Impact of uterine artery embolization on ovarian function and pregnancy outcome after uterine-fibroids treatment: A prospective study.

BACKGROUND: Uterine fibroids are benign tumors that originate from smooth muscle cells of the uterus. It is the most common gynecological disorder, affecting up to 80% of women of reproductive age. Uterine fibroids can cause various symptoms such as abnormal uterine bleeding, pelvic pain, infertility, and pregnancy complications. The treatment options for uterine fibroids include medical therapy, surgical intervention, and minimally invasive techniques. AIM: To compare ovarian function of women with uterine fibroids who did or did not undergo uterine artery embolization (UAE). METHODS: This prospective cohort study enrolled 87 women with symptomatic uterine fibroids who underwent UAE, and 87 women with the same symptoms who did not undergo UAE but received conservative management or other treatments. The two groups were matched for age, body mass index, parity, and baseline characteristics of uterine fibroids. The primary outcome was ovarian function that was evaluated by serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and anti-Müllerian hormone (AMH), as well as ovarian reserve tests, such as antral follicle count (AFC) and ovarian volume (OV). The secondary outcome was fertility that was evaluated based on the menstrual cycle, ovulation, conception, pregnancy, and delivery. The participants were followed-up for 36 months and assessed at 1, 3, 6, 12, 24, and 36 months after treatment. RESULTS: The study found that the most common minor complication of UAE was postembolization syndrome in 73.6% of women, resolving within a week. No significant differences were observed between the UAE group and the control group in serum levels of reproductive hormones (FSH, LH, E2, AMH) and ovarian reserve indicators (AFC, OV) at any point up to 36 months post-treatment. Additionally, there were no significant differences in conception, pregnancy, or delivery rates, with the average time to conception and gestational age at delivery being similar between the two groups. Birth weights were also comparable. Finally, there was no significant correlation between ovarian function, fertility indicators, and the type or amount of embolic agent used or the change in fibroids post-treatment. CONCLUSION: UAE resulted in significantly positive pregnancy outcomes, no adverse events post-treatment, and is a safe and effective treatment for uterine fibroids that preserves ovarian function and fertility.

Surgical Treatment of Cervical Kyphosis and Atlantoaxial Dislocation in a Child With Loeys-Dietz Syndrome: A Case Report and Literature Review.

We report the case of a 3-year-old child with Loeys-Dietz syndrome, a rare genetic connective tissue disorder. The young girl had concurrent cervical kyphosis, atlantoaxial dislocation (AAD), and spinal cord compression. Posterior occipitocervical fusion was performed. Postoperative examination and clinical manifestations confirmed that all pedicle screws were satisfactorily placed, cervical kyphosis and AAD were corrected, and spinal cord compression was relieved. At the 1-year postoperative follow-up, the patient had recovered well, indicating that our operation was successful. To the best of our knowledge, this is the first reported surgical case of cervical kyphosis and AAD caused by Loeys-Dietz syndrome.

Comparison of structural occipital and iliac bone grafts for instrumented atlantoaxial fusions in pediatric patients: Radiologic research and clinical outcomes.

Background: Structural autografts harvested from the iliac bone have been used in atlantoaxial fusion; they have been the gold standard for years. However, emerging occipital bone grafts have the advantage of avoiding donor-site morbidity and complications. Thus, we compared the clinical outcomes of structural autografts from the occipital bone or iliac crest and discussed the clinical significance of occipital bone grafts in pediatric patients. Methods: Pediatric patients who underwent posterior fusion using occipital bone grafts (OBG) or iliac bone grafts (IBG) between 2017 and 2021 were included in this study. Data on clinical outcomes, including operation time, estimated blood loss, length of hospitalization, complications, fusion rate, and fusion time, were collected and analyzed. Additionally, 300 pediatric patients who underwent cranial computed tomography scans were included in the bone thickness evaluation procedure. The central and edge thicknesses of the harvested areas were recorded and analyzed. Results: Thirty-nine patients were included in this study. There were no significant differences in patient characteristics between the OBG and IBG groups. Patients in both groups achieved a 100% fusion rate; however, the fusion time in the OBG group was significantly longer than that in the IBG group. Estimated blood loss, operation time, and length of hospitalization were significantly lower in the OBG group than those in the IBG group. The surgery-related complication rate was lower, but not significantly, in the OBG group than that in the IBG group. For occipital bone thickness evaluation, a significant difference in the central part of the harvesting area was found between the young and old groups, with no significant sex differences. Conclusion: The use of OBG for atlantoaxial fusion is acceptable for pediatric patients with atlantoaxial dislocation, avoiding donor-site morbidity and complications.

Posterior Atlantoaxial Fusion With C1-2 Pedicle Screw Fixation for Atlantoaxial Dislocation in Pediatric Patients With Mucopolysaccharidosis IVA (Morquio a Syndrome): A Case Series.

OBJECTIVE: To investigate the efficacy and safety of posterior atlantoaxial fusion (AAF) with C1-2 pedicle screw fixation for atlantoaxial dislocation (AAD) in pediatric patients with mucopolysaccharidosis IVA (MPS IVA). METHODS: This study included 21 pediatric patients with MPS IVA who underwent posterior AAF with C1-2 pedicle screw fixation. Anatomical parameters of the C1 and C2 pedicle were measured on preoperative computed tomography (CT). The American Spinal Injury Association (ASIA) scale was used to evaluate the neurological status. The fusion and accuracy of pedicle screw was assessed on postoperative CT. Demographic, radiation dose, bone density, surgical, and clinical data were recorded. RESULTS: Patients reviewed included 21 patients younger than 16 years with an average age of 7.4 ± 4.2 years and an average of 20.9 ± 7.7 months follow-up. Fixation of 83 C1 and C2 pedicle screws was performed successfully and 96.3% of them were identified as being safe. One patient developed postoperative transient disturbance of consciousness and one developed fetal airway obstruction and died about 1 month after the surgery. Out of the remaining20 patients, fusion was achieved, symptoms were improved, and no other serious surgical complications were observed at the latest follow-up. CONCLUSIONS: Posterior AAF with C1-2 pedicle screw fixation is effective and safe for AAD in pediatric patients with MPS IVA. However, the procedure is technically demanding and should be performed by experienced surgeons with strict multidisciplinary consultations.

Naringin Inhibits Apoptosis Induced by Cyclic Stretch in Rat Annular Cells and Partially Attenuates Disc Degeneration by Inhibiting the ROS/NF-κB Pathway.

Oxidative stress and apoptosis play important roles in the pathogenesis of various degenerative diseases. Previous studies have shown that naringin can exert therapeutic effects in multiple degenerative diseases by resisting oxidative stress and inhibiting apoptosis. Although naringin is effective in treating degenerative disc disease, the underlying mechanism remains unclear. This study is aimed at investigating the effects of naringin on oxidative stress, apoptosis, and intervertebral disc degeneration (IVDD) induced by cyclic stretch and the underlying mechanisms in vitro and in vivo. Abnormal cyclic stretch was applied to rat annulus fibrosus cells, which were then treated with naringin, to observe the effects of naringin on apoptosis, oxidative stress, mitochondrial function, and the nuclear factor- (NF-) κB signaling pathway. Subsequently, a rat model of IVDD induced by dynamic and static imbalance was established to evaluate the effects of naringin on the degree of degeneration (using imaging and histology), apoptosis, and oxidative stress in the serum and the intervertebral disc. Naringin inhibited the cyclic stretch-induced apoptosis of annulus fibrosus cells, reduced oxidative stress, improved mitochondrial function, enhanced the antioxidant capacity, and suppressed the activation of the NF-κB signaling pathway. Additionally, it reduced the degree of IVDD (evaluated using magnetic resonance imaging) and the level of oxidative stress and inhibited apoptosis and p-P65 expression in the intervertebral discs of rats. Thus, naringin can inhibit cyclic stretch-induced apoptosis and delay IVDD, and the underlying mechanism may be related to the inhibition of oxidative stress and activation of the NF-κB signaling pathway. Naringin may be an effective drug for treating degenerative disc disease.

Cervical spine involvement in pediatric mucopolysaccharidosis patients: Clinical features, early diagnosis, and surgical management.

Mucopolysaccharidosis (MPS) is a progressive genetic disease that causes a deficiency in lysosomal enzymes, which play an important role in the degradation pathway of glycosaminoglycans. As a result of enzyme defects, mucopolysaccharides cannot be metabolized and thus accumulate. The cervical spine is one of the most commonly involved sites; thus, prompt surgical management before the onset of severe neurological deterioration is critical. However, because of the rarity of the disease, there is no standard treatment. In this review, we characterize the cervical spinal involvement in pediatric patients with MPS, describe the useful imaging technologies for diagnosis, and provide screening procedure for children with MPS. Surgical managements, including indications, surgical methods, possible difficulties, and solutions, are reviewed in detail.

TGF-ß1 can be regulated by NDRG2 via the NF-κB pathway in hypoxia-induced liver fibrosis.

BACKGROUND: The identification of the important elements that control hepatic stellate cell (HSC) activation will expand our understanding of the mechanism of liver fibrosis induced by hypoxia and affect the outcome of clinical treatment. A previous research demonstrated that N-Myc downstream-regulated gene 2 (NDRG2) is a potential regulator of fibrosis and a downstream target gene of hypoxia-inducible factor 1 (HIF-1). In this research, we studied the expression and function of NDRG2 in liver fibrosis induced by hypoxia. METHODS: LX-2 cells/NF-κB-silenced LX-2 cells were exposed to hypoxic conditions (1% O2) to activate HSCs in vitro. The protein and mRNA expression levels of NDRG2, α-SMA and transforming growth factor beta 1 (TGF-ß1) were evaluated by western blotting and real-time polymerase chain reaction (RT-PCR), respectively. Functional studies were performed using adenovirus-mediated gene upregulation. RESULTS: The NDRG2 mRNA and protein levels were reduced under hypoxic conditions in LX-2 cells and overexpression of NDRG2 resulted in a decrease in the expression of TGF-ß1 and α-SMA. Interestingly, no relationship was observed between NDRG2 and TGF-ß1 when the NF-κB pathway was blocked, which indicates that NDRG2 can regulate the expression of TGF-ß1 in LX-2 cells via the NF-κB pathway under hypoxic conditions. CONCLUSIONS: NDRG2 may regulate the expression of TGF-ß1 via the NF-κB pathway and may be a novel therapeutic target for liver fibrosis induced by hypoxia.

Serum DKK-1 level in the development of ankylosing spondylitis and rheumatic arthritis: a meta-analysis.

To explore the association of serum Dickkopf-1 (DKK-1) levels with the development of ankylosing spondylitis (AS) and rheumatic arthritis (RA) in humans, databases including PubMed, EBSCO, Springerlink, Ovid, WANFANG and China National Knowledge Infrastructure (CNKI) were searched to identify relevant studies. On the basis of rigorous inclusion and exclusion criteria, case-control studies of the relationships between serum DKK-1 levels and AS and RA published before December 2014 were enrolled. Statistical analyses were performed using Comprehensive Meta-analysis 2.0 (CMA 2.0). Seven case-control trials with a total of 300 AS patients, 136 RA patients and 232 healthy controls were included in this study. Meta-analysis results revealed that DKK-1 serum levels were significantly higher in AS patients than in normal controls (standard mean differences (s.m.d.)=0.301, 95% confidence interval (CI)=0.094-0.507, P=0.004), whereas no significant difference in DKK-1 serum levels was observed between RA patients and healthy controls (s.m.d.=0.798, 95% CI=-2.166-3.763, P=0.598). Serum DKK-1 level may be closely related to the development of AS but not of RA.

Transvenous treatment of complex cavernous dural arteriovenous fistulae with Onyx and coils.

Transvenous embolization has become the treatment of choice for such lesions We evaluated Onxy for patients with cavernous dural arteriovenous fistulae (CDAVFs) who underwent transvenous embolization via different transvenous approaches. Case records of six patients with symptomatic CDAVFs, treated between October 2006 and November 2007 were reviewed. A total of seven transvenous procedures were performed in the six patients with CDAVFs. All the patients with CDAVFs of the cavernous sinus were symptom free following embolization. The approach via the internal jugular vein and the inferior petrosal sinus was possible in four of the six patients, with complete occlusion of the fistula. In the remaining two patients, the approach was via the facial vein. Transient bradyarrythmia without morbidity was the only complication in two patients.

Tumor cell-released TLR4 ligands stimulate Gr-1+CD11b+F4/80+ cells to induce apoptosis of activated T cells.

Gr-1(+)CD11b(+)F4/80(+) cells play important roles in tumor development and have a negative effect on tumor immunotherapy. So far, the mechanisms underlying the regulation of their immunosuppressive phenotype by classical and alternative macrophage activation stimuli are not well elucidated. In this study, we found that molecules from necrotic tumor cells (NTC-Ms) stimulated Gr-1(+)CD11b(+)F4/80(+) cells to induce apoptosis of activated T cells but not nonstimulated T cells. The apoptosis-inducing capacity was determined by higher expression levels of arginase I and IL-10 relative to those of NO synthase 2 and IL-12 in Gr-1(+)CD11b(+)F4/80(+) cells, which were induced by NTC-Ms through TLR4 signaling. The apoptosis-inducing capacity of NTC-Ms-stimulated Gr-1(+)CD11b(+)F4/80(+) cells could be enhanced by IL-10. IFN-gamma may reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells only if their response to IFN-gamma was not attenuated. However, the potential of Gr-1(+)CD11b(+)F4/80(+) cells to express IL-12 in response to IFN-gamma could be attenuated by tumor, partially due to the existence of active STAT3 in Gr-1(+)CD11b(+)F4/80(+) cells and NTC-Ms from tumor. In this situation, IFN-gamma could not effectively reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells. Tumor immunotherapy with 4-1BBL/soluble programmed death-1 may significantly reduce, but not abolish the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells in local microenvironment. Blockade of TLR4 signaling could further reduce the apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells and enhance the suppressive effect of 4-1BBL/soluble form of programmed death-1 on tumor growth. These findings indicate the relationship of distinct signaling pathways with apoptosis-inducing capacity of Gr-1(+)CD11b(+)F4/80(+) cells and emphasize the importance of blocking TLR4 signaling to prevent the induction of T cell apoptosis by Gr-1(+)CD11b(+)F4/80(+) cells.

Heated lipiodol as an embolization agent for transhepatic arterial embolization in VX2 rabbit liver cancer model.

PURPOSE: To evaluate the therapeutic effect of heated (60 degrees C) lipiodol via hepatic artery administration in a rabbit model of VX2 liver cancer. MATERIALS AND METHODS: Thirty male New Zealand white rabbits were randomly divided into three groups with 10 rabbits assigned to each group. VX2 carcinoma cells were surgically implanted into the left hepatic lobe. The tumors were allowed to grow for 2 weeks, and studies were performed until the diameter of the tumors detected by ultrasonograph reached 2-3cm. Under anesthesia, trans-catheter hepatic arterial embolization was performed and doxorubicin-lipiodol (37 degrees C) (1mL), lipiodol (60 degrees C) (1mL) or control (physiological saline (37 degrees C) (1mL)) solution was injected into the hepatic arteries of animals in the three groups. One week later, the volume of the tumor was measured by ultrasonograph again. The serum of all rabbits was collected before injection and at 4 and 7 days after injection, and the level of aspartate aminotransferase (AST) was checked. The survival period of the three groups of rabbits after treatment was also recorded. During the last course of their disease, the rabbits were given analgesics to relieve suffering. RESULTS: The tumor growth rate in the lipiodol (60 degrees C) group (0.92+/-0.21, tumor volume from 1811+/-435 to 1670+/-564mm(3)) was significantly lower than that in the control group (3.48+/-1.17, tumor volume from 1808+/-756 to 5747+/-1341mm(3)) (P<0.05) and in the doxorubicin-lipiodol (37 degrees C) group (1.69+/-0.26, tumor volume from 1881+/-641 to 2428+/-752mm(3)) (P<0.05). Consequently, the survival period of the animals in the lipiodol (60 degrees C) group (41.0+/-3.0 days) was significantly greater than that in the doxorubicin-lipiodol (37 degrees C) group (38.0+/-2.5 days) (P<0.05). On the other hand, there was no statistically significant difference in serum AST levels between the lipiodol (60 degrees C) group (148.2+/-11.3UL(-1)) and the doxorubicin-lipiodol (37 degrees C) group (139.7+/-12.3UL(-1)) (P>0.05). However, the serum AST level in the lipiodol (60 degrees C) group was significantly higher at 4 days after injection (P<0.05) than in the control group (68.6+/-6.6UL(-1)). CONCLUSIONS: Treatment with lipiodol (60 degrees C) resulted in an effect on serum AST levels similar to that caused by treatment with doxorubicin-lipiodol (37 degrees C). Thus, lipiodol (60 degrees C) treatment could greatly prolong the survival period of rabbits with VX2 cancer by inhibiting tumor growth.

Dendritic cells modified with 6Ckine/IFNgamma fusion gene induce specific cytotoxic T lymphocytes in vitro.

BACKGROUND AND OBJECTIVE: [corrected] Dendritic cells play an important role in initiation and regulation of immune responses. Previous studies demonstrated that intratumoral administration of 6Ckine-modified DCs enhanced local and systemic antitumor effects. Herein we report the investigation of the specific CTL responses elicited by adenoviral 6Ckine/IFNgamma fusion gene-modified DCs in vitro. METHODS: Human monocyte-derived DCs were modified with an adenoviral vector encoding 6Ckine/IFNgamma fusion protein (Ad-6Ckine/IFNgamma), and then investigated the effect of 6Ckine/IFNgamma fusion protein on the maturation, cytokine and chemokine secretion of DCs, and their activities of recruiting and activating T cells in vitro were investigated. RESULTS: 6Ckine/IFNgamma fusion protein induced DC maturation characterized with the upregulation of CD83 and CCR7. And it up-regulated the expression of RANTES and IL-12p70, down-regulated that of IL-10 in DCs. Additionally, 6Ckine/IFNgamma markedly increased DC's recruiting ability for naive T cells, benefiting from the enhanced expression of chemokines 6Ckine and RANTES in DCs. Fusion gene-modified DCs significantly promoted the proliferation of autologous T cells, induced Th1 differentiation by upregulating the expression of IL-2 and T-bet in T cells, and increased specific cytotoxicity of CTLs against specific tumor cells, HepG2 or LoVo cells, respectively. CONCLUSION: Combining the effects of 6Ckine and IFNgamma, Ad-6Ckine/IFNgamma modified DCs induced enhanced CTL responses in vitro, which indicated that Ad-6Ckine/IFNgamma modified DCs might be used as an adjuvant to trigger an effective antitumor immune response.

Long-Term toxicity studies in Canine of E10A, an adenoviral vector for human endostatin gene.

E10A, a recombinant adenovirus type 5 vector carrying the human endostatin gene, may be a promising gene therapy drug in the treatment of solid tumors by antiangiogenesis, but a preclinical safety evaluation of E10A has not yet been performed. With high and low doses equivalent to 30 and 7.5 times the human curative dose, respectively, intramuscular injections of E10A were given once daily, 6 days/week, for 3 months, followed by a 1-month recovery period. As of 4 months, all experimental animals appeared generally healthy: normal behavior and eating habits, no nausea, vomiting, or salivation, no abnormal changes in urination or defecation, and increased body weight with the time of experiment. Urinalysis, hemogram, blood biochemistry, electrocardiogram, macroscopic and microscopic studies of organs and tissues were done before treatment, at month 3 of treatment, and 1 month posttreatment. At all time points, no significant abnormal toxic effects were noted. Preliminary investigation of E10A immunotoxicity in dogs indicated that anti-adenoviral antibodies were generated, in a dose- and time-independent manner, after E10A injection. Our data demonstrated that, long term, high-dose intramuscular administration of recombinant human endostatin-carrying adenovirus (E10A) was not notably toxic and might be safe for clinical therapeutic use, although additional long-term toxicity studies by other administration routes are still necessary.

Uric acid enhances T cell immune responses to hepatitis B surface antigen-pulsed-dendritic cells in mice.

AIM: To study the induction of T cellular immune responses in BALB/c mice immunized with uric acid and dendritic cells (DCs) pulsed with hepatitis B virus surface antigen (HBsAg). METHODS: DCs were generated from bone-marrow cells of BABL/c mice, and then pulsed or unpulsed with HBsAg protein (HBsAg-pulsed-DCs or unpulsed-DCs) in vitro. BABL/c mice were immunized with HBsAg-pulsed-DCs (1 x 10(6)) and uric acid, injected through the tail vein of each mouse. The mice in control groups were immunized with HBsAg-pulsed-DCs alone, unpulsed-DCs alone or 200 microg uric acid alone or PBS alone. The immunization was repeated 7 d later. Cytotoxic T lymphocytes (CTLs) in vivo were determined by the CFSE labeled spleen lysis assay. Spleen cells or spleen T cells were isolated, and re-stimulated in vitro with HBsAg for 120 h or 72 h. Production of IFN-gamma and IL-4 secreted by spleen cells were determined by ELISA method; proliferation of spleen T cells were detected by flow cytometry. RESULTS: The cytotoxicities of HBsAg-specific-CTLs, generated after immunization of HBsAg-pulsed-DCs and uric acid, were 68.63% +/- 11.32% and significantly stronger than that in the control groups (P < 0.01). Compared with control groups, in mice treated with uric acid and HBsAg-pulsed-DCs, the spleen T cell proliferation to HBsAg re-stimulation was stronger (1.34 +/- 0.093 vs 1.081 +/- 0.028, P < 0.01), the level of IFN-gamma secreted by splenocytes was higher (266.575 +/- 51.323 vs 135.223 +/- 32.563, P < 0.01) , and IL-4 level was lower (22.385 +/- 2.252 vs 40.598 +/- 4.218, P < 0.01). CONCLUSION: Uric acid can strongly enhance T cell immune responses induced by HBsAg-pulsed-DCs vaccine. Uric acid may serve as an effective adjuvant of DC vaccine against HBV infection.

[Effect of T-bet on biological functions of mouse macrophage Raw264.7].

BACKGROUND & OBJECTIVE: T-bet (T box expressed in T cells), a Th1-specific T box transcription factor, controls many kinds of immune cells, such as Th1, NK, CD8+, dendritic cells, and B cells. This study was to explore potential effects of T-bet gene on biological functions of mouse macrophage Raw264.7 cells in vitro. METHODS: The eukaryotic expression vector carrying mouse T-bet (pcDNA3.0-mT-bet) was constructed and identified by consequence analysis, double restrictive endonucleases digestion and polymerase chain reaction (PCR). The gene expression in Raw264.7 cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. PBS, pcDNA3.0, and pcDNA3.0-mT-bet were transiently transfected into Raw264.7 cells respectively; cell cycle, MHC I/II expression levels, phagocytic activity of FITC-dextran, nitric oxide (NO) secretion level, and the cytotoxicity of Raw264.7 cells to mouse leukemia cell line L1210 were evaluated 48 hours after transfection. RESULTS: Eukaryotic expression vector which could express T-bet protein in Raw264.7 cells was successfully constructed. There was no difference in cell cycle between pcDNA3.0 group and pcDNA3.0-mT-bet group. There was significant difference in MHC I expression level between pcDNA3.0 group (20.8+/-0.7) and pcDNA3.0-mT-bet group (24.8+/-0.6, P<0.05), but not in MHC II expression level; there was also difference in mean fluorescence intensity of phagocytized dextran between pcDNA3.0 group (28.2+/-0.4) and pcDNA3.0-mT-bet group (32.8+/-0.8, P<0.05); there was also significant difference in NO secretion level between pcDNA3 group (0 pmol) and pcDNA3.0-mT-bet group [(1.7+/-0.6) pmol, P<0.05] without lipopolysaccharide (LPS) stimulation; meanwhile, significant difference was also observed between pcDNA3.0 group [(10.5 +/-1.3) pmol] and pcDNA3.0-mT-bet group [(15.6+/-1.6) pmol, P<0.05] under the stimulation of LPS (10 microg/ml) for 20 h; there was also difference in cytotoxicity of Raw264.7 cells to L1210 cells in vitro between pcDNA3 group [(35.6+/-2.1)%] and pcDNA3.0-mT-bet group [(51.9+/-3.5)%, P<0.05]. CONCLUSION: T-bet up-regulates MHC I expression and NO secretion level in Raw264.7 cells, increases their cytotoxicity to L1210 cells, but has no influences on the cell cycle and MHC II expression.

Induction of the Epstein-Barr virus latent membrane protein 2 antigen-specific cytotoxic T lymphocytes using human leukocyte antigen tetramer-based artificial antigen-presenting cells.

Cytotoxic T lymphocytes (CTLs) specific for the Epstein-Barr virus (EBV) latent membrane protein 2 (LMP2) antigen are important reagents for the treatment of some EBV-associated malignancies, such as EBV-positive Hodgkin's disease and nasopharyngeal carcinoma. However, the therapeutic amount of CTLs is often hampered by the limited supply of antigen-presenting cells. To address this issue, an artificial antigen-presenting cell (aAPC) was made by coating a human leukocyte antigen (HLA)-pLMP2 tetrameric complex, anti-CD28 antibody and CD54 molecule to a cell-sized latex bead, which provided the dual signals required for T cell activation. By co-culture of the HLA-A2-LMP2 bearing aAPC and peripheral blood mononuclear cells from HLA-A2 positive healthy donors, LMP2 antigen-specific CTLs were induced and expanded in vitro. The specificity of the aAPC-induced CTLs was demonstrated by both HLA-A2-LMP2 tetramer staining and cytotoxicity against HLA-A2-LMP2 bearing T2 cell, the cytotoxicity was inhibited by the anti-HLA class I antibody (W6/32). These results showed that LMP2 antigen-specific CTLs could be induced and expanded in vitro by the HLA-A2-LMP2-bearing aAPC. Thus, aAPCs coated with an HLA-pLMP2 complex, anti-CD28 and CD54 might be promising tools for the enrichment of LMP2-specific CTLs for adoptive immunotherapy.

[HLA-G1 molecule expressed by ECV304 cells inhibit cytotoxic activity of allogeneic NK cells].

AIM: To study the effect of HLA-G1 molecule expressed by an endothelial cell line (ECV304) on the cytotoxic activity of allogeneic NK cells. METHODS: ECV304 cells were transfected with recombinant plasmid pcDNA3-HLA-G1 by the liposome transfection, and the expressed HLA-G1 on the cell surface was detected by indirect immunofluorescent assay and flow cytometry. The cytotoxic activity of allogeneic NK cells against ECV304 cells was analyzed by the MTT method. RESULTS: HLA-G1 was expressed on the surface of the transfected ECV304 cells. The specific lysis of NK cells against plasmid pcDNA3 transfected ECV304 was (50.6+/-18.1)%, while the specific lysis against pcDNA3-HLA-G1 transfected ECV304 was (29.7+/-11.4)%, which was significantly lower than the former (P<0.001). CONCLUSION: HLA-G1 expressed by the ECV304 cells can inhibit cytotoxicity of allogeneic NK cells.

[Prokaryotic expression of BirA enzyme and identification of the bioactivity of expressed product].

AIM: To construct the expression vector of biotin-protein ligase (BirA enzyme) gene and express the BirA enzyme with bioactivity in E.coli BL-21 (DE3). METHODS: The BirA gene was amplified from E.coli genome by PCR and cloned into pGEX-4T-2 to construct the recombinant plasmid pGEX-BirA. After being verified by DNA sequencing, the fusion protein was expressed under IPTG induction in the E.coli BL-21 (DE3). The expressed product was purified through Glutathione-agarose chromatography column. The enzyme activity of the expressed product was identified by ELISA and Western blot. RESULTS: The recombinant prokaryotic expression vector pGEX-BirA was constructed and the fusion protein GST-BirA was expressed successfully. The results of ELISA and Western blot showed that the purified BirA enzyme biotinylated HLA-A2 peptide complex. CONCLUSION: BirA enzyme with bioactivity is prepared successfully, which can be used for studying the interaction between protein and protein.

Dynamic distribution and expression in vivo of human endostatin gene delivered by adenoviral vector.

Endostatin, a 20-kDa carboxyl-terminal fragment of collagen XVIII, is a potent inhibitor of endothelial cell proliferation and tumor angiogenesis. We have constructed replication-deficient recombinant adenovirus (Ad-rhE), which encoded secreted human endostatin, and our previous studies showed that Ad-rhE had a potent suppression of tumor growth in vivo. In the present study, we investigated the dynamic distribution and expression of human endostatin gene in vivo using fluorogenic real-time quantitative PCR and enzyme-linked immunosorbent assay(ELISA), respectively, with an injection of 2.0 x10(9)pfu of Ad-rhE. After injection, the Ad-rhE DNAs decreased sharply, but lasted a relative long-term at low concentration (10,000--20,000 copies/mg tissues). Whereas the expressed endostatin rose up rapidly, and reached to the top on day 5 after injection of Ad-rhE, and then decreased sharply, but endostatin in tumors sustained to over 9 days at a certain level. Both Ad-rhE DNAs and endostatin mainly enriched in tumors in vivo, and then in livers. These results suggest that endostatin gene delivered by adenoviral vector can generate a high expression in vivo, and both the metabolism pathways of Ad-rhE DNAs and endostatin in vivo are through the systems of livers.