria and Nucleus Dual Delivery System To Overcome DOX Resistance.

Doxorubicin (DOX) is a broad-spectrum chemotherapy drug to treat tumors. However, severe side effects and development of DOX resistance hinder its clinical application. In order to overcome DOX resistance, DOX/TPP-DOX@Pasp-hyd-PEG-FA micelles were prepared by using newly synthesized comb-like amphiphilic material Pasp-hyd-PEG-FA. Drug released in vitro from micelles showed a pH-dependent manner. DOX/TPP-DOX@Pasp-hyd-PEG-FA induced more apoptosis in KB cell and MCF-7/ADR cell than DOX@Pasp-hyd-PEG-FA. Confocal laser scanning microscopy experiment indicated that DOX/TPP-DOX@Pasp-hyd-PEG-FA delivered TPP-DOX and DOX to the nucleus and mitochondria of the tumor cell simultaneously. Thus, DOX/TPP-DOX@Pasp-hyd-PEG-FA could significantly damage the mitochondrial membrane potential. DOX/TPP-DOX@Pasp-hyd-PEG-FA markedly shrinked the tumor volume in tumor-bearing nude mice grafted with MCF-7/ADR cell as compared with the same dose of free DOX. DOX was mainly accumulated in tumor tissue after DOX/TPP-DOX@Pasp-hyd-PEG-FA was injected to tumor-bearing nude mice by tail vein. After free DOX was injected to tumor-bearing nude mice by tail vein, DOX widely distributed through the whole body. Therefore, mitochondria and nucleus dual delivery system has potential in overcoming DOX resistance.

pH-Triggered Surface Charge Reversed Nanoparticle with Active Targeting To Enhance the Antitumor Activity of Doxorubicin.

PLGA nanoparticles are widely used in tumor targeting drug delivery systems. However, the naked PLGA nanoparticles (NNPs) not only have low drug loading but also can be rapidly removed from blood circulation by the immune system. The aim of this study was to prepare pH-triggered surface charge reversed lipid hybrid PLGA nanoparticles (LNPs) to enhance drug loading and drug delivery efficiency. CHO-Arg-His-OMe and FA-PEG-DSPE were synthesized to modify PLGA nanoparticles to prepare LNPs. The drug loading and encapsulation rate of LNPs were greatly improved as compared with NNPs. In pH 7.4 medium, doxorubicin (DOX)-loaded LNPs showed negative charge and released DOX slowly. In pH 5.0 medium, DOX-loaded LNPs exhibited positive charge and released DOX quickly. DOX-loaded LNPs delivered more DOX to the nucleus of KB cells and MBA-MD-231/ADR cells than did free DOX. In addition, DOX-loaded LNPs significantly inhibited the proliferation of KB cells and MBA-MD-231/ADR cells. Compared with free DOX, the same dose of the DOX-loaded LNPs delivered more DOX to tumor tissue. Thus, DOX-loaded LNPs significantly inhibited the growth of tumor in tumor-bearing nude mice and obviously reduced the systemic toxicity of DOX. In conclusion, pH-triggered surface charge reversed DOX-loaded LNPs significantly enhanced the antitumor activity of DOX in vitro and in vivo. DOX-loaded LNPs had great potential in tumor targeted chemotherapy.

Effects of pulsed electromagnetic field on differentiation of HUES-17 human embryonic stem cell line.

Electromagnetic fields are considered to potentially affect embryonic development, but the mechanism is still unknown. In this study, human embryonic stem cell (hESC) line HUES-17 was applied to explore the mechanism of exposure on embryonic development to pulsed electromagnetic field (PEMF) for 400 pulses at different electric field intensities and the differentiation of HUES-17 cells was observed after PEMF exposure. The expression of alkaline phosphatase (AP), stage-specific embryonic antigen-3 (SSEA-3), SSEA-4 and the mRNA level and protein level of Oct4, Sox2 and Nanog in HUES-17 cells remained unchanged after PEMF exposure at the electric field intensities of 50, 100, 200 or 400 kV/m. Four hundred pulses PEMF exposure at the electric field intensities of 50, 100, 200 or 400 kV/m did not affect the differentiation of HUES-17 cells. The reason why electromagnetic fields affect embryonic development may be due to other mechanisms rather than affecting the differentiation of embryonic stem cells.

Role of tumor necrosis factor-alpha in the anti-HBV activity of tetracycline / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the role of tumor necrosis factor-alpha (TNFalpha) in the anti-replication effects of tetracycline (Tet) on hepatitis B virus (HBV).</p><p><b>METHODS</b>The Tet-dependent regulatory fragment (TO) was PCR amplified from the pcDNA4TM/TO vector, inserted into the pUC118 cloning vector, and verified by sequencing. The counterpart fragment in the pVITRO3 expression vector, which contains two multiple cloning sites (MCSs), was replaced with the confirmed TO to generate a pVITRO3-TO vector. The Tet repressor (TR) gene from the pcDNA6/TR regulatory vector was incorporated into one MCS of pVITRO3-TO and the TNFalpha gene was subsequently incorporated into the other MCS. The resultant vector, pVITRO3-TOTR-TNFalpha, was transiently transfected into HepG2 cells. TNFalpha expression from the vector was induced by exposure to various concentrations of Tet and measured by enzyme-linked immunosorbent assay to determine the appropriate Tet concentration for experimentation. To investigate whether Tet inhibits TNFalpha expression as a mechanism of its anti-replication activity against HBV, the HepG2.2.15 cell line stably transfected with pVITRO3-TOTR-TNFalpha was used as an HBV replication model. Levels of hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) were detected by immunoassay. HBV DNA level was detected by fluorescence quantitative PCR.</p><p><b>RESULTS</b>The TNFalpha expression from the newly constructed pVITRO3-TOTR-TNFalpha vector was Tet-controllable in the eukaryotic cells examined. The optimal concentration of Tet for the experimental system was 1.0 mug/ml. HBsAg and HBeAg expression was down-regulated in the HepG2.2.15 cells stably transfected with the pVITRO3-TO-TR-TNFalpha vector. After incubation with Tet for 1, 3 and 5 days, the inhibition rate of HBsAg was 2%, 1.1% and 0, compared to 14.8%, 11.5% and 28.4% in the non-Tet control group. The corresponding inhibition rates of HBeAg were 50.0%, 26.7% and 47.9%, compared to 0.3%, 1.6% and 0.0%, in the control group. HBV DNA levels in the cells and the cell culture supernatants exposed to Tet were decreased by 70.3% and 79.9%, respectively. TNFalpha inhibited production of HBsAg mRNA.</p><p><b>CONCLUSION</b>A Tet-dependent regulatory fragment double-expressing TNFalpha single vector system was constructed successfully, achieving controllable TNFalpha expression in both transiently transfected eukaryotic cells and stable cell lines. In this HBV cell model system, Tet-induced overexpression of human TNFalpha inhibited HBV DNA replication and reduced HBsAg and HBeAg expression. Inhibition of HBV transcription may be a key role of TNFalpha against HBV replication.</p>

Role of serum procalcitonin assay for diagnosis of spontaneous bacterial peritonitis in end-stage liver diseases / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the clinical value of serum procalcitonin (PCT) for predicting spontaneous bacterial peritonitis (SBP) in end-stage liver diseases.</p><p><b>METHODS</b>The clinical data of 362 ascitic inpatients with end-stage liver diseases who had underwent serum PCT assay in our department from March 2011 to June 2013 were analyzed retrospectively. These patients were then divided into SBP group (n=178) and non-SBP group (n=184). The dynamic changes of the PCT values upon admission and after antibiotic treatment were compared. The receiver operating characteristic curve was drawn to identify the optimal cut-off value of serum PCT in diagnosing SBP.</p><p><b>RESULTS</b>The positive rate of bacteria culture in ascites was only 4.6% (4/87) in SBP group. The median value of serum PCT was 0.73 and 0.15 ng/ml in SBP group and non-SBP group (Z=-11.9, U=0.000), respectively, before antibiotic treatment. In the SBP group, the median value of serum PCT was 1.73 ng/ml in 13 patients with positive culture findings, which was higher than the overall median value in SBP group. Among patients who were responsive to the antibiotic therapy, the median values of serum PCT were 0.40(n=46), 0.32(n=19), and 0.33 ng/ml(n=25), respectively, 3, 5, and 7 days after the effective antibiotics treatment, which were significantly lower than the pre-treatment levels [0.86(Z=-5.91, U=0.000), 0.72(Z=-3.10, U=0.002), and 0.79 ng/ml(Z=-4.37, U=0.000), respectively]. ROC analysis showed that a serum PCT value of more than 0.462 ng/ml had a sensitivity of 83.7% and a specificity of 94.9%(AUC:0.95, 95%CI:0.93-0.97, P=0.00) in diagnosing SBP in patients with end-stage liver diseases.</p><p><b>CONCLUSIONS</b>Ascitic fluid positive rate is low in SBP patients. Serum PCT is a sensitive and specific marker for predicting peritoneal bacteria infection in end-stage liver disease patients with ascites. Higher serum PCT can be expected in these patients with heavier infections, it can also be used to evaluate the effectiveness of anti-bacteria therapies.</p>

Synthesis of a new pH-sensitive folate-doxorubicin conjugate and its antitumor activity in vitro.

Folate-aminocaproic acid-doxorubicin (FA-AMA-DOX) was synthesized and characterized by H NMR spectroscopy and mass spectrometry. Cytotoxicity and cellular uptake experiments were performed in KB and HepG2 cells, which express folic acid receptor, and the cell line A549, which does not express folic acid receptor. Cytotoxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and cellular uptake was monitored using fluorescence microscopy. The amount of DOX released from FA-AMA-DOX was much greater at pH 5.0 than that at pH 6.5 or 7.4. The cytotoxicity of FA-AMA-DOX toward KB and HepG2 cells was greater than that of DOX or AMA-DOX at the same concentrations, and cytotoxicity could be attenuated by FA in a dose-dependent manner. On the contrary, the cytotoxicity of FA-AMA-DOX and AMA-DOX toward A549 cells was lower than that of DOX at the same concentration, and cytotoxicity could not be reduced by FA. Compared with FA-AMA, FA-AMA-DOX increased the intracellular accumulation of DOX in KB cells. These results suggested that FA-AMA-DOX have suitable attributes for the active targeting of folate-receptor-positive tumor cells and for releasing the chemotherapeutic agent, DOX, in situ; it therefore has potential as a novel cancer therapeutic.

Co-transfection of dendritic cells with AFP and IL-2 genes enhances the induction of tumor antigen-specific antitumor immunity.

Dendritic cells (DCs) are highly efficient, specialized antigen-presenting cells and DCs transfected with tumor-related antigens are regarded as promising vaccines in cancer immunotherapy. The aim of the present study was to investigate whether DCs co-transfected with the α-fetoprotein (AFP) and human interleukin-2 (IL-2) genes were able to induce stronger therapeutic antitumor immunity in transfected DCs. In this study, DCs from hepatocellular carcinoma (HCC) patients were co-transfected with the IL-2 gene and/or the AFP gene. The reverse transcription-PCR (RT-PCR) data revealed that the DCs transfected with the adenovirus AdAFP/IL-2 expressed AFP and IL-2. The DCs co-transfected with IL-2 and AFP (AFP/IL-2-DCs) enhanced the cytotoxicities of cytotoxic T lymphocytes (CTLs) and increased the production of IL-2 and interferon-γ significantly compared with their AFP-DC, green fluorescent protein (GFP)-DC, DC or phosphate-buffered saline (PBS) counterparts. In vivo data suggested that immunization with AFP-DCs enhances antigen-specific antitumor efficacy more potently than immunization with IL-2-DCs or AFP-DCs. These findings provide a potential strategy to improve the efficacy of DC-based tumor vaccines.

Metabolism and pharmacokinetic study of ardipusilloside I in rats.

Ardipusilloside I, extracted from ARDISIA PUSILLA A.DC, effectively inhibits the progression of several cancers in animal models and is a potential anti-cancer drug candidate. However, the metabolism and pharmacokinetic characteristics of ardipusilloside I remain unknown. In this study, we developed a highly sensitive liquid chromatography-tandem MS method to determine the ardipusilloside I concentration in rat plasma using ginsenoside Re (whose structure is similar to ardipusilloside I) as the internal standard. After oral administration of ardipusilloside I, its four possible metabolites (M1, M2, M3, and M4, whose structures were determined by MS) were detected in the content from rat small intestine. In rat plasma, however, only M3 and M4 were detected after oral administration of ardipusilloside I. None of the metabolites were detected in plasma samples after intravenous administration of ardipusilloside I to rats. These results indicated that the metabolites, but not the drug itself, were absorbed into plasma after oral administration of ardipusilloside I to rats and that M3 and M4 may be responsible for the antitumor activity of orally administered ardipusilloside I in rat models of cancer.

Wortmannin potentiates roscovitine-induced growth inhibition in human solid tumor cells by repressing PI3K/Akt pathway.

Roscovitine has been reported to have anti-tumor effects in some cancer cell lines. The phosphatidylinositol-3-kinase (PI3K) signaling, which activates protein kinase B (PKB)/Akt, is known to mediate cell survival. The current study examined the role of wortmannin, a PI3K inhibitor, as a chemosensitizer for roscovitine and its proposed mechanism of action. The results showed that wortmannin significantly chemosensitized three human tumor cell lines (A549, HCT116 and HeLa cells). In A549 cells, wortmannin increased roscovitine-induced apoptosis in a dose-dependent manner, which was correlated with the inhibition of phosphorylated PKB/Akt level. Wortmannin enhanced the effects of roscovitine by causing pronounced reduction of mitochondrial transmembrane potential (MMP) and increases of cytochrome c release and active caspase-3, as well as enhanced activation of Bax and Bad, including Bax oligomerization and mitochondrial translocation of Bax and Bad. Taken together, these results provide evidence for the potential application of roscovitine/wormannin combination in clinical treatment for solid tumors.

Sensitization to gamma-irradiation-induced cell cycle arrest and apoptosis by the histone deacetylase inhibitor trichostatin A in non-small cell lung cancer (NSCLC) cells.

Histone deacetylase (HDAC) inhibitors (HDIs) play an important role in the regulation of gene expression associated with cell cycle and apoptosis and have emerged as promising anticancer agents. In addition to their intrinsic anticancer properties, some studies have demonstrated that HDIs can modulate cellular responses to ionizing radiation (IR). Here we show evidence that co-treatment with the HDI trichostatin A (TSA) radiosensitizes human non-small cell lung cancer (NSCLC) A549 cells and H1650 cells. Cells were exposed to gamma-irradiation with or without TSA co-treatment. Clonogenic survival was significantly reduced in cells with TSA co-treatment. In A549 cells, TSA enhanced IR-induced accumulation of cells in G(2)/M phase, with upregulated expression of p21(waf1/cip1). In addition, TSA co-treatment caused pronounced apoptosis in irradiated cells, which was accompanied with p21(waf1/cip1) cleavage to a 15 kDa protein. The enhanced apoptotic effect was via mitochondrial pathway, as indicated by the increased dissipation of mitochondrial transmembrane potential (MMP) and release of cytochrome c from the mitochondria to the cytoplasm. Caspase-3 activation was also significantly increased, with accordingly more cleavage of PARP, associated with the repression of X-linked inhibitor of apoptosis protein (XIAP). Furthermore, TSA co-treatment impaired DNA repair capacity after IR by downregulation of Ku70, Ku80 and DNA-PKcs, reflected by enhanced and prolonged expression of gammaH2AX. Taken together, our results demonstrate that TSA acts as a powerful radiosensitizer in NSCLC cells by enhancing G(2)/M cell cycle arrest, promoting apoptosis through multiple pathways and interfering with DNA damage repair processes.

Conjugation with alpha-linolenic acid improves cancer cell uptake and cytotoxicity of doxorubicin.

The synthetic DOX-LNA conjugate was characterized by proton nuclear magnetic resonance and mass spectrometry. In addition, the purity of the conjugate was analyzed by reverse-phase high-performance liquid chromatography. The cellular uptake, intracellular distribution, and cytotoxicity of DOX-LNA were assessed by flow cytometry, fluorescence microscopy, liquid chromatography/electrospray ionization tandem mass spectrometry, and the tetrazolium dye assay using the in vitro cell models. The DOX-LNA conjugate showed substantially higher tumor-specific cytotoxicity compared with DOX.

Effects of in vitro conditions on release behavior of different types of sustained and controlled release formulations of breviscapin / 药学学报

Insoluble breviscapin was chosen as the model drug. Bi-layer osmotic pump technology and gel matrix technology were used to prepare the breviscapin sustained and controlled release preparations. Dissimilarity factors (f1) and similarity factors (f2) were applied as similar judgment index to compare the effects of in vitro conditions on the release behavior of different types of breviscapin sustained and controlled release preparations. The tolerance of in vitro release conditions of bi-layer osmotic pump technology and gel matrix technology were studied. The results showed that in vitro release conditions have a greater impact on the gel matrix sustained release formulations, while have almost no effects on the osmotic pump controlled release formulations. Therefore, osmotic pump controlled release technology is less affected by the drug release environment. And it has a very good application prospect.

Quantitation assay for absorption and first-pass metabolism of emodin in isolated rat small intestine using liquid chromatography-tandem mass spectrometry.

Emodin has numerous biochemical and pharmacological activities, though information about its intestinal absorption and first-pass metabolism is scarce. The purpose of this study was to evaluate intestinal absorption and metabolism of luminally administered emodin in an isolated rat small intestine using the method of LC/MS/MS. About 22.55% of the administered emodin appeared at the vascular side, chiefly as free emodin (12.01%), but some emodin glucuronide (8.69%) and sulfate (1.84%) were also detected. Free glucuronide (5.23%) and sulfate (1.08%) moieties were found in the luminal perfusate. This model serves as a valuable tool for understanding intestinal handling of emodin, and our results confirm absorption and first-pass metabolism of emodin in the rat small intestine. Phase II metabolic enzymes such as glucuronyl transferase or sulfate transferase may also play an important role in the first-pass metabolism of emodin in the small intestine, which may ultimately reduce the bioavailability (and thus the efficacy) of orally administered emodin.

Cellular absorption of anthraquinones emodin and chrysophanol in human intestinal Caco-2 cells.

The intestinal absorption characteristics of anthraquinones emodin and chrysophanol were observed by measuring the intracellular accumulation across Caco-2 cells by the reverse-phase high performance liquid chromatography. The intracellular accumulation of chrysophanol was much greater than that of emodin, the maximum absorption of emodin and chrysophanol being 414.02+/-15.28 and 105.56+/-11.57 nmol/l x mg x protein, respectively. The absorption of each anthraquinone was significantly lower at 4 degrees C than that of 37 degrees C. The effects of the transport inhibitors, verapamil, cyclosporine and phloridzin, on the intracellular accumulation were also examined. Verapamil and cyclosporine increased the absorption of emodin and chrysophanol, while phloridzin inhibited their absorption, all in a dose-dependent manner. These results suggest that the absorption characteristics of emodin and chrysophanol were closely related to their special structure with the hydroxy groups. It is also likely that a specific transport system mediated the intracellular accumulation of emodin and chrysophanol across the Caco-2 cells.

alpha-fetoprotein and interleukin-18 gene-modified dendritic cells effectively stimulate specific type-1 CD4- and CD8-mediated T-Cell response from hepatocellular carcinoma patients in Vitro.

The T-helper 1 (Th1) immune reaction is most important in dendritic cell (DC)-based immunotherapy. Interleukin (IL)-18, a Th1-biasing cytokine, plays a pivotal role in inducing cytotoxic T lymphocyte (CTL) responses. In this study, we analyzed whether dendritic cells (DCs) from patients with hepatocellular carcinoma (HCC) can be transduced with the IL-18 gene and/or alpha-fetoprotein (AFP) gene, and we examined whether vaccinations using these genetically engineered DC can induce stronger therapeutic antitumor immunity. The results showed that DC transfected with AdIL-18/AFP can expressed IL-18 and AFP by reverse transcriptase-polymerase chain reaction and enzyme-linked immunoassay. Compared with those before transfection, the expressions of membrane molecules were increased dramatically. Specific T cells generated by DC transfected with AdIL-18/AFP recognized HLA-matched HepG2 cell lines specifically. Most importantly, The cytotoxic activity of CTLs against HepG2 with DC expressing AFP(AFP-DC) was significantly augmented by co-transduction with the IL-18 gene. Administration with such vaccine also significantly increased the production of interleukin-12p70 and interferon-gamma. These results indicate that a vaccination therapy using DC co-transduced with the TAA gene and IL-18 genes is effective strategy for immunotherapy in terms of the activation of DCs, CD4+ T, cells and CD8+ T cells, and may be useful in the clinical application of a cancer vaccine therapy.

[Immune response of HBsAg gene-modified dendritic cell-based vaccine in HepG2. 2. 15 hepatocellular carcinoma cells].

OBJECTIVE: To study the cytotoxic T lymphocyte (CTL) response induced by dendritic cells (DC) transduced with recombinant adenovirus vector bearing hepatitis B virus surface antigen (HBsAg) gene in hepatocellular carcinoma HepG2. 2. 15 cells in vitro. METHODS: Full length HBsAg cDNAs were subcloned into pIND vector, followed by being cloned into pShuttle vector. The HBsAg gene fragments resulted from the pShuttle-S digested with PI-Sce and I-Ceu were linked to the linear adeno-X virus DNA. After packaged with HEK293 cells, the adenovirus expression vector was obtained. Then the recombinant adenovirus expression plasmid AdVHBsAg was transfected into human monocyte-derived dendritic cells, to construct AdVHBsAg hepatocarcinoma tumor vaccine. The effectiveness of transfection was detected by Western blot. Surface molecules of AdVHBsAg-DC were detected by FACS. Autologous T cell proliferation stimulated by AdVHBsAg-DC was detected by 3H-TdR assay. Cytotoxic CTL activity induced by AdVHBsAg-DC in vitro was detected by LDH assay. RESULTS: HBsAg gene in the inserted DNA of AdVHBsAg was confirmed by PCR, and predictive fragments proved by restriction enzyme digestion analysis were exhibited. Cell pathological changes appear after 10 days HEK293 cells transfected AdVHBsAg. Western blot analysis showed that HBV surface antigen gene was expressed in transfected DC, indicating that the transfection was effective. AdVHBsAg-DC was able to upregulate CD1a, CD11c, CD80, CD86 and HLA-DR. Autologus T cell proliferation induced by AdVHBsAg-DCs was significantly higher than that in DC control group and LacZ-DC group (P < 0.05). AdVHBsAg-DC activated CTL presented the specific killer ability to the hepatocellular carcinoma cells expressing HBsAg. CONCLUSION: DC transduced with recombinant adenovirus HBsAg can express HBV-related hepatocellular carcinoma antigen (HBsAg), and AdVHBsAg-DC can induce potent immune response against HBsAg-positive hepatocellular carcinoma cells in vitro.