The research on intracellular trafficking of adenovirus has been described mainly through observations of subgroup C adenoviruses in transformed cell lines. The basic elements of the trafficking pathway include binding to receptors at the cell surface, internalization by endocytosis, lysis of the endosomal membrane, escape to the cytosol, intracellular trafficking along microtubules, nuclear pore docking, and viral genome translocation into the nucleus. More than 80% of the adenovirus genome is delivered to the nucleus in a highly efficient manner in approximately 1 h. However, exceptions to this trafficking pattern have been noted, including: variations based on target cell type, cell physiology, and adenovirus serotype. This review summarizes mechanism of adenovirus infection pathway and intracellular trafficking, providinging a foundation for the development of clinical adenoviral vector.
Adenoviridae ; physiology ; Cell Membrane ; virology ; Cell Nucleus ; virology ; Cytoplasm ; virology ; Endocytosis ; Endosomes ; virology ; Genetic Vectors ; Humans ; Microtubules ; Virus Internalization

AIM:To explore the role of survivin-2B in the process of tumor cell apoptosis .METHODS:The survivin-2B gene was cloned into pcDNA3.1 vector and the recombinant plasmid pcDNA3.1-survivin-2B was obtained.Hu-man breast cancer MCF7 cells were transfected with pcDNA3.1 and pcDNA3.1-survivin-2B using Lipofectamine 2000.The cell cycle was determined by propidium iodide staining , and the apoptosis was detected by annexin V/7-AAD staining 48 h after transfection.Meanwhile, tatal RNA was extrated and multiplex polymerase chain reaction based on GenomeLab GeXP Genetic Analysis System was performed to detect the expression of 21 tumor-related genes .RESULTS: Flow cytometry analysis indicated that over-expression of survivin-2B promoted the apoptosis and cell cycle arrest of MCF 7 cells.Compared with control group , totally 10 differential expressed genes were related to the over-expressed survivin-2B, among which 2 were up-regulated and 8 were down-regulated. The expression of aldehyde dehydrogenase 4 family member A1 (ALDH4A1) was 48%down-regulated, and the expression of protein regulator of cytokinesis 1 (PRC1) was 1.08 folds up-regulated.CONCLUSION:Survivin-2B induces the expression changes of some tumor-related genes, which results in the apoptosis and G 2/M arrest of MCF7 cells.

Objective To investigate the subtype distribution and changing trend of human immunodeficiency virus (HIV)-1 strains among men who have sex with men (MSM) during 2005-2011 in Beijing.Methods Five serial cross-sectional surveys of MSM were conducted in the year of 2005-2006,2007,2008,2009,and 2010-2011 in Chaoyang district of Beijing.Whole blood samples were collected and then RNA was extracted.HIV-1 gag gene was characterized by reverse transcriptase and nested polymerase chain reaction (RT-PCR) amplification,DNA sequencing,and phylogenetic analysis of viral sequences to determine the HIV-1 subtypes.Results Phylogenetic analysis of the sequences revealed that the predominant subtypes of HIV-1 gag gene included subtype B,CRF01_AE and CRF07_BC.And CRF15_01B was detected from the year of 2008.In addition,significant changes of the distributions of subtypes and CRFs occurred from 2005 to 2011 in HIV+ MSM.Subtype B showed a significant decreased trend,while the proportions of CRF01 _AE and CRF07_BC significantly increased in the 7-year period,particularly that of CRF01_AE.Conclusions The substantial changes are observed in the diversity of HIV-1 strains circulating among MSM in Beijing during a 7-year period.

Objective:To investigate effects of tumor specific TCR gene Vα12.2-Vβ7.1 modification on recognition of tumor antigen and activation of anti-tumor reactivity of T cells.Methods: T cells were transduced using recombinant Ad 5F35-TRAV-TRBV adenovirus ,and multiplicity of infection was optimized.Specific lysis of T cells was evaluated by calcein release assay.The frequency of apoptotic cells in target cells was detected by Annexin V /PI double-labeled FACS.The expression of FasL on T cells was analyzed by FACS.The secretion of cytokine IFN-γand IL-2 of T cells was determined by ELISA assays.Results: The highest tranduce efficiency was obtained at MOI 100 by recombinant Ad5F35-TRAV-TRBV adenovirus.The frequency of TCRVα12+Vβ7+cells reached above 25%3 days after transduction.TCR gene modification enhanced the ability of T cells to lyse HLA-A2+AFP+target cells(P<0.001), the ability of T cells to induce HepG-2 apoptosis(P<0.001),and expression of FasL on T cells(P<0.001).TCR gene modification also enhanced T cells to secret IFN-γafter coculture with antigen positive tumor cells ( P<0.001 ).Conclusion: Specific TCR gene modification by recombinant adeno virus effectively promotes T cells to recognize antigen positive tumor cell and exert anti -tumor reac-tivity.

Objective To investigate the anti-hepatocarcinoma(anti-HCC) function of HLA-A2-restricted point-mutated Survivin peptide induced CTLs.Methods The HLA-A2-restricted Survivin nonapeptides were evaluated using bioinformatics software.The binding affinity of Survivin peptide to HLA-A2 molecular was determined with flow cytometry analysis.After peptide-induced CTLs were generated in vitro,flow cytometry and ELISA were performed to detect the levels of IFN-γ,which were secreted by reactive CTLs.Peptide-induced CTLs were co-cultured with hepatoma cell lines HepG2 and BEL-7402.The rates of tumor cells lysis were assayed using CytoTox 96(R) and the morphological changes of tumor cells were observed with inverted phase contrast microscope.Results Point-mutated Survivin nonapeptide Sur79M2 (KMSSGCAFL) was filtered out,which was shown higher scores compared with the wild-type peptide Sur79.Consistent with the results of software analysis,Sur79M2 showed higher binding ability in T2 binding assays.At the same time,Sur79M2-induced CTLs could release a large number of IFN-γ after incubated with target cells rather than Sur79.When co-cultured with HCC cell lines HepG2 and BEL-7402,Sur79M2-induced CTLs effectively lysis HepG2 on HLA-A2-restricted manner without killing effect on BEL-7402 that do not express HLA-A2 molecules.Conclusion Sur79M2 could elicited specific cytotoxic T lymphocytes in vitro,which were able to specifically kill HCC cell lines on HLA-A2-restricted manner.

Objective:To investigate the impact of human umbilical cord-derived mesenchymal stem cells on the activation ,the survival of human peripheral blood mononuclear cell ( hPBMC) and the proportions of each human lymphoid subgroup .Methods:PB-MC were isolated from healthy donors by density gradient centrifugation , then cultured in MSC-CM as treatment group after being acti-vated by OKT3.Each lymphoid subgroup proportion was analyzed by flow cytometry to observe the difference between treatment and control group .The effect of MSC-CM on activated PBMC for the production of IFN-γand IL-10 were tested by ELISA .The level of ap-optosis was assessed by flow cytometry with Annexin-V/PI as fluorescent marker .Results:Compared with the control group , MSC-CM down-regulated the ratio of CD4 +T cell to CD8 +T cell, and increased the proportion of CD4 +CD25 +CD127low Treg cell, thus other subgroup had no significant difference .MSC-CM inhibited the production of IFN-γby PBMC, but promoted the secretion of IL-10, and protected PBMCs from apoptosis when activated with OKT 3.Conclusion:hUC-MSC may play a role of immunosuppression by promo-ting the proliferation and activation of Treg cell .This kind of inhibitory activity is neither relied direct or indirect contact with the lym -phocytes , nor influenced by inducing immune cells apoptosis .

Objective:To develop and optimize a novel assay for determination of cytotoxicity based Calcein -AM release.Methods:The target cells stained by Calcein-AM dye,then effectors and targets were incubated at E/T ratios from 30∶1-1∶1 for 4 h at 37℃,and the supernatant of reactions were detected by Fluorescence-Measurement to analyze specific cytotoxity.Results:The optimal excitation and emission wave lengths of Calcein were 485 nm and 515 nm.Dilutions of target cells stained by Calcein-AM had a linear relationship with measured fluorescence values.The Calcein-AM dye used to stain the living cells was shown to have a low spontaneous leakage rate-less than 15% in 4 hours at 37℃.Cytotoxicity activity of CIK showed a significant and positive correlation with E/T ratio when incubated at 4 h.Conclusion:The developed cytotoxicity test by Calcein-AM release is accurate and can avoid the application of radioactive reagents.

Objectiv e:To investigate the effect of different culture conditions on the differentiation of Treg and Th17 to lay a foundation for exploring the methods to reverse the immune tolerance induced by tumor microenvironment.Methods:The IL-6 gene was cloned and stablely transferred into the tumor cell line expressing TGF-β.The conditioned mediums ( CM) were prepared by collecting the culture supernatants of tumor cell lines with or without IL-6 expression and used in the in vitro culture of peripheral blood mononuclear cells ( PBMC ) .The changes of Treg and Th17 in PBMC treated with different CM were detected with flow cytometry ( FCM) .Results:The expression of TGF-βin BEL-7402 was higher than that in HepG2.Thus the BEL-7402 was selected for preparation of cell line stablely transfected with IL -6 gene.ELISA detection confirmed the effective expression of IL -6 by the identified cell lines.It was showed that the Treg increased in PBMC treated with culture supernatants of tumor cells .However,the presence of IL-6 reversed the increase of Treg and promoted the differentiation of Th 17.Conclusion: The culture supernatants of tumor cells increases the proportion of Treg.However,the presence of IL-6 in this CM can reverse the increase of Treg and raise the proportion of Th 17.

ObjectiveTo discuss the central modulating mechanisms while acupuncturing the Stomach 36[ST36(Zusanli)]by brain functional imaging with positron emission tomography (PET).MethodsPET imaging of whole brain was performed in a group of six healthy subjects during two stimulation paradigms: pseudo acupuncture and real acupuncture at acupoint ST36(Zusanli). The data on cerebral glycometabolism,obtained by using PET,was analyzed by using statistical parametric mapping (SPM).ResultsThere was certain increase of glycometabolism in ipsilateral hypothalamus,back of medulla oblongata;bilateral insular lobe; contralateral paracentral lobule,superior part of precentral and postcentral gyrus,opercular part of frontal and temporal lobe,middle part of cingulate gyrus,head of caudate nucleus,middle part of the back of midbrain and pons,and deep part of cerebellum,whereas decrease in ipsilateral superior part of precentral and postcentral gyrus and lateral part of ipsilateral anterior cerebellar lobe,while acupuncturing at acupoint ST36(Zusanli on the right leg).ConclusionsThe central modulating mechanisms of acupuncturing ST36 are realized by neural and neuroendocrine network modulation mechanisms of vegetative nerve center in cortex and subcortex.

Objective:To explore the effect of acute and chronic glycemic ratio on the prognostic assessment in vulnerable phase of patients with acute heart failure (AHF).Methods:The clinical data of 98 AHF patients who treatment in Nanjing Pukou Hospital of Traditional Chinese Medicine from May 2019 to May 2022 were collected retrospectively, the patients were followed up for 3 months, according to whether adverse events occurred in the vulnerable phase, the patients were divided into adverse prognosis group (31 cases) and non-adverse prognosis group(67 cases). The acute and chronic glycemic ratio was calculated based on the intravenous blood glucose and glycosylated hemoglobin (HbA 1c). The influencing factors of adverse prognosis of AHF patients in vulnerable phase was analyzed by Cox risk proportion model, the predictive value of acute and chronic glycemic ratio on adverse prognosis was evaluated by receiver operating characteristic (ROC) curve. Kaplan-Meier method was used to draw the survival curve and compared the risk of adverse prognosis in patients with different acute and chronic glycemic ratio. Results:The severity of cardiac function grading as well as total cholesterol, urea nitrogen, blood glucose, acute and chronic glycemic ratio, highly sensitive C-reactive protein (hs-CPR), left ventricular anteroposterior diameter, right atrial anteroposterior diameter in the adverse prognosis group were higher than those in the non-adverse prognosis group: (3.88 ± 0.18)mmol/L vs. (3.76 ± 0.24) mmol/L, (9.39 ± 1.07) mmol/L vs. (8.68 ± 1.79) mmol/L, (10.49 ± 2.20) mmol/L vs. (7.64 ± 1.57)mmol/L, 1.37 ± 0.47 vs. 1.04 ± 0.35, (3.85 ± 0.36) mg/L vs. (3.68 ± 0.28) mg/L, (48.47 ± 7.86) mm vs. (45.37 ± 3.56) mm, (47.18 ± 5.04) mm vs. (44.05 ± 6.11) mm, there were statistical differences ( P<0.05). Cox multivariate analysis showed that hypertension, white blood cell count, blood sodium, low density lipoprotein cholesterol, acute and chronic glycemic ratio were the risk factors for adverse prognosis in vulnerable phase ( P<0.05). The area under the curve of acute and chronic glycemic ratio for predicting adverse prognosis in vulnerable phase was 0.718 (95 CI: 0.618 - 0.805, P<0.01), with specificity of 62.7%, sensitivity of 77.4%, and cut-off value of 1.07. According to the cut-off value of acute and chronic glycemic ratio, the patients were divided into acute and chronic glycemic ratio>1.07 group (43 cases), acute and chronic glycemic ratio≤ 1.07 group (55 cases), there was a statistically significant difference in the event free survival between the two groups ( P<0.01). Conclusions:AHF patients who with high acute and chronic glycemic ratio have a high risk of adverse prognosis in the vulnerable phase, which can be used as a predictor of the prognosis patients.