Rare primary squamous cell carcinoma of the intrahepatic bile duct: A case report and review of literature.

BACKGROUND: Cholangiocarcinoma is the most common malignancy of the biliary tree and has a poor prognosis. Adenocarcinoma is the most common pathological type of cholangiocarcinomas, but rare squamous, adenosquamous, and mucinous variants have been reported without adequate clinical data. CASE SUMMARY: This report describes a rare case of primary squamous cell carcinoma (SCC) of the intrahepatic bile duct. The patient was admitted with a tumor in the hepatic caudate lobe with no obvious clinical symptoms. Examination revealed hepatitis B surface antigen positivity, a slight increase in alfa-fetoprotein to 16.34 ng/mL, and an irregular slightly heterogeneous enhancing lesion in the hepatic caudate lobe, which was initially thought to be hepatocellular carcinoma. Laparoscopic resection was performed, and the final pathology suggested a rare primary SCC of the intrahepatic bile duct. Immunohistochemistry indicated positivity for villin, partial positivity for p63, and negativity for hepatocyte, CK7, CK8, CK19, and CK20. The Ki-67 index was approximately 60%. The patient received six cycles of Tegio chemotherapy. A new lesion was detected in the liver after 15 months. The surgery was performed, and the patient was followed-up at a local hospital. To date, no new lesions have been observed. CONCLUSION: Surgery is the first choice for resectable lesions, and combined chemotherapy based on pathology is essential for increasing overall survival.

Effect of sevoflurane and halothane anesthesia on cognitive function and immune function in young rats.

In the current study, we scrutinized the effect of sevoflurane and halothane on cognitive and immune function in young rats. The rats were divided into following groups: sevoflurane, halothane and sevoflurane + halothane groups, respectively. The rats were regularly treated with the pre-determined treatment. We also scrutinized the serum proinflammatory cytokines including IL-10, IL-4 and IL-2; brain level IL-1ß; hippocampal neuronal apoptosis concentration were estimated. The water maze test was performed in rats for the estimation of cognitive ability. During the water maze test, on the 1st day the sevoflurane group showed the latency; sevoflurane and sevoflurane + halothane group demonstrated the declined latency gradually as compared to the control group rats after the 3 days. The latency of the control, halothane, sevoflurane + halothane group rats showed the reduced latency and also showed the reduced crossing circle times. The hippocampal neuron apoptosis was significantly increased in halothane and sevoflurane + halothane group as compared to control group rats, respectively. Control group rats demonstrated the increased neuron apoptosis. The proinflammatory cytokines including IL-10 and IL-4 was significantly higher in sevoflurane, halothane and sevoflurane + halothane group rats after anesthesia and the whole brain IL-1ß was significantly decrease in the sevoflurane, halothane and sevoflurane + halothane as compared to control group. Sevoflurane can inhibit the anesthesia effect of halothane on the immune and cognitive function of rats.

Langerhans cell histiocytosis with multiple spinal involvement.

To stress the clinical and radiologic presentation and treatment outcome of Langerhans cell histiocytosis (LCH) with multiple spinal involvements. A total of 42 cases with spinal LCH were reviewed in our hospital and 5 had multifocal spinal lesions. Multiple spinal LCH has been reported in 50 cases in the literature. All cases including ours were analyzed concerning age, sex, clinical and radiologic presentation, therapy and outcome. Of our five cases, three had neurological symptom, four soft tissue involvement and three had posterior arch extension. Compiling data from the eight largest case series of the spinal LCH reveals that 27.2% multiple vertebrae lesions. In these 55 cases, there were 26 female and 29 male with the mean age of 7.4 years (range 0.2-37). A total of 182 vertebrae were involved including 28.0% in the cervical spine, 47.8% in thoracic and 24.2% in the lumbar spine. Extraspinal LCH lesion was documented in 54.2% cases, visceral involvement in 31.1% and vertebra plana in 50% cases. Paravertebral and epidural extension were not documented in most cases. Pathological diagnosis was achieved in 47 cases including 8 open spine biopsy. The treatment strategy varied depending on different hospitals. One patient died, two had recurrence and the others had no evidence of the disease with an average of 7.2 years (range 1-21) of follow-up. Asymptomatic spinal lesions could be simply observed with or without bracing and chemotherapy is justified for multiple lesions. Surgical decompression should be reserved for the uncommon cases in which neurologic compromise does not respond to radiotherapy or progresses too rapidly for radiotherapy.

Differential proteomic profiling of chordomas and analysis of prognostic factors.

BACKGROUND: The recurrence rate of chordoma is high, and the prognosis is poor. METHODS: Differential proteomic analysis was performed on chordomas and adjacent normal tissues, with verification by Western blot. Protein expression was evaluated by immunohistochemistry of 37 chordomas. Association of candidate protein expression with clinical parameters, disease-free survival, and overall survival were analyzed. RESULTS: We identified 14 up-regulated and 5 down-regulated proteins in chordomas. Expression of alpha enolase (ENO1), pyruvate kinase M2 (PKM2), and gp96 was higher in recurrences than in primary tumors. Univariate analysis showed that significantly adverse factors for disease-free survival were overexpression of ENO1 and PKM2, involvement of contiguous vertebral levels, and inadequate surgical margin at initial surgery. Inadequate surgical margin without radiotherapy, involvement of contiguous vertebral levels, and cervical spine location were adverse factors for overall survival. By multivariate analysis, independent adverse prognostic factors were inadequate surgical margin and involvement of multiple contiguous vertebral levels for recurrence; upper cervical spine location and involvement of contiguous vertebral levels for tumor-related death. Multivariate analysis failed to show the significance of the proteins. CONCLUSIONS: Involvements of multiple contiguous vertebral levels and upper cervical spine, rather than overexpression of ENO1, PKM2, or gp96, are independent prognostic indicators for chordomas.

Langerhans cell histiocytosis of the cervical spine: a single Chinese institution experience with thirty cases.

STUDY DESIGN: A retrospective study of cervical Langerhans cell histiocytosis (LCH). OBJECTIVE: To evaluate the safety and efficiency of the present diagnosis and treatment strategy. SUMMARY OF BACKGROUND DATA: The diagnosis and treatment protocols are still controversial for the rarity of cervical LCH. METHODS: Thirty patients with cervical LCH were diagnosed in the past 10 years. Biopsy was routinely performed to establish the final diagnosis before treatment. Immobilization was usually the first choice. Low-dose radiotherapy was suggested for cases with solitary marked bony erosion and/or soft tissue extension, and chemotherapy for cases with multiple lesions. Surgery was preserved for suspected malignancy, neurologic deficits, severe deformity, and/or instability. RESULTS: The mean age at diagnosis was 14.2 (range: 1.5-41) years old. Neck pain (96.7%) was the most common symptom, followed by restricted motion (70%), neurologic symptoms (36.7%), and torticollis (30%). Four cases had multiple lesions. Fourteen cases had atlantoaxial lesion and 16 cases were subaxial. The lesion extended to paravertebral soft tissue in 40% cases, to epidural space in 30%, to pedicle and/or transverse process in 56.3%. One case had endplate destruction. The accuracy of percutaneous needle biopsy under CT guidance was 91.2%. Eighteen patients had conservative treatment and 12 underwent operation. Three cases involving C2 vertebral body had fixed atlantoaxial anterior dislocation. Another 3 cases with atlantoaxial lateral mass destruction had spontaneous fusion. Eighteen patients had conservative treatment (1 only by immobilization, 13 by radiotherapy, 2 by chemotherapy, and 2 by combined chemotherapy and radiotherapy) and 12 underwent operation. All the initial symptoms were resolved, and there was no recurrence. From retrospective view, the surgical procedure might be avoided in 60% cases. Twenty-five cases had an average 61.6-month follow-up. In cases with severe bony collapse, the vertebral height ratio increased from 20.0% to 44.9% and the lateral mass height ratio from 22.2% to 56.8%. CONCLUSION: Cervical LCH lesions often extend to paravertebral soft tissue, epidural space, pedicles, and even to the endplate and lamina. Needle biopsy under CT guidance is safe and effective. The prognosis of cervical LCH is generally fair. Conservative treatment is usually enough and surgery should be reserved for major neurologic defects like myelopathy or monoparesis.

Langerhans cell histiocytosis of the atlas in an adult.

Langerhans cell histiocytosis (LCH), formerly known as histiocytosis X, is a rare disorder (approximately 1:1,500,000 inhabitants) characterized by clonal proliferation and excess accumulation of pathologic Langerhans cells causing local or systemic effects. The exact etiology of LCH is still unknown. LCH could affect patients of any age, although most present when they are children. The most frequent sites of the bony lesions are the skull, femur, mandible, pelvis and spine. A variety of treatment modalities has been reported, but there was no evidence suggesting that any one treatment was more advantageous than another. We present an adult with LCH of the atlas. A 26-year-old young man presented with a 2-month history of neck pain and stiffness. CT revealed osteolytic lesion in the left lateral mass of atlas with compression fracture. Histopathological diagnosis was Langerhans cell histiocytosis by percutaneous needle biopsy under CT guidance. The patient underwent conservative treatment, including Halo-vest immobilization and radiotherapy. At 7-year follow-up, the patient was asymptomatic except for mild motion restriction of the neck. CT revealed a significant reconstruction of the C1 lateral mass.

[Study on interaction between SARS-CoV N and MAP19].

AIM: Severe acute respiratory syndrome coronavirus (SARS-CoV) is the etiological agent of SARS, an emerging disease characterized by atypical pneumonia. Using a yeast two-hybrid screen with the nucleocapsid (N)protein of SARS-CoV as a bait, the N protein was found to interact with MAP19, a non-enzymatic protein of MASP(mannan-associated serine protease). The interaction between SARS-CoV N and MAP19 would be further tested in cells in this article. METHODS: The interaction between SARS-CoV N and MAP19 was demonstrated by immuno- coprecipitation, and the amount of MAP19 influenced by SARS-CoV N was investgated by Western blot. RESULTS: The interaction between SARS-CoV N and MAP19 was already demonstrated by immuno-coprecipitation. SARS-CoV N greatly increased the amount of MAP19. CONCLUSION: SARS-CoV N can bind with MAP19 in cells. Our study may be conductive to further research into the molecular mechanism of action between SARS-CoV N and MAP19.

Results of surgical treatment of cervical dumbbell tumors: surgical approach and development of an anatomic classification system.

STUDY DESIGN: A retrospective study of a new classification and surgical approach of cervical dumbbell tumors. OBJECTIVE: To evaluate PUTH classification. SUMMARY OF BACKGROUND DATA: The high recurrence rate and postoperative deformity are unsolved problems. Asazuma's landmark classification could not cover all cases and could not provide clear suggestion for the surgical approach. The ideal classification should be comprehensive, easily understood and of practical value. METHODS: PUTH classification for cervical dumbbell tumors includes 7 categories (types 1-7) and 2 foraminal modifiers. Posterior approach is appropriate for type 1, 2 and 5 tumors, anterior and anterolateral approach is an ideal choice for type 4 and 6 tumors. Type 7 tumors need combined anterior and posterior approach. RESULTS: Forty-four consecutive patients with cervical dumbbell tumor were surgically treated. The pathology included schwannoma in 31 cases, neurofibroma in 9 and ganglioneuroma in 4. Based on PUTH classification, type 3 was diagnosed in 13 cases, type 5 in 17, type 6 in 8, and type 7 in 6. Tumors were unilateral in 41 cases, and bilateral in 3 cases. Five were tumor revision cases. Thirty patients underwent posterior approach, 7 had anterior approach, 1 had anterolateral approach, and 6 had combined approach. Gross total resection was achieved in all the patients. Tumors involved nerve roots were transected in 12 cases. Single vertebral artery was ligated in 3. The complications included cerebrospinal fluid leakage in 18 cases, esophagus injury in 1, Horner syndrome in 1, dysphagia in 2, dyspnea in 1 and deep infection in 1. Thirty-six cases (81.1%) had an average 61-month follow-up. Recurrence was found in only one case (2.8%). CONCLUSION: PUTH classification covers all tumor types and is easier to remember. It is practical and useful for determining the surgical approach. The recurrence rate decreases significantly after radial tumor resection. Revision surgeries are associated with more complications.

Upper cervical spine chordoma of C2-C3.

Chordoma is a rare low-grade malignant neoplasm derived from the remnants of the embryonic notochord. This locally invasive neoplasm is subject to recurrence after treatment. The median survival time is estimated to be 6.3 years. Various treatment approaches have been attempted, including radical excision, radiotherapy and chemotherapy. Treatment outcome is significantly influenced by the size and site of the chordoma. Recently, Imatinib, a molecular-targeted agent, has been shown to have antitumor activity in chordoma. Proton radiotherapy, stereotactic radiotherapy and intensity-modulated radiotherapy have also been used. Surgical treatment is still the primary choice for chordoma. It has become more aggressive in recent years, evolving from intralesional or partial excision to en bloc resection. However, upper cervical localizations make such en bloc resection in most cases not possible. We present and discuss the therapeutic challenges of a young female with large retropharyngeal chordoma who presented to our institution after conventional photon beam radiotherapy. This C2/3 tumor was classified IB according to the Enneking classification. It distributed to layers A-D and sectors 1-6 according to the Weinstein Boriani Biagini Classification. The left vertebral artery (VA) was encapsulated and displaced. One stage intralesional extracapsular tumor excision and reconstruction was achieved by combined bilateral high anterior cervical approaches and posterior approach. No recurrence or metastasis was observed 3 years after the operation. She returned to her previous occupation as office worker.

Ataxia telangiectasia-mutated-Rad3-related DNA damage checkpoint signaling pathway triggered by hepatitis B virus infection.

AIM: To explore whether acute cellular DNA damage response is induced upon hepatitis B virus (HBV) infection and the effects of the HBV infection. METHODS: We incubated HL7702 hepatocytes with HBV-positive serum, mimicking a natural HBV infection process. We used immunoblotting to evaluate protein expression levels in HBV-infected cells or in non-infected cells; immunofluorescence to show ATR foci ands Chk1 phosphorylation foci formation; flow cytometry to analyze the cell cycle and apoptosis; ultraviolet (UV) radiation and ionizing radiation (IR)-treated cells to mimic DNA damage; and Trypan blue staining to count the viable cells. RESULTS: We found that HBV infection induced an increased steady state of ATR protein and increased phosphorylation of multiple downstream targets including Chk1, p53 and H2AX. In contrast to ATR and its target, the phosphorylated form of ATM at Ser-1981 and its downstream substrate Chk2 phosphorylation at Thr-68 did not visibly increase upon infection. However, the level of Mre11 and p21 were reduced beginning at 0.5 h after HBV-positive serum addition. Also, HBV infection led to transient cell cycle arrest in the S and the G2 phases without accompanying increased apoptosis. Research on cell survival changes upon radiation following HBV infection showed that survival of UV-treated host cells was greatly increased by HBV infection, owing to the reduced apoptosis. Meanwhile, survival of IR-treated host cells was reduced by HBV infection. CONCLUSION: HBV infection activates ATR DNA damage response to replication stress and abrogates the checkpoint signaling controlled by DNA damage response.

Cellular DNA repair cofactors affecting hepatitis B virus infection and replication.

AIM: To investigate whether hepatitis B virus (HBV) infection activates DNA damage response and DNA repair cofactors inhibit HBV infection and replication. METHODS: Human hepatocyte cell line HL7702 was studied. Immunoblotting was performed to test the expression of ataxia telangiectasia-mutated (ATM)-Rad3-related protein (ATR), p21 and the level of phosphorylation of Chk1, p53, H2AX, ATM in HBV-infected or non-infected-cells. Special short RNAi oligos was transfected to induce transient ATR knockdown in HL7702. ATR-ATM chemical inhibitors caffeine (CF) and theophylline (TP), or Chk1 inhibitor 7-hydroxystaurosporine (UCN01) was studied to determine whether they suppress cellular DNA damage response and MG132 inhibits proteasome. RESULTS: The ATR checkpoint pathway, responding to single-strand breaks in DNA, was activated in response to HBV infection. ATR knockdown cells decreased the HBV DNA yields, implying that HBV infection and replication could activate and exploit the activated DNA damage response. CF/TP or UCN01 reduced the HBV DNA yield by 70% and 80%, respectively. HBV abrogated the ATR-dependent DNA damage signaling pathway by degrading p21, and introduction of the p21 protein before HBV infection reduced the HBV DNA yield. Consistent with this result, p21 accumulation after MG132 treatment also sharply decreased the HBV DNA yield. CONCLUSION: HBV infection can be treated with therapeutic approaches targeting host cell proteins by inhibiting a cellular gene required for HBV replication or by restoring a response abrogated by HBV, thus providing a potential approach to the prevention and treatment of HBV infection.

Icariine stimulates proliferation and differentiation of human osteoblasts by increasing production of bone morphogenetic protein 2.

BACKGROUND: Icariine is a flavonoid isolated from a traditional Chinese medicine Epimedium pubescens and is the main active compound of it. Recently, Epimedium pubescens was found to have a therapeutic effect on osteoporosis. But the mechanism is unclear. The aim of the study was to research the effect of Icariine on the proliferation and differentiation of human osteoblasts. METHODS: Human osteoblasts were obtained by inducing human marrow mesenchymal stem cells (hMSCs) directionally and were cultured in the presence of various concentrations of Icariine. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test was used to observe the effect of Icariine on cell proliferation. The activity of alkaline phosphatase (ALP) and the amount of calcified nodules were assayed to observe the effect on cell differentiation. The expression of bone morphogenetic protein 2 (BMP-2) mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Icariine (20 microg/ml) increased significantly the proliferation of human osteoblasts. And, Icariine (10 microg/ml and 20 microg/ml) increased the activity of ALP and the amount of calcified nodules of human osteoblasts significantly (P < 0.05). BMP-2 mRNA synthesis was elevated significantly in response to Icariine (20 microg/ml). CONCLUSIONS: Icariine has a direct stimulatory effect on the proliferation and differentiation of cultured human osteoblast cells in vitro, which may be mediated by increasing production of BMP-2 in osteoblasts.

[Single-stage combined anterior-posterior instrumentation for multiple level cervical spine fractures].

OBJECTIVE: To investigate the efficacy of single-stage combined anterior-posterior instrumentation for treatment of multiple level cervical spine fractures. METHODS: Nine patients with multiple-level fractures of the cervical spine, 8 males and 1 female aged 24 - 63, underwent ingle-stage combined anterior-posterior instrumentation. Seven patients with multiple contiguous fractures of the cervical spine were treated with anterior decompression and plating combined with posterior cervical lateral mass screw fixation; and 2 patients with non-contiguous cervical fractures, both with type II odontoid fracture and lower cervical fracture, were treated with both anterior odontoid screw and posterior cervical lateral mass screw fixation. Cranioskeletal traction with a weight of 5 kg was done before the operation. Follow-up was conducted for 31 months. ASIA motor scores were used to evaluate the neural function. RESULTS: Satisfactory reduction and fusion were obtained without any complication, and the neural function was improved. Nerve root function recovered in two patients and one patient's spinal cord function became normal. The average ASIA score was 63.8. CONCLUSION: A feasible option in the treatment of multiple level cervical spinal fractures, single-stage combined anterior-posterior instrumentation provides decompression and stabilization in a short time and helps the neural function to recover.

[The effect of early diagnosis of recurrence and revision after resection of primary spine tumors].

OBJECTIVE: To explore the effect of early diagnosis of recurrence and early revision after resection of primary spine tumors. METHODS: From March 1989 to September 2005, the relate clinic data of 55 patients with giant cell tumors, osteoblastomas, chondrosarcomas and chordomas in spine in big piecemeal and current fashion was analysed. RESULTS: In 55 cases, 43 patients were followed up and had complete materials. The follow-up time ranged from 1.6 to 16.5 years, averagely 5.8 years. Thirty-four patients followed up regularly, and 12 were found recurrent, in which one C(1) giant cell tumor was found extensively large 3 months after initial surgery and was undertaken palliatory curet. The other eleven lesions were small and re-operated with wide margin. As a result, six patients lived without tumors during the 1 approximately 9.5 years follow-up, one patient gave up revision when found recurred again for economic reason, another four patients recurred repeatedly, but they persisted in regular follow-up and took revision surgeries whenever the recurred lesion were found. As a result, 3 of them lived without tumor and the other one died of other disease without sign of recurrence. In contrast, there were another nine patients who came to follow up until they had symptoms and were confirmed recurrent extensively. Two of them were excised radically for the tumors located in the relatively easily exposed segments of spine and lived without tumor now. While the other seven patients only received palliatory curet and all died of tumors. CONCLUSIONS: Regular follow-up, early diagnosis of recurrence and early revision need to be regarded as part of radical excision and are very important of surgical treatment of spinal tumors, which can prolong the patients' survival time.

[SCT-OGP fusion protein expression and its potential role as osteoporosis therapy].

Osteoporosis is a disease of bone metabolic disorder, the incidence of which increases sharply in old people. Calcitonin (CT) is a peptide hormone containing 32 amino acid that can inhibit osteoclasts activity. Osteogenic Growth Peptide (OGP) is a peptide hormone with 14 amino acid. It is an autocrine mitogen for osteoblastic and firbroblastic cells which has anabolic activiy. Six SCT and OGP DNA segments were chemically synthesized and ligated into a yeast expression vector pPIC9. The recombinant plasmid was transformed into pichia pastoris GS115. Finally, we got two stable SCT-OGP high expression clones after screening. Purifed protein can stimulate the proliferation of osteoblastic and fibroblastic cells, and also stimulate serum ALP activity and decrease serum calcium level using mice as animal models, demonstrating its potential role in oteoporosis therapy.

[Clinical characteristics and treatment of flexion-distraction stage I injuries in subaxial cervical spine].

OBJECTIVE: To study the clinical characteristics and treatment of flexion-distraction stage I injuries in subaxial cervical spine. METHODS: Twelve cases of flexion-distraction stage I injuries with delayed symptoms, admitted in our hospital between January 1995 and December 2004, were studied retrospectively. In acute phase, all of 12 cases had neck pain and limited neck movements, neurological deficits were found in 6 of 12 cases. Eight cases had a correct diagnosis, and 2 cases had a error diagnosis, 2 cases missed. All cases were satisfactory by the primary conservative treatment. After 274 days average asymptomatic intervals, all of 12 cases had recurrence of neck pain, delayed neurological deficits were found in 10. MRI showed that all of 12 cases were unstable injuries. RESULTS: All of the 12 patients were treated operatively. Decompression, fusion and fixation were performed by anterior approach in 9 cases, and by combined anterior and posterior approach in 3 cases. The average follow-up period was 33.1 months. Neck pain had great recovery in all cases, 10 cases with neurological deficits, 7 returned normal. Radiographic evidences of intervertebral bony fusion and good cervical alignment were observed in all of 12 cases. CONCLUSIONS: Flexion-distraction stage I injuries is often caused by ligament and disc injuries, and often missed with subtle symptoms and radiographic changes. Inadequate primary treatment options are often due to failure to recognize the instability, and maybe result in delayed injuries. MRI is helpful for the early accurate evaluation of spinal stability. Unstable injury require early surgical treatment. The anterior approach operation is recommended to most of these patients with acute and old injuries. Combined anterior and posterior approach operation should be considered in these patients who have old injuries with stiff kyphosis.

[Expression in Pichia pastoris and properties of human serum albumin-interferon alpha2b chimera].

To reduce the serum clearance of interferon alpha2b, a chimeric gene encoding an human serum albumin(HSA)--human interferon alpha2b(IFNalpha2b) fusion protein was overexpressed in Pichia pastoris. After fermentation in a 5L bioreactor, the fusion protein, capable of cross-reacting with anti-IFN alpha and anti-HSA antibody, was purified from the culture of the recombinant yeast by ultrafiltration, blue Sepharose affinity, phenyl hydrophobic interaction and Q ion exchange chromatography. Its IFNa2b moiety exhibits antiviral activity similar to that of recombinant human IFNa2b. In Cynomolgus monkeys model, The fusion protein was detectable in plasma, even 336h after a single does of 90 microg/kg injection intravenously or subcutaneously. The elimination phase half-life of the fusion protein was 101h after intravenous injection and 68.2h after subcutaneous injection. Its Subcutaneous bioavailability was 67.9%. The enhanced pharmacokinetics of interferon a2b fused to human serum albumin suggest its promissing application in clinic medicine.

[Enhancement of immune responses to hepatitis B DNA vaccine by superantigen SEA in mice].

To investigate the adjuvant effect of plasmid DNA encoding superantigen SEA (D227A) (pmSEA) on immune responses induced by HBV DNA vaccine containing HBV preS2 and S antigen in BABL/c (H-2d). BALB/c mice were immunized intramuscular injection with HBV DNA vaccine (pHBVS2S) mixed with or without pmSEA plasmid. Antibodies againat HBV PreS2 and S antigen in the sera were accessed by Anti-HBs ELISA, and the HBsAg specific cytotoxic T lymphocytes (CTLs) activity was determined by 5 Chromium Release Assay. The HBs peptide-specific IFN-gamma secreting T cells were detected by ELISPOT. Anti-HBs antibody titers and CTLs activity in mice immunized with pmSEA + pHBVS2S group were significant higher (P < 0.05) than pHBVS2S DNA vaccine group. The ratio of IgG1/IgG2a (0.282) was apparently different from the group immunized with peptide (10). Mice immunized with HBV DNA vaccine plus adjuvant produce higher titer of IgG1 and IgG2a antibodies against HBV S antigen 1.36 and 1.73 time higher than that without adjuvant respectively. HBs peptide--specific IFN-gamma secreting T cells increased 2 - 3 times by the pmSEA adjuvant, compared to DNA vaccine group. HBV DNA vaccine (pHBVS2S) induces humoral and cellular immuno-responses in BALB/c mice, and the responses could be significantly boasted by the plasmid encoding mSEA. Therefore the pmSEA was a potential adjuvant for DNA vaccines.

Adeno-associated virus-mediated bone morphogenetic protein-7 gene transfer induces C2C12 cell differentiation into osteoblast lineage cells.

AIM: To investigate the effects of bone morphogenetic protein-7 (BMP7)-expressing recombinant adeno-associated virus (AAV) vector on the differentiation of C2C12 cells. METHODS: AAV-BMP7 was packaged by infecting the stable cell clone BHK-21 (integrated with recombinant AAV vector plasmid pSNAV-BMP7) with recombinant herpes simplex virus type 1, which expresses AAV-2 Rep and Cap and possesses AAV packaging functions. Following infection with AAV-BMP7 at multiplicities of infection of 1 x 10(5) vector genomes per cell and subsequent culture, C2C12 cells were assessed qualitatively for BMP7 production, alkaline phosphatase activity, osteocalcin production and Cbfal and MyoD expression. RESULTS: C2C12 cells transduced with AAV-BMP7 could produce BMP7 protein until d 28. Alkaline phosphatase in the cultured C2C12 cell lysate was elevated. Secreted osteocalcin in the culture medium was detectable at d 12 and Cbfal mRNA expression level was upregulated, coinciding with downregulation of MyoD in a temporal manner. CONCLUSION: The present in vitro study demonstrated that AAV-BMP7 could infect and efficiently convert C2C12 cells from myoblasts into osteoblast lineage cells.