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Lymphocyte recovery is a probable indictor of response.

Clinical response of primary-refractory HL in patients receiving CART-30 cell therapy.

The adverse event after the CART-30 cell infusion.

Case selection criteria;Methods;Figure Legends

Maximum reduction in target lesion size.

Progression-free survival by Kaplan-Meier method.

Purpose: Relapsed or refractory Hodgkin lymphoma is a challenge for medical oncologists because of poor overall survival. We aimed to assess the feasibility, safety, and efficacy of CD30-targeting CAR T cells in patients with progressive relapsed or refractory Hodgkin lymphoma.Experimental Design: Patients with relapsed or refractory Hodgkin lymphoma received a conditioning chemotherapy followed by the CART-30 cell infusion. The level of CAR transgenes in peripheral blood and biopsied tumor tissues was measured periodically according to an assigned protocol by quantitative PCR (qPCR).Results: Eighteen patients were enrolled; most of whom had a heavy treatment history or multiple tumor lesions and received a mean of 1.56 × 107 CAR-positive T cell per kg (SD, 0.25; range, 1.1–2.1) in total during infusion. CART-30 cell infusion was tolerated, with grade ≥3 toxicities occurring only in two of 18 patients. Of 18 patients, seven achieved partial remission and six achieved stable disease. An inconsistent response of lymphoma was observed: lymph nodes presented a better response than extranodal lesions and the response of lung lesions seemed to be relatively poor. Lymphocyte recovery accompanied by an increase of circulating CAR T cells (peaking between 3 and 9 days after infusion) is a probable indictor of clinical response. Analysis of biopsied tissues by qPCR and immunohistochemistry revealed the trafficking of CAR T cells into the targeted sites and reduction of the expression of CD30 in tumors.Conclusions: CART-30 cell therapy was safe, feasible, and efficient in relapsed or refractory lymphoma and guarantees a large-scale patient recruitment. Clin Cancer Res; 23(5); 1156–66. ©2016 AACR.

Supplementary Table S1. Phonotypical characteristics of PBMCs and expanded T Cells as well as transduction efficiency of infused cell products. Supplementary Table S2. Detailed prior chemotherapy regimens for patients before CD30-CART treatment. Supplementary Table S3. Statistics of patient characteristics. Supplementary Table S4. The absolute doses of conditioning regimens for all enrolled patients. Supplementary Table S5. Toxicities possibly related to CD30-CART infusion-containing protocol. Supplementary Table S6. Maximum change from baseline of target measurable lesions. Supplementary Table S7. Clinical response in CD30-CAR T cell- treated patients.

The change of cytokine levels within three months after CART-30 cell infusion.

Background The National Comprehensive Cancer Network's Rectal Cancer Guideline Panel recommends American Joint Committee of Cancer and College of American Pathologists (AJCC/CAP) tumor regression grading (TRG) system to evaluate pathologic response to neoadjuvant chemoradiotherapy for locally advanced rectal cancer (LARC). Yet, the clinical significance of the AJCC/CAP TRG system has not been fully defined. Materials and Methods This was a multicenter, retrospectively recruited, and prospectively maintained cohort study. Patients with LARC from one institution formed the discovery set, and cases from external independent institutions formed a validation set to verify the findings from discovery set. Overall survival (OS), disease-free survival (DFS), local recurrence-free survival (LRFS), and distant metastasis-free survival (DMFS) were assessed by Kaplan-Meier analysis, log-rank test, and Cox regression model. Results The discovery set (940 cases) found, and the validation set (2,156 cases) further confirmed, that inferior AJCC/CAP TRG categories were closely /ccorrelated with unfavorable survival (OS, DFS, LRFS, and DMFS) and higher risk of disease progression (death, accumulative relapse, local recurrence, and distant metastasis) (all p < .05). Significantly, pairwise comparison revealed that any two of four TRG categories had the distinguished survival and risk of disease progression. After propensity score matching, AJCC/CAP TRG0 category (pathological complete response) patients treated with or without adjuvant chemotherapy displayed similar survival of OS, DFS, LRFS, and DMFS (all p > .05). For AJCC/CAP TRG1–3 cases, adjuvant chemotherapy treatment significantly improved 3-year OS (90.2% vs. 84.6%, p < .001). Multivariate analysis demonstrated the AJCC/CAP TRG system was an independent prognostic surrogate. Conclusion AJCC/CAP TRG system, an accurate prognostic surrogate, appears ideal for further strategizing adjuvant chemotherapy for LARC. Implications for Practice The National Comprehensive Cancer Network recommends the American Joint Committee of Cancer and College of American Pathologists (AJCC/CAP) tumor regression grading (TRG) four-category system to evaluate the pathologic response to neoadjuvant treatment for patients with locally advanced rectal cancer; however, the clinical significance of the AJCC/CAP TRG system has not yet been clearly addressed. This study found, for the first time, that any two of four AJCC/CAP TRG categories had the distinguished long-term survival outcome. Importantly, adjuvant chemotherapy may improve the 3-year overall survival for AJCC/CAP TRG1–3 category patients but not for AJCC/CAP TRG0 category patients. Thus, AJCC/CAP TRG system, an accurate surrogate of long-term survival outcome, is useful in guiding adjuvant chemotherapy management for rectal cancer.

T cells engineered with the chimeric antigen receptor (CAR) are rapidly emerging as an important immunotherapy for hematologic malignancies. The anti-cluster of differentiation (CD)19 CAR-T cell therapy has been remarkably successful against refractory/relapsed acute lymphoblastic leukemia (ALL), and a complete remission rate as high as 90% was observed, in both children and adults. Although the achievement of clinical efficacy using CAR-T cell therapy for solid tumors has encountered several obstacles that were associated with the multiple mechanisms contributing to an immunosuppressive microenvironment, investigators are exploring more optimized approaches to improve the efficiency of CAR-T in solid tumors. In addition, cytokine release syndrome (CRS) and neurotoxicity following CAR-T cell therapy can be severe or even fatal; therefore, the management of these toxicities is significant. Herein, we briefly review the structure of CAR-T and some novel CAR designs, the clinical application of CAR-T cell therapies, as well as the assessment and management of toxicities. Keywords: Chimeric antigen receptor T-cell, Hematologic malignancies, Solid tumor, Toxicities

Objective To investigate the effect of human umbilical cord mesenchymal stem cells (hUC-MSCs) on macrophage polarization under hypoxia. Methods hUC-MSCs were obtained by explants adherent culture and cultured under normoxia (21% O2) and hypoxia (5% O2). The multi-directional differentiation of hUC-MSCs was observed by osteogenic and adipogenic differentiation induction. Live/death staining was performed to detect the cell viability, and ELISA was executed to detect the protein content in supernatant of hUC-MSCs. Transwell chamber was employed to co-culture the hUC-MSCs cultured under normoxia and hypoxia and macrophage (THP-1) stimulated by lipopolysaccharide (IPS), then the polarization of THP-1 was detected by immunofluorescence, and the secretions of inflammatory factor and anti-inflammatory factor of THP-1 were detected by ELISA. Results hUC-MSCs cultured under hypoxia showed the ability of osteogenic and adipogenic multi-directional differentiation. Live/death staining showed the high cell viability of hUC-MSCs cultured under normoxia and hypoxia. The expression levels of prostaglandin E2 (PGE2) and indoleamine 2,3-dioxygenase (IDO) were significantly higher in the hUC-MSCs cultured under hypoxia than in those cultured under normoxia. hUCMSCs cultured under hypoxia promoted the polarization of THP-1 to M2, obviously reduced the expression of TNF-α and IL-1β, and increased the expression of IL-10 significantly. Conclusion hUC-MSCs cultured under hypoxia may promote the polarization of THP-1 to M2 and improve the viability of anti-inflammatory. DOI: 10.11855/j.issn.0577-7402.2016.07.01

Objective To study the therapeutic effect of exosome derived from lipopolysaccharides (LPS) priming mesenchymal stem cells (MSCs) for diabetic wound healing. Methods Human umbilical cord MSCs were treated with LPS (100ng/ml) for 2 days, the supernatant were then collected, and exosomes were harvested by density gradient centrifugation and identified. Diabetic cutaneous wounds were prepared and the animals were divided into the following three groups: control group, untreated MSCs derived exosome (un-exosome) treatment group and LPS primed MSCs derived exosome (LPS-exosome) treatment group. Exosomes (60μg) were injected dispersively into the wound edge daily for 10 days. After treatment, the therapeutic results were evaluated by gross observation of the wounds, the expression levels of inflammation related factors and macrophage subtype markers in the injured sites were detected by qRT-PCR at day 3, 7 and 14 after treatment. Results Compared with control group, the diabetic wound healing was obviously improved in LPS-exosome treatment group after treatment for 7 and 14 days, with faster wound close, depressed expression of pro-inflammatory factors IL -1, IL -12 and M1 macrophage surface marker iNOS, and up-regulation of anti-inflammatory factors IL-10, TGF-βand M2 macrophage surface marker CD163, the differences were significant (P

Objective To evaluate the temporary hypoglycemic effect of adipose mesenchymal stem cells (AD-MSCs) on type 2 diabetic rats, and further discuss the mechanisms of AD-MSCs regulating blood glucose through hepatic glycometabolism. Methods The type 2 diabetic rat model was established by intraperitoneal injection of a single small dose of streptozotocin (STZ, 25mg/kg) after a high-fat diet for 8 weeks. The content of blood glucose was determined by tail vein bleeding at indicated time points (0h, 3h, 6h, 12h, 24h) after infusion of AD-MSCs, concurrently the rats were executed and the liver tissues were collected (diabetes group, n=5, normal group, n=3) for detecting the mRNA and protein expressions of glucokinase (GK), pyruvate kinase (PK) and phosphofructokinase (PFK) (the key enzyme of glycolysis) by real-time qPCR and Western blotting. Results The infusion of AD-MSCs significantly decreased blood glucose in a short period [30.55±1.49mmol/L(0h), 18.90±0.85mmol/ L(3h), 23.70±1.13mmol/L(6h), 21.90±1.00mmol/L(12h), respectively]. PCR results showed that the expressions of GK and PFK increased in the liver tissue of type 2 diabetic rats 3h after AD-MSCs infusion, and the expression of PK increased significantly 6h after AD-MSCs infusion (P

Diabetes is a chronic metabolic disorder caused by relative or absolute insulin deficient or reduced sensitivity of target cells to insulin. Mesenchymal stem cells (MSCs) are adult stem cells with multiple differentiation potential, self-renewable and immunoregulatory properties. Accumulating evidences from clinic or animal experiments recent years showed that MSCs infusion could ameliorate hyperglycemia in diabetes. The research progress of MSCs in diabetes treatment is summarized and a corresponding perspective is herewith proposed in present paper. DOI: 10.11855/j.issn.0577-7402.2016.07.16

Objective To investigate the effect and mechanism of bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) on pro-inflammatory cytokines-induced apoptosis of insulinoma cells (INS-1). Methods The conditioned medium containing various cytokines and growth factors was collected and condensed from the supernatants of culture medium of BMSC of rats at passage three. After exposed to the medium with a pro-inflammatory cytokines cocktail containing TNF-α, IL-1β and IFN-γ for 48 hours, INS-1 cells were cultured with BMSC-CM in different concentrations for another 12 hours. INS-1 cells were divided into the following five groups: control group, pro-inflammatory cytokines-treated group, 1× BMSC-CM-treated group, 2× BMSC-CM-treated group and 4× BMSC-CM-treated group. The survival rate of INS-1 cells was assessed by CCK8 assay. The incidence of cell apoptosis was measured using Annexin V/PI staining. Intracellular reactive oxygen species (ROS) production was determined with dichlorofluorescein diacetate (DCFH-DA). Results Compared with the control group, prolonged exposure to pro-inflammatory cytokines induced significant decrease in the viability of INS-1 cells (75.84%±1.53%) and a great increase in the incidence of cell apoptosis (17.23%±1.77%), and also in intracellular ROS level (34.3%±0.80%) with statistically significant difference (P

Objective To explore the association between high-risk human papillomavirus (HR-HPV) viral load and pathological grades of cervical intraepithelial neoplasia (CIN) and cervical cancer. Methods A total of 1248 patients from General Hospital of PLA, who underwent colposcopy and surgery due to cervical lesions between Jan. 2006 and Aug. 2011 were enrolled in this study, and they were divided five groups: cervicitis, CIN Ⅰ, CIN Ⅱ-Ⅲ, stage Ⅰ cervical cancer and stage Ⅱ cervical cancer. HR-HPV viral load (RLU/CO) was determined by the Hybrid Capture Ⅱ (HCⅡ) system, and they were categorized into five groups: 0-0.99, 1.00-9.99, 10.00-99.99, 100.00-999.99, ≥1000.00. The mean value and standard deviation of different HR-HPV viral load in the patients with cervicitis or with CIN Ⅰ, CINⅡ-Ⅲ, stage Ⅰ cervical cancer or stage Ⅱ cervical cancer were compared, and the correlation of HR-HPV viral load and pathogenesis of cervical lesions was analyzed. Results HPV viral loads were significantly higher in CINⅠ(842.1±983.9), CINⅡ-Ⅲ (690.1±795.0), stage Ⅰ cervical cancer (893.1±974.2) and stage Ⅱ cervical cancer (699.5±908.3) patients than in cervicitis patients (274.2±613.6, P < 0.05), and the HPV viral loads in CINⅠ(842.1±983.9) and stage Ⅰ cervical cancer patients were higher than those in CINⅡ-Ⅲ patients (P < 0.05). When HR-HPV viral load was ≥100RLU/CO, the risk of CIN and cervical cancer increased with the increase in viral load, but there was no correlation between the viral load and pathological grades of cervical lesions. In the patients with stage ⅠB-Ⅱ cervical squamous cell carcinoma, when the HR-HPV viral load was ≥100RLU/CO, the risk of lymph node metastasis increased (P < 0.05), and the number of patients with maximum diameter of the cervical tumor ≥4cm also increased (P < 0.05). However, the HR-HPV viral load was not correlated with patient age, pathological type of the lesion, depth of cancer cell invasion into cervical muscle layer and lymphatic invasion. Conclusions HR-HPV viral load is closely correlated with genesis of CIN and cervical carcinoma. The higher the viral load, the greater the risk of CIN and cervical cancer is, and it is correlated with the clinical features of cervical squamous cell carcinoma.

Leizhou Peninsula, located at southern end of mailand China, has 9 284.3 ha mangrove distributed more than 100 sites along its inlets and open coastlines. This paper presents the surveys on mangrove area in Leizhou Peninsula during 2000 and 2008, especially the survey in the eight major mangrove areas in 2002. The flora recorded in mangrove systems includes 69 large algea species, 13 native true mangrove species, nine native semi-mangrove species, and another seven intoduced true mangrove species with Sonneratia apetala as the quickest growing exotic mangrove species dominatant in the mangrove plantations, and more than 100 land flora species, consisting of 17 main mangrove associations. The plant biodiversity habitats remained mainly small patches and diverse, and were becoming worse under the intensive disturbance of human acitivities and coastal pollutions, which decreased the value of mangrove coasts as the important sites for flying-by migrating birds in Leizhou Peninsula. The effective mangrove conservation measures should be implemented.

In the present study, we have analyzed the regulation of Wnt/β-catenin signaling in lung adenocarcinoma stem cells (CSCs), that are responsible for tumor recurrence.Lung cancer samples were studied for the presence of cancer stem like cells and analyzed by flow cytometry. Then, the sorted cells were analyzed for the stem cell surface markers and Wnt/β-catenin signaling pathways. Moreover, the sorted side population (SP) and non-SP cells were also subjected to drug resistance assay.Western blot analysis showed that the protein level of β-catenin was highly upregulated in fluorescence activated cells (FACs) sorted SP cells which led to elevated expression of stem cell protein Oct-4 that is responsible for SP cells' self-renewal. RT-PCR revealed that the relative mRNA expression level of Wnt target gene cyclin D was significantly higher (p0.01) in SP cells, enhancing thus the cell proliferation rate and clone formation efficiency. In addition, the matrigel invasion assay revealed that SP cells were highly invasive than non-SP cells.In the present study we demonstrated that lung adenocarcinoma samples contain a small population of tumor-initiating SP cells which possess the characteristic features of CSCs. Wnt/β-catenin mediated increased expression of β-catenin, Oct-4 and cyclin D in SP cells but not in non-SP cells was also observed. FACs-purified SP cells are resistant to a number of chemotherapeutic drugs. Our data suggest that the use of novel anticancer drugs, targeting Wnt/β-catenin signaling pathways, may help eradicate the lung cancers stem cells.

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 (P0.05), The therapy of CIK could significantly reduce the incidence of infection (P0.05) and shorten the time of high fever in AML patients (P0.05). CIK also could reduce the volume of erythrocyte infusion to maintenance hematoglobin level (P0.05). 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 rate95%) 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.

Id3 plays a key role in the progression of breast cancer. Previously, four and a half LIM protein (FHL2) was identified as a repressor of Id family proteins by interacting with them. This study aimed to investigate the effects of FHL2 on the transcriptional regulation and oncogenic activities of Id3 in human breast cancer cells.Cell transfection was performed with SuperFect reagent. Stable transfectants that overexpressed Id3 were obtained by selection on G418. The level of Id3 protein was determined by Western blotting analysis. Dual luciferase assays were used to measure the effect of Id3 and FHL2 on E47-mediated transcriptional activity in MCF-7 human breast cancer cells. The MTT assay was used to measure cell proliferation. The transwell assay was used to measure the invasive capacity of MCF-7 cancer cells.Id3 markedly repressed transcription mediated by the basic helix-loop-helix (bHLH) factor E47 in MCF-7 cells. This Id3-mediated repression was effectively antagonized by FHL2. Overexpression of Id3 markedly promoted the proliferation and invasive capacity of MCF-7 cells; however, these effects were significantly suppressed by the overexpression of FHL2.FHL2 can inhibit the proliferation and invasive growth of human breast cancer cells by repressing the functional activity of Id3. The functional roles of FHL2-Id3 signaling in the development of human breast cancer need further research.

We demonstrate the structure from AlGaN-based multiple quantum well light emitting diodes at very short wavelength. Using software APSYS to simulate, 280nm wavelength can be achieved.

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 (P0.05). 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 (P0.05), the serum level of β2 microglobulin was significantly declined (P0.05), and the frequency and degree of infection was also decreased (P0.05). 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 (p0.05). 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.

The elderly population is susceptible to haematological malignancies, and these elderly patients are intolerant to cytotoxic drugs. Therefore, the exploration of a safe and reliable strategy exclusive of chemotherapy is critical in improving the prognosis of elderly patients with haematological malignancies. We evaluated the safety and the efficacy of autologous cytokine-induced killer (CIK) cells combined with recombinant human interleukin 2 (rhIL-2) in the treatment of haematological malignancies in elderly patients. Peripheral blood mononuclear cells were isolated from 20 elderly patients with haematological malignancies, then augmented by priming with interferon gamma, rhIL-2 and CD3 monoclonal antibody. The autologous CIK cells (2-3 × 10(9)) were transfused back to patients, followed by a subcutaneous injection of IL-2 (1 mU/day) for 10 consecutive days. The regimen was repeated every 4 weeks. The host cellular immune function, tumour-related biological parameters, imaging characteristics, disease condition, quality of life and survival time were assessed. Fourteen patients received 8 cycles of transfusion and 6 received 4 cycles. No adverse effects were observed. The percentages of CD3(+), CD3(+) CD8(+) and CD3(+) CD56(+) cells were significantly increased (p0.05), and the levels of serum β2 microglobulin and lactate dehydrogenase (LDH) were markedly decreased (p0.05) after autologous CIK cell transfusion. Cancer-related symptoms were profoundly alleviated, as demonstrated by the improved quality of life (p0.01). Complete remission was observed in 11 patients, persistent partial remission in 7 patients and stable disease in 2 patients. At the end of follow-up, the mean survival time was 20 months. Transfusion with autologous CIK cells plus rhIL-2 treatment is safe and effective for treating haematological malignancies in elderly patients.

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 (p0.05), and serum levels of β2-microglobulin and LDH markedly decreased (p0.05) after autologous CIK cells transfusion. The tumor-related symptoms were relieved, QOL obviously improved (p0.01) 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.

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 (p0.05), and serum levels of β2-microglobulin and LDH were markedly decreased (p0.05) after autologous CIK cell transfusion. The lymphoma symptoms were relieved with quality of life obviously elevated (p0.01) 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.

Cytokine-induced killer (CIK) cells have been shown to be effective in the treatment of advanced cancer and minimal residual diseases. We report a multiple myeloma (MM) patient with concomitant lung cancer and paraneoplastic dermatoses, who received cellular immunotherapy with CIK cells which were derived from peripheral blood mononuclear cells (PBMCs) after being primed with anti-cluster of differentiation 3 (CD3) monoclonal antibody, interleukin-2 (IL-2), interferon-γ (IFN-γ) and IL-1. After treatment MM and lung cancer remained stable and no progression or recurrence was observed. Paraneoplastic dermatoses were obviously improved after treatment, which was first reported. No evident side effects were observed. These findings suggested that cellular immunotherapy with CIK cells was safe and effective in this patient with MM and lung cancer, and it might be a potent therapeutic option for paraneoplastic dermatoses.

AIM: To explore the expression of leukemia related protein 16 (LRP16) in colorectal carcinoma, and analyze its correlation with clinicopathologic features and prognosis. METHODS: Immunohistochemistry for LRP16 was performed in 201 cases of colorectal carcinoma and 60 cases of distal normal mucosa. Medical records were reviewed and clinicopathological analysis was performed. RESULTS: LRP16 expression was detected in 117 of 201 cases of the colorectal carcinoma and in 21 cases of 60 distal normal mucosa. The expression of LRP16 in carcinoma was significantly higher than that in normal mucosa (χ2 = 9.999, P = 0.002). LRP16 protein expression was found in 43.3% (52/120) of carcinoma at stage I and II, and 80.2% (65/81) of carcinoma at stage III and IV (χ2 =27.088, P = 0.001). Correlation between LRP16 expression and clinicopathological factors was significant in differentiation (P = 0.010), tumor size (P = 0.001), infiltrative depth (P = 0.000) and distant metastasis (P = 0.027). The difference of median survival time between cancer patients with LRP16 expression (38.0 mo) and those without was statistically significant (105.0 mo, Log rank = 41.455, P = 0.001). The multivariate survival analysis revealed that LRP16 expression was correlated significantly (Cox’s regression: P = 0.001, relative risk = 2.082) with shortened survival in the patients with colorectal cancer. CONCLUSION: The expression of LRP16 is related to the degree of differentiation, invasiveness, metastasis and prognosis of colorectal carcinoma.

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 (p0.001). 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.

Estrogen directly up-regulates LRP16 gene expression via activating its receptor (ER), and the overexpression of LRP16 promotes the proliferation of human breast cancer cells. This study was to detect the mRNA level of LRP16 gene in breast cancer, and investigate its correlation to the clinicopathologic features.The mRNA level of LRP16 in carcinoma and matched peritumor tissues from 22 breast cancer patients was detected by Northern blot, and that in the tissues from 30 patients was detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The expression of Ki-67, ER, and progesterone receptor (PR) in the carcinoma tissues was detected by immunohistochemistry.According to the results of Northern blot, compared with that in peritumor tissues, LRP16 was overexpressed by 2 folds in 9 (40.9%) out of 22 breast cancer samples. Of the 9 samples with LRP16 overexpression, 7 were ER-positive, and 8 were PR-positive; of the 13 samples without LRP16 overexpression, 6 were ER-positive, and 5 were PR-negative. The positive rates of ER and PR were significantly higher in the samples with LRP16 overexpression than in the samples without LRP16 overexpression (P0.05). Only 1 of the 9 samples with LRP16 overexpression was negative for both ER and PR, but 7 of the 13 without LRP16 overexpression were negative for both of them. The proportion of the tumors with diameters of 3.0-4.5 cm was significantly higher in the patients with LRP16 overexpression than in those without LRP16 overexpression (8/9 vs. 5/13, P=0.031). Axillary lymph node metastasis was detected in 12 out of 22 patients, including 8 of the 9 patients with LRP16 overexpression and 4 of the 13 without LRP16 overexpression (P=0.011). In addition, LRP16 overexpression was detected in 6 of the 8 patients with Ki-67 overexpression, and 2 of the 14 patients without Ki-67 overexpression (P=0.026). According to the results of RT-PCR, LRP16 was overexpressed in 9 (30%) out of 30 breast cancer samples. All of the 9 samples with LRP16 overexpression were positive for both ER and PR, with Ki-67 overexpression, tumor diameters of more than 3.5 cm and axillary lymph node metastasis. The differences between the patients with or without LRP16 overexpression were significant (P0.05).LRP16 overexpression is closely correlated to the positive rates of ER and PR, Ki-67 level, tumor diameter, and axillary lymph node metastasis of breast cancer, and might be involved in the proliferation and metastasis of human breast cancer.

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 analyse the WT1 expression and its DNA methylation status of its promoter domain.The expression of WT1 gene and its DNA methylation status were assayed in leukemia cell lines and normal peripheral blood mononuclear cells (PBMNC) by RT-PCR and MS-PCR.WT1 was overexpressed in HL60, K562 and KG1 leukemia cell lines, but not in U937 and PBMNC. Methylation of WT1 promoter was not observed in HL60 cells.DNA methylation of WT1 gene promotor did not inhibit its expression. Other mechanisms may appear to regulate the WT1 expression.

Telomerase has been thought to play an important role in the carcinogenesis in recent years. Human telomerase reverse transcriptase (hTERT) is a limiting component for telomerase activity. This study was designed to explore the effect of transfection of the full-length cDNA of antisense hTERT on the malignant phenotype of human pulmonary giant cell carcinoma cell line (PLA-801D) and its potential role in the gene therapy for cancers.An antisense hTERT cDNA eukaryotic expression vector pcDNA3.1(-)-hTERT including the full length of hTERT cDNA sequence was constructed using recombinant DNA technique and transfected into human pulmonary giant cell carcinoma cells (PLA-801D) with liposome. The effect of antisense hTERT on the cellular proliferation capacity of PLA-801D cells was analyzed by the growth curve. The expression of hTERT mRNA was examined by reverse transcription polymerase chain reaction (RT-PCR). The telomerase activity was determined by telomeric-repeat amplification protocol enzyme-linked immunoassay (TRAP-ELISA).Antisense pcDNA3.1 (-)-hTERT eukaryotic expression have been constructed and was successfully transfected into the PLA-801D cells. The growth speed of PLA-801D transfected with antisense hTERT was significantly inhibited compared with the control cells, and the hTERT mRNA expression was inhibited, the relatively expression was only 15.7% of control cells, and telomerase activity was down-regulated about 82.4%.Full-length antisense hTERT cDNA can suppress hTERT mRNA expression and telomerase activity, and restrict the growth speed of tumor cells.

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.