Interactions of interleukin-12A and interleukin-12B polymorphisms on the risk of intracranial aneurysm

Several lines of evidence indicate that inflammatory processes play pivotal role in the development of intracranial aneurysm (IA). Recently, polymorphisms in the interleukin-12 (IL-12) gene were shown to be associated with immune-mediated inflammatory disease. The aim of this study was to investigate the interactions of IL-12A and IL-12B polymorphisms on the risk of IA in a Chinese population. A total of 422 individuals (including 164 patients with IA and 258 controls) were involved in the study. The polymorphisms (i.e., rs2243115 and rs568408 in IL-12A and rs3212227 in IL-12B) were genotyped by polymerase chain reaction–restriction fragment length polymorphism assay and DNA sequencing. We found an association of the AC/CC genotypes and C allele of IL-12B rs3212227 with an increased risk of IA, compared with the AA genotype and A allele (AC/CC vs. AA: OR?=?2.09, 95?% CI: 1.29–3.38; C vs. A: OR?=?1.45, 95?% CI: 1.10–1.91). Moreover, a significant gene interaction of IL-12A and IL-12B was evident on the risk of IA, and subjects carrying variant genotypes of IL-12B rs3212227 had an increased risk of IA. In the stratified analysis by gender, the IL-12B rs3212227 AC/CC genotypes had an increased risk of IA compared with the AA genotype in male patients (AC/CC vs. AA: OR?=?4.63, 95?% CI: 1.92–11.16). These findings suggest that the IL-12A and IL-12B independently and jointly be involved in the susceptibility to IA.     

Activation of the murine interleukin-12 p40 promoter by functional interactions between NFAT and ICSBP

Interleukin (IL)-12 is a heterodimeric cytokine that is critical for the development of a T-helper-1 immune response and immunity against intracellular pathogens. The IL-12 p40 gene product, expressed specifically in macrophages and dendritic cells, heterodimerizes with p35 to form bioactive IL-12, and heterodimerizes with p19 to comprise the cytokine IL-23. Regulation of the murine IL-12 p40 promoter is complex. Multiple cis-acting elements have been characterized that are involved in activation by bacterial products. However, molecular mechanisms through which interferon (IFN)-gamma and bacterial products synergistically activate IL-12 p40 gene expression are less clear. In this study, a composite NFAT/ICSBP binding site at -68 to -54 is identified that is functionally important for p40 promoter activation by lipopolysaccharide (LPS) and LPS plus IFN-gamma. DNA binding of NFAT and ICSBP is demonstrated on the endogenous promoter by chromatin immunoprecipitation. NFAT is required for ICSBP binding to this region. Overexpression of NFAT and ICSBP synergistically activates the p40 promoter. A dominant negative NFAT molecule attenuates LPS- and IFN-gamma-activated endogenous IL-12 p40 mRNA expression. A physical association between NFAT and ICSBP in the absence of DNA is detected by co-immunoprecipitation of endogenous proteins. Three NFAT domains are required for ICSBP interaction. Finally, in LPS- and IFN-gamma-activated RAW-264.7 cells, the association between NFAT and ICSBP is abrogated by IL-10 priming.     

CXCL12 mediates trophic interactions between endothelial and tumor cells in glioblastoma

Emerging evidence suggests endothelial cells (EC) play a critical role in promoting Glioblastoma multiforme (GBM) cell proliferation and resistance to therapy. The molecular basis for GBM-EC interactions is incompletely understood. We hypothesized that the chemokine CXCL12 and its receptor CXCR4 could mediate direct interactions between GBM cells and tumor-associated endothelial cells and that disruption of this interaction might be the molecular basis for the anti-tumor effects of CXCR4 antagonists. We investigated this possibility in vivo and in an in vitro co-culture model that incorporated extracellular matrix, primary human brain microvascular ECs (HBMECs) and either an established GBM cell line or primary GBM specimens. Depletion of CXCR4 in U87 GBM cells blocked their growth as intracranial xenografts indicating that tumor cell CXCR4 is required for tumor growth in vivo. In vitro, co-culture of either U87 cells or primary GBM cells with HBMECs resulted in their co-localization and enhanced GBM cell growth. Genetic manipulation of CXCL12 expression and pharmacological inhibition of its receptors CXCR4 and CXCR7 revealed that the localizing and trophic effects of endothelial cells on GBM cells were dependent upon CXCL12 and CXCR4. These findings indicate that the CXCL12/CXCR4 pathway directly mediates endothelial cell trophic function in GBMs and that inhibition of CXCL12-CXCR4 signaling may uniquely target this activity. Therapeutic disruption of endothelial cell trophic functions could complement the structural disruption of anti-angiogenic regimens and, in combination, might also improve the efficacy of radiation and chemotherapy in treating GBMs.     

Oxidized low density lipoprotein inhibits interleukin-12 production in lipopolysaccharide-activated mouse macrophages via direct interactions between peroxisome proliferator-activated receptor-gamma and nuclear factor-kappa B

Lipopolysaccharide (LPS) increases the production of interleukin-12 (IL-12) from mouse macrophages via a kappaB site within the IL-12 p40 promoter. In this study, we found that oxidized low density lipoprotein (oxLDL) inhibited this LPS-stimulated production of IL-12 in a dose-dependent manner while native LDL did not. OxLDL inhibited p40 promoter activation in monocytic RAW264.7 cells transiently transfected with p40 promoter/reporter constructs, and the repressive effect mapped to a region in the p40 promoter containing a binding site for nuclear factor-kappaB (NF-kappaB) (p40-kappaB). Activation of macrophages by LPS in the presence of oxLDL resulted in markedly reduced binding to the kappaB site, as demonstrated by the electrophoretic mobility shift assays. In contrast, native LDL did not inhibit the IL-12 p40 promoter activation and NF-kappaB binding to the kappaB sites, suggesting that oxidative modification of LDL was crucial for the inhibition of NF-kappaB-mediated IL-12 production. 9-Hydroxyoctadecadienoic acid, a major oxidized lipid component of oxLDL, significantly inhibited IL-12 production in LPS-stimulated mouse macrophages and also suppressed NF-kappaB-mediated activation in IL-12 p40 promoter. The NF-kappaB components p50 and p65 directly bound peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in vitro. In cotransfections of CV-1 and HeLa cells, PPAR-gamma inhibited the NF-kappaB transactivation in an oxLDL-dependent manner. From these results, we propose that oxLDL-mediated suppression of the IL-12 production from LPS-activated mouse macrophages may, at least in part, involve both inhibition of the NF-kappaB-DNA interactions and physical interactions between NF-kappaB and PPAR-gamma.     

Antitumor effects of the combination immunotherapy with interleukin-12 and tumor necrosis factor α in mice

 There is strong evidence that antitumor activity of interleukin-12 (IL-12) in vivo is mediated, in part, through interferon (IFNγ) produced by IL-12-stimulated natural killer and T cells. Since IFNγ and tumor necrosis factor α (TNFα) have been reported to synergize in antitumor effects in a number of models, we decided to examine whether the combined treatment with recombinant mouse IL-12 and recombinant human TNFα would produce similar effects. The efficacy of the combined IL-12/TNFα immunotherapy was evaluated in three tumor models in mice: B16F10 melanoma, Lewis lung (LL/2) carcinoma and L1 sarcoma. Intratumoral daily injections of 1 μg IL-12 in combination with 5 μg TNFα into B16F10-melanoma-bearing mice resulted in a significant retardation of the tumor growth as compared with that in controls and in mice treated with either cytokine alone. Similar effects were obtained using 0.1 μg IL-12 and 5 μg TNFα in LL/2 carcinoma and L1 sarcoma models. Antitumor activity against L1 sarcoma was still preserved when TNFα at a low dose (1 μg) was combined with 0.1 μg IL-12 and applied for a prolonged time. Potentiation of antitumor effects, which was observed in IL-12/TNFα-based immunotherapy, could result from at least three different mechanisms, partly related to stimulation of IFNγ and TNFα production in treated mice: (a) direct cytostatic/cytotoxic effects on tumor cells, (b) induction of antitumor activity of macrophages, and (c) inhibition of blood vessel formation in the tumor. Our studies demonstrate that combination tumor immunotherapy with IL-12 and TNFα may be more effective than single-cytokine treatment, and suggest possible mechanisms by which IL-12 and TNFα may exert potentiated therapeutic effects against locally growing tumors. Received: 17 February 1997 / Accepted: 5 August 1997     

Exogenous interleukin-12 protects against lethal infection with coxsackievirus B4

Infections with the group B coxsackieviruses either can be asymptomatic or can lead to debilitating chronic diseases. To elucidate the mechanism by which these viruses cause chronic disease, we developed a mouse model of chronic pancreatitis by using a virulent variant of coxsackievirus B4, CVB4-V. Infection with CVB4-V results in an early, severe pancreatitis, which can lead to mortality or progress to chronic pancreatitis. Chronic pancreatitis, in this model, is due to immunopathological mechanisms. We investigated whether interleukin-12 (IL-12) could modulate the outcome of CVB4-V infection. Eighty-five percent of the infected mice treated with 500 ng of IL-12 survived, whereas all untreated mice succumbed. To understand the mechanism underlying the beneficial effect of IL-12, we investigated the role of gamma interferon (IFN-gamma). Three lines of evidence suggest that the protective effect of IL-12 is due to IFN-gamma. First, administration of IL-12 increased the production of endogenous IFN-gamma in CVB4-V-infected mice. Both NK and NKT cells were identified as the source of IFN-gamma. Second, IFN-gamma knockout mice treated with IL-12 succumbed to infection with CVB4-V. Third, wild-type mice treated with IFN-gamma survived infection with CVB4-V. Due to the antiviral effects of IFN-gamma, we examined whether IL-12 treatment affected viral replication. Administration of IL-12 did not decrease viral replication in the pancreas, but it did prevent extensive tissue damage and the subsequent development of chronic pancreatitis. The data suggest that IL-12 treatment during CVB4-V infection is able to suppress the immunopathological mechanisms that lead to chronic disease.     

Mutualistic interactions between vitamin B(12) -dependent algae and heterotrophic bacteria exhibit regulation

Many algae are auxotrophs for vitamin B₁₂ (cobalamin), which they need as a cofactor for B₁₂‐dependent methionine synthase (METH). Because only prokaryotes can synthesize the cobalamin, they must be the ultimate source of the vitamin. In the laboratory, a direct interaction between algae and heterotrophic bacteria has been shown, with bacteria supplying cobalamin in exchange for fixed carbon. Here we establish a system to study this interaction at the molecular level. In a culture of a B₁₂‐dependent green alga Chlamydomonas nivalis, we found a contaminating bacterium, identified by 16S rRNA analysis as Mesorhizobium sp. Using the sequenced strain of M. loti (MAFF303099), we found that it was able to support the growth of B₁₂‐dependent Lobomonas rostrata, another green alga, in return for fixed carbon. The two organisms form a stable equilibrium in terms of population numbers, which is maintained over many generations in semi‐continuous culture, indicating a degree of regulation. However, addition of either vitamin B₁₂ or a carbon source for the bacteria perturbs the equilibrium, demonstrating that the symbiosis is mutualistic and facultative. Chlamydomonas reinhardtii does not require B₁₂ for growth because it encodes a B₁₂‐independent methionine synthase, METE, the gene for which is suppressed by addition of exogenous B₁₂. Co‐culturing C. reinhardtii with M. loti also results in reduction of METE expression, demonstrating that the bacterium can deliver the vitamin to this B₁₂‐independent alga. We discuss the implications of this for the widespread distribution of cobalamin auxotrophy in the algal kingdom.     

Physical and functional interactions of the Arf tumor suppressor protein with nucleophosmin/B23

The Arf tumor suppressor inhibits cell cycle progression through both p53-dependent and p53-independent mechanisms, including interference with rRNA processing. Using tandem-affinity-tagged p19(Arf), we purified Arf-associated proteins from mouse NIH 3T3 fibroblasts undergoing cell cycle arrest. Tagged p19(Arf) associated with nucleolar and ribosomal proteins, including nucleophosmin/B23 (NPM), a protein thought to foster the maturation of preribosomal particles. NPM is an abundant protein, only a minor fraction of which binds to p19(Arf); however, a significant proportion of p19(Arf) associates with NPM. The interaction between p19(Arf) and NPM requires amino acid sequences at the Arf amino terminus, which are also required for Mdm2 binding, as well as the central acidic domain of NPM and an adjacent segment that regulates NPM oligomerization. The interaction between p19(Arf) and NPM occurs in primary mouse embryonic fibroblasts, including those lacking both Mdm2 and p53. In an NIH 3T3 derivative cell line (MT-Arf) engineered to conditionally express an Arf transgene, induced p19(Arf) associates with NPM and colocalizes with it in high-molecular-weight complexes (2 to 5 MDa). An NPM mutant lacking its carboxyl-terminal nucleic acid-binding domain oligomerizes with endogenous NPM, inhibits p19(Arf) from entering into 2- to 5-MDa particles, and overrides the ability of p19(Arf) to retard rRNA processing.     

Prenatal exposure to medicines and the risk of childhood brain tumor

Background: Childhood brain tumors are associated with high mortality and morbidity but little is known about its causes. About half of women use medicines when pregnant and some of the medicines commonly used might be carcinogenic. Objective: The aim with this population-based case–control study was to analyze associations between specific groups of medicines taken during pregnancy and the risk of brain tumor in the offspring. Methods: All children, up to 15 years of age, born in Sweden between 1975 and 1984 were eligible for the study. Cases (N = 512) were children diagnosed with brain tumor and controls (N = 525) were randomly selected from the Medical Birth Register. Exposure data on medicines was extracted blindly from antenatal medical records and grouped according to Anatomical Therapeutic Chemical (ATC) code. Information on maternal reproductive history was received from the Medical Birth Register. We used logistic regression to estimate associations between fetal exposure to medicines and childhood brain tumor. Results: No significant changes in risk were noted after exposure to iron supplementation, antiemetics, analgesics, antibiotics or any other main ATC group. A tendency of protective effect was seen for prenatal exposure to folic acid (adjusted OR 0.6, 95% CI 0.3–1.1). Ten children with a diagnosis of brain tumor had been exposed to β-blocking agents in fetal life as compared to two children without brain tumor (adjusted OR 5.3, 95% CI 1.2–24.8). Conclusions: In this case–control study, an increased risk of brain tumor was seen in children exposed to β-blocking agents during fetal life. However, due to the low number of exposed the interpretation of this finding should be made with caution.     

Serum levels of interleukin-12 in various clinical states with hepatitis B virus infection

Hepatitis B virus (HBV) infection involves various clinical states. Interleukin-12 (IL-12) has been identified as a crucial cytokine in the development of cellular immunity. But the association between HBV infection clinical states and cellular immunity response remains unclear. The aim was to explore the association by investigating serum levels of IL-12. Observed in acute hepatitis B, the highest serum levels of IL-12 was accompanied by HBeAg seroconversion. Serum levels of IL-12 was associated with alanine transaminase (ALT) levels and significant more in chronic hepatitis B patients with ALT over five times upper limit of normal and a minority of immune-tolerance patients than controls. Serum levels of IL-12 may be an available marker to evaluate cellular immunity for HBV infection. Elevation in IL-12 levels may be a factor to promote HBeAg seroconversion and an opportunity to be given antivirus treatment for immune-tolerance carriers.     

A mathematical analysis of the interactions between immunogenic tumor cells and cytotoxic T lymphocytes.

Recent developments of biotechnology have enabled us to use immunotherapy against certain kinds of tumors in patients. However, it is reasonable to doubt if the immunotherapy can completely aid the rejection of tumors that have escaped from the immune system. In this paper, we propose a new mathematical model of tumor immunity by tumor-specific cytotoxic T lymphocytes (CTLs), since tumor-specific CTLs play an important role in tumor immunity. Using this model, we have mathematically investigated the interactions between immunogenic tumor cells (TCs) and tumor-specific CTLs and evaluated the availability of immunotherapies for tumors. The findings herein demonstrate that three kinds of dynamics of tumor immunity exist: i.e. (1) TCs continue to proliferate with CTLs; (2) TCs are rejected by CTLs; and (3) TCs equilibrate with CTLs, but with little possibility of the equilibrium. The findings also demonstrate that a sufficient increase in CTLs by immunotherapy can aid the rejection of TCs, but an insufficient increase in CTLs by immunotherapy causes only a transient regression of TCs. Clinically the findings mean that increasing tumor-specific CTLs, e.g., by vaccination or adoptive transfer of tumor-specific CTLs expanded ex vivo, can theoretically aid the rejection of TCs.     

Biophysical interactions between components of the tumor microenvironment promote metastasis

During metastasis, tumor cells need to adapt to their dynamic microenvironment and modify their mechanical properties in response to both chemical and mechanical stimulation. Physical interactions occur between cancer cells and the surrounding matrix including cell movements and cell shape alterations through the process of mechanotransduction. The latter describes the translation of external mechanical cues into intracellular biochemical signaling. Reorganization of both the cytoskeleton and the extracellular matrix (ECM) plays a critical role in these spreading steps. Migrating tumor cells show increased motility in order to cross the tumor microenvironment, migrate through ECM and reach the bloodstream to the metastatic site. There are specific factors affecting these processes, as well as the survival of circulating tumor cells (CTC) in the blood flow until they finally invade the secondary tissue to form metastasis. This review aims to study the mechanisms of metastasis from a biomechanical perspective and investigate cell migration, with a focus on the alterations in the cytoskeleton through this journey and the effect of biologic fluids on metastasis. Understanding of the biophysical mechanisms that promote tumor metastasis may contribute successful therapeutic approaches in the fight against cancer.     

Productive interactions between B and natural killer cells

In this report, we have established that natural killer (NK) cells can increase IgG2a secretion by B lymphocytes as well as alter the distribution of the remaining immunoglobulin isotypes. The effect of NK cells on B cell differentiation is similar to that obtained by the direct addition of recombinant interferon-gamma (IFN-gamma) and, therefore, most likely results from the elaboration of IFN-gamma by NK cells, this is a clear demonstration that NK cells can regulate cell function(s) via a mechanism other than cytotoxicity. In addition, we have shown that the induction of NK cells by B lymphocytes requires close interactions between the two cell types. Further, while only low-density B lymphocytes activated in vivo are effective inducers of NK cells, high-density, resting B cells can be rendered effective by preactivation with either interleukin-4 or anti-mu.     

Immune gene therapy of experimental mouse brain tumor with adenovirus-mediated gene transfer of murine interleukin-4

 We examined the antitumor activity of replication-deficient adenoviral vectors carrying the murine interleukin-4 (IL-4) gene (AdCIL4) using a syngeneic brain tumor model in mice. Mice implanted with malignant astrocytoma cells infected with AdCIL4 survived significantly longer than those in the control groups. Immunocytochemical analysis of the tumors showed that AdCIL4 caused the strong up-regulation of MHC class II antigen expression by the tumor cells and macrophages, and consequent infiltration by CD8⁺ T lymphocytes. This study demonstrates the efficacy of IL-4 gene transfection mediated by adenoviral vectors for intracerebral tumor and characterizes the immunoreaction caused by AdCIL4. Received: 27 August 1999 / Accepted: 12 November 1999