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Abstract: IL-23 is a novel cytokine composed of an IL-12 p40 and a p19 subunit. We analyzed human peripheral blood mononuclear cells (PBMC) and hematopoietic cell lines for constitutive expression of IL-23 and IL-12 subunits and expression following exposure to various stimuli, and investigated the mechanisms of their induction by LPS and SAC. IL-23 p19 and IL-12 p35 mRNAs were expressed in fresh PBMC, and expression of all IL-23 and IL-12 subunits was up-regulated by LPS and SAC. LPS-induced increase of IL-23 and IL-12 subunits expression was CD14-dependent, while CD14 was not involved in SAC-induced p19 transcription. Both LPS- and SAC-induced subunits expression required p38 MAPK pathway. PHA effected an increase of p19 mRNA in both CD4+ and CD8+ T cells, whereas p35 was minimally regulated by PHA. IFN-¿ primed monocytes for LPS stimulation of p19, p35 and p40 expression. p19 mRNA was detectable in most hematopoietic cell lines tested but p35 distribution was more restricted. A phorbol diester enhanced p19, p35 and p40 expression in EBV-positive cell lines. Our results suggest that both the subunits of IL-23 are tightly regulated; the expression pattern and regulation mode of IL-23 p19 is similar to as well as distinct from that of IL-12 p35. [Copyright &y& Elsevier]

Abstract: Nucleotides are new players in intercellular communication network. P2X family receptors are ATP-gated plasma membrane ion channels with diverse biological functions. Their distribution patterns and significance in pediatric leukemias have not been established. Here we investigated the expression of P2X receptors in BMMC samples from Chinese pediatric acute leukemias. Real-time PCR and Western blot results showed that P2X1, P2X4, P2X5 and P2X7 receptors were simultaneously over expressed in leukemias compared with controls, whereas P2X2, P2X3 and P2X6 were absent or marginally expressed in both groups. It was worth noting that the co-expression feature of them, especially between P2X4 and P2X7, could be observed and the highest expression of P2X7 was detected in relapsed patients. Moreover, concomitant decrease of P2X4, P2X5 and P2X7 expressions was observed at CR stage in a follow-up study. Functional P2X7 was also verified. These results suggested that P2X1, P2X4, P2X5 and P2X7 were hematopoiesis-related P2X receptors, and their signaling, especially for P2X7, might play important roles in pediatric leukemias. P2X receptors might co-operatively contribute to the malignant phenotype in human pediatric leukemias. [Copyright &y& Elsevier]

Abstract: The cDNA encoding N-terminal three immunoglobin-like domains of human M-CSFR was linked to His-tag and endoplasmic reticulum retention sequence (KDEL) before being inserted into the genome of tobacco plant, Nicotiana tabacum cv. NC-89, by Agrobacterium tumefaciens-mediated transformation. The insertion and expression of target gene were confirmed by PCR, ELISA, and Western blot. The recombinant M-CSFsR reached a maximum expression level of 1.92% of total soluble protein in transgenic tobacco plant leaf tissues. The recombinant M-CSFsR could be purified through a one-step IMAC process and its bioactivity was confirmed by the inhibition of colony formation of J6-1 cells. The results suggested that we successfully expressed a high level of bioactive human M-CSFsR in tobacco plants. [Copyright &y& Elsevier]

Abstract: DNA vaccine against M-CSFRJ6-1 (macrophage colony-stimulating factor receptor cloned from the J6-1 leukemic cell line) has shown both protective and therapeutic effects. In this study, to explore the adjuvant effects of LL-37 to M-CSFRJ6-1 DNA vaccines, we constructed genetically fused vaccines encoding M-CSFRJ6-1 and LL-37(pF). After immunizing BALB/c mice, specific humoral and cellular immune responses were detected. Compared with pR (encoding the extracellular region of M-CSFRJ6-1), pF was more effective in inducing humoral and cytotoxic immune response, prolonging survival of mice challenged with SP2/0-CSFRJ6-1 tumor cells, and inducing IFN-γ and IL-4 release by splenocytes. In this study, we also constructed pLL37 (encoding the mature LL-37) and coadministrated pLL37 and pR to see whether the genetic fusion was necessary. We found that compared with pR alone, pLL37+pR could not prolong survival of mice challenged with SP2/0-CSFRJ6-1 tumor cells. Our results suggest that when genetically fused with M-CSFRJ6-1, LL-37 could enhance adaptive immune response against M-CSFRJ6-1 in a murine model challenged with tumor cells bearing M-CSFRJ6-1. [Copyright &y& Elsevier]

The human cationic antimicrobial peptide hCAP-18/LL-37 is the unique cathelicidin identified in human to date. It has broad spectrum of antimicrobial activities and LPS-neutralizing activity and is involved in angiogenesis. Both purified and synthetic LL-37 or its derivatives were used in the study on LL-37. However, production of LL-37 in Escherichia coli has not been established. In this study, its precursor instead of the mature peptide was adopted for expression to avoid the lethal effect of recombinant LL-37 on host cells. A thrombin recognition site was introduced between the cathelin-like domain and LL-37 domain by overlap PCR to construct fragment encoding modified precursor (mhCAP-18) to facilitate the final release of the recombinant peptide. Then mhCAP-18 was fused in-frame to thioredoxin gene under the control of inducible T7 promoter to construct expression vector pET-mhCAP-18. The soluble form fusion protein was expressed in E. coli and purified by Chelating Sepharose column chromatography. Thrombin digestion of the fusion protein yielded recombinant GSLL-39, which was then purified by cation-exchange chromatography. Recombinant GSLL-39, which has two extra residues on its N-terminus when compared with its native counterpart, showed similar antimicrobial activities against both Gram-negative and Gram-positive bacteria. [Copyright &y& Elsevier]

The theory of evolution of tumor cell population has been established for nearly 40 years. It was widely accepted for research and clinical anti-tumor treatment. Recently, it was suggested that cancer stem cells are the unit of evolution. Considering recent advances on genesis of tumor and leukemia with ecological and evolutionary views, this article reviews origin and evolution of leukemia stem cells. Over the last few years, clinical and experimental data suggest there are two paths for the origin of leukemia stem cells: from a transformed hematopoietic stem cell or progenitor. The mechanisms of leukemia stem cell formation and clonal evolution were elucidated. Sub-clonal mutations and clonal architectures in leukemia were studied and a mosaic evolution pattern is described. Random evolution or non-inherited mutations of leukemia cells would accelerate the progression of malignant disease. Finally, the mosaic or network mechanism for leukemogenesis is also discussed.

Nucleotides are new players in the intercellular communication network. P2X7 is a member of the P2X family of receptors, which are ATP-gated plasma membrane ion channels with diverse biological functions. Abnormal expression and dysfunction of P2X7 have been reported in leukemias. Here, we report a new P2X7 mutant (an A(559)-to-G substitution causing N187D P2X7) cloned from J6-1 leukemia cells. The characteristics of N187D P2X7 were studied by establishing stably transfected K562 cell lines. Our results show that N187D P2X7 required a higher concentration of agonist for its activation, leading to Ca(2+) influx (EC(50) = 293.3 ± 6.6 μm for the mutant and 93.6 ± 2.2 μm for wild-type P2X7) and ERK phosphorylation, which were not caused by differential cell-surface expression or related to high ATPase activity on the cell surface and in the extracellular space. K562 cells expressing this N187D mutant showed a proliferative advantage and reduced pro-apoptosis effects in vitro and in vivo. Furthermore, elevated angiogenesis and CD206-positive macrophage infiltration were found in tumor tissues formed by K562-M cells. In addition, higher expression of VEGF and MCP1 could be detected in tumor tissues formed by K562-M cells. Our results suggest that N187D P2X7, representing mutants hyposensitive to agonist, might be a positive regulator in the progression of hematopoietic malignancies.

The membrane form of macrophage colony-stimulating factor (mM-CSF) is an alternative splicing variant of this cytokine. Although its high expression was detected in hematopoietic malignancies, its physiologic and pathologic roles in hematopoietic system have not been established. In this report, stable transfectant clones expressing mM-CSF (Namalwa-M and Ramos-M) were obtained, which showed reduced proliferation potential in vitro. Moreover, the in vivo study showed that Namalwa-M and Ramos-M exhibited enhanced oncogenicity in tumor size in nude mice model, which could be inhibited by M-CSF monoclonal antibody. A remarkable increase in infiltrating macrophage and the vessel densities was found in tumor tissues formed by lymphoma cell lines that stably expressed mM-CSF, which suggested the involvement of macrophages in this process. The in vitro results using coculture system showed that macrophages could promote Namalwa-M and Ramos-M proliferation and activate extracellular signal-regulated kinase/mitogen-activated protein kinase signal pathway. In addition, the expression of murine origin vascular endothelial growth factor, basic fibroblast growth factor, and hepatocyte growth factor was elevated in Namalwa-M formed tumor tissues. These results suggested that mM-CSF should be a positive regulator in the development of hematopoietic malignancies by abnormally activating infiltrating macrophages, which in turn promote the malignant development. Thus, mM-CSF may be a critical linker between macrophages and malignant cells in the development of hematopoietic malignancies.

Objective: To clone the entire coding sequence and analyze the function of P2X7 receptor of J6-1 human leukemia cells., Methods: The entire coding sequence of P2X7 receptor was amplified by RT-PCR and then inserted into pTARGET plasmid to construct an eukaryotic expressing plasmid followed by DNA sequencing. HEK293 cells stably expressing P2X7 receptor were obtained after transfection and screening, and confirmed by RT-PCR and Western blotting. The bleb formation upon agonist stimulation was observed under phase contrast microscope., Results: The entire coding sequence of P2X7 receptor of J6-1 cells was successfully cloned. DNA sequencing analysis revealed a substitution of G559, for A559, causing a substitution of Glu187 for Gln187. The P2X7 receptor derived from J6-1 cells could be functionally expressed in HEK293 cells, in which bleb formation could be detected upon stimulation., Conclusions: The entire coding sequence of P2X7 receptors was successfully cloned from J6-1 leukemia cells. Other unknown mechanism may contribute to the dysfunction of P2X7 receptor in these cells.

Murine beta-defensin 2 (MBD2) is a small antimicrobial peptide of the innate immune system. Recent study showed that MBD2 could not only recruit immature dendritic cells but also activate them by Toll-like receptor 4 and thus may provide a critical link between the innate immune system and the adaptive immune response. In this report, we examined the antileukemia activity of MBD2 in a murine model of acute lymphoid leukemia (ALL) L1210. L1210 cells were engineered to secrete biologically functional MBD2. MBD2-modified L1210 (L1210-MBD2) showed significantly reduced leukemogenecity, resulting in a 80% rate of complete leukemia rejection. Inoculation of mice with L1210-MBD2 induced enhanced CTL and natural killer (NK) activity and augmented interleukin-12 and IFN-gamma production. All the recovered mice from the inoculation showed a protective immunity to the following challenge with parental L1210 cells and generate leukemia-specific memory CTL. Vaccines with irradiated L1210-MBD2 cells could cure 50% leukemia-bearing mice. Depletion of CD8+ T cells but not CD4+ T cells completely abrogated the antileukemia activity of MBD2. Interestingly, NK cells were also required for the MBD2-mediated antileukemia response, although ALL generally display a high degree of resistance to NK-mediated lysis. Our results suggest that MBD2 can activate both innate and adaptive immunity to generate potent antileukemia response, and MBD2 immunotherapy warrants further evaluation as a potential treatment for ALL.

Regulation of P2X7 receptor expression is of interest because activation of this receptor by extracellular ATP triggers a wide variety of cell functions in leukocytes. However, its expression and modulation in human peripheral blood mononuclear cells (PBMC) and monocytes remain unclear. RT-PCR was used to detect the constitutive level of P2X7 receptor and the levels upon stimulation with bacteria, bacterial product, mitogen and various cytokines in human PBMC and monocytes. P2X7 receptor mRNA was detected in PBMC and monocytes. P2X7 receptor expression in PBMC was up-regulated by interleukin-2, -4, -6 (IL-2, IL-4, IL-6) tumour necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS) and heat-inactivated Staphylococcus aureus Cowan strain I (SAC). However, interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and phytohemagglutinin-M (PHA-M) had little effect on the expression of P2X7 receptor. Furthermore, LPS and M-CSF could up-regulate P2X7 receptor expression in monocytes, while IFN-gamma, TNF-alpha and GM-CSF had weak effects, but pretreatment with these inducers could not further enhance LPS-stimulated P2X7 receptor expression in monocytes. The results obtained demonstrate that inflammatory stimuli drive P2X7 expression, thus supporting the hypothesis that P2X7 receptor may play a role in the inflammatory responses against bacteria infection, which need further verification.

Objective: To study the expression of gelatinases, including matrix metalloproteinase-9 (MMP-9, gelatinase B) and matrix metalloproteinase-2 (MMP-2, gelatinase A), in CD(34)(+) cells and leukemic cell lines, and explore the significance of gelatinase in migrating and homing capacity of CD(34)(+) cells, as well as the role of gelatinase in leukemia pathogenesis., Methods: CD(34)(+) cells were isolated from umbilical cord blood and normal bone marrow by Mini MACS system. By zymogram analysis, MMP-2 and MMP-9 were detected in the serum free condition medium of CD(34)(+) cells and cell lines., Results: One brilliant band with molecular weight of 92 x 10(3) was detected in condition medium of cord blood CD(34)(+) cells. No band was detected in condition medium of bone marrow CD(34)(+) cells. Brilliant bands with molecular weight of 92 x 10(3) and 72 x 10(3) were detected in the condition medium of U937, KG-1a and HL-60 cell lines, but not in that of HEL, Namalva, CEM, K562 and LCL-H cell lines. In the condition media of J6-1 and J6-2 cells only the 92 x 10(3) band was detected., Conclusions: Cord blood CD(34)(+) cells produced MMP-9, but bone marrow CD(34)(+) cells did not, partly explains the fact that cord blood CD(34)(+) cells possessed higher migrating capacity in comparison with bone marrow CD(34)(+) cells. The expression of MMP-9 and MMP-2 in leukemic cell lines varied.

Background and Objectives: DNA vaccine against macrophage colony-stimulating factor receptor (M-CSFR) has shown both protective and therapeutic effects. In this study, we explore the possibility of using DNA vaccines against both M-CSFR and membrane-bound macrophage colony-stimulating factor (mM-CSF) to achieve better effects., Design and Methods: Three plasmids were constructed by inserting either extracellular and transmembrane region of mM-CSF (pM), or extracellular region of M-CSFR (pR), or extracellular region of M-CSFR linked with extracellular and transmembrane regions of mM-CSF by a (Gly Gly Ser)2 flexible linker (pF), into pcDNA3.1. A SP2/0 cell line stably expressing pF (SP2/0-F) was established to evaluate humoral and cytotoxic immune responses as well as therapeutic and preventive effects induced by pM, pR, pF or pM+pR vaccination in BALB/c mice. The mechanisms of these vaccinations were also studied by monitoring the release of interleukin (IL)-4 and interferon (IFN)-g by splenocytes upon activation., Results: Vaccination against two epitopes had better effects than against a single epitope while vaccination by pM+pR had the greatest effects on inducing humoral and cytotoxic immune responses, prolonging survival of mice challenged with SP2/0-F, and inducing IL-4 and IFN-g release by splenocytes., Interpretation and Conclusions: Our results suggest that co-immunization of M-CSFR and mM-CSF DNA vaccines is better than M-CSFR-mM-CSF fusion DNA vaccine.