Disappearance of perihepatic lymph node enlargement after hepatitis C viral eradication with direct-acting antivirals.

Perihepatic lymph node enlargement (PLNE) which has been shown to be negatively associated with hepatocellular carcinoma (HCC) occurrence is frequently observed in chronic liver disease; however, changes in the state of perihepatic lymph nodes after eradication of hepatitis C virus (HCV) have not been investigated yet. We aimed to evaluate this issue. We enrolled 472 patients with chronic HCV infection who achieved viral eradication with direct-acting antivirals (DAA). We investigated whether the status of perihepatic lymph nodes changed before and after HCV eradication (primary endpoint). We also evaluated the association between PLNE and clinical findings such as liver fibrosis or hepatocellular injury before HCV eradication (secondary endpoint). Perihepatic lymph node enlargement was detected in 164 of 472 (34.7%) patients before DAA treatment. Surprisingly, disappearance of PLNE was observed in 23.8% (39 patients) of all PLNE-positive patients after eradication of HCV. Disappearance of PLNE was not associated with baseline clinical parameters or changing rates of clinical findings before and after DAA treatment. At baseline, presence of PLNE was significantly associated with a lower serum HCV-RNA level (P = .03), a higher serum AST level (P = .004) and a higher ALT level (P < .001) after adjustment for sex and age. In conclusion, PLNEs became undetectable after DAA treatment in 23.8% of PLNE-positive patients. Further study with a longer follow-up period is needed to clarify the clinical importance of this phenomenon especially in relationship with the risk of HCC development.

Nerve growth factor derived from bronchial epithelium after chronic mite antigen exposure contributes to airway hyperresponsiveness by inducing hyperinnervation, and is inhibited by in vivo siRNA.

BACKGROUND: Bronchial asthma is a chronic allergic airway inflammatory disease. Neurotrophins, including nerve growth factor (NGF), play an important role in the pathogenesis of asthma. However, the effects of NGF derived from epithelium on airway hyperresponsiveness (AHR) after antigen sensitization/exposure remain uncertain. OBJECTIVE: In this study, we examined the role of NGF on AHR after chronic antigen exposure and the effect of inhibiting NGF by in vivo siRNA on AHR exacerbation. METHODS: We generated chronic mouse models of bronchial asthma using house-dust mite antigen (Dermatophagoides pteronyssinus; Dp). NGF concentrations in bronchoalveolar lavage fluid (BALF), lung histopathology, hyperresponsiveness, and related neuronal peptides and cytokines in supernatants of lung homogenates were determined. RESULTS: NGF in BALF was increased in a dose- and time-dependent manner, and was expressed primarily in bronchial epithelium. Nerve fibres and substance P-positive fibres were detected in subepithelium of Dp-sensitized and challenged mice over 4 weeks of mite antigen exposure. AHR was positively correlated with NGF concentration and nerve fibre innervation. AHR, modulation of innervation, and increased substance P were inhibited by in vivo administration of siRNA that targeted NGF, although the inhibition of NGF did not affect allergic inflammation and subepithelial fibrosis. CONCLUSION AND CLINICAL RELEVANCE: These findings suggest that NGF derived from bronchial and alveolar epithelium plays an important role in AHR after chronic exposure to mite antigen. NGF inhibition could potentially manage bronchial asthma, including AHR.

Blockade of Th1 chemokine receptors ameliorates pulmonary granulomatosis in mice.

Sarcoidosis is a granulomatous disease of unknown aetiology. We identified immunological targets for the treatment of pulmonary granulomatosis using a murine model generated with Propionibacterium acnes. Sensitisation and challenge using heat-killed P. acnes and dendritic cells (DCs) were performed to produce pulmonary granulomatosis in C57BL/6 mice. Immunological analyses using ELISA as well as cDNA microarray analysis were used to search for cytokines or chemokines associated with the formation of granulomas in the lungs. Co-administration of P. acnes and DCs reproducibly induced the formation of pulmonary granulomas, which resembled sarcoid granulomas. The cDNA microarray assay demonstrated that the gene expression of CXCL9 and CXCL10, ligands for CXCR3, and of CCL4, a ligand for CCR5, was strongly upregulated during granulomatosis. ELISA confirmed that levels of CXCL9 and CXCL10 as well as T-helper (Th)1 cytokines and chemokines including tumour necrosis factor-α and interferon-γ were elevated in bronchoalveolar lavage fluid (BALF). The blockade of Th1 chemokine receptors using TAK-779, a dual blocker for CXCR3 and CCR5, led to reduced numbers of CXCR3+CD4+ and CCR5+CD4+ T-cells in BALF. Furthermore, administration of TAK-779 ameliorated the granulomatosis. The targeted inhibition of Th1 chemokines might be useful for inhibiting Th1-biased granulomatous diseases, including sarcoidosis.

IκB kinase ß inhibitor IMD-0354 suppresses airway remodelling in a Dermatophagoides pteronyssinus-sensitized mouse model of chronic asthma.

BACKGROUND: Nuclear factor (NF)-κB is a transcription factor that regulates cytokine and chemokine production in various inflammatory diseases, including bronchial asthma. IκB kinase (IKK) ß is important for NF-κB activation in inflammatory conditions, and is possibly related to airway remodelling. Thus, inhibition of the IKKß-NF-κB pathway may be an ideal strategy for the management of airway remodelling. OBJECTIVE: We examined the effects of a newly synthesized IKKß inhibitor, IMD-0354, in a chronic allergen exposure model of bronchial asthma in mice. METHODS: A chronic mouse model was generated by challenge with house dust mite antigen (Dermatophagoides pteronyssinus). IMD-0354 was administrated intraperitoneally in therapeutic groups. Lung histopathology, hyperresponsiveness and the concentrations of mediators and molecules in supernatants of lung homogenates were determined. RESULTS: NF-κB activation was inhibited by prolonged periods of IMD-0354 administration. IMD-0354 reduced the numbers of bronchial eosinophils. IMD-0354 also inhibited the pathological features of airway remodelling, including goblet cell hyperplasia, subepithelial fibrosis, collagen deposition and smooth muscle hypertrophy. Inhibition of these structural changes by IMD-0354 was the result of the suppressing the production and activation of remodelling-related mediators, such as TGF-ß, via inhibition of IKKß. IMD-0354 inhibited IL-13 and IL-1ß production, and it restored the production of IFN-γ. It also ameliorated airway hyperresponsiveness. CONCLUSION: IKKß plays crucial roles in airway inflammation and remodelling in a chronic mouse model of asthma. A specific IKKß inhibitor, IMD-0354, may be therapeutically beneficial for treating airway inflammation and remodelling in chronic asthma.

Protein biomarker analysis by mass spectrometry in patients with rheumatoid arthritis receiving anti-tumor necrosis factor-alpha antibody therapy.

OBJECTIVE: To investigate the mechanism of action of anti-tumor necrosis factor-alpha (TNF-alpha) antibody in patients with rheumatoid arthritis (RA), we analyzed serum or plasma proteins by mass spectrometry system. METHODS: Ten RA patients who received treatment with anti-TNF-alpha antibody were studied. Samples obtained before and after therapy were analyzed by a two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) system after pretreatment by a recently developed method to remove high molecular weight proteins. RESULTS: Using this system, certain proteins were identified after treatment with anti-TNF-alpha antibody, including proteins related to the TNF-alpha-mediated pathway for nuclear factor kappa B (NF-kappaB) activation and/or to the metabolism (including regeneration) of articular cartilage. CONCLUSION: Our mass spectrometry system appears to be useful for proteomic analysis. The efficacy of anti-TNF-alpha antibody therapy for RA may be related to various consequence of the inhibition of TNF-alpha activity.

Differential effects of recombinant interferons alpha, beta, and gamma on induction of human lymphokine (IL-2)-activated killer activity.

The effects of three classes of recombinant interferons (IFN-alpha A, IFN-beta, and IFN-gamma) on maximal induction of lymphokine (IL-2)-activated killer (LAK) activity were studied. Highly purified lymphocytes (greater than 99%) were obtained by counter-flow centrifugal elutriation from peripheral blood of healthy donors. After incubation for 4 days with IL-2 (1 U/ml), purified lymphocytes showed maximal LAK activity against NK cell-resistant target (Daudi) cells, as assessed by 4-hour 51Cr release assay. Addition of exogenous IFN-alpha A or IFN-beta to cultures of lymphocytes plus IL-2 resulted in significant inhibition of LAK activity, but addition of IFN-gamma had no effect on LAK induction by IL-2. IFN-alpha A caused greatest inhibition of LAK activity when added at the start of culture of lymphocytes with IL-2, and was less inhibitory when added 1 day later. Similar inhibition by IFN-alpha A or IFN-beta was observed with nine lines of human tumorigenic cells as targets of LAK activity. IFN-alpha A and IFN-beta also inhibited the proliferative responses of lymphocytes to IL-2 stimulation, and the expression of IL-2 receptors on their surface, whereas IFN-gamma did not. These results suggest that IFN-alpha A and IFN-beta may be important in in situ regulation of LAK cell induction against neoplasms.

Kinetics and function of tumor cytotoxic factor(s) produced by human blood monocytes activated to the tumoricidal state.

Human monocytes obtained from healthy volunteers and isolated by centrifugal elutriation were not cytotoxic to allogeneic tumorigenic cells. These freshly isolated monocytes were rendered tumoricidal following interaction in vitro for 24 hours with greater than 0.01 micrograms lipopolysaccharide (LPS)/ml or over 1 microgram nor-muramyl dipeptide/ml. Monocytes activated by this procedure produced a soluble factor that lysed tumor cells. Full expression of tumor cell lysis required a minimum of 18 hours' exposure of tumor cells to the factor. The degree of tumor cytotoxic factor (TCF) production was closely related to the intensity of monocyte activation to become tumoricidal. Significant production of TCF by monocytes was detected in the supernatants after treatment for 3 hours with LPS. TCF was also released by activated monocytes when cocultivated with tumorigenic cells. Similarly, the level of TCF production correlated with the monocyte density. TCF destroyed human allogeneic tumor cell lines (melanoma, glioblastoma, colon carcinoma, prostatic carcinoma, and breast carcinoma), but it did not affect nontumorigenic cell lines (lung and skin fibroblasts). TCF activity was not blocked by superoxide dismutase, catalase, or protease inhibitors; it was destroyed by being heated at 100 degrees C for 2 minutes. The ability of activated monocytes to release TCF could enhance host defense against cancer.

Lack of production of interleukin 1 by human blood monocytes activated to the antitumor state by liposome-encapsulated muramyl tripeptide.

Previously human blood monocytes were shown to become tumoricidal when treated in vitro with lipopolysaccharide, muramyl dipeptide analogue, or liposomes containing muramyl dipeptide analogue. In this study the ability of human blood monocytes activated to the antitumor state by these macrophage activators to produce interleukin 1 (IL-1) was examined. Blood monocytes separated by centrifugal elutriation did not release IL-1 into the culture supernatant but elaborated IL-1 maximally within 24 h after treatment with lipopolysaccharide or desmethyl muramyl dipeptide. In contrast, they did not elaborate IL-1 when rendered tumoricidal by muramyl tripeptide phosphatidylethanolamine (MTP-PE) encapsulated in multilamellar vesicle liposomes composed of phosphatidylcholine and phosphatidylserine in a molar ratio of 7:3. IL-1 rich supernatants that induced thymocyte proliferation were not adsorbed or destroyed by the liposomes, and addition of supernatants from cultures of monocytes treated with liposome-MTP-PE to IL-1 rich supernatants did not inhibit thymocyte proliferation. MTP-PE in liposomes composed of phosphatidylserine or phosphatidylcholine or both in various molar ratios also did not induce IL-1 production by monocytes. These results indicate that MTP-PE encapsulated in liposomes may be useful in in situ activation of human blood monocytes to the antitumor state for destruction of clinical micrometastases because MTP-PE encapsulated in liposomes does not stimulate production of IL-1, which is responsible for undesirable side effects such as fever and granulomatous reactions.

Augmentation of 1-beta-D-arabinofuranosylcytosine resistance in human KB epidermoid carcinoma cells upon induction of a second resistance to vincristine.

Human KB epidermoid carcinoma cells (KB/p) and their variant cells (KB/ara-C) resistant to 1-beta-D-arabinofuranosylcytosine (ara-C) that were not cross-resistant to vincristine (VCR) were exposed to increasing concentrations of VCR to establish VCR resistant cells (KB/VCR) and doubly resistant variant cells (KB/ara-C/VCR). KB/VCR and KB/ara-C/VCR cells showed a similar increase in resistance to VCR (about 337- to 460-fold) over that of the parent KB/p cells. KB/ara-C showed a 911-fold increase in resistance to ara-C, while KB/ara-C/VCR showed a 60,837-fold increase in resistance to ara-C compared with the parent cells. The concentration of ara-C required for 50% inhibition of KB/ara-C/VCR cells was much higher (67-fold) than that for KB/ara-C cells, although the concentration of ara-C required for 50% inhibition of KB/VCR cells was 12-fold that for KB/p cells. Thus the acquisition of resistance to a second drug by these human KB cells augmented their resistance to the first drug. Induction of resistance to ara-C or VCR resulted in marked decrease in cellular uptake of the respective drugs, but there was no difference in cellular uptakes of ara-C by KB/ara-C and KB/ara-C/VCR or of VCR by KB/VCR and KB/ara-C/VCR. KB/VCR and KB/ara-C/VCR cells were cross-resistant to vinblastine, colchicine, vindesine, etoposide, mitomycin C, and Adriamycin. Cells of the two groups (KB/p and KB/ara-C; KB/VCR and KB/ara-C/VCR) differed in sizes and doubling times. In the absence of ara-C, the resistance of KB/ara-C cells to ara-C was stable for at least 1 year, whereas in the absence of VCR resistance to VCR was almost completely lost within 1 month.

Activation of rat alveolar macrophages to the tumoricidal state in the presence of progressively growing pulmonary metastases.

The purpose of this study was to determine whether the presence of progressively growing pulmonary metastases influences the number and function of alveolar macrophages (AM). Female F344 rats were given i.v. injections of cells from a metastatic variant line of the syngeneic adenocarcinoma MADB-105. At Days 7, 14, 21, and 28 after injection, normal and tumor-bearing animals (3/group) were killed, and their AM were harvested by lavage. The functional integrity of AM was determined by their capacity to phagocytose opsonized erythrocytes and by their ability to respond to a variety of activating agents in vitro. Normal and metastasis-bearing rats were given i.v. injections of Nocardia rubra cell wall skeleton to determine whether the presence of large pulmonary metastases would interfere with AM activation in situ. The data demonstrated that the presence of progressively growing lung metastases led to a slight increase in the number of harvested AM and that these cells from tumor-bearing rats were as phagocytic as AM obtained from normal rats. Also, AM harvested from rats with metastases became tumoricidal against syngeneic tumor target cells in response to activation stimuli in vitro and in vivo. We conclude that, in this tumor system, the presence of progressively growing lung metastases doses not suppress the number or function of AM. The use of agents known to activate AM could, therefore, be of benefit in the therapy of pulmonary metastases.

Membrane-associated interleukin 1 alpha as a mediator of tumor cell killing by human blood monocytes fixed with paraformaldehyde.

Human blood monocytes isolated by centrifugal elutriation from healthy donors were tested for ability to produce membrane-associated antitumor monokine(s) in response to activation stimuli such as various types of interferon (IFN) and/or synthetic desmethyl muramyl dipeptide (norMDP). IFNs (alpha, beta, and gamma) and norMDP rendered blood monocytes cytotoxic to allogeneic A375 melanoma cells, as assayed by measuring release of [125I]iododeoxyuridine in 72 h. When monocytes were treated with any type of IFN for 16 h, and then fixed with paraformaldehyde, they did not show cytotoxicity to A375 cells, but when they were fixed after treatment with norMDP or lipopolysaccharide they showed significant cytotoxicity to A375 melanoma cells. This membrane-associated antitumor monokine induced by the synergistic actions of suboptimal concentrations of IFN-gamma and norMDP, was cytotoxic to HT-29 colon cancer cells as well as A375 melanoma cells, but not to actinomycin D-treated L-929 cells. The fixed monocyte-mediated cytotoxicity against A375 melanoma cells was completely inhibited by a specific anti-interleukin 1 alpha antiserum, but not by a specific anti-interleukin 1 beta antiserum or monoclonal anti-TNF antibody. These results suggest that membrane-associated interleukin 1 alpha is involved through cell-to-cell contact in the host defense mechanism against cancer.

Macrophage colony-stimulating factor gene transduction into human lung cancer cells differentially regulates metastasis formations in various organ microenvironments of natural killer cell-depleted SCID mice.

We investigated whether local production of macrophage colony-stimulating factor (M-CSF), responsible for migration and activation of monocytes/macrophages at a tumor growth site, affected the metastatic pattern of lung cancer. For this, highly metastatic human squamous (RERF-LC-AI) or small (H69/VP) cell lung carcinoma cells were transduced with the human M-CSF gene inserted into pRc/CMV-MCSF to establish M-CSF-producing clones (MCSF-AI-9-18, MCSF-AI-9-24, and MCSF-VP-5). M-CSF gene transduction had no effect on the expression of surface antigen or on in vitro proliferation. After s.c. injection into SCID mice, the growth rates of M-CSF-producing cells were slower than those of parent or mock-transduced cells. In the metastatic model in SCID mice depleted of natural killer cells, RERF-LC-AI cells formed metastases mainly in the liver and kidneys, whereas H69/VP cells metastasized mainly to the liver and systemic lymph nodes. The numbers of metastatic colonies of MCSF-AI-9-18 and MCSF-AI-9-24 cells in the liver but not the kidneys were significantly reduced. The development of lymph node metastases of MCSF-VP-5 cells was also less than that of parent or mock-transduced cells. Treatment of SCID mice with anti-human M-CSF antibody resulted in a significant increase in liver metastases of their M-CSF gene transfectants. No significant differences were observed in the distributions in mice or in the in vitro invasive potentials of MCSF-AI-9-18 cells and Neo-AI-3 cells. These findings indicate that the antimetastatic effect of M-CSF may be specific to particular organs, suggesting the influence of heterogeneity of organ microenvironments on the metastasis of lung cancer.

Interferon gamma-dependent induction of thymidine phosphorylase/platelet-derived endothelial growth factor through gamma-activated sequence-like element in human macrophages.

Thymidine phosphorylase (TP), an enzyme involved in the reversible conversion of thymidine to thymine, is identical to platelet-derived endothelial cell growth factor. TP expression in cancer cells and/or infiltrated macrophages is associated with microvessel density and poor clinical prognosis in patients with various tumor types. However, how TP expression is up-regulated in human tumors is unclear. Of various inflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha), interleukin 1 alpha (IL-1alpha), and interferon gamma (IFN-gamma), we observed that IFN-gamma most effectively increased the expression of TP in cultured human monocytic U937 cells. Transient transfection of the various deletion constructs of the TP promoter showed that the presence of the -474 to -355 sequence containing gamma-activated sequence-like element was essential for IFN-gamma-dependent activation of the TP gene. Furthermore, the IFN-gamma-dependent transcriptional activity of the promoter construct containing mutations in the gamma-activated sequence-like element was significantly decreased. An electrophoretic mobility shift assay showed that IFN-gamma increased signal transducers and activators of transcription 1 binding to gamma-activated sequence-like element in the TP promoter. IFN-gamma could be a mediator of TP expression in infiltrated monocyte/macrophages, and those monocyte/macrophages expressing TP might play an important role in malignancy and angiogenesis in various human tumors.

Down-regulation by interleukin 4 of activation of human alveolar macrophages to the tumoricidal state.

The effect of recombinant human interleukin 4 (IL-4) on the expression of antitumor activity of human alveolar macrophages (AM) obtained by bronchoalveolar lavage from healthy donors was examined. AM were incubated for 16 h in medium with various macrophage activators [lipopolysaccharide, des-methyl muramyldipeptide, Nocardia rubra cell wall skeleton, and heptanoyl-gamma-D-Glu-(L)-meso-alpha,epsilon-A2pm(L)-D-Al aOH] in the presence or absence of IL-4, and then their tumoricidal activity was assayed by measuring 125I-UdR release from human melanoma (A375) cells. The spontaneous tumoricidal activity of AM was slightly suppressed by IL-4 in 3 of 7 donors. Addition of IL-4 to cultures of AM with the activators resulted in dose-dependent suppression of AM-mediated cytotoxicity against A375 cells. IL-4 also inhibited AM-mediated cytotoxicity against A375-R cells, which are resistant to interleukin 1 (IL-1) and tumor necrosis factor alpha, HT-29 colon cancer cells, and KB cells. IL-4 inhibited the early induction phase of AM activation. Pretreatment of AM with IL-4 also suppressed their expression of antitumor activity in response to lipopolysaccharide. IL-4 inhibited the production of monokines (IL-1 and tumor necrosis factor alpha) by AM at the protein and mRNA levels. These findings suggest that IL-4 may be important in vivo in the down-regulation of antitumor expression of AM in the lung by inhibiting the production of monokines and other killing mechanisms.

Production of a tumor cytolytic factor(s) by activated human alveolar macrophages and its action.

When human alveolar macrophages (AM) lavaged from healthy donors were incubated in medium with or without lipopolysaccharide (LPS) or muramyl dipeptide, they released a factor(s) responsible for tumor cell killing. The activity of the tumor cytolytic factor(s), called TCF, was determined by radioactive release assay. Human AM released variable amounts of TCF into the culture medium without any stimulation, but the release was stimulated significantly by LPS (0.1 micrograms/ml) or muramyl dipeptide (1 micrograms/ml). Maximal production of TCF by the AM was detected in the supernatant after treatment for 3 hr with LPS, and the extent of TCF release correlated with the density of AM. In cultures with LPS, the ability of activated AM to secrete TCF was maintained for 48 hr but was lost by 96 hr. After its loss, the ability to produce TCF could be restored by a second treatment with LPS. Full expression of lysis by TCF to lyse tumor cells required its interaction with tumor cells for at least 24 hr. TCF destroyed human allogeneic tumor cell lines but did not affect nonneoplastic cell lines. TCF activity was resistant to treatment with protease inhibitors, superoxide dismutase, or catalase and to heating at 70 degrees for 1 hr, but it was labile on heating at 100 degrees for 10 min. The tumoricidal activity in the supernatant of activated human AM indicates a potential effector mechanism by which AM kill neoplastic cells.

In vitro generation of tumoricidal properties in human alveolar macrophages following interaction with endotoxin.

Human alveolar macrophages (AM) obtained by bronchoalveolar lavage from healthy nonsmoking donors exhibited primarily low levels of cytolytic activity against allogeneic tumor target cells. These AM acquired enhanced capacity to kill tumor cells following a 24-hr incubation in vitro with endotoxin [lipopolysaccharide (LPS)]. Maximal tumoricidal activity of LPS-activated AM as measured by lysis of tumor target cells was obtained after incubation with tumor cells for 72 hr. LPS-activated AM lysed allogeneic tumor cell lines of different origins but did not affect normal, nonneoplastic cells. We conclude that LPS induces human AM to become tumoricidal. This method should be useful in studies on therapeutic agents enhancing AM-mediated cytotoxicity in situ.

Natural killer cell-dependent suppression of systemic spread of human lung adenocarcinoma cells by monocyte chemoattractant protein-1 gene transfection in severe combined immunodeficient mice.

Monocyte chemoattractant protein-1 (MCP-1) is a chemokine with various biological activities, including augmentation of cytotoxic activity of monocytes and natural killer (NK) cells. The present study was undertaken to determine whether transfection of the MCP-1 gene into lung cancer cells affected their tumorigenicity and metastatic potential by the NK cell-mediated mechanism. The human MCP-1 gene inserted into an expression vector (BCMGSNeo) was transfected into human lung adenocarcinoma (PC-14) cells. There was no difference in in vitro proliferation between MCP-1 gene-transfected PC-14 cells and the parent cells or mock-transfected cells. The tumorigenicity and in vivo tumor growth of MCP-1 gene-transfected PC-14 cells were similar to those of the parent cells or mock-transfected cells when tumor cells were injected into the s.c. space of NK cell-intact severe combined immunodeficient (SCID) mice. Although parent cells and mock-transfected cells inoculated i.v. formed lung metastatic colonies and pleural effusion, MCP-1 gene transfectants reduced the systemic spread in NK cell-intact SCID mice. Interestingly, these modulations in a systemic spread by MCP-1 gene transfection were not observed in NK cell-depleted SCID mice. Decreased survival of MCP-1 gene transfectants in the lung was observed in NK cell-intact SCID mice but not in NK cell-depleted SCID mice. Recombinant MCP-1 or the supernatant of MCP-1 gene transfectants enhanced the cytotoxicity of human CD56+ NK cells and spleen cells of SCID mice against PC-14 cells. These findings suggest that locally produced MCP-1 suppresses tumor progression by a NK cell-mediated mechanism, depending on organ microenvironment.

Nuclear factor-kappaB-dependent expression of metastasis suppressor KAI1/CD82 gene in lung cancer cell lines expressing mutant p53.

KAI1/CD82 has been shown to be a metastasis suppressor for several human cancers, and a recent study revealed that wild-type tumor suppressor p53 can directly activate KAI1/CD82 gene expression. However, the response of KAI1/CD82 expression in cancer cells to exogenous stimulants has not been investigated. The present study examined whether tumor necrosis factor (TNF), which mediates many of the cellular responses associated with inflammatory reactions or cancer progression, can affect the KAI1/CD82 expression in lung cancer cells and, if so, whether nuclear factor (NF)-kappaB, a key molecule in TNF-mediated gene expression, is involved in the mechanism of KAI1/CD82 induction. Our results demonstrated that expression of KAI1/CD82 in PC-14 cells expressing mutant p53 could be augmented by TNF-alpha, and that transfer of the gene for a specific inhibitor of NF-kappaB, IkappaB alphaSR (mutant IkappaB alpha; NF-kappaB super-repressor), into PC-14 cells could inhibit this augmentation. The amount of NF-kappaB in the nucleus of PC-14/IkappaB alphaSR cells correlated well with KAI1/CD82 mRNA and protein expression. In addition, IkappaB alphaSR gene transfer inhibited the spontaneous expression of KAI1/CD82 protein in KAI1/CD82-high-expressing RERF-LC-OK cells, which contain a mutant-type p53. These observations indicate that NF-kappaB activation may play a role in the regulation of KAI1/CD82 expression in lung cancer cells independently of wild-type p53, and suggest that KAI1/CD82 expression may be regulated by interaction with the host microenvironment.

Synergism between human recombinant gamma-interferon and muramyl dipeptide encapsulated in liposomes for activation of antitumor properties in human blood monocytes.

Highly purified human blood monocytes, isolated by centrifugal elutriation under endotoxin-free conditions, were activated in vitro by combining subthreshold amounts of human recombinant gamma-interferon (r-IFN-gamma) and muramyl dipeptide (MDP) to become tumor cytotoxic against allogeneic A375 melanoma cells. Only intact r-IFN-gamma and MDP produced synergism for human monocyte activation. Neither pH 2-treated r-IFN-gamma and intact MDP nor heat-treated IFN-gamma and intact MDP, nor intact IFN-gamma and the biologically inactive stereoisomer of MDP, N-acetylmuramyl-D-alanyl-D-isoglutamine, produced activation of blood monocytes. The encapsulation of intact r-IFN-gamma and MDP within the same preparation of multilamellar liposomes was synergistic for monocyte activation. These data show that synergism for monocyte activation can be produced by human r-IFN-gamma and MDP produced synthetically can be simultaneously delivered to monocytes. Because both r-IFN-gamma and MDP can now be produced in large standardized quantities their synergism for activation of tumoricidal properties in human monocytes could be of clinical significance.