Search

Abstract: The role of interleukin 17 (IL-17) to epilepsy-associated cortical tubers of tuberous sclerosis complex (TSC) is unknown. We investigated the expression patterns of the IL-17 and IL-17 receptor (IL-17R) in cortical tubers of TSC compared with normal control cortex (CTX). We found that IL-17 and IL-17R were clearly upregulated in cortical tubers at the protein levels. Immunostaining indicated that IL-17 was specifically distributed in the innate immunity cells (DNs, GCs, astrocytes, and microglia) and adaptive immunity cells (T-lymphocytes) as well as the endothelial cells of blood vessels. The overexpression and distribution patterns of IL-17 may be involved in the epileptogenicity of cortical tubers in TSC. [Copyright &y& Elsevier]

Similar to matrix metalloproteinases (MMP-9/-2), IL-18 was overexpressed in some hematologic malignancies such as acute myeloid leukemia (AML), which is associated with a poor clinical outcome. To establish a possible functional relationship between IL-18 and MMPs in myeloid leukemia, we used semi-quantitative PCR and zymographic analysis to examine whether IL-18 stimulates human myeloid leukemia cell line HL-60 to produce MMPs and/or specific tissue inhibitors (TIMPs), and to degrade extracellular matrix (ECM) gel in vitro. In the ECM invasion assay IL-18 significantly up-regulated transmigration of HL-60 cells, which in turn was inhibited by a synthetic MMP inhibitor: O-phenanthroline (o-PE), anti-MMP-9, anti-MMP-2 as well as anti-IL-18 monoclonal antibody (McAb), respectively, suggesting that induction of gelatinases by IL-18 leads to ECM degradation by these cells. Moreover, IL-18 could significantly increase MMP-9 but not MMP-2 production at both mRNA and/or protein level, slightly up-regulate TIMP-1 mRNA, and clearly induce TIMP-2 mRNA secretion. We postulate that IL-18 may in part play a role in the clinical aggressiveness of human myeloid leukemia by stimulating MMP-9 production. [Copyright &y& Elsevier]

Macrophage colony-stimulating factor (M-CSF) plays important roles in hematopoietic and immunologic systems. Some isoforms or mutations have been demonstrated including membrane-bound and cellular M-CSF, which associated with some leukemia, lymphoma and other solid tumors. We previously reported that the M-CSF-like membrane-associated factor (MAF-J6-1) and its receptor was found from human leukemic cell line J6-1. In this report, the cDNA of MAF-J6-1 and its receptor were cloned. The cDNA sequence of MAF-J6-1 shows a 768 bp open reading frame (ORF) with 99.2% homology to m-M-CSF, but six site mutations, including two synonymous mutations and four missense mutations. The cDNA of MAF-J6-1-R has a 2916 bp ORF shared 99.6% homology with M-CSF-R, but 13 site mutations, including six synonymous mutations and seven missense mutations. At the same time, a 1662 bp mutant s-M-CSF cDNA, which has 10 site mutations including three synonymous mutations and seven missense mutations, was cloned from J6-1 cells. The cDNAs of MAF-J6-1 and MAF-J6-1-R were inserted into a mammalian expression plasmid pTARGET and were expressed in COS-7 cells that demonstrated by their specific MAb. COS-7 cells transfected with MAF-J6-1-R show obvious protein tyrosine kinase (PTK) activity. Our present work shows that MAF-J6-1 and its receptor are mutations of M-CSF and its receptor. [Copyright &y& Elsevier]

Abstract: Tuberous sclerosis complex (TSC) and focal cortical dysplasia type IIb (FCDIIb) are recognized as causes of intractable epilepsy. Transient receptor potential vanilloid receptor 1 (TRPV1), a member of the transient receptor potential family, is the capsaicin receptor and is known to be involved in peripheral nociception. Recent evidence suggested that TRPV1 may be a contributing factor in epileptogenicity. Here, we evaluated the expression of TRPV1 in the cortical lesions of TSC and FCDIIb relative to normal control cortex. TRPV1 was studied in epilepsy surgery cases with TSC (cortical tubers; n =12) and FCDIIb (n =12) using immunocytochemistry, confocal analysis, and Western blotting (WB). Immunohistochemical location of the TRPV1 was predominately detected in the abnormal cell types, such as dysmorphic neurons, balloon cells (BCs) and giant cells. Co-localization assays further revealed that cells expressing TRPV1 mainly had a neuronal lineage, apart from some BCs in FCDIIb, which obviously were of astrocytic lineage. The increased TRPV1 expression within the dysplastic cortex of TSC and FCDIIb was confirmed by WB. Interestingly, both immunohistochemical and WB data indicated that TRPV1 might have both cytoplasm and nuclear distribution, suggesting a potential nuclear role of TRPV1. The over-expression of TRPV1 in cortical lesions of TSC and FCDIIb suggested the possible involvement of TRPV1 in the intrinsic and increased epileptogenicity of malformations of cortical development associated epilepsy diseases and may represent a potential antiepileptogenic target. However, the current data are merely descriptive, and further electrophysiological investigation is needed in the future. [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: The P2X7 nucleotide receptor is an adenosine 5′-triphosphate (ATP)-gated ion channel, which induces cation channel opening imparting significant permeability to Ca2+, and is widely expressed in cells of hematopoietic origin. Our previous report showed that P2X7-mediated calcium response was absent in three Epstein-Barr virus (EBV)-positive and P2X7 positive cell lines. In this report, we detected the cell surface ATPase activity, which contributes to the hydrolysis of extracellular ATP, and the expression of CD39, which is the main source of ATPase on hematopoietic cells, in these cell lines. Then, we tried to restore the P2X7-mediated calcium response in LCL-H and J6-1 cells by either increasing the concentration of agonist or suppressing the ATPase activity by βγMeATP, a synthetic poorly metabolizable ATP analogue. The results showed that LCL-H and J6-1 cells had higher levels of ATPase activity and CD39 expression. The treatment of 300μM βγMeATP efficiently inhibited the ATPase activity on LCL-H and J6-1 cells. Both elevation of agonist concentration (10mM ATP or 1mM BzATP) and pretreatment with 300μM βγMeATP followed by stimulation with normal concentration of agonists (1mM ATP or 0.1mM BzATP) could cause P2X7-mediated calcium response in LCL-H but neither in J6-1 cells. These results suggested that multiple mechanisms contributed to the loss of the P2X7-mediated calcium response. CD39-associated high ATPase activity contributed to the loss of the P2X7-mediated calcium response in LCL-H cells, while additional mechanism(s) existed in J6-1 cells. [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 P2X7 nucleotide receptor is an adenosine 5′-triphosphate (ATP) -gated ion channel, which is widely expressed in cells of hematopoietic origin and functions as a non-selective cation channel permeable to Na+, Ca2+, etc upon stimulation. Here, we investigated P2X7 expression in 11 human hematopoietic cell lines, representing different lineages, as well as bone marrow mononuclear cells (BMMC) samples from 87 leukemia and 10 myelodysplastic syndrome (MDS) patients. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry results showed that both P2X7 mRNA and protein were detected in eight cell lines with a non-lineage-specific manner. Samples from 69 leukemia and 9 MDS patients were P2X7 positive at mRNA level. Moreover, both positive rates and relative expression levels were significantly higher in acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), and MDS groups than that in normal donor group. The expression levels varied among AML subtypes with higher levels being observed in M4, M5, and M6 groups but not in M1 or M2 group. Furthermore, after one course of standard induction therapies, the remission rate in high P2X7 expression group was lower than that in either P2X7 negative group or low P2X7 expression group. Cytoplasmic free calcium increase was detected in five of eight P2X7+ cell lines as well as P2X7+ normal donor and patient samples tested, but not in three Epstein–Barr virus (EBV) positive cell lines (J6-1, Namalwa, and LCL-H) in Locke’s solution upon stimulation by extracellular ATP or the more potent and specific agonist, 2′,3′-O-(4-benzoyl)benzoyl-ATP (BzATP). The possible mechanisms causing the loss of P2X7 function were discussed. [Copyright &y& Elsevier]

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]

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]

LL-37/hCAP-18 is an important part of host defense. Several diseases in human are characterized by impairment in the function of LL-37/hCAP-18 peptide. We examined the expression of LL-37/hCAP-18 in a panel of hematopoietic cell lines representing multiple cell lineages. LL-37/hCAP-18 expression at mRNA level was detected and varied among six of nine cell lines. The level of Raji cells was about eight folds higher than that of Ramos cells. However, only two cell lines, J6-1 and U937, expressed protein products. We also investigated LL-37/hCAP-18 protein in nine leukemia and three idiopathic thrombocytopenic purpura (ITP) patients via immunocytochemical staining. The rate of LL-37/hCAP-18 positive cells ranged from 60 (ITP) to 0.5% (M5). These data suggested that the low translation efficiency of LL-37/hCAP-18 expresses in some leukemia cells might be one of the reasons that leukemia patients were susceptible to infection. [Copyright &y& Elsevier]

The importance of IL-18, although clearly established in solid tumors, has not been fully elucidated in human hematopoietic neoplasms. Here we examined the mRNA and protein for IL-18 in eight human hematopoietic cell lines representing different lineages and neoplasms including leukemia, lymphoma and others. Our results revealed that IL-18 mRNA was expressed in these cells and that the corresponding protein was found in the cytoplasm. Seven of eight cell lines were also found to express two subunits of the IL-18 receptor (IL-18R) at varied levels. Furthermore, 29 out of 51 leukemia patients tested were observed to express IL-18R with 18/29 (62%) co-expression of both receptor and ligand. By blocking the IL-18 loop using specific antisense oligodeoxynucleotide (ASON) for IL-18 mRNA or anti-human IL-18R monoclonal antibody (McAbR), we were not able to demonstrate a marked inhibition on the most leukemic cell lines growth. Moreover, the potential proliferation in vitro of primary AML cells co-expressing IL-18 and its receptor was not significantly enhanced by recombinant human IL-18, suggesting that IL-18 is not apparently implicated in the proliferation of the leukemia cells via an autocrine loop. Additionally, we also found the effective modulating effect of M-CSF, IFN-α and TNF-α on IL-18R expression, implying an important in vivo effect of cytokines on IL-18-induced reaction. Moreover, the modulation of IL-18R expression was possibly irrelevant to IFN-γ secretion induced by these cytokines. [Copyright &y& Elsevier]

Little is known about the clinical significance of interleukin (IL)-18, a novel immunoregulatory cytokine, in acute myeloid leukemia (AML). Using reverse transcriptase polymerase chain reaction (RT-PCR) analysis, levels of IL-18 mRNA were assessed in bone marrow mononuclear cells (BMMC) from 47 adult patients with de novo or CR AML in order to explore the clinical significance of IL-18. The relationship between expression levels and the established prognostic factors such as age, cytogenetic aberrations, CD34 expression and FAB subtypes was investigated. Either disease status, age or CD34 expression were found to significantly correlate with the expression of IL-18. With respect to FAB cytotypes, expression of IL-18 gene in M4/M5 (n=15) was statistically higher than that in other subtypes (n=32, P<0.001). Moreover, a significant difference in IL-18 gene expression was obtained between the high risk group and the intermediate risk group (0.5627 versus 0.3111, P=0.038). In addition, a relationship between IL-18 expression of BMMC and initial white blood cell (WBC) was clearly demonstrated by a statistical analysis (r=0.806, P<0.001). These observations suggest that IL-18 gene over-expression might reflect the convergence of several important unfavorable prognostic factors in AML. [Copyright &y& Elsevier]