The Viral Load of Epstein-Barr Virus in Blood of Children after Hematopoietic Stem Cell Transplantation.

Objective: To detect the Epstein-Barr virus (EBV) viral load of children after hematopoietic stem cell transplantation (HSCT) using chip digital PCR (cdPCR). Methods: The sensitivity of cdPCR was determined using EBV plasmids and the EBV B95-8 strain. The specificity of EBV cdPCR was evaluated using the EBV B95-8 strain and other herpesviruses (herpes simplex virus 1, herpes simplex virus 2, varicella zoster virus, human cytomegalovirus, human herpesvirus 6, and human herpesvirus 7). From May 2019 to September 2020, 64 serum samples of children following HSCT were collected. EBV infection and the viral load of serum samples were detected by cdPCR. The epidemiological characteristics of EBV infections were analyzed in HSCT patients. Results: The limit of detection of EBV cdPCR was 110 copies/mL, and the limit of detection of EBV quantitative PCR was 327 copies/mL for the pUC57-BALF5 plasmid. The result of EBV cdPCR was up to 121 copies/mL in the EBV B95-8 strain, and both were more sensitive than that of quantitative PCR. Using cdPCR, the incidence of EBV infection was 18.75% in 64 children after HSCT. The minimum EBV viral load was 140 copies/mL, and the maximum viral load was 3,209 copies/mL using cdPCR. The average hospital stay of children with EBV infection (184 ± 91 days) was longer than that of children without EBV infection (125 ± 79 days), P = 0.026. Conclusion: EBV cdPCR had good sensitivity and specificity. The incidence of EBV infection was 18.75% in 64 children after HSCT from May 2019 to September 2020. EBV cdPCR could therefore be a novel method to detect EBV viral load in children after HSCT.

LMP2-DC Vaccine Elicits Specific EBV-LMP2 Response to Effectively Improve Immunotherapy in Patients with Nasopharyngeal Cancer.

OBJECTIVE: To evaluate the safety and effectiveness of a vaccine based on latent membrane protein 2 (LMP2) modified dendritic cells (DCs) that boosts specific responses of cytotoxic T lymphocytes (CTLs) to LMP2 before and after intradermal injection in patients with nasopharyngeal carcinoma (NPC). METHODS: DCs were derived from peripheral blood monocytes of patients with NPC. We prepared LMP2-DCs infected by recombinant adenovirus vector expressing LMP2 (rAd-LMP2). NPC patients were immunized with 2 × 10 5 LMP2-DCs by intradermal injection at week 0 and after the second and fourth weeks. Specific responses to LMP2 were detected by enzyme-linked immunospot (ELISPOT) assay at week 0 and at the fifth and eighth weeks. Local clinicians performed the follow-up and tracking of patients. RESULTS: We demonstrated that DCs derived from monocytes displayed typical DC morphologies; the expression of LMP2 in the LMP2-DCs vaccine was confirmed by immunocytochemical assay. Twenty-nine patients with NPC were enrolled in this clinical trial. The LMP2-DCs vaccine was well tolerated in all of the patients. Boosted responses to LMP2 peptide sub-pools were observed in 18 of the 29 patients with NPC. The follow-up data of 29 immunized patients from April, 2010 to April 2015 indicated a five-year survival rate of 94.4% in responders and 45.5% in non-responders. CONCLUSION: In this pilot study, we demonstrated that the LMP2-DCs vaccine is safe and effective in patients with NPC. Specific CTLs responses to LMP2 play a certain role in controlling and preventing the recurrence and metastasis of NPC, which warrants further clinical testing.

[Construction and immunological responses of recombinant adenovirus containing Epstein-Barr nuclear antigen 1 in mice].

This study aimed to construct recombinant adenovirus expressing Epstein-Barr nuclear antigen 1 (EBNA1) against nasopharyngeal carcinoma (NPC). The C-terminal region fragment of the ebna1 gene of Epstein-Barr virus was amplified from the standard strain B95-8 by polymerase chain reaction (PCR). The gene fragment was inserted into the pDC316 shuttle plasmid using the EcoRI and BgIII restriction enzyme sites. The pDC316-ebna1 shuttle plasmid and pBHG helper plasmid were cotransfected into HEK293 cells after sequencing. The soluble protein was extracted from HEK293 cells, which caused apparent cytopathic effects. The transcription and expression of the ebna1 gene were confirmed using flow cytometry and Western blotting. rAd-ebna1 titers were measured by the TCID50. rAd-ebna1 was injected into BALB/c mice at a dose of 2 x 10(8) VP per mouse, EBNA1 epitope-specific responses were measured at 1st, 2nd, 4th and 8th weeks post-immunization. The target fragment of ebna1 (939 bp) was obtained by PCR, and was in consensus with the sequence from the standard strain B95-8. Cytopathic effects were observed after the pDC316-ebna1 shuttle plasmid and pBHG helper plasmid were cotransfected into HEK293 cells. rAd-ebna1 was successfully recombined in HEK293 cells. EBNA1 protein was detected in HEK293 cells, rAd-ebna1 titers reached 10(8) TCID50/mL. Specific responses to CD4+ epitopes of EBNA1 were detected in the immunized mice. In conclusion, rAd-ebna1 was successfully constructed and induced specific responses to CD4+ epitopes of EBNA1 in immunized mice.

[Expression and purification of EBV-LMP2 protein].

OBJECTIVE: To obtain a second Epstein-Barr virus membrane protein (LMP2) in insect cells. METHODS: The full length EBV-LMP2 gene was inserted into baculovirus expression transfer vector pFastBac HT B to obtain the recombinant baculoviruses Bac-LMP2. And generation of recombinant baculoviruses was followed by transfection of the recombinant Bac-LMP2 into insect cells, then the recombinant LMP2 protein was recognized by SDS-PAGE and western blot. The expressed LMP2 protein was purified by one step with Ni-NTA metal chelation chromatography. RESULTS: The expressed LMP2 protein was confirmed by SDS-PAGE and western blot. The purity of purified LMP2 protein is up to 86% by HPLC analysis. CONCLUSION: The EBV-LMP2 was expressed in insect cells, and the purified LMP2 protein was obtained.

[Expression of peroxiredoxin III in cervical lesions].

OBJECTIVE: To investigate the expression feature of peroxiredoxin III in cervical lesions and to further understand the mechanism for cervical cancer development/progression. METHODS: Expression of peroxiredoxin III was immunohistochemically detected in cervical cancer. In addition, cervical epithelia were transfected with recombinant adeno-associated virus vector containing human papillomavirus 16 E6/E7 and peroxiredoxin III expression was detected by quantitative real time PCR and Western blotting. RESULTS: Peroxiredoxin III was significantly up-regulated in cervical cancer tissues. Nevertheless, expression of peroxiredoxin III remained unchanged in cervical epithelial cells after transfection. CONCLUSION: It seems that Prx III is not related to cervical cancer initiation. Up-regulation of peroxiredoxin III in cervical cancer might be an active response to oxidative stress in malignant cells, which protects against oxidatiton-induced apoptosis.

[A serological survey of Epstein-Barr virus infection in children in Beijing].

OBJECTIVE: To understand the prevalence of Epstein-Barr virus (EBV) infection in urban and rural areas of Beijing using the serological method. METHODS: Totally 589 serum samples were collected from children in Beijing urban and rural areas who were 0--14 years old and tested with Viron-Seron ELISA classic EBV virus capsid antigen IgG antibody (EBV VCA IgG) kit. Optical density of serum samples was obtained at the wavelength of 405 nanometers. Sero-positive or negative samples were determined according to standard curve and cut-off attached in ELISA classic EBV VCA IgG kits. The activity of EBV VCA IgG was calculated by using special formula. The percentage and activity of EBV VCA IgG from Beijing children were compared with SPSS 13.0 between the urban and rural areas. RESULTS: The percentage of EBV VCA IgG seropositive samples was 83.6%, and 80.8% in those from urban and 86.2% in those from rural areas. The peak value of EBV infection was 71% seen among children under the age of 3 years, and in urban area the rate was 67.7%, which was lower than that in the rural area (75.3%), and was 82.5% by the age of 6, which was lower than the data (up to 90%) reported 30 years ago. There was a significant difference in EBV infection rate and VCA IgG activities in children at different ages between urban and rural areas (P < 0.05). CONCLUSION: The rate of EBV infection in children living in urban area was lower by the age of 6 years. The primary infection of EBV occurred late in part of children lived in urban area.

[Immune responses induced by recombinant adenovirus Ad5F35-LMP2 in rhesus monkeys].

OBJECTIVE: To observe the specific cellular and humoral immune responses after immunization with recombinant adenovirus Ad5F35-LMP2 in rhesus monkeys. METHODS: Sixteen rhesuses were immunized with Ad5F35-LMP2 through intra-muscular injection in three groups: high dosage group (1.5 x 10(10) TCID(50)/rhesus), medium dosage group (1.5 x 10(9)TCID(50)/rhesus), low dosage group (1.5 x 10(8)TCID50/rhesus) and the last group was control (PBS 4 ml/rhesus). They were totally immunized three times at intervals of one month. The EBV-LMP2 specific cellular immune responses were tested during the 0, 4, 8, 12 weeks by Elispot after immunization respectively. And the titers of anti-LMP2 antibody were tested by EIA at the same time. RESULTS: EBV-LMP2 specific cellular and humoral immune responses which were induced by recombinant adenovirus Ad5F35-LMP2 can be found in all the three dosage groups. The potency of immune responses was related with the dosage of immunization. Higher dosage elicited more potent immune response. CONCLUSION: The recombinant adenovirus Ad5F35-LMP2 could elicit LMP2 specific cellular and humoral immune responses in rhesus.

[Effect of Nocardia rubra cell wall skeleton on the growth of HeLa cell line infected with HPV].

OBJECTIVE: To investigate the effects of Nocardia rubra cell wall skeleton (Nr-CWS) on the HeLa cell line, one of the cell lines of human cervical cancer, infected with HPV. METHODS: HPV-infected HeLa (HPV 18-positive cells) cultured in vitro were divided into two groups: the experiment group and control group. Nr-CWS was added to the experiment group and PBS to the control. The growth and proliferation of HeLa cells were detected with MTT and flow cytometry technology. Inhibitive effect of HeLa transplanted tumor was investigated in Scid mice. RESULTS: The growth of HeLa cells in the experimental group was apparently decreased compared with that of the control. The results of flow cytometry demonstrated that more HeLa cells were transferred into quiescent phase in the experimental group than that in the control. While less in the proliferative phase, both of the volume and weight of HeLa transplanted tumor with drug-added group were less than those of control group. CONCLUSION: The Nocardia rubra cell wall skeleton is a potiental growth inhibitor and inducer of apoptosis of cervical cancer cells in vitro and may provide a new way in prevention or supplementary management of anti-human papilloma virus.

[Transfection of human hepatic stimulator substance gene could protect BEL-7402 cells against hepatotoxins].

OBJECTIVE: To investigate protective effects of hHSS transfection against CCl4 or H2O2. METHODS: cDNA coding for hHSS was constructed into eukaryotic vector of pcDNA3.1 and transfected into BEL-7402 hepatoma cells. The expression of hHSS was analyzed with Northern blot. RESULTS: The growth of the hepatoma cells was remarkably enhanced 24 to 144h after hHSS gene transfection, which suggesting hHSS gene expression could stimulate cells activity. Meantime, incubation of both wild-type and vector-transfected as well as hHSS-transfected cells with CCl4 or H2O2 resulted in severe damage as marked by cell mortality and the rate of apoptosis. However, it appeared that the transfection of hHSS enabled the hepatoma cells to raise obvious resistance against CCl4 and H2O2 injury. Compared the vector cells to the vector-transfected cells, apoptosis ratio were (32.44+/-0.52)% and (25.60+/-0.66)% in which treated with CCl4, while (47.78+/-0.45)% and (37.40+/-0.69)% in which treated with H2O2, t value is 16.82 and 25.20, P<0.01. MAPK phosphorylation was also activated after HSS transfected. CONCLUSION: The function of hHSS gene expression could be related to proliferation of cell and protection against free radical damage.

[Induction of human oral carcinoma by human papillomavirus 16 E6/E7 and TPA].

BACKGROUND: To study the effect of human papillomavirus (HPV) 16 E6/E7 and TPA (12-O-tetradecanog-1-phorbol-13-acetate) on malignant transformation of human embryo oral tissue. METHODS: Recombinant plasmid with HPV 16 E6/E7 was constructed and transfected into human embryo oral tissue. The oral tissue with HPV 16 E6/E7 gene or without the gene was inoculated into the hypophloeodal of right shoulder in scid mice, respectively. The study was conducted in four groups: the first group was the oral tissue transfected plasmid with HPV 16 E6/E7 plus TPA, which were inoculated into 8 scid mice; the second group was only oral tissue transfected with plasmid with HPV 16 E6/E7 into 6 scid mice; the third group was normal oral tissue plus TPA inoculated into 6 scid mice, and the final group was only normal oral tissue inoculated into 5 scid mice. Three days after inoculation, TPA was injected at the left shoulder of the mice once a week. Twelve weeks after inoculation, tumor was found in 7 scid mice from the first group. HPV 16 E6/E7 gene in tumor tissues was analyzed by PCR. RESULTS: The rate of tumor formation was 7/8 in the first group; no tumor was found in the other groups. Pathological diagnosis of the tumor was fibrohistiocytoma. HPV 16 E6/E7 gene was detected by PCR in tumor tissues. CONCLUSION: With the cooperating action of TPA, human oral tissue containing HPV 16 E6/E7 gene could cause malignant transformation in scid mice.

[Increased resistance against oxidant-induced injury in the rat vascular smooth muscle cells transfected with human heme oxygenase-1 gene].

The heme oxygenase-1 (HO-1), a rate-limiting enzyme in heme metabolism, has been recently defined as a novel stress-stimulated protein, since the intracellular expression of HO-1 in response to various stimuli as oxidation, ischemia and endotoxin injury has been proved to be able to protect the cells from damage. In this study, a retroviral vector containing human HO-1 gene was constructed and transfected to rat vascular smooth muscle cells (VSMCs). Using Southern and Northern blot analyses, the integration and mRNA expression of HO-1 gene in the transfected cells were confirmed. The profound protein expression of HO-1 as well as HO enzyme activity in the transfected cells increased by 1.8-fold and 2.0-fold respectively as compared with the non-transfected cells. It was found that the HO-1 transfected-VSMCs presented dominant resistance to toxicity produced by exposure to H2O2, as a significant protective effect of HO-1 marked by cell survival and LDH leakage was observed when 200, 400 and 600 micromol/L of H2O2 were used. The protection of HO-1 rapidly declined after the transfected-VSMCs were pretreated 24 h with an HO-1 specific inhibitor (ZnPP-IX). The results of this investigation suggest that the functional expression of HO-1 gene within VSMCs raises an alternative ability to protect the vascular cells against active oxygen injury.

[Prokaryotic expression and purification of human hepatic stimulator substance].

To explore the possibility of prokaryotic expression of human hepatic stimulator substance (hHSS), hHSS gene was inserted in the downstream of glutathion S-transferase (GST) in a pET-42a expression vector and recombinant GST-hHSS fusion protein was expressed under IPTG induction in BL-21(DE3) cells. The recombinant HSS was purified with His.Tag affinity chromatography, and its bioactivity was analyzed. The results showed that GST-hHSS fusion protein was expressed both as a soluble or a inclusive body in bacterial cytosol. The soluble GST-hHSS expression reached up to 30% of the whole soluble protein of bacteria as determined by densitometry. The cleavage of GST-hHSS fusion protein with Factor Xa produced two fragments of the protein, which sized 33 and 15 kD, respectively. The molecular weight of recombinant HSS protein was identical to theoretical deduction based on the DNA sequences. The protein homology of 15 kD hHSS could be efficiently eluted out after Factor Xa cleavage. It is further indicated that the recombinant hHSS is able to proliferate hepatoma cells of BEL-7402 in the preliminary experiments.