Objective: To explore the differences in the biological effects of different expansion systems on natural killer (NK) cells, as well as the safety and preliminary clinical efficacy in the treatment of patients with recurrence after allogeneic hematopoietic stem cell transplantation (allo-HSCT) . Methods: Peripheral blood cells from healthy donors were stimulated with either CD3 combined with CD52 or K562 feeder cells loaded with IL-21/4-1BB to induce NK cell expansion. Changes in the NK cell phenotype, cytokine secretion, and cytotoxicity before and after expansion were detected. We also evaluated the safety and clinical efficacy of two different expansion strategies for patients received NK infusion. Results: Compared with the CD3/CD52 monoclonal antibody amplification system, the feeder cell expansion group had a higher purity of NK cells and higher expression ratios of NK cell surface activation receptors such as DNAM-1 and NKp30, while inhibitory receptor CTLA-4 expression was low and NKG2D/CD25/CD69/ Trail/PD-1/TIM-3/TIGIT had no statistically significant differences between the groups. Further functional results showed that the expression level of KI67 in NK cells after expansion in the two groups increased significantly, especially in the feeder cell expansion group. Simultaneously, the perforin and granzyme B levels of NK cells in the feeder cell expansion group were significantly higher than in the CD3/CD52 expansion group. A retrospective analysis of eight patients who received monoclonal antibody-expanded NK cell reinfusion and nine patients with trophoblast cell-expanded NK cell reinfusion was done. The disease characteristics of the two groups were comparable, NK cell reinfusion was safe, and there were no obvious adverse reactions. Clinical prognostic results showed that in the CD3/CD52 monoclonal antibody amplification group, the MRD conversion rate was 50% (2/4) , and the feeder cell expansion group was 50% (3/6) . After 5 years of follow-up from allo-HSCT, three patients in the monoclonal antibody expansion group had long-term survival without leukemia, and the remaining five patients had died; two patients died in the feeder cell expansion group, and the other six patients had long-term survival. Six cases had GVHD before NK cell reinfusion, and GVHD did not aggravate or even relieved after NK cell reinfusion. Conclusions: Preliminary results show that the biological characteristics of NK cells with diverse expansion strategies are significantly different, which may affect the clinical prognosis of patients with recurrence or persistent minimal residual disease after HSCT. The two groups of patients treated with NK cells from different expansion strategies had no obvious adverse reactions after NK cell infusion, but efficacy still needs to be further confirmed.
Antibodies, Monoclonal/pharmacology* ; Graft vs Host Disease/metabolism* ; Hematopoietic Stem Cell Transplantation ; Humans ; Killer Cells, Natural ; Retrospective Studies ; Treatment Outcome

OBJECTIVETo investigate the effects of inhibiting TIM-4 function in Kupffer cells (KCs) on liver graft rejection in mice and explore the underlying mechanism.

METHODSMouse models of orthotopic liver transplantation were treated with a control mAb group and TIM-4 mAb. The activated KCs were assayed with immunohistochemistry after operation. The expression of TIM-4 in KCs were assayed with Western blotting and RT-PCR and the levels of AST, ALT, TBIL, TNF-α, IFN-γ and CCL2 were assayed detected. The expression of TIM-4 in KCs was observed with laser confocal microscopy. HE staining was used to observe the microstructure of the liver tissues, and the number of CD25Foxp3T cells was determined using with flow cytometry; the proteins levels of p-P65and p-P38 were assayed with Western blotting. The donor mice were treated with clodronate liposomes to destroy the KCs in the liver before transplantation, and the liver grafts were examined for graft rejection.

RESULTSThe number of activated KCs in the liver graft increased progressively over time. Compared with the sham-operated group, the liver graft showed significantly increased TIM-4 protein and mRNA levels at 1, 3, and 7 days after transplantation (P<0.05) and increased levels of AST, ALT, TBIL, TNF-α, IFN-γ and CCL2 at 7 days (P<0.05). The graft in TIM-4 mAb group showed mild pathological changes with a mean RAI score of 2.67∓0.75, which was significantly lower than that in control mAb group (P<0.05). The mean survival time of the recipient mice was 53.8∓6.4 days in TIM-4 mAb group, significantly longer than that in the control mAB group (14.5∓2.9 days, P<0.05). Donor treatment with clodronate liposomes resulted in comparable RAI scores in TIM-4 mAb and control mAb groups (8.01∓0.64 vs 7.93∓0.56, P>0.05). The protein levels of p-P65 and p-P38 in TIM-4 mAb group were significantly lower than those in control mAb group (P<0.05), and CD25Foxp3T cells in the liver graft increased significantly in TIM-4 mAb group.

CONCLUSIONInhibition of TIM-4 function in KCs reduces the production of inflammatory factors after liver transplantation possibly by inhibiting the NF-κB and MAPK signaling pathways and promoting the proliferation of Foxp3Treg cells to induce allograft tolerance.

Animals ; Antibodies, Monoclonal ; pharmacology ; Graft Rejection ; Immunohistochemistry ; Kupffer Cells ; drug effects ; metabolism ; Liver ; surgery ; Liver Transplantation ; Male ; Membrane Proteins ; antagonists & inhibitors ; Mice ; NF-kappa B ; metabolism ; T-Lymphocytes, Regulatory ; immunology

PURPOSE: Reduced brain glucose metabolism and basal forebrain cholinergic neuron degeneration are common features of Alzheimer's disease and have been correlated with memory function. Although regions representing glucose hypometabolism in patients with Alzheimer's disease are targets of cholinergic basal forebrain neurons, the interaction between cholinergic denervation and glucose hypometabolism is still unclear. The aim of the present study was to evaluate glucose metabolism changes caused by cholinergic deficits. MATERIALS AND METHODS: We lesioned basal forebrain cholinergic neurons in rats using 192 immunoglobulin G-saporin. After 3 weeks, lesioned animals underwent water maze testing or were analyzed by 18F-2-fluoro-2-deoxyglucose positron emission tomography. RESULTS: During water maze probe testing, performance of the lesioned group decreased with respect to time spent in the target quadrant and platform zone. Cingulate cortex glucose metabolism in the lesioned group decreased, compared with the normal group. Additionally, acetylcholinesterase activity and glutamate decarboxylase 65/67 expression declined in the cingulate cortex. CONCLUSION: Our results reveal that spatial memory impairment in animals with selective basal forebrain cholinergic neuron damage is associated with a functional decline in the GABAergic and cholinergic system associated with cingulate cortex glucose hypometabolism.
Acetylcholine/metabolism ; Alzheimer Disease ; Animals ; Antibodies, Monoclonal/*pharmacology ; Basal Forebrain/*drug effects/metabolism ; Cholinergic Agents/administration & dosage/*pharmacology ; Cholinergic Neurons/*drug effects/metabolism ; Fluorodeoxyglucose F18 ; GABAergic Neurons/*drug effects/metabolism ; Glucose/*metabolism ; Gyrus Cinguli/*drug effects/metabolism ; Humans ; Injections ; Maze Learning ; Motor Activity/physiology ; Positron-Emission Tomography ; Rats ; Ribosome Inactivating Proteins, Type 1/*pharmacology

OBJECTIVETo investigate the role of epidermal growth factor receptor (EGFR) expression level in cetuximab cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) effect against A549 lung cancer cell line.

METHODSA549 cell line and NKTm cells were used as the target cell and the effector cell, respectively. pEGFR-EGFP plasmids were transfected into A549 cells by nucleofector method. EGFR expression levels were measured by immunohistochemistry. The ADCC activity induced by cetuximab was assessed by cell counting kit-8 assay.

RESULTSA549 cells transfected with pEGFR-EGFP plasmids expressed higher level of EGFR protein on membrane and were more sensitive to ADCC activity mediated by cetuximab (P<0.05). The inhibition rate of A549 cells showed no significant difference between transfection group and wild-type group when treated with cetuximab alone (P> 0.05).

CONCLUSIONEGFR expression level influences the sensitivity of A549 lung cancer cell line to ADCC activity mediated by cetuximab but not to cetuximab alone.

Adenocarcinoma ; pathology ; Antibodies, Monoclonal, Humanized ; pharmacology ; Antineoplastic Agents ; pharmacology ; Cetuximab ; Humans ; Immunohistochemistry ; Lung Neoplasms ; pathology ; Receptor, Epidermal Growth Factor ; metabolism ; Tumor Cells, Cultured

FXYD6, FXYD domain containing ion transport regulator 6, has been reported to affect the activity of Na(+)/K(+)-ATPase and be associated with mental diseases. Here, we demonstrate that FXYD6 is up-regulated in hepatocellular carcinoma (HCC) and enhances the migration and proliferation of HCC cells. Up-regulation of FXYD6 not only positively correlates with the increase of Na(+)/K(+)-ATPase but also coordinates with the activation of its downstream Src-ERK signaling pathway. More importantly, blocking FXYD6 by its functional antibody significantly inhibits the growth potential of the xenografted HCC tumors in mice, indicating that FXYD6 represents a potential therapeutic target toward HCC. Altogether, our results establish a critical role of FXYD6 in HCC progression and suggest that the therapy targeting FXYD6 can benefit the clinical treatment toward HCC patients.
Animals ; Antibodies, Monoclonal ; pharmacology ; Antineoplastic Agents ; pharmacology ; Carcinoma, Hepatocellular ; drug therapy ; metabolism ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Female ; HEK293 Cells ; Humans ; Ion Channels ; antagonists & inhibitors ; metabolism ; Liver Neoplasms ; drug therapy ; metabolism ; Mice, Inbred BALB C ; Mice, Nude ; Sodium-Potassium-Exchanging ATPase ; metabolism ; Tumor Burden ; drug effects ; Xenograft Model Antitumor Assays

PURPOSE: Hypoxia-inducible factor-1alpha (HIF-1alpha) increases transcription of the vascular endothelial growth factor (VEGF) gene. Inhibition of VEGF abolishes VEGF mediated induction of HIF-1alpha. Recent reports suggested that HIF-1alpha also mediated the induction of class III beta-tubulin (TUBB3) in hypoxia. TUBB3 confers resistance to taxanes. Inhibition of VEGF may decrease the expression of HIF-1alpha and TUBB3. This study was undertaken to investigate the roles of vascular endothelial growth factor receptor (VEGFR) in gastric cancer cell behavior and to identify methods to overcome paclitaxel resistance in vitro. MATERIALS AND METHODS: The protein expression levels of HIF-1alpha and TUBB3 were measured in human gastric cancer cell lines (AGS) under normoxic and hypoxic conditions. The relationship between TUBB3 and paclitaxel resistance was assessed with small interfering TUBB3 RNA. AGS cells were treated with anti-VEGFR-1, anti-VEGFR-2, placental growth factor (PlGF), bevacizuamb, and paclitaxel. RESULTS: Hypoxia induced paclitaxel resistance was decreased by knockdown of TUBB3. Induction of HIF-1alpha and TUBB3 in AGS is VEGFR-1 mediated and PlGF dependent. Hypoxia-dependent upregulation of HIF-1alpha and TUBB3 was reduced in response to paclitaxel treatment. Expressions of HIF-1alpha and TUBB3 were most decreased when AGS cells were treated with a combination of paclitaxel and anti-VEGFR-1. AGS cell cytotoxicity was most increased in response to paclitaxel, anti-VEGFR-1, and anti-VEGFR-2. CONCLUSION: We suggest that blockade of VEGFR-1 and VEGFR-2 enhances paclitaxel sensitivity in TUBB3-expressing gastric cancer cells.
Angiogenesis Inhibitors/pharmacology ; Antibodies, Monoclonal, Humanized/pharmacology ; Antineoplastic Agents, Phytogenic/*pharmacology ; Cell Hypoxia ; Cell Line, Tumor ; *Drug Resistance, Neoplasm ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Knockdown Techniques ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism ; Paclitaxel/*pharmacology ; Pregnancy Proteins/pharmacology ; Stomach Neoplasms/drug therapy/genetics ; Tubulin/genetics/metabolism ; Vascular Endothelial Growth Factor Receptor-1/antagonists & inhibitors/*physiology ; Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors/*physiology

OBJECTIVEThis study was designed to evaluate the role of interleukin (IL)-1β in the development of fibrosis in mice exposed to silica.

METHODSThe total of 96 Male C57BL/6 mice were divided into four groups. (1) blank control group, (2) PBS group in which mice were instilled with PBS only, (3) silica + IL-1β mAb group in which mice were instilled with 2.5 mg silica dust and 40 µg anti-IL-1β mAb, (4) silica group in which mice were instilled with 2.5 mg silica dust and 40 µg IgG. The final volume of suspension or PBS instilled into the mouse was 50 µl. At 7, 28 and 84 days after treatment, 8 mice were sacrificed in each group. Then BALF was collected for the count of inflammatory cells and cytokines determination. The lung tissues were collected for the detecting of mRNA levels of fibrogenic molecules.

RESULTSThe collagen deposition induced by silica in the lung tissues was partly inhibited by anti-IL-1β. A intensely pulmonary cytokines such as IL-1β, TNF-α, MCP-1 were induced by crystalline silica exposure, and partly inhibited by anti-IL-1β. The levels of TGF-β and fibronectin in silica exposed mice were significantly elevated than those in control mice at days 28 and 84 after treatment (P < 0.01). And the mRNA levels of TGF-β, collagen I and fibronectin were significantly decreased in silica+IL-1β mAb group when compared with those in silica group at days 7, 28 and 84 (P < 0.01). There was a significant decrease of the ratios of IFN-γ/IL-4 in both silica+anti-IL-1β mAb and silica groups when compared with those in control mice at the above three time points (P < 0.01). However, the IFN-γ/IL-4 ratios in silica+anti-IL-1β group were significantly higher than those in silica group at 7, 28 and 84 days (P < 0.05 or P < 0.01).

CONCLUSIONIL-1β may promote the pulmonary fibrosis in mice exposed to silica.

Animals ; Antibodies, Monoclonal ; pharmacology ; Bronchoalveolar Lavage Fluid ; chemistry ; Collagen Type I ; metabolism ; Disease Models, Animal ; Fibronectins ; metabolism ; Interferon-gamma ; metabolism ; Interleukin-1beta ; metabolism ; physiology ; Interleukin-4 ; metabolism ; Lung ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Pulmonary Fibrosis ; chemically induced ; metabolism ; pathology ; Silicon Dioxide ; toxicity ; Transforming Growth Factor beta ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

Adult ; Antibodies, Monoclonal ; pharmacology ; Bronchoalveolar Lavage Fluid ; cytology ; Cells, Cultured ; Cytokines ; biosynthesis ; blood ; secretion ; Fas Ligand Protein ; antagonists & inhibitors ; metabolism ; Humans ; Macrophages, Alveolar ; immunology ; metabolism ; Middle Aged ; Occupational Exposure ; analysis ; Signal Transduction ; Silicon Dioxide ; adverse effects ; Silicosis ; blood ; immunology ; fas Receptor ; antagonists & inhibitors ; metabolism

This study was purposed to investigate the molecular mechanism of 4-1BBL reverse signals in the human acute monocytic leukemia cell line of U937. The U937 cell line was used as target cells, and stimulated by the mouse anti-human 4-1BBL monoclonal antibody 1F1. The nuclear translocation of NF-κB and the co-location of 4-1BBL and CD28i molecules in U937 cells were observed with confocal laser scanning microscopy. The protein and m-RNA expression levels of 4-1BBL and CD28i were detected by flow cytometry and RT-PCR respectively. The results showed that the significant nuclear translocation of NF-κB and co-localization of 4-1BBL and CD28i on membrane of U937 cells appeared after being stimulated by mAb1F1. It is concluded that the 4-1BBL reverse signals transduction mediating the growth of U937 cells relates with the nuclear translocation of NF-κB. CD28i may be involved in intracellular 4-1BBL reverse signaling pathways.
4-1BB Ligand ; immunology ; metabolism ; Antibodies, Monoclonal ; pharmacology ; CD28 Antigens ; metabolism ; Coculture Techniques ; Humans ; NF-kappa B ; genetics ; Signal Transduction ; U937 Cells

This study was purposed to investigate the CIK cell cytotoxicity to hematological malignant cell lines by interaction NKG2D receptors and corresponding ligands. The CIK cells was expanded from healthy individual with interferon (IFN)γ, CD3 monoclonal antibodies (mAb) and interleukin-2 (IL-2). The subset of lymphocyte and the expression of NK cell receptors on CIK cells was detected by flow cytometry; NKG2D ligand expression on hematological malignant cell lines was also analyzed by flow cytometry, the calcein acetoxymethyl ester (CAM) was used for labeling target cells, then the cytotoxicity of CIK cells to hematological malignant cell lines was detected by flow cytometry. The results showed that most of CIK cells expressed CD3 (97.85 ± 1.95%) , CD3(+)CD8(+) cells and CD3(+)CD56(+) cells increased significantly as compared with un-cultured cells (P < 0.001;P = 0.033). About 86% CIK cells expressed NKG2D receptor but no other NK receptors such as CD158a, CD158b and NCR. Different levels of NKG2D ligands were detected in hematological malignant cell lines U266, K562 and Daudi. CIK cells showed high cytotoxicity to these three different cell lines, and this cytotoxicity was partially blocked by treating CIK cells with anti-NKG2D antibody (U266 52.67 ± 4.63% vs 32.67 ± 4.81%, P = 0.008;K562 71.67 ± 4.91% vs 50.33 ± 4.91%, P = 0.007;Daudi 68.67 ± 5.04 vs 52.67 ± 2.60%, P = 0.024) . It is concluded that most of CIK cells express NKG2D receptor, interaction of NKG2D-NKG2D ligands may be one of the mechanisms, by which CIK cells kill hematological malignant cells.
Antibodies, Monoclonal ; pharmacology ; Cell Line, Tumor ; Culture Media ; chemistry ; Cytokine-Induced Killer Cells ; metabolism ; Humans ; Interferon-gamma ; pharmacology ; Interleukin-2 ; pharmacology ; Ligands ; Monocytes ; cytology ; metabolism ; NK Cell Lectin-Like Receptor Subfamily K ; metabolism