Animals ; Drugs, Chinese Herbal ; therapeutic use ; Humans ; In Vitro Techniques ; Mice ; Myocarditis ; drug therapy ; virology ; Phytotherapy ; methods ; Virus Diseases ; drug therapy

Using Sepharose CL-6B as support, 3-Chloro-1, 2-epoxypropane as activated agent, carboxymethylated aspartate (CM-Asp) as chelating ligand, A chelate affinity chromatographic medium based on Co2+, named Co-CM-Asp-Sepharose, was prepared and used to purify 6 x His-tagged fusion proteins. The amount of Co-CM-Asp-Sepharose reacted with 200 microL of lysate, the incubation time, wash condition and the imidazole concentration in the elution buffer were optimized. The purification results using Co-CM-Asp-Sepharose and Ni-NTA-Agarose (product of Qiagen) were compared. The CD155D1 fusion protein was also purified from 5mL of lysate and the amount of protein was determined by Bradford method. The results show that 60 microL of Co-CM-Asp-Sepharose (50% suspension) was suitable for the protein purification from 200 microL of lysate, the optimal incubation time of medium and lysate was 30 min, the optimal imidazole concentration in the eluting buffer was 200 mmol/L, and 200 microg of fusion protein was obtained. In a big scale experiment, 4.6 mg of fusion protein was obtained from 5 mL of lysate using 1.5 mL of Co-CM-Asp-Sepharose (50% suspension). Compared with Ni-NTA-Agarose, the Co-CM-Asp-Sepharose medium exhibits higher selectivity and the protein possesses higher purity.
Aspartic Acid ; chemistry ; Chelating Agents ; chemistry ; Chromatography, Affinity ; methods ; Epoxy Compounds ; chemistry ; Histidine ; biosynthesis ; chemistry ; genetics ; Polymers ; chemistry ; Recombinant Fusion Proteins ; isolation & purification ; Sepharose ; chemistry

OBJECTIVETo Summarize the clinical experience of individual immunosuppressive regime in heart transplantation with high risk.

METHODSFrom September 2001 to December 2006, 51 cases with the complication of Hepatitis B viruses (HBV) infection, diabetes mellitus, renal dysfunction or pulmonary infection in perioperative period were analyzed retrospectively. All cases received daclizumab (Zenapax) induction therapy, and baseline triple immunosuppressive regime was consist of cyclosporine (CsA), azathioprine (Aza) or mycophenolate mofetil (MMF) and prednisone (Pred). Ten cases received HBV infection in preoperative period, the immunosuppressive protocol was emphasized on the use of MMF and the withdraw of Pred one month later in postoperation. Nine cases received diabetes mellitus in pre-operation, 4 cases had post-transplant diabetes mellitus. The immunosuppressive protocol was emphasized on the use of CsA rather than FK506, the use of Pred was less dosage, and the therapy of insulin was necessary. Sixteen cases had renal dysfunction in pre-operation, the use of MMF was routine but the use of CsA was delayed to the time 5 to 19 d postoperative. Twelve cases received pulmonary infection after allograft transplantation. The immunosuppressive agent was to be taped or suspended in therapy time.

RESULTSThe liver function of the 10 cases with HBV infection was stable in 1 year follow-up, and 1 case received acute rejection after 13 months allograft transplantation. In the 6 months follow-up, the blood glucose level of the 13 cases with diabetes mellitus was stable, none of the cases suffered from acute rejection. In the one month follow-up, none of the 16 cases with renal dysfunction suffered from acute rejection, and the renal function was normal. Two of the 12 cases with the pulmonary infection were died of serious infection, others were survival. One case received acute rejection on the 17th day in postoperation.

CONCLUSIONSLow mortality can be realized by selecting appropriately individual immunosuppressive regime and the episode of acute rejection is rare.

Adult ; Female ; Follow-Up Studies ; Graft Rejection ; prevention & control ; Heart Transplantation ; Humans ; Immunosuppressive Agents ; administration & dosage ; therapeutic use ; Male ; Perioperative Care ; Retrospective Studies ; Risk Factors

OBJECTIVETo analyze the clinical features of mid- and long-term acute cardiac allograft rejection to improve the long-term clinical outcomes of the patients.

METHODSFourteen recipients (11 males and 3 females) underwent orthotopic heart transplantation with standard immunosuppressive therapy protocols (3 cases) or induction therapy protocols (11 cases). Cyclosporine, azathioprine or mycophenolate mofetil, and prednisolone were applied as the maintenance immunosuppressive regimen. Acute graft rejection episodes occurred within 3 to 6 months in 1 case, within 6 months to 1 year in 3 cases, within 1 to 2 years in 3 cases, within 2 to 5 years in 6 cases, and above 5 years in 1 case.

RESULTSNo significant difference was found in the incidence of late heart rejection between the patients receiving the two immunosuppressive therapy protocols. Immunosuppressants were withdrawn or spared in 8 recipients due to different causes. Nine recipients with steroid-sensitive acute cardiac allograft rejection were treated with steroid-pulse therapy, while the other 5 were treated with a short course of polyclonal antithymocyte antibodies because of steroid-resistant acute rejection; in 11 cases, azathioprine was converted to mycophenolate mofetil. Four of the 5 late deaths occurred in the recipients with steroid-resistant rejection. The surviving recipients had a good quality of life, and no recurrent episodes of rejection or infection were observed in the follow-up period.

CONCLUSIONSLate acute cardiac allograft rejection is associated mainly with patient compliance but not with early immunosuppressive therapy protocols. The episodes are rather severe and should be timely treated with steroid pulses or polyclonal antithymocyte antibodies.

Adolescent ; Adult ; Cyclosporine ; Female ; Graft Rejection ; etiology ; prevention & control ; Heart Transplantation ; adverse effects ; Humans ; Immunosuppression ; methods ; Male ; Middle Aged ; Mycophenolic Acid ; analogs & derivatives ; Young Adult

OBJECTIVETo discuss the clinical characteristics, diagnosis and treatment of the clinical characteristics and prognosis of asbestosis complicated with malignant mesothelioma patients.

METHODSIn 3 cases of asbestosis complicated with malignant mesothelioma were retrospectively analyzed.

RESULTSIn the 3 patients, 2 cases of pleural mesothelioma, with chest tightness, chest and back pain as initial symptom; 1 case of peritoneal mesothelioma, with abdominal distention, abdominal pain, dysuria as initial symptom. One case of the pleural mesothelioma misdiagnosed as tuberculous pleurisy. 3 patients were in CT or B ultrasound guided biopsy pathology confirmed to be malignant mesothelioma. 2 patients received systemic chemotherapy, another received symptomatic and supportive treatment. Up till now, 3 patients have died.

CONCLUSIONThe disease is a high degree of malignant, the early clinical manifestations are not specific, easily missed diagnosis and misdiagnosis. The treatment effect is not ideal, the prognosis is poor. Biopsy is a reliable method for diagnosis of MM.

Abdominal Pain ; Asbestosis ; complications ; diagnosis ; Back Pain ; Biopsy ; Diagnostic Errors ; Humans ; Lung Neoplasms ; complications ; diagnosis ; Mesothelioma ; complications ; diagnosis ; Peritoneal Neoplasms ; Pleural Neoplasms ; Prognosis ; Retrospective Studies ; Tuberculosis, Pleural

OBJECTIVETo explore the feasibility and efficiency of immunotherapy with dendritic cell (DC) in leukemic mice model after allogeneic bone marrow transplantation (allo-BMT).

METHODSMature DC were expanded from mice bone marrow mononuclear cells (MNC) by adding mouse granulocyte-macrophage colony stimulating factor (mGM-CSF) and interleukin-4 (mIL-4). Three days later they were pulsed with frozen thawing L7212 leukemia-related antigen. Mice bearing leukemia received allo-BMT at d 0, and then were divided into control group (A), T cells group (B) and DC + T cells group (C) to receive respective immune therapy at d 14. The survival rate, survival time, occurrence of graft-versus-host disease (GVHD), cytotoxicity of spleen cells and serum cytokine level were observed. The survivors in each group were rechallenged with L7212 cells to observe the immune response to the leukemia.

RESULTSMature DC were successfully induced from bone marrow MNC. In groups B and C, the relapse rates were 30% and 0%, while the long term survival rates after BMT was 30% and 70% respectively. Both of the differences were statistically significant (P < 0.05). However, the incidence of GVHD in these two groups were similar. The mean survival times were (32.95 +/- 13.29) days and (41.15 +/- 13.88) days, respectively (P < 0.01). MTT assay indicated that spleen cells from group C had specific killing activity to L7212 cells. Enzyme-labeled immunosorbent assay (ELISA) showed that the serum IL-2 level in group C was (419.75 +/- 26.66) pg/ml, being significantly higher than that in the other two groups (P < 0.01). When the survivors were rechallenged with L7212 cells, there was difference between the survival rates of groups C and B (85.7% vs 33.3%, P < 0.05).

CONCLUSIONImmunotherapy with leukemia related antigen-pulsed DC in combination with donor lymphocyte infusions is an effective approach to reinforce GVL effect and decrease relapse after allo-BMT.

Animals ; Bone Marrow Cells ; cytology ; drug effects ; immunology ; Bone Marrow Transplantation ; Cancer Vaccines ; immunology ; Cell Differentiation ; Dendritic Cells ; immunology ; Female ; Graft vs Leukemia Effect ; Immunotherapy ; Leukemia, Experimental ; immunology ; surgery ; therapy ; Male ; Mice ; Mice, Inbred BALB C ; Survival Rate ; Transplantation, Homologous

OBJECTIVETo evaluate the cytogenetic and molecular genetic features of chronic myeloid leukemia (CML) in Chinese.

METHODSA total of 1193 CML patients were retrospectively studied. Chromosome preparation of bone marrow cells was made using direct and short-term culture. Karyotype and bcr-abl fusion genes were analyzed by R-banding, RT-PCR, respectively.

RESULTSIn the 1193 cases, 98.07% was Ph chromosome positive (Ph+) and 1.93% negative (Ph-). In the Ph+ patients, 95.64% was classical Ph and 4.36% variant rearrangements. Additional genetic changes were demonstrated in 11.88% of classical Ph cases. Cytogenetic clonal evolution was found in 7.94% of patients in chronic phase (CP), 27.78% in accelerated phase (AP), and 49. 04% in blast crisis (BC). Among the classical Ph cases, +Ph, +8, -21 were found in 14.62%, 10.77% and 7.69% of them respectively. In patients in BC and AP, the most common additional chromosome changes were + Ph (28.57%), +8 (16.67%) and +19 (7.14%), while in CP, -21 (10.26%), +Ph (8.97%), and +8 (8.97%). The combination of +Ph and +8 (3.60%) was the most frequent of combination pattern. 524 cases were investigated for bcr-abl fusion gene, and 54.01% was b3a2 (+) and 27.67% b2a2 (+).

CONCLUSIONIn Chinese CML patients seem to have their unique features in terms of cytogenetic clonal evaluation.

Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; Female ; Fusion Proteins, bcr-abl ; genetics ; Humans ; Karyotyping ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Male ; Middle Aged ; Retrospective Studies

OBJECTIVETo observe clinical therapeutic effects of acupuncture for pattern of liver-stagnation and spleen-deficiency in diarrhea-predominant irritable bowel syndrome (D-IBS) and its impact on cell factors.

METHODSForty cases were selected and divided into an acupuncture group (21 cases) in which acupuncture was applied and a medicine group (19 cases) in which oral administration of dicetel and bifidobacterium lactobacillus triple viable capsules were applied. The symptom scores, level of Th1-type cytokine (IFN-gamma, IL-2) and Th2-type cytokine (IL-4, IL-10) and ratio of IFN-gamma to IL-4 were compared in two groups before and after treatment to analyze acupuncture effect.

RESULTSThe clinical symptoms were improved after one-week treatment in the acupuncture group (P<0.05), which had faster onset than the medicine group (P<0.05). The total effective rate was 90.48% (19/21) in the acupuncture group, which was superior to 78.95% (15/19) in the medicine group (P<0.05). Compared with medicine treatment, imbalanced condition of Th1/Th2 was turning towards the direction of Th2 after acupuncture, indicating a tendency to recover the balance.

CONCLUSIONThe clinical efficacy of acupuncture for D-IBS has close relationship with effectively improving balance of Th1/Th2 in patients with liver-stagnation and spleen-deficiency.

Acupuncture Therapy ; Adult ; Aged ; Cytokines ; immunology ; Female ; Humans ; Irritable Bowel Syndrome ; immunology ; physiopathology ; therapy ; Liver ; physiopathology ; Male ; Middle Aged ; Spleen ; physiopathology ; Th1 Cells ; immunology ; Th2 Cells ; immunology ; Young Adult

To establish lentivirus-mediated system of double suicide genes and explore its killing effects on K562 cells, lentivirus transfer vector for double suicide genes was constructed using molecular methods, three plasmids of lentivirus gene transfer vector system were transferred into packaging cell line 293T using lipofectine method, the transfer effect was observed through fluorescence microscopy, the lentivirus particles were observed by means of electron microscopy. High titer of lentivirus was harvested from the supernatant of virus-producing cell culture and concentrated by high-speed centrifugation with Poly-L-Lysine (PLL). The K562 cells were infected with the concentrated supernatant containing the virus with the double suicide genes. Fluorescence microscopy and RT- PCR confirmed the integration and expression of extraneous gene. The cytotoxicity to these transgenic cells treated with 5-FC and GCV was measured by MTT assays. The growth inhibition ratio (GIR) of cells and inhibition concentration 50 (IC(50)) were counted. After administration of GCV and 5-FC, the changes of those cells were observed through scanning electron microscope. The results showed that lentivirus transfer vector with double suicide genes was constructed successfully. The above-mentioned plasmids were effectively transferred into 293T cells. So much green fluorescence was observed through fluorescence microscope. A lot of lentivirus particles were observed through transmission electron microscope. Double suicide genes mediated by lentivirus were stably integrated and expressed in K562 cells after infection with the concentrated virus using fluorescence microscopy and RT-PCR. The GIR of K562 cells using GCV or 5-FC was 48.73% or 50.69% respectively and it was apparently higher than that of untransfected cells (P < 0.01). When using GCV and 5-FC together, the GIR was 87.69%, which was apparently higher than that of group using GCV or 5-FC alone (P < 0.01). In conclusion, lentivirus-mediated gene transfer system could transfer CD and TK double suicide genes into K562 cells with high efficiency and it had strong killing effects when giving 5-FC and/or GCV. The cytotoxic effects of double suicide genes were superior to that of single suicide gene. The lentivirus-mediated double suicide gene transfer system is a high-efficiency gene transfer vector.
Cytosine Deaminase ; genetics ; Flucytosine ; pharmacology ; Ganciclovir ; pharmacology ; Genetic Therapy ; Genetic Vectors ; genetics ; HIV-1 ; genetics ; Humans ; K562 Cells ; Thymidine Kinase ; genetics

OBJECTIVETo explore the feasibility and efficiency of cytosine deaminase (CD)/thymidine kinase (TK) gene-modified donor T cells used in allogeneic bone marrow transplantation (allo-BMT) as an approach to mitigate GVHD without compromising engraftment.

METHODSThe pseudotyped lentivirus vectors containing CD and TK double suicide genes were transfected with lipofectine to donor T cells. Lethally irradiated 615 leukemia mice were transplanted with BALB/c bone marrow plus CD(+)TK(+)T cells. GVHD prophylaxis was by administration of ganciclovir (GCV) and 5-Fluoride cytosine (5-FC).

RESULTSThe pseudotyped lentivirus-mediated gene transfer system could efficiently transfer CD and TK double suicide genes into donor T cells. Administration of GCV and 5-FC to the mice could markedly potentiate the CFU-S and CFU-GM yields and raise the number of peripheral white blood cells. 1 x 10(7) CD(+)TK(+) allogeneic T cells caused GVHD of a similar magnitude and time course to that of fresh, naive T cells after allo-BMT. Administration of GCV and 5-FC in mice received CD(+)TK(+)T cells reduced the severity of GVHD and resulted in significantly longer survival as compared with non-administration mice, and the effect was stronger than that of administration of GCV or 5-FC alone.

CONCLUSIONAdministration of CD + TK gene-modified donor T cells to recipient in allo-BMT might be an approach to mitigate GVHD without compromising alloengraftment.

Animals ; Body Weight ; Bone Marrow Transplantation ; Cytosine Deaminase ; genetics ; Female ; Genetic Therapy ; Graft vs Host Disease ; pathology ; therapy ; Lentivirus ; genetics ; Mice ; Mice, Inbred BALB C ; Thymidine Kinase ; genetics ; Transplantation, Homologous