OBJECTIVETo investigate the effects of immunologic effector cells to enhance apoptosis induced by adriamycin (ADR) in multi-drug resistant human breast cancer cell line MCF7/ADR.

METHODSThe immunologic effector cells were induced and expanded by IFN-gamma, McAb CD3, IL-1 and IL-2. The expression of P-glycoprotein (P-gp) and its relation to apoptosis in target cells were detected by TUNEL technique and immunohistochemical staining. Flow cytometry (FCM) was carried out to determine the expression level of human breast cancer related P185 antigen and the positive rate of Annexin V-FITC/PI expression. The subcellular distribution of ADR and Annexin V expression in the target cells were detected by fluorescence microscopy.

RESULTSThe immunologic effector cells down-regulated the expression of P185 and P-gp in MCF7/ADR cells. The accumulation and subcellular distribution of ADR in MCF7/ADR cells were increased after co-culture with the immunologic effector cells. After treatment with the immunologic effector cells in combination with ADR, apoptosis rate of the target cells was 10 times higher than that induced by ADR alone, and 13 times higher than that induced by the immunologic effector cells alone.

CONCLUSIONImmunologic effector cells can simultaneously down-regulate the expression of P185 and P-gp in MCF7/ADR cell line, and increase the apoptosis rate of MCF7/ADR cells in combination with ADR.

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Antibiotics, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Breast Neoplasms ; immunology ; metabolism ; pathology ; Cell Line, Tumor ; Down-Regulation ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Female ; Humans ; Killer Cells, Lymphokine-Activated ; immunology ; Receptor, ErbB-2 ; metabolism

In order to investigate whether cytokine-induced killer (CIK) cells can induce apoptosis of bcr-abl(+) K562 cells, apoptosis of K562 cells and CEM cells induced by CIK cells, etoposide or camptothecin was detected with flow cytometry DNA assay. RT-PCR showed that K562 cells expressed the bcr-abl fusion gene, K 562 cells, K562 cells/etoposide or K562 cells/camptothecin groups showed no sub-G(1) peak. K562 cells/CIK cells group showed sub-G(1) peak (38.1%). CEM cells showed no sub-G(1) peak. CEM cells/camptothecin or CEM cells/etoposide groups showed sub-G(1) peak (23.5% or 32.3% respectively). CEM cells/CIK cells group showed sub-G(1) peak (45.4%). Etoposide or camptothecin did not induce apoptosis of K562 cells. CIK cells induce apoptosis of K562 cells. Bcr-abl fusion gene prevented apoptosis induced by etoposide or camptothecin, but did not prevent apoptosis induced by CIK cells. This property may support the observed adoptive immunologic effect of allogeneic bone marrow transplantation and donor lymphocyte transfusions of CML case relapsing after allogeneic bone marrow transplantation.
Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; immunology ; Camptothecin ; pharmacology ; Coculture Techniques ; Cytotoxicity, Immunologic ; Etoposide ; pharmacology ; Flow Cytometry ; Fusion Proteins, bcr-abl ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; K562 Cells ; drug effects ; immunology ; metabolism ; Killer Cells, Lymphokine-Activated ; cytology ; immunology

OBJECTIVETo summarize the incidence of various adverse reactions in the clinical application of L-asparaginase (L-Asp), and to analyze the cause of hepatic encephalopathy in three cases.

METHODSThe complete data of 23 patients in our department from December 2009 to December 2010 were collected. Their blood ammonia levels, transaminase, serum albumin and blood coagulation function before, during and after the L-Asp application were assayed.

RESULTS(1) All patients had elevated blood ammonia level after the L- Asp application. This occurred 2 days after the beginning of treatment and the median time to reach peak level (ranged from 194 to 446 μmol/L, with a median value of 300 μmol/L) was 4 days. It returned to normal level after a median time of 5 days (ranged 3-7 days) with drug withdrawal. Of the 23 patients studied, 3 developed hepatic encephalopathy. (2) All patients appeared lower blood fibrinogen, 10 cases (43.5%) with lower fibrinogen only, while 13 cases (56.5%) with both prolonged APTT and lower fibrinogen. The lowest level of fibrinogen was detected at 1 week after drug application. Of the 23 patients, 14 (60.9%) had mild lower blood fibrinogen (1-2 g/L), and 9 (39.1%) had significantly lower fibrinogen (0-1 g/L). (3) Six cases (26.1%) had slightly elevated level of transaminase (<2 times the upper limits of normal), 8 (34.8%) appeared hypoalbuminemia.

CONCLUSIONAs the incidence of elevated blood ammonia levels was high in the application of L-Asp, the level of blood ammonia should be closely monitored to avoid the occurrence of hepatic encephalopathy, especially in elderly patients and patients with previous liver disease or long-term heavy drinking. L-Asp can also lead to low fibrinogen level, hypoalbuminemia and abnormal transaminase. Monitoring the blood coagulation function and liver function is required and, if necessary, plasma infusion and liver protection therapy are required.

Adolescent ; Adult ; Aged ; Ammonia ; blood ; Asparaginase ; adverse effects ; therapeutic use ; Female ; Hepatic Encephalopathy ; chemically induced ; Humans ; Male ; Middle Aged ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; blood ; drug therapy ; Young Adult

To investigate the relationship between the plasma levels of chemokine CXCL9/Mig and acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The plasma levels of CXCL9/Mig of 35 patients who received all-HSCT were detected by using ELISA assay, these patients included 13 patients with grade 0-I, 12 patients with grade II and 10 patients with grade III - IV aGVHD, respectively. The four different time points including prior to allo-HSCT, one week before aGVHD onset, the plateau of aGVHD and time after completely controlled, were studied. The results showed that the plasma levels of CXCL9/Mig in the patients with serious aGVHD (grade II - IV) were significantly increased during aGVHD than those in the patients without aGVHD or with slight aGVHD (P < 0.001). It was found that CXCL9/Mig levels were significantly correlated with the severity of grade aGVHD (P < 0.001). Another important finding was that CXCL9/Mig levels obviously increased at one week before aGVHD was diagnosed. CXCL9/Mig level was not obviously correlated with CMV infection or other infectious complication (P > 0.05). It is concluded that the plasma level of CXC19/Mig significantly correlated with the severity of aGVHD and plays a critical role in pathogenesis of aGVHD, the changes in plasma level of CXCL9/Mig after allo-HSCT may be used as a valuable indicator for early diagnosis of aGVHD, finally, provide a early therapeutic approach to reduce aGVHD severity and improve the outcome for patients after allo-HSCT.
Chemokine CXCL9 ; blood ; Graft vs Host Disease ; blood ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Humans

OBJECTIVETo explore early diagnosis of hemophagocytic syndrome (HPS) and effective treatment.

METHODSA multicenter retrospective study was carried out to analyze the causes, clinical features, laboratory findings, treatment and clinical outcomes of 72 patients with HPS.

RESULTSAmong the 72 patients, EBV infection and T lymphoma were the most common initiating diseases. The most common clinical features were persistent fever (100%) and splenomegaly (83.3%). The diagnostic sensitivity was persistent fever (100%), peripheral cytopenia in two or more lineages (97.2%), high concentration of serum soluble CD25 (93.1%) and low NK cell activity (94.4%). The median percentage of serum glycosylated ferritin was significantly lower in patients in HPS group \[(17.4 +/- 16.0)%\] than in control group \[(53.6 +/- 13.3)%\] (P < 0.01). And the median level of serum TNF-alpha was significantly higher in patients group \[(143.2 +/- 64.8) microg/L\] than in controls \[(66.9 +/- 19.4) microg/L\] (P < 0.01). Hepatic dysfunction was seen in most patients (83.6%) mainly manifested as elevated liver enzymes and hypoalbuminemia. The 15-week total survival rate was 46.8% in 47 treated patients, and was 63% in 27 treated with fludarabine in combination with high dose methylprednisolone. The platelet count and fibrinogen level were significantly lower in death group than in survival group.

CONCLUSIONSThe diagnostic sensitivities of presistent fever, peripheral cytopenia in two or more lineages, high concentration of serum soluble CD25 and low NK cell activity are relatively high and lacking hemophagocytosis does not exclude the diagnosis. Low percentage of glycosylated ferritin and high concentration of TNF-alpha would be helpful to the diagnosis. High dose methylprednisolone combined with fludarabine is an effective therapy. Platelet count and fibrinogen level are poor prognostic factors for HPS.

Humans ; Lymphohistiocytosis, Hemophagocytic ; diagnosis ; Methylprednisolone ; Retrospective Studies ; Treatment Outcome ; Tumor Necrosis Factor-alpha

The aim of this study was to investigate the incidence, risk factors of acute renal failure (ARF) after allogeneic hematopoietic stem cell transplantation (allo-HSCT), and evaluate its effect on the prognosis of patients after allo-HSCT. A retrospective analysis was performed in 86 patients undergoing allo-HSCT at Peking University First Hospital from June 2003 to April 2007. ARF is defined as a doubling of baseline serum creatinine at any time during the first 100 days post-transplant. The risks of ARF and mortality after ARF were examined using univariate analysis and multivariate unconditional logistic regression. The correlation of ARF and survival was examined using Cox regression. The results indicated that 27 patients (31.40%) developed ARF at a median of 59.5 days after transplant (range 1 to 93 days). The univariate analysis showed that elevated risks were severe acute GVHD (OR 6.196; 95% CI 1.121 - 34.249, p = 0.033), sepsis or septic shock (OR 4.184; 95% CI 1.314 - 13.325, p = 0.018) and hyperbilirubinemia (OR 3.709; 95% CI 1.428 - 9.635, p = 0.006). Renal disease before transplant (OR 6.711; 95% CI 1.199 - 37.564, p = 0.027), hypertension (OR 2.067; 95% CI 0.739 - 5.782, p = 0.165), the use of vancomycin (OR 2.133; 95% CI 0.844 - 5.392, p = 0.106) or foscarnet sodium (OR 2.133; 95% CI 0.844 - 5.392, p = 0.106) may be potential risks. Multivariate logistic regression analysis showed that renal disease before transplant (OR 6.288; 95% CI 1.218 - 32.455, p = 0.028), sepsis or septic shock (OR 3.614; 95% CI 1.040 - 12.544, p = 0.043) and hyperbilirubinemia (OR 4.448; 95% CI 1.563 - 12.665, p = 0.005) appear to be independently associated with an increased risk of ARF. Age, gender, baseline serum creatinine level, advanced malignant disease, unrelated-donor, total body irradiation (TBI) and cyclosporine levels were not associated with the development of ARF. Cox regression showed that ARF (RR 2.124; 95% CI 1.016 - 4.441, p = 0.045) was independently associated with survival of patients after allo-HSCT. The mortality of patients with ARF within 6 months post-transplant was significantly higher than that of those without ARF (44.4% vs 8.47%, p < 0.001). It is concluded that the cumulative incidence of ARF after allo-HSCT remains high. Renal disease before transplant, hyperbilirubinemia and sepsis or septic shock are all related factors which can increase the risk of ARF. ARF appears to be independent factor influencing survival of patients after allo-HSCT.
Acute Kidney Injury ; etiology ; Adolescent ; Adult ; Child ; Child, Preschool ; Female ; Graft vs Host Disease ; etiology ; Hematopoietic Stem Cell Transplantation ; adverse effects ; methods ; Humans ; Incidence ; Male ; Middle Aged ; Retrospective Studies ; Risk Factors ; Transplantation, Homologous ; Young Adult

OBJECTIVETo investigate the specific anti-breast cancer immune response induced by dendritic cells (DC) loaded with trastuzumab and apoptotic Her-2+ breast cancer cells.

METHODSDCs were generated from healthy peripheral blood mononuclear cells (PBMCs) in the presence of recombinant cytokines GM-CSF, IL-4 and TNF-alpha. Mature DCs were harvested after 7 days' co-culture of PBMCs and trastuzumab-treated apoptotic SKBr3 cells. The morphologic characteristics and ultrastructure of the DC were observed under the inverted phase-contrast microscope and transmission electron microscope (TEM), respectively. Flow cytometry (FCM) was used to check the expression of several DC specific markers: CD14, CD1a, CD64, CD80, CD83, CD86, HLA-ABC and HLA-DR. DC-cytokine induced killer (DC-CIK) cells were prepared by co-culture of DCs and peripheral blood lymphocytes in the presence of anti-CD3 antibodies and human IL-2 at an appropriate concentration. The number of antigen-specific T cells was analyzed by human interferon gamma enzyme linked immunospot (ELISPOT) assay. MTT assay was employed to assess the lysis of breast cancer cell line induced by DC-CIK cells.

RESULTS5 minutes after the adding of DCs to SKBr3 cells pretreated with trastuzumab, the apoptotic SKBr3 cells were found to be circled by DCs. 48 hours later, many membrane-wrapped organelles of the apoptotic target cells in the cytoplasm of DCs were found by TEM. The majority of the organelles were degraded. Fewer organelles from the apoptotic cells were found in DCs without Herceptin. More than 60% in every group of DCs expressed a high-affinity receptor for IgG (FcgammaRI or CD64). CD14 expression on the mature DCs were comparatively lower, and HLA-DR and HLA-ABC expressions were higher in the trastuzumab group. The expression of CD1a, CD80, CD83 and CD86 in trastuzumab group were higher than those in immature DCs group (P < 0.05). ELISPOT assay suggests that the spot number of antigen-specific T cells were higher in trastuzumab group than that in the antigen unloaded DCs group (P < 0.05). The lysis of SKBr3 cells induced by the SKBr3 antigen loaded DC-CIK cells were 1.7 times higher than that of CIK.

CONCLUSIONThe lysis of SKBr3 cells induced by DC-CIK was increased after that DCs were combined with trastuzumab to capture antigen from SKBr3 cells. These findings support further investigation into the use of combination immunotherapy of the humanized monoclonal antibody, DC vaccines and immunological effector cells.

Antibodies, Monoclonal ; pharmacology ; Antibodies, Monoclonal, Humanized ; Apoptosis ; Cell Line, Tumor ; Coculture Techniques ; Cytokine-Induced Killer Cells ; immunology ; Cytokines ; metabolism ; Cytotoxicity, Immunologic ; immunology ; Dendritic Cells ; cytology ; immunology ; metabolism ; ultrastructure ; Humans ; Receptor, ErbB-2 ; metabolism ; Receptors, IgG ; metabolism ; Trastuzumab

OBJECTIVETo explore the therapeutic feasibility of allogeneic hematopoietic stem cell transplantation (allo-HSCT) from partially HLA-mismatched related donors for hematologic diseases.

METHODSThirty patients with hematologic diseases received allo-HSCT from 1 - 3 loci mismatched related donors conditioning regimen consisting of ATG (thymoglobulin, total dose of 10 mg/kg, intravenously on - 4 d to - 1 d), and only 5 (18%) of 28 recipients from HLA-identical sibling donors were treated with regimen containing ATG. Donors were given G-CSF prior to hematopoietic stem cell harvest and CsA, short-term MTX and mycophenolate mofetil (MMF) were used for GVHD prophylaxis in both group.

RESULTSAll patients were successfully engrafted. There was no significant difference in the incidence of grade II to IV acute graft-versus-host disease (aGVHD) and grade III to IV aGVHD between the mismatched and matched groups (34% vs 32%, and 13% vs 11%, respectively). 3-year overall survival (OS) and disease-free survival (DFS) in mismatched and matched groups were 57% vs 77% (P = 0.14) and 57% vs 69% (P = 0.28), respectively. Multivariate analysis showed that advanced disease pre-transplant (P = 0.006) and CMV infection (P = 0.04) were risk factors for OS. OS for patients with stable disease in mismatched and matched groups were 87% vs 81% (P = 0.65) respectively, and for those with advanced disease were 21% vs 71% (P = 0.02).

CONCLUSIONSIt is feasible to perform allo-HSCT from 1 -3 loci HLA-mismatched related donors for patients with stable disease who lack HLA-identical sibling donors. Nevertheless, for patients with advanced disease optimized conditioning regimen and intensive supporting therapy should be administered to obtain better clinical outcomes.

Graft vs Host Disease ; prevention & control ; HLA Antigens ; Hematopoietic Stem Cell Transplantation ; methods ; Humans ; Siblings ; Tissue Donors ; Transplantation Conditioning

Histologic transformation (HT) is a frequent event in the clinical course of patients with indolent lymphoma with dismal outcome. The diagnosis of HT is based on clinical manifestation, PET-CT and pathologic biopsy, and the latter is a golden standard for HT. There are contradictory data about the impact of initial management on the risk of transformation. Patients who present with HT did not receive R-CHOP or chemotherapy-naive, should receive this regimen. For the subset of patients received R-CHOP prior to HT, the second line chemotherapy for DLBCL should be adopted. Consolidation with HDT-ASCT should be considered for the suitable young patients. The radio-immunotherapy and novel drugs showed a bright perspective for the patients with HT.
Antineoplastic Combined Chemotherapy Protocols ; Humans ; Lymphoma, Non-Hodgkin

The latent membrane protein 2 (LMP2) is a kind of protein expressed by EBV-infected cells. This study was aimed to investigate whether the stimulation of peripheral blood mononuclear cells with peptides induces EBV-specific cytotoxic T lymphocytes (CTL). The peptides were mixture of LMP2 protein and available for people with different HLA types. Peripheral blood sample was collected from a patient with EBV-associated hemophagocytic syndrome. The mononuclear cells were isolated and cultured to obtain dendritic cells (DCs). Immature DCs were pulsed with MIX-LMP2 and added with different maturation-promoting factors. The auto-T lymphocytes were stimulated weekly with the harvested mature DCs loaded with MIX-LMP2, and totally for two times. Part of isolated lymphocytes was cultured without any stimulation as control. T-cell receptor (TCR) beta spectratyping was used to analyze the distribution of different T cell subgroups before and after culture. The phenotype of T lymphocytes was determined by flow cytometry. The IFN-gamma assay was used to estimate specific cytoxic activity of the cultured T cells. The results showed that the distribution of TCRbeta was changed according to analysis of TCR spectratypes. From the distribution of gene families of TCRbeta, the T lymphocytes were oligoclonal before culture, but shifted to a polyclonal after culture in vitro like the normalization of TCR diversity, suggesting the subgroups of lymphocyte could return to normal. The percentage of CD3+, CD3+CD8+ CD3+ CD45RA- CD45 RO+ on T lymphocytes from freshly isolated mononuclear cells were 70.73%, 42.99%, 27.56% respectively. After being stimulated twice with DC loaded with MIX-LMP2, they further increased to 95.17%, 52.54% and 81.41%. The percentages of CD3-CD56+ NK cells and CD4+CD35+ FOXP3+ regulation T cells seldom changed, from 2.12%, 0.03% to 2.35%, 0.02% respectively. The increase of CD3+CD45RA-CD45RO+ cells obviously indicated that most naive T cells could be activated. ELISA for IFN-gamma showed that when DCs loaded with LMP2 peptide were used as target cells, IFN-gamma level secreted by the T cells stimulated with LMP2 peptide-pulsed DCs was 805+/-16 pg/ml and 1729+/-49 pg/ml, the IFN-gamma level secreted by T cells stimulated twice with LMP2 peptide-pulsed DCs was 956+/-23 pg/ml and 2325+/-58 pg/ml respectively at effector-target ratios of 10:1 and 10:2. They were both significantly higher than that secreted by T cells without any stimulation (441+/-27 pg/m and 557+/-19 pg/ml) (p<0.05). But DCs unpulsed with LMP2 peptide were used as target cells, there were no significant differences between the T cells stimulated with LMP2 peptide-pulsed DCs and the T cells without stimulation (p>0.05). It is concluded that the antigen specific T cells recognizing EBV epitopes can be obtained by using DCs pulsed with MIX-LMP2 peptide in vitro, meanwhile the distribution of T cell subgroups can be changed and normalized.
Antigens, Viral ; immunology ; Cells, Cultured ; Coculture Techniques ; Cysteine Endopeptidases ; immunology ; metabolism ; Dendritic Cells ; immunology ; metabolism ; Epitopes, T-Lymphocyte ; immunology ; Epstein-Barr Virus Infections ; immunology ; Herpesvirus 4, Human ; immunology ; Humans ; Leukocytes, Mononuclear ; cytology ; T-Lymphocytes ; cytology ; immunology