Screening and identification of HLA-A0201 restricted cytotoxic T lymphocyte epitopes from hepatitis B virus E antigen in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To identify HLA-A0201 restricted cytotoxic T lymphocyte (CTL) epitopes derived from the hepatitis B virus e (HBe) antigen, for future use in a specific immunotherapy based on the identified epitope(s).</p><p><b>METHODS</b>HBe gene sequences from the hepatitis B virus serotypes with the highest frequencies in China were analyzed by bioinformatic web-based interfaces for quantitative motif prediction, extended motif prediction, and peptide super-motif prediction. Four candidate peptides were identified: HBe1, HBe2, HBe3, and HBe4. The affinities of each were tested in vitro with T2 cells, which lack the transporter-associated with antigen transport (TAP) protein but express low levels of the MHC class I surface molecule, and measured by the T2 binding assay and DC50 assay. Flow cytometry was used to detect the fluorescence index of control and experimental groups.</p><p><b>RESULTS</b>The peptides HBe1 (LLWFHISCL), HBe2 (YLVSFGVWI), HBe3 (CLTFGRETV), and HBe4 (DLLDTASAL) were identified and tested as candidate targets. HBe2 and HBe3 showed higher HLA-A0201 affinity. HBe1, HBe2, and HBe3 showed better binding stability.</p><p><b>CONCLUSION</b>Two peptides based on HBe antigen, YLVSFGVWI and CLTFGRETV, possess both sufficient binding affinity and stability and may represent useful HLA-A0201-restricted CTL epitopes. Further study is needed to determine the immunogenic properties of these two peptides in vivo.</p>

Protective effects of human bone marrow mesenchymal stem cells on hematopoietic organs of irradiated mice / 中国实验血液学杂志

The objective of this study was to explore the protective effects of human bone marrow mesenchymal stem cells (MSC) on hematopoietic organs of irradiated mice. Human bone marrow MSC were isolated, ex vivo expanded, and identified by cell biological tests. Female BALB/c mice were irradiated with (60)Co γ-ray at a single dose of 6 Gy, and received different doses of human MSC and MSC lysates or saline via tail veins. The survival of mice was record daily, and the femurs and spleens were harvested on day 9 and 16 for pathologic examination. The histological changes were observed and the cellularity was scored. The results showed that the estimated survival time of MSC- and MSC lysate-treated mice was comparable to that of controls. The hematopoiesis in the bone marrow of mice that received high-dose (5×10(6)) of MSC or MSC lysates was partially restored on day 9 and the capacity of hemopoietic tissue and cellularity scorings were significantly elevated as compared with that of controls (P < 0.05). Proliferative nudes were also obviously observed in the spleens of mice that received high-dose of MSC or MSC lysates on d 9 after irradiation. The histological structures of the spleen and bone marrow of the mice that received high-doses (5×10(6)) of MSC or MSC lysates were restored to normal, the cell proliferation displayed extraordinarily active. Further, the cellularity scores of the bone marrow were not significantly different between the high-dose MSC and MSC lysate-treated mice. It is concluded that the bone marrow MSC can promote the hematopoietic recovery of the irradiated mice, which probably is associated with the bioactive materials inherently existed in bone marrow cells.

Investigating the treatment of silicosis with autologous bone marrow-derived mesenchymal stem cells / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To explore the safety and curative effects of autologous bone marrow-derived mesenchymal stem cells (BMSCs) in the treatment of silicosis.</p><p><b>METHODS</b>The protocol was approved by the Ethics Committee of the hospital, and ten patients with silicosis who had given written consent were enrolled in this study. BMSCs isolated from 100 ml of bone marrow for each case were purified and cultured. In each case the 3rd generation of qualified BMSCs (5 × 10(7)) were intravenously administered weekly for 3 weeks. Three cases among 10 patients were treated with BMSCs modified by hepatocyte growth factor (HGF) gene. The clinical symptoms, chest films, chest CT, pulmonary functions, T cells, serum IgG and ceruloplasmin (CP) were observed in 6 or 9 months after treatment.</p><p><b>RESULTS</b>No obvious sub-effect was observed in cases treated with BMSCs, the clinical symptoms (such as cough, sputum and chest tightness) basically disappeared in 9 months after treatment. Pulmonary function tests showed that FVC increased from 71.2% ± 17.0% to 84.0% ± 10.9% (P < 0.01) and FEV1.0 increased from 67.5% ± 17.7% to 80.6% ± 14.9% (P < 0.01). The levels of serum CP and IgG significantly decreased (P < 0.01). Further, the chest films and CT in cases treated with autologous BMSCs modified by HGF gene were improved to different extent.</p><p><b>CONCLUSION</b>Treatment with autologous BMSCs modified by HGF gene exhibit a beneficial effect on silicosis.</p>

Therapy of aplastic anemia with autologous peripheral mononuclear cells treated by IL-2 and GM-CSF in culture: a long-term follow-up report on 49 patients / 中国实验血液学杂志

This study was purposed to evaluate the long-term outcome and the safety of autologous peripheral blood mononuclear cells (PBMNC) treated by interleukin 2 (IL-2) and granulocyte-macrophage colony stimulating factor (GM-CSF) in the therapy of patients with aplastic anemia (AA). The therapy of 49 patients admitted BG in hospital from April 2001 to December 2007 were analyzed retrospectively. PBMNC were isolated and cultured for 48 hours in presence of IL-2 and GM-CSF. Cells were collected, and 6 × 10(6) - 1 × 10(8) PBMNC were intravenously injected weekly for 4 - 22 months. Hematopoietic recovery was evaluated by examinations of peripheral blood, bone marrow aspirates and bone marrow biopsy. Flow cytometry was used to assess the peripheral T cell subsets before and after treatment. Polymerase chain reaction was performed to observe the clonal diversity of T cell receptor variable β-chain (TCR-Vβ) recombination. The results showed that 37 cases were cured and none of them relapsed during the follow-up, 5 cases were in partial remission, 3 cases got improvement, and 4 cases showed no response. The total efficiency reached up to 91.8%. The ratios of CD4(+)/CD8(+) subsets were abnormal in 39 patients prior to the treatment, and 31 cases restored to the normal range after cell transfusions. Analysis on the clonal diversity of TCR-Vβ recombination in 11 patients showed the transition from monoclonal or biclonal spectratype to polyclonal one. No long-term side effects were documented. It is concluded that the treatment with PBMNC treated by IL-2 and GM-CSF is generally safe and effective. The underlying mechanisms may be in relation to the restoration of cell immunity.

Changes of T lymphocyte subsets in workers with long-term benzene exposure / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To observe the changes of T-lymphocyte subsets in workers with long-term benzene exposure, and further understand the benzene's lymphotoxicity.</p><p><b>METHODS</b>Blood was sampled from 44 patients with chronic benzene poisoning of different degrees, (mild 22 patients, moderate 14, severe 8) respectively. Twenty-two health benzene exposed workers, and 94 health unexposed workers served as normal control. A total of the phenotype (CD4, CD8) of T lymphocyte in peripheral blood was analyzed by indirect immunofluorescence assay.</p><p><b>RESULTS</b>Lymphocyte subset analysis showed significantly decreased CD4(+) T lymphocytes, CD4(+)/CD8(+) ratio, except CD8(+) T lymphocytes in benzene exposed groups (P<0.05). Among the four benzene-exposed groups, CD4(+) T lymphocytes and CD4(+)/CD8(+) ratio showed no difference (P>0.05).</p><p><b>CONCLUSION</b>The primary changes of T-lymphocyte subsets in workers following benzene long-term exposure are the decrease of CD4(+)%, but the changes are not correlated with haematopoietic injury.</p>

Synthesis and immunosuppressive activity of new artemisinin derivatives containing polyethylene glycol group / 药学学报

<p><b>AIM</b>To search for new artemisinin derivatives with higher immunosuppressive activity.</p><p><b>METHODS</b>Two kinds of new artemisinin derivatives containing polyethylene glycol group were synthesized from dihydroartemisinin via condensation and esterification. These compounds were assayed for their inhibitory activity on ConA-induced T cell proliferation and LPS-induced B cell proliferation.</p><p><b>RESULTS</b>Twenty three new compounds (2a - 2f, 3a - 3d, 4a - 4f, 6a, 6b and 7a - 7g) were synthesized and identified by 1H NMR and elemental analysis.</p><p><b>CONCLUSION</b>These compounds had immunosuppressive activity in vitro. Among them, the symmetrical substituted compound 2 and 6 had higher activity than mono-substituted compound 3, 4 and 7. Especially, compounds 2a - 2f remarkably exhibited higher inhibition in comparison with artemisinin and artesunate.</p>

Synthesis of a novel L-nucleoside, beta-L-D4A and its inhibition on the replication of hepatitis B virus in vitro / 药学学报

<p><b>AIM</b>Nucleoside analogues have become the most promising candidates of anti-HBV drugs. In this study, beta-L-D4A was synthesized and explored its inhibitiory action against hepatitis B virus (HBV) in 2. 2. 15 cells derived from HepG2 cells transfected with HBV genome.</p><p><b>METHODS</b>beta-L-D4A was stereo-controlled synthesized from D-glutamic acid, and the structure was identified by IR, 1H NMR and MS. 2. 2. 15 Cells were placed at a density of 5 x 10(4) per well in 12-well tissue culture plates, and treated with various concentrations of beta-L-D4A for 6 days. At the end, medium was processed to obtain virions by a polyethlene glycol precipitation method. At the same time, intracellular DNA was also extracted and digested with Hind III. Both of the above DNA were subjected to Southern blot, hybridized with a 32P-labeled HBV probe and autoradiographed. The intensity of the autoradiographic bands was quantitated by densitometric scans of computer and EC50 was calculated. 2. 2. 15 cells were also seeded in 24-well tissue culture plates, and cytotoxicity with different concentrations was examined by MTT method. IC50 was calculated.</p><p><b>RESULTS</b>The synthesized compound structure conformed with beta-L-D4A; Autoradiographic bands showed similar for supernatant and intracellular HBV DNA. Episomal HBV DNA was inhibited in a dose-dependent manner. EC50 0.2 micromol x L(-1). The experiment of cytotoxicity gained IC50 200 micromol x L(-10.</p><p><b>CONCLUSION</b>beta-L-D4A has been synthesized successfully. beta-L-D4A possessed potent inhibitory effect on replication of HBV in vitro with low cytotoxicity, TI value was 1 000. It is expected to be developed clinically into a new anti-HBV drug.</p>

Evaluation of recent thymic output function in benzene-exposed workers / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To analyze the content of signal joint T-cell receptor excision DNA circles signal joint T-cell receptor excision DNA circles (sjTRECs) within peripheral blood mononuclear cells (PBMCs), thereby to infer the level of naive T cells and the recent thymic output function in benzene-exposed workers.</p><p><b>METHODS</b>Quantitative detection of sjTRECs in DNA of peripheral blood mononuclear cells from 11 normal individuals and 62 benzene-exposed workers were performed by real-time polymerase chain reaction (PCR) and TaqMan technique.</p><p><b>RESULTS</b>The median value of sjTRECs copies/1,000 PBMCs was 7.81 in normal individuals whereas it was 2.56 copies/1 000 PBMCs in age-unadjusted benzene-exposed workers (P < 0.01). And its levels were obviously different between two different age groups: that in 30-year-old group (1.76 copies/1,000 PBMCs, n = 23) was less than <or= 30-years-old group (3.19 copies/1,000 PBMCs, n = 39, P < 0.05).</p><p><b>CONCLUSIONS</b>Benzene exposure may influence the recent thymic output function, and the sjTRECs levels may relate with the age of benzene-exposed workers.</p>

Establishment and identification of highly expressing and replicating hepatitis B virus genome transgenic mouse models / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To establish a highly expressing and replicating hepatitis B virus (HBV) genome transgenic mouse models for screening anti-HBV drugs and investigating the pathogenesis of hepatitis B.</p><p><b>METHODS</b>Elongated HBV genome as the investigated gene was transducted into the pronuclei of the fertilized eggs of mice by the technique of microinjection, then the eggs were transplanted into the oviducts of the pseudopregnant mice. All the newborn mice were screened and identified by PCR and Southern blot detecting genomic DNA in tail tissue, then the positive mice were examined plasma HBsAg, HBeAg by ELISA and plasma HBV DNA by Southern blot.</p><p><b>RESULTS</b>Among the 61 offsprings, 18 were positive for tail tissue HBV DNA examination, 7 of which were positive for replication and expression detection.</p><p><b>CONCLUSION</b>Transgenic mice with elongated HBV genome possess high efficiency of replication and expression, which can be used for further investigation.</p>

Effect and mechanism of beta-L-D4A (a novel nucleoside analog) against hepatitis B virus / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the effect and the molecular targets of anti-hepatitis B virus (HBV) by beta-L-D4A in vitro.</p><p><b>METHODS</b>2.2.15 cells were cultured and treated with various concentrations of beta-L-D4A for 6 hours, then the effect of anti-HBV was examined by Southern blot and the replicating core particles from the cells were isolated. The endogenous polymerase reaction and activity gel experiment were performed to monitor the activities of the DNA polymerase and reverse transcriptase.</p><p><b>RESULTS</b>The replication of HBV DNA was inhibited in a dose-dependent manner. The endogenous polymerase reaction showed both the two enzymatic activities were irreversibly inactivated in a concentration -dependent manner, with IC50 at 0.51 micromol/L and 0.55 micromol/L, respectively. But the activities of DNA polymerase and reverse transcriptase were found to remain active by activity gel with exogenous templates.</p><p><b>CONCLUSIONS</b>The mechanism of inhibiting HBV replication by beta-L-D4A may be in that either the DNA replication priming is blocked or the elongation of DNA chain is terminated irreversibly.</p>

The treatment with allogeneic peripheral blood stem cell transplantation for benzene-induced severe aplastic anemia / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To evaluate the efficacy of treatment with allogeneic peripheral blood stem cell transplantation for benzene-induced severe aplastic anemia.</p><p><b>METHODS</b>HLA-compatible sibling (pregnancy) allogeneic peripheral blood stem cell transplantation (Allo-PBSCT) was successfully performed for a patient with severe aplastic anemia caused by benzene poisoning. 9.41 x 10(8) nucleated cells/kg, 12.49 x 10(6) CD(34) positive cells/kg and CFU-GM 8.2 x 10(5)/kg were infused. The patient was treated with cyclophosphamide (120 mg/kg), total body radiation (8 Gy) and anti-lymphocyte globulin (60 mg/kg) before transplantation. Donor buffy coat cells (9.02 x 10(8) nucleated cells/kg, 10.62 x 10(6) CD(34) positive cells/kg, 6.3 x 10(5) CFU-GM/kg) were infused again on day 18 after transplantation to prevent from graft failure. Graft versus host disease prophylaxis consisted of both methotrexate and cyclosporin A.</p><p><b>RESULTS</b>The lowest ANC was 0, the lowest platelet was 3 x 10(9)/L after transplantation. The patient achieved an ANC of greater than 0.5 x 10(9)/L from 21st day, and the platelet of greater than 50 x 10(9)/L from 28th day after transplantation. Grade I cGVHD was found the fourth month after grafting. Examination of recipient's bone marrow cells showed a normal 46, XX (presumably marrow donor) karyotype. Blood group changed from B to O.</p><p><b>CONCLUSION</b>This is the first case reported in China showing a successful treatment of benzene-induced severe aplastic anemia with allo-PBSCT. Allo-PBSCT may be an effective remedy for this kind of patients.</p>

Effect of immunocyte therapy on benzene-induced bone marrow haemopoietic dysfunction / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To explore the effect of treatment with immunocyte therapy on benzene-induced haemopoietic dysfunction.</p><p><b>METHODS</b>Mono-nuclear cells (MNC) were separated from 40 - 50 ml peripheral blood in patients and mixed with interleukin-2 and granulocyte macrophage colony stimulating factor (GM-CSF) for six day cultivation. The new formed immunocytes were collected and transfused into the patients. Bone marrow aspiration and biopsy were taken before and after therapy for all patients with severe benzene poisoning. Blood samples were stained by flow cytometry for detecting CD(4) and CD(8) positive cells.</p><p><b>RESULTS</b>Of 20 patients with chronic benzene poisoning, 9 were severe benzene poisoning. All examination including blood count, bone marrow biopsy and T cell subpopulation restored to normal after immunocyte therapy. Laboratory tests (liver and kidney function, and myocardial enzymes) were observed periodically and showed normal during therapy. Follow-up study (the longest time was more than 15 months) showed that bone marrow haemopietic function of all treated patients were in normal range.</p><p><b>CONCLUSION</b>Bone marrow haemopoietic dysfunction caused by benzene poisoning may be closely related to disorder of immune function. Immunocyte therapy may significantly improve bone marrow haemopoietic dysfunction induced by benzene poisoning.</p>