A water-soluble derivative of propolis augments the cytotoxic activity of natural killer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Propolis, a resinous material collected from numerous plants by honeybees, has historically been used as a health-promoting food. Recently, due to its potential anti-tumor effects, use of propolis has been proposed as an adjuvant therapy to chemotherapy; however, the effects of propolis on immune responses remain unclear. AIM OF THE STUDY: In this study, we examined the effects of the oral ingestion of propolis on natural killer (NK) cell activity, which is important in immune surveillance against cancer and viral infections. In addition, we assessed the effects of the major components of the water-soluble powder derivative of propolis (WPP). MATERIALS AND METHODS: C57BL/6 (B6) wild-type (WT) and RAG 2-deficient (RAG-/-) mice and BALB/c WT, interferon (IFN)-γ-deficient (IFN-γ-/-), IFN-γ receptor-deficient (IFN-γR-/-) and RAG-/- mice were orally administered WPP or its major components. NK cell populations and cytotoxic activity were then examined by flow cytometry and 51Cr release assay, respectively. RESULTS: While the cytotoxic activity of NK cells was increased following administration of 100 mg/kg/day of WPP for 7 days or 200 or 500 mg/kg/day of WPP for 4 days in WT mice, the proportions of NK cell populations were unaltered. Similar activation of NK cell cytotoxicity was observed when RAG-/-, but not IFN-γ-/- or IFN-γR-/-, mice were orally administered 200 mg/kg/day of WPP for 4 days. Oral ingestion of artepillin C or p-coumaric acid, but not drupanin, augmented NK cell cytotoxicity in a manner similar to WPP and to the mixture of these three components. CONCLUSION: These results suggest that oral ingestion of WPP enhances NK cell cytotoxic activity, but not proliferation, in a manner dependent on IFN-γ and without the contribution of acquired immune responses. Further, artepillin C or p-coumaric acid, but not drupanin, may be the components responsible for this augmentation of NK cell cytotoxicity. These findings suggest the possible utility of WPP as a therapeutic for prevention of cancer development and against viral infection through NK cell activation.

N-3 polyunsaturated fatty acids inhibit IFN-γ-induced IL-18 binding protein production by prostate cancer cells.

Prostate cancer cells can produce IL-18 binding protein (IL-18BP) in response to interferon-γ (IFN-γ), which may function to neutralize IL-18, an anti-tumor factor formerly known as IFN-γ inducing factor. The consumption of n-3 polyunsaturated fatty acids (PUFAs) has been associated with a lower risk of certain types of cancer including prostate cancer, although the precise mechanisms of this effect are poorly understood. We hypothesized that n-3 PUFAs could modify IL-18BP production by prostate cancer cells by altering IFN-γ receptor-mediated signal transduction. Here, we demonstrate that n-3 PUFA treatment significantly reduced IFN-γ-induced IL-18BP production by DU-145 and PC-3 prostate cancer cells by inhibiting IL-18BP mRNA expression and was associated with a reduction in IFN-γ receptor expression. Furthermore, IFN-γ-induced phosphorylation of Janus kinase 1 (JAK1), signal transducers and activators of transcription 1 (STAT1), extracellular signal-regulated kinases 1/2 (ERK1/2), and P38 were suppressed by n-3 PUFA treatment. By contrast, n-6 PUFA had no effect on IFN-γ receptor expression, but decreased IFN-γ-induced IL-18BP production and IFN-γ stimulation of JAK1, STAT1, ERK1/2, and JNK phosphorylation. These data indicate that both n-3 and n-6 PUFAs may be beneficial in prostate cancer by altering IFN-γ signaling, thus inhibiting IL-18BP production and thereby rendering prostate cancer cells more sensitive to IL-18-mediated immune responses.

cDNA microarray analysis of serially sampled cervical cancer specimens from patients treated with thermochemoradiotherapy.

PURPOSE: To elucidate changes in gene expression after treatment with regional thermochemoradiotherapy in locally advanced squamous cell cervical cancer. METHODS AND MATERIALS: Tru-Cut biopsy specimens were serially collected from 16 patients. Microarray gene expression levels before and 24 h after the first and second trimodality treatment sessions were compared. Pathway and network analyses were conducted by use of Ingenuity Pathways Analysis (IPA; Ingenuity Systems, Redwood City, CA). Single gene expressions were analyzed by quantitative real-time reverse transcription-polymerase chain reaction. RESULTS: We detected 53 annotated genes that were differentially expressed after trimodality treatment. Central in the three top networks detected by IPA were interferon alfa, interferon beta, and interferon gamma receptor; nuclear factor kappaB; and tumor necrosis factor, respectively. These genes encode proteins that are important in regulation cell signaling, proliferation, gene expression, and immune stimulation. Biological processes over-represented among the 53 genes were fibrosis, tumorigenesis, and immune response. CONCLUSIONS: Microarrays showed minor changes in gene expression after thermochemoradiotherapy in locally advanced cervical cancer. We detected 53 differentially expressed genes, mainly involved in fibrosis, tumorigenesis, and immune response. A limitation with the use of serial biopsy specimens was low quality of ribonucleic acid from tumors that respond to highly effective therapy. Another "key limitation" is timing of the post-treatment biopsy, because 24 h may be too late to adequately assess the impact of hyperthermia on gene expression.

Chitosan monomer accelerates alkaline phosphatase activity on human osteoblastic cells under hypofunctional conditions.

Chitosan is a natural polyaminosaccharide that is extensively applied as an antitumor and antirheumatic drug. However, there are few reports about its effects on hypofunctional osteoblasts in vitro. We investigated the biological characteristics of a human osteoblastic cell line (NOS-1 cells) that was cultured with a chitosan monomer-containing medium under simulated microgravity conditions. After 7 days of cell incubation under the conventional conditions, the flasks were transferred to a microgravity simulator for 3 days. In the 0.005% chitosan monomer supplemented group, the marker enzyme of biological mineralization, the alkaline phosphatase (ALP) activity, was significantly higher compared with the control group (p<0.05). A cDNA microarray was performed to investigate the effects on the mRNA level by chitosan monomer, and the fluorescent signal was analyzed. The interferon gamma (IFN-gamma) receptor gene was detected with a signal ration of 2.2. The slight increase of IFN-gamma receptor expression was confirmed after 3 days of incubation according to RT-PCR analysis. Western blot analysis also showed the increased expression of IFN-gamma receptor. These results suggest that a supra-low concentration of chitosan monomer may increase the ALP activity of osteoblastic cells through the IFN-gamma receptor at the early phase of cell culture and recover the activity for biological mineralization under the hypofunctional condition.

Chronic polyarthritis caused by mammalian DNA that escapes from degradation in macrophages.

A large amount of chromosomal DNA is degraded during programmed cell death and definitive erythropoiesis. DNase II is an enzyme that digests the chromosomal DNA of apoptotic cells and nuclei expelled from erythroid precursor cells after macrophages have engulfed them. Here we show that DNase II-/-IFN-IR-/- mice and mice with an induced deletion of the DNase II gene develop a chronic polyarthritis resembling human rheumatoid arthritis. A set of cytokine genes was strongly activated in the affected joints of these mice, and their serum contained high levels of anti-cyclic citrullinated peptide antibody, rheumatoid factor and matrix metalloproteinase-3. Early in the pathogenesis, expression of the gene encoding tumour necrosis factor (TNF)-alpha was upregulated in the bone marrow, and administration of anti-TNF-alpha antibody prevented the development of arthritis. These results indicate that if macrophages cannot degrade mammalian DNA from erythroid precursors and apoptotic cells, they produce TNF-alpha, which activates synovial cells to produce various cytokines, leading to the development of chronic polyarthritis.

Effects on gene expression and viral load of a medicinal extract from Agaricus blazei in patients with chronic hepatitis C infection.

Extracts from the mushroom Agaricus blazei Murill (AbM) are used extensively as a non-prescription remedy against cancer and infections, including hepatitis. We previously demonstrated a potent immunomodulating effect of a particular preparation on monocytes in vitro, and a protective effect on bacterial infections in mice. Here we report the effect on gene expression in peripheral blood cells from four chronic hepatitis C patients, using global (29 k) oligo-based, single channel microarrays. The viral load was slightly, but not significantly, decreased after 1 week of AbM treatment. The cytokine genes most strongly induced in vitro were not induced in vivo. The more notable changes in mRNA levels were related to genes involved in the G-protein coupled receptor signalling pathway, in cell cycling, and in transcriptional regulation. The results suggest that the beta-glucans of the extract, which presumably are responsible for cytokine induction, did not readily enter the blood, while other components, such as substances proposed to have anticancer effects, were active in the blood.

Mice lacking the IFN-gamma receptor or fyn develop severe experimental autoimmune uveoretinitis characterized by different immune responses.

Endogenous interferon (IFN)-gamma negatively regulates experimental autoimmune uveoretinitis (EAU), a Th1-mediated disease. Although it is well known that IFN-gamma exerts its effects by binding to the IFN-gamma receptor (IFN-gammaR), the role that IFN-gammaR plays in the development of EAU has not been investigated. Fyn has been reported to inhibit Th2 differentiation. We aimed to investigate how endogenous IFN-gammaR and fyn, which influence Th1/Th2 differentiation, participate in the development of EAU. Sex-matched 6- to 10-week-old C57BL/6 wild-type (WT), IFN-gammaR knockout (GRKO) and fyn knockout (fyn KO) mice were compared. Mice were immunized subcutaneously with human interphotoreceptor retinoid-binding protein peptide 1-20 emulsified in Freund's complete adjuvant together with an intraperitoneal injection of Bordetella pertussis toxin. Three weeks later, mice were sacrificed, and their eyes and spleens were harvested for histopathologic analyses and examination of cellular immune responses, respectively. Cellular immune responses were evaluated by measuring the proliferative responses and cytokine production [interleukin (IL)-4, IL-5, IL-6, IL-13, IFN-gamma and tumor necrosis factor (TNF)-alpha] of splenocytes. The incidence of EAU was 40.0% in WT mice, 59.3% in GRKO mice and 78.6% in fyn KO mice. The average EAU score was 0.294 in WT mice, 0.917 in GRKO mice and 1.063 in fyn KO mice. Upon EAU induction, significant infiltration of eosinophils into the eyes was observed in GRKO and fyn KO mice compared to WT mice. Splenocytes from GRKO mice proliferated against the antigen and a mitogen more vigorously than those from WT and fyn KO mice. Stimulation of splenocytes with the antigen induced a higher production of IL-4, IL-6, IL-13 and IFN-gamma in GRKO mice compared to WT and fyn KO mice. In contrast, IL-5 and TNF-alpha were most abundantly produced by splenocytes from fyn KO mice compared to WT and GRKO mice. The incidence and mean severity of EAU were significantly higher in GRKO and fyn KO mice than in WT mice, suggesting that endogenous IFN-gammaR and fyn negatively regulate the development of EAU. The different cytokine production patterns by the GRKO and fyn KO mice indicate that the negative regulatory mechanism mediated by IFN-gammaR and fyn may differ.

Differential expression of various cytokine receptors in the brain after stimulation with LPS in young and old mice.

The effect of lipopolysaccharides (LPS) on the expression of cytokine receptors was examined in the spleen, brain and pituitary gland, and compared in young and old mice. The level of mRNA for various cytokine receptors (IL-1RI, IL-2Ralpha, IL-3Ralpha, IL-6R, TNFalphaR and IFNgammaR) was found to be increased in the spleen of young but not in old mice within 2-6h of stimulation with LPS. Similar enhancement of cytokine receptor mRNA was also observed in the brain after LPS stimulation, but the magnitude varied according to the type of cytokine receptor, the site of brain and the age of the mice. In the hypothalamus, the level of mRNA for IL-1R, IL-3R, IL-6R and IFNgammaR increased in young but not in old mice. Reciprocally, in the cerebral cortex, mRNA for TNFalphaR and IFNgammaR increased in old but not in young mice. In the hippocampus, TNFalphaR mRNA expression, increased in young but not in old mice, and expression of the other cytokine receptors did not change greatly in either. In the pituitary gland, mRNA for IL-6R, TNFalphaR and IFNgammaR increased in both young and old mice, but IL-2Ralpha increased only in young mice.Thus, various cytokines produced by immune cells might directly or indirectly influence brain functions through the various cytokine receptors expressed in the brain. Moreover, interactions between the immune system and the brain at the time of infection would be expected to be different in young and old mice, because cytokine production changes with age, as does the expression of their receptors in the brain.

Lethal autoimmune myocarditis in interferon-gamma receptor-deficient mice: enhanced disease severity by impaired inducible nitric oxide synthase induction.

BACKGROUND: Interferon-gamma (IFN-gamma) is an essential cytokine in the regulation of inflammatory responses in autoimmune diseases. Little is known about its role in inflammatory heart disease. METHODS AND RESULTS: We showed that IFN-gamma receptor-deficient mice (IFN-gammaR(-/-)) on a BALB/c background immunized with a peptide derived from cardiac alpha-myosin heavy chain develop severe myocarditis with high mortality. Although myocarditis subsided in wild-type mice after 3 weeks, IFN-gammaR(-/-) mice showed persistent disease. The persistent inflammation was accompanied by vigorous in vitro CD4 T-cell responses and impaired inducible nitric oxide synthase expression, together with evidence of impaired nitric oxide production in IFN-gammaR(-/-) hearts. Treatment of wild-type mice with the nitric oxide synthetase inhibitor N:-nitro-l-arginine-methyl-ester enhanced in vitro CD4 T-cell proliferation and prevented healing of myocarditis. CONCLUSIONS: Our data provide evidence that IFN-gamma protects mice from lethal autoimmune myocarditis by inducing the expression of inducible nitric oxide synthase followed by the downregulation of T-cell responses.

The effects of NO synthase inhibitors on murine collagen-induced arthritis do not support a role of NO in the protective effect of IFN-gamma.

DBA/1 mice deficient in expressing the interferon-gamma (IFN-gamma) membrane receptor (IFN-gammaR KO mice) are more susceptible to collagen-induced arthritis (CIA) than wild-type mice, indicating that endogenous IFN-gamma plays a protective role in the pathogenesis of CIA. In IFN-gammaR KO mice, nitric oxide (NO) production during CIA is impaired. Because NO is known to exert immunosuppressive and anti-inflammatory effects in certain model systems, the protective effect of IFN-gamma might be mediated by NO. Here, we tested in wild-type mice whether inhibition of NO production by metabolic inhibitors, aminoguanidine (AG) and L-N-(1-iminoethyl)lysine (L-NIL), could mimic the ablation of the IFN-gamma receptor. A high-dose regimen of AG supplied in the drinking water inhibited NO production, disease development, and anticollagen antibody production but was also associated with transient body weight loss. At a dose and time regimen that still inhibited NO production but did not cause body weight loss, AG failed to affect disease scores. Treatment with L-NIL, which more specifically than AG affects inducible NO production, caused a slight increase in anticollagen antibody production although not significantly affecting disease occurrence. These data indicate that the diminished capacity of the IFN-gammaR KO mice to produce NO following immunization with collagen is unlikely to account for their higher susceptibility to CIA.

The human interferon alpha/beta receptor: characterization and molecular cloning.

We describe a universal ligand-binding receptor for human interferons alpha and interferon beta (type I IFNs). A soluble 40 kDa IFN-alpha/beta receptor (p40) that blocks the activity of type I IFNs was purified from urine and sequenced. Antibodies raised against p40 completely block the activity of several type I IFNs and immuno-precipitate both a cellular 102 kDa IFN-alpha/beta receptor and its cross-linked complexes with IFN-alpha 2. The receptor is a disulfide-linked dimer, consisting of 51 kDa subunits. We isolated and expressed a 1.5 kb cDNA, coding for the IFN-alpha/beta receptor. Its 331 amino acid sequence includes a leader and a transmembrane region, while its ectodomain corresponds to p40. IFN-alpha/beta receptor is physically associated with the cytoplasmic Tyr kinase JAK1, hence, in addition to ligand binding, it is directly involved in signal transduction.