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This study was purposed to evaluate the effectiveness and safety of autologous cytokine induced killer (CIK) cells combined with chemotherapy in treatment of elderly patients with acute myeloid leukemia. Peripheral blood mononuclear cells (PBMNC) were isolated from 5 elderly patients with acute myeloid leukemia, and then augmented by priming with interferon gamma (IFN-γ) followed by IL-2 and monoclonal antibody (mAb) against CD3. The autologous CIK cells thus obtained were infused back to individual patients, 28 days as one cycle. The changes in cellular immune function, incidence of infection, independence of hematoglobin or blood transfusion, and progression of disease were observed and assessed before and after therapy. The results showed that the 46 cycles of CIK cell infusion were performed for 5 patients, no adverse reaction was observed in these patients. The percentages of CD3(+), CD3(+)CD8(+) and CD3(+)CD56(+) increased significantly (P < 0.05), The therapy of CIK could significantly reduce the incidence of infection (P < 0.05) and shorten the time of high fever in AML patients (P < 0.05). CIK also could reduce the volume of erythrocyte infusion to maintenance hematoglobin level (P < 0.05). We found that although CIK could not change the outcome of AML, the combination of CIK and chemotherapy could control patients' condition and prolong their survival during the development and end stage of AML. It is concluded that autologous CIK cells combined with chemotherapy is safe and efficacious for the elderly patients with acute myeloid leukemia.

This study was aimed to evaluate the effectiveness and safety of low methylation drug decitabine combined with autologous cytokine induced killer cells (CIK) to treat the elderly patients with acute myeloid leukemia (AML). Two AML patients aged over 80 years old were diagnosed and treated in our department from 2006 to 2012; both company with MDS history, and one case was M4-type, another case was M6-type according to FAB classification. The changes in lymphocyte subsets, hematologic response, transfusion frequency, leukemic gene expression, obtaining CR or PR, quality of life and survival time of the patients with different treatment regimen (decitabine alone; CIK alone; decitabine combined with CIK) were systematically observed. The results showed that therapy of decitabine combined with CIK cells could reduce bone marrow suppression extent, decrease the frequency and volume of blood transfusion, and prolong the duration of partial remission, compared with the single use of CIK cell infusion and single use of decitabine treatment. Meanwhile, the kinds of expressed genes associated with leukemia decreased and the survival time was prolonged obviously. The patients' life quality significantly improved. It is concluded that decitabine combined with CIK for treatment of elderly patients with AML is safe and effective.

The aim of this study was to observe the curative effects and safety of autologous cytokine induced killer (CIK) cells in treatment of aged patients with orbital diffuse large B cell lymphoma after rituximab therapy. The patient was given rituximab three times with low dose COP chemotherapy one time when he was diagnosed with orbital diffuse large B cell lymphoma. Two months later, the patient began to receive five cycles CIK cells infusion. One course of therapy was defined as follows: about (2-3)×10(9) of CIK cells (survival rate > 95%) was transfused twice and then rhIL-2 (1 MU daily) was subcutaneously administered for 10 consecutive days. Efficacy and adverse effect was observed during or after CIK cells infusion. The results showed that the peripheral blood mononuclear cells of the patient could be cultured and expanded into CIK cells. The majority of CIK cells was positive for CD3 and CD8 after culture. The CD3(+)CD56(+) cells markedly increased after culture. After two cycles of CIK cell infusion, the orbital lymphoma and possible involvement of the kidney disappeared. The patient obtained complete remission after five cycles of CIK cells infusion. The side effects of CIK cell treatment were minor. It is concluded that CIK cell infusion may prevent recurrence, prolong progression-free survival and improve quality of life after rituximab (alone or with chemotherapy) for aged patients with orbital diffuse large B-cell lymphoma.

The function of immune system degenerates in an aging-dependent manner and this results in immunosenescence. Human immune system includes two parts: genetic/innate immunity and adaptive immunity. The former is involved in monocytes, nature killer cells, and dendritic cells, the later is involved in acquired B and T lymphocytes. During the aging of immunity system, the both parts of immunity are damaged to some degree. Generally, innate immunity seems well-retained and the acquired immunity is degenerative seriously with aging. Immunocyte senescence is closely related to the elderly decreased ability to control infectious disease, cancer and to their generally poor response to vaccination. This review summarized the research progress on immunosenescence characteristics in aged phase.

This study was purposed to evaluate the safety and curative effect of autologous cytokine induced killer cells (CIK) combined with low-dose IL-2 regimen containing immune enhancement of thymic peptide on elderly patients with B-cell chronic lymphocytic leukemia (B-CLL). Thymic peptide α1 was subcutaneously given as the immunoenhancement agent at a dose of 1.6 mg/d, 14 days as one cycle. Peripheral blood mononuclear cells (PBMNC) from 5 patients with B-CLL were isolated once a week to induce ex vivo CIK cells through culture in the context of interferon (IFN)-γ, interleukin (IL)-2 and anti-CD3 monoclonal antibody. The PBMNC were separated from patients before and after 14 days as one cycle of thymic peptide α1 administration. Parameters of amplification ability, effector cells quantity, lymphocyte subgroups percentage and antitumor cytotoxicity were compared before and after thymic peptide administration. The 5 patients were treated with CIK cells combined with low-dose IL-2 regimen immediately after injection of thymic peptide α1. The CIK cells plus low-dose IL-2 regimen containing thymic peptide enhancement was defined as: thymic peptide α1 1.6 mg/d was subcutaneously administered once every other day; (4 - 6) ×10(9) of CIK cells were transfused followed by IL-2 subcutaneous administration of 1 mU/d on days 1-10, 28 days as one cycle. Clinical evaluation parameters including cellular immunity function, CLL related biomarkers, disease state and infectious frequency and degree were investigated before and after CIK cells infusion puls IL-2. The results showed that the amount of amplified CIK cells, the percentage and amplification times of effector cells and antitumor cytotoxicity more significantly increased after thymic peptide α1 treatment than before its use (P < 0.05). The total 46 cycles of CIK cells infusion plus IL-2 were completed in the 5 CLL patients. No adverse reaction was observed. After treatment of CIK cells plus IL-2, the general conditions of 5 CLL patients were to different extent improved. Simultaneously, percentages of CD3(+), CD3(+)CD8(+), and CD3(+)CD56(+) cells in peripheral blood remarked by raised (P < 0.05), the serum level of β2 microglobulin was significantly declined (P < 0.05), and the frequency and degree of infection was also decreased (P < 0.05). Following CIK cells plus IL-2 therapy, the transformation of disease state from partial remission (PR) to complete remission was seen in 3 patients, from stable disease (SD) to PR in 1 patient, and from progress of disease to SD in 1 patient. It is concluded that the regimen of autologous CIK cells combined with low-dose IL-2 containing immune enhancement of thymic peptide is safety and effective for the treatment of elderly patients with B-CLL.

Objective of this study was to evaluate the effectiveness and safety of autologous cytokine induced killer (CIK) cells combined with IL-2 in treatment of elderly patients with myelodysplastic syndromes (MDS). Peripheral blood mononuclear cells were isolated from 6 elderly MDS patients and were stimulated by cytokines in vitro to form CIK cells. The autologous CIK cells were then infused back into the corresponding patients. The regimen was repeated every 4 weeks. Effector cell proportion changes, adverse effects, effects on inflammation, hemoglobin level and blood transfusion were assessed after treatment. The results showed that after autologous CIK cell infusion, the percentages of CD3(+), CD3(+)CD8(+) and CD3(+)CD56(+) increased significantly (p < 0.05). No severe adverse effects were observed in all patients. It also significantly reduced inflammation frequency and shortened high fever duration. During stable stage of disease, the CIK cell infusion could reduce the red blood cell infusion amount and stabilize hemoglobin level. However, the natural course of transformation from myelodysplastic syndromes to high-risk subtypes could not be changed by CIK cell treatment. It is concluded that the autologous CIK cell infusion is a safe and effective therapy for geriatric myelodysplastic syndrome.

Objective of this study was to evaluate the effectiveness and safety of autologous cytokine induced killer (CIK) cells combined with IL-2 in treatment of elderly patients with B-cell malignant lymphoma. Peripheral blood mononuclear cells (PBMNC) were isolated from 9 elderly patients with B-cell malignant lymphoma, and then induced into CIK cells by IFN-γ, IL-2 and monoclonal antibody (mAb) against CD3. The autologous CIK cells [(2-3)×10(9)] thus obtained were infused back to individual patients, then followed by subcutaneous injection of IL-2 at single daily dose of 1×10(4) U/day for 10 consecutive days. The regimen was repeated every 4 weeks and total 64 cycles of CIK cell transfusion were completed. The changes in cellular immune function, tumor-related biological parameters, imaging characteristics, the condition of remission, quality of life and survival time were assessed. 7 patients received 8 cycles of CIK cell infusion, and 4 cycles were completed in 2 patients. The results showed that no adverse reaction was observed in all above mentioned patients. The percentages of CD3+, CD3+CD8+ and CD3+CD56+ increased significantly (p<0.05), and serum levels of β2-microglobulin and LDH were markedly decreased (p<0.05) after autologous CIK cell transfusion. The lymphoma symptoms were relieved with quality of life obviously elevated (p<0.01) in all patients. Complete remission was seen in 8 patients. Though one patient received 8 cycles of CIK cell transfusion therapy and achieved transient very good partial remission, but he died of acute large-area myocardial infarction and persistent progression of lymphoma. In conclusion, regimen of autologous CIK cells combined with IL-2 is safe and effective for the therapy of elderly patients with B-cell malignant lymphoma.

The aim of study was to explore the efficacy of cytokine induced autologous killer (CIK) cell infusion as an immune therapy for elderly patients with hematological malignancies. Peripheral blood mononuclear cells (PBMNC) were isolated from 20 elderly patients with hematological malignancies, and then augmented by priming with human recombinant interferon gamma (rhIFN-γ) followed by human recombinant interleukin 2 (rhIL-2) and monoclonal antibody (mAb) against CD3. The obtained autologous CIK cells [(2-3)×10(9)] were infused back to individual patients, then followed by subcutaneous injection of IL-2 at single daily dose of 1×10(6) U for 10 consecutive days. The regimen was repeated every 4 weeks and total 136 cycles of CIK cells transfusion were completed. The changes in cellular immune function, tumor-related biological parameters, imaging characteristics, the condition of remission, quality of life (QOL) and survival were assessed. The results indicated that 14 patients received 8 cycles of CIK cells infusion, and 4 cycles were completed in 6 patients. No adverse reaction was observed in all patients. The percentages of CD3+, CD3+CD8+ and CD3+CD56+ cells increased significantly (p<0.05), and serum levels of β2-microglobulin and LDH markedly decreased (p<0.05) after autologous CIK cells transfusion. The tumor-related symptoms were relieved, QOL obviously improved (p<0.01) in all patients. Complete remission was seen in 11 patients, and partial remission was observed in 7 patients. It is concluded that the autologous CIK cell infusion can improve immunity in elderly patients of hematological malignancies and displays its effectiveness and safety for elderly patients.

The study was aimed to investigate the promotive effect of LRP16 gene on K562 cell proliferation. Open reading frame of LRP16 gene was amplified using reverse transcription-polymerase chain reaction (RT-PCR) and ligated to pGEM-T plasmid to construct LRP16 ORF-pGEM-T recombinant vector. Then, LRP16 ORF identified by sequencing was inserted into pcDNA3.1+ plasmid to construct LRP16 ORF-pcDNA3.1+ recombinant expression plasmid which was transfected into K562 cell lines to make overexpression of LRP16 gene in K562 cells. Survival of cells was determined by MTT assay and growth curve of cells was drawn, the cell cycle was detected by flow cytometry. The results showed that LRP16 ORF was successfully amplified, then the LRP16 ORF-pcDNA3.1+ recombinant plasmid was constructed. The K562 cell line with overexpression of LRP16 gene was established. The promotive effect of LRP16 gene overexpression on proliferation of K562 cells was observed and the effect partially related to the enhancement of cells from G0 to S phase induced by LRP16 gene. It is concluded that LRP16 gene overexpression shows a promotive effect on proliferation of K562 cells.

This study was purposed to investigate lrp16 gene expression in leukemia cell lines and bone marrow cells of leukemia patients and explore the relationship between lrp16 gene expression and development of leukemia. Reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to test the lrp16 mRNA expression in 4 leukemia cell lines, including K562 (CML), HL-60 (APL), MOLT4 (ALL) and U937 cell lines, as well as in bone marrow-derived cells from 115 patients with leukemia. The effect of lrp16 gene expression on genesis and progression of leukemia was analyzed according to clinicopathological features. The results indicated that positive expression of lrp16 mRNA was found in all 4 leukemia cell lines. For leukemia patients, the positive expression rate of lrp16 mRNA in all AML patients was 38% (16/42), in which the positive rates in AML patients with complete remission (CR) and AML patients without remission were 13% (4/30) and 100% (12/12) respectively. The positive expression rate of lrp16 mRNA in ALL patients was 38% (10/26), in which the positive rate in ALL patients with CR and ALL patients without remission were 16% (3/18) and 87% (7/8) respectively. The positive expression rate of lrp16 mRNA in CML patients was 36% (9/25), in which the positive rates in CML patients with CR and CML patients without remission were 20% (4/20) and 100% (5/5) respectively. The positive rate of lrp16 mRNA in CLL patients was 31% (7/22), in which the positive rate in CLL patients with CR and CLL patients without remission were 11% (2/17) and 100% (5/5) respectively. There was no difference of lrp16 gene expression between leukemia subtypes, but there was statistical significant difference in lrp16 gene expression between CR patients and non CR patients (p < 0.001). It is concluded that the lrp16 gene is a leukemic oncogene and closely relates to genesis and progression of leukemia, which may be an indicator for evaluating clinical efficacy of leukemia therapy.

The study was aimed to analyze the characteristics of LRP16 gene promoter and its activity in order to explore the possible regulation mechanism of LRP16 gene expression. A 2.6 kb genomic DNA sequence of LRP16 5'-end was obtained from NCBI by BLAST software. The 7 target sequences between 0.2 - 2.6 kb from a healthy blood donor DNA sample were amplified by PCR, then identified by DNA sequencing and semi-nest PCR. The verified sequences were analyzed on-line. The results showed that the 7 target sequences were about 400 bp different from each other. All 7 sequences were the same to these GenBank described. At last, all 7 promoter sequences were ligated with luciferase vector, and then the luciferase activity was analyzed in HeLa cells. A known gene promoter sequence can be freely obtained from NCBI database. It is concluded that LRP16 promoter is a standard type II promoter and its activity is strongest in the region from -200 to -600 bp.

To clone the full length cDNA of a novel leukemia relapse-associated candidate gene (LRP15), the human ESTs (Expression Sequence Tags) fragments obtained from electronic hybridization were assembled by a 1.8 kb DNA probe, which was only methylated in relapsed leukemia. Then the primers were designed for rapid amplification of cDNA end (RACE). Bioinformatic data of High Throughout Genomic Sequences (HTGS) and Serial Analysis of Gene Expression (SAGE) were used for chromosome localization and tissue expression analysis. The results showed that the full-length cDNA of the novel gene had an open reading frame of 780 bp encoding a 259 amino acid protein of unkown functions. LRP15 gene expressed in many different tissues was localized in chromosome 3p24. It is concluded that RACE is an effective method to clone novel genes and LRP15 may be a leukemia relapse-associated candidate gene playing an important role in carcinogenesis.

LRP16 is a novel gene which was found in our laboratory by using methylation-sensitive restriction landmark genomic scanning (RLGS) technique. In order to clone the full-length cDNA of this leukemia relapse associated gene, the method of rapid amplification of cDNA end (RACE) was employed. By optimizing some procedures of RACE method, the 5'- and 3'-untranslated region of LRP16 cDNA was successfully sequenced. Then, the full length of LRP16 cDNA and open reading frame (ORF) was constructed and was registered in GenBank. The above-mentioned procedure demonstrated RACE technique is a rapid and sensitive method for cloning unknown gene. Especially, it is very useful to cloning the 5'- and 3'-untranslated region of a novel gene.