Isolation of adipose tissue-derived stem cells by direct membrane migration and expansion for clinical application.

Mesenchymal stem cells (MSCs) have recently made significant progression in multiple clinical trials targeting several clinical disorders and in the modulation of immune responses. In the present study, we isolated human adipose tissue-derived stem cells (ADSCs) by direct membrane migration method without using enzymatic digestion via collagenase, and tried to extract adequate number of cells for clinical application. Hydroxyapatite-treated nonwoven fabric membrane made up of synthetic macromolecular fiber materials, polyethylene and polyester terephthalene was used. Expansion culture of ADSCs having plastic flask adherent characteristic in serum-free condition was successfully established, and adequate number of cells were obtained for clinical application. They were found to be positive for CD44, CD73, CD90 and CD105 and negative for CD11b, CD34, CD45, CD80 and HLA-DR. The resulting immunological marker profile satisfied the immunophenotype of previously reported MSCs. Also, microscopic findings demonstrated trilineage differentiation into adipogenic, osteogenic and chondrogenic cells as the characteristics of MSCs. The isolation by nonwoven fabric membrane and expanded cells under serum-free condition satisfied the criteria of MSCs, as proposed by the International Society for Cellular Therapy. Our direct membrane migration method without enzyme digestion is useful as ADSCs can be obtained from small pieces of adipose tissue and expanded under serum-free culture condition. This method was considered to be feasible for clinical application.

Effects of bergamot ( Citrus bergamia (Risso) Wright & Arn.) essential oil aromatherapy on mood states, parasympathetic nervous system activity, and salivary cortisol levels in 41 healthy females.

BACKGROUND: Bergamot essential oil (BEO) is commonly used against psychological stress and anxiety in aromatherapy. The primary aim of the present study was to obtain first clinical evidence for these psychological and physiological effects. A secondary aim was to achieve some fundamental understanding of the relevant pharmacological processes. METHODS: Endocrinological, physiological, and psychological effects of BEO vapor inhalation on 41 healthy females were tested using a random crossover study design. Volunteers were exposed to 3 experimental setups (rest (R), rest + water vapor (RW), rest + water vapor + bergamot essential oil (RWB)) for 15 min each. Immediately after each setup, saliva samples were collected and the volunteers rested for 10 min. Subsequently, they completed the Profile of Mood States, State-Trait Anxiety Inventory, and Fatigue Self-Check List. High-frequency (HF) heart rate values, an indicator for parasympathetic nervous system activity, were calculated from heart rate variability values measured both during the 15 min of the experiment and during the subsequent 10 min of rest. Salivary cortisol (CS) levels in the saliva samples were analyzed using ELISA. RESULTS: CS of all 3 conditions R, RW, and RWB were found to be significantly distinct (p = 0.003). In the subsequent multiple comparison test, the CS value of RWB was significantly lower when compared to the R setup. When comparing the HF values of the RWB setup during the 10 min of rest after the experiment to those of RW, this parameter was significantly increased (p = 0.026) in the RWB setup for which scores for negative emotions and fatigue were also improved. CONCLUSION: These results demonstrate that BEO inhaled together with water vapor exerts psychological and physiological effects in a relatively short time.

Spiritual care of cancer patients by integrated medicine in urban green space: a pilot study.

BACKGROUND: Psycho-oncological care, including spiritual care, is essential for cancer patients. Integrated medicine, a therapy combining modern western medicine with various kinds of complementary and alternative medicine, can be appropriate for the spiritual care of cancer because of the multidimensional characteristics of the spirituality. In particular, therapies that enable patients to establish a deeper contact with nature, inspire feelings of life and growth of plants, and involve meditation may be useful for spiritual care as well as related aspects such as emotion. The purpose of the present study was to examine the effect of spiritual care of cancer patients by integrated medicine in a green environment. METHODS: The present study involved 22 cancer patients. Integrated medicine consisted of forest therapy, horticultural therapy, yoga meditation, and support group therapy, and sessions were conducted once a week for 12 weeks. The spirituality (the Functional Assessment of Chronic Illness Therapy-Spiritual well-being), quality of life (Short Form-36 Health Survey Questionnaire), fatigue (Cancer Fatigue Scale), psychological state (Profile of Mood States, short form, and State-Trait Anxiety Inventory) and natural killer cell activity were assessed before and after intervention. RESULTS: In Functional Assessment of Chronic Illness Therapy-Spiritual well-being, there were significant differences in functional well-being and spiritual well-being pre- and postintervention. This program improved quality of life and reduced cancer-associated fatigue. Furthermore, some aspects of psychological state were improved and natural killer cell activity was increased. CONCLUSIONS: It is indicated that integrated medicine performed in a green environment is potentially useful for the emotional and spiritual well-being of cancer patients.

Cyclic AMP responsive element binding proteins are involved in 'emergency' granulopoiesis through the upregulation of CCAAT/enhancer binding protein ß.

In contrast to the definitive role of the transcription factor, CCAAT/Enhancer binding protein α (C/EBPα), in steady-state granulopoiesis, previous findings have suggested that granulopoiesis during emergency situations, such as infection, is dependent on C/EBPß. In this study, a novel lentivirus-based reporter system was developed to elucidate the molecular switch required for C/EBPß-dependency. The results demonstrated that two cyclic AMP responsive elements (CREs) in the proximal promoter region of C/EBPß were involved in the positive regulation of C/EBPß transcription during granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced differentiation of bone marrow cells. In addition, the transcripts of CRE binding (CREB) family proteins were readily detected in hematopoietic stem/progenitor cells. CREB was upregulated, phosphorylated and bound to the CREs in response to GM-CSF stimulation. Retroviral transduction of a dominant negative CREB mutant reduced C/EBPß mRNA levels and significantly impaired the proliferation/differentiation of granulocyte precursors, while a constitutively active form of CREB facilitated C/EBPß transcription. These data suggest that CREB proteins are involved in the regulation of granulopoiesis via C/EBPß upregulation.

C/EBPß is involved in the amplification of early granulocyte precursors during candidemia-induced "emergency" granulopoiesis.

Granulopoiesis is tightly regulated to meet host demands during both "steady-state" and "emergency" situations, such as infections. The transcription factor CCAAT/enhancer binding protein ß (C/EBPß) plays critical roles in emergency granulopoiesis, but the precise developmental stages in which C/EBPß is required are unknown. In this study, a novel flow cytometric method was developed that successfully dissected mouse bone marrow cells undergoing granulopoiesis into five distinct subpopulations (#1-5) according to their levels of c-Kit and Ly-6G expression. After the induction of candidemia, rapid mobilization of mature granulocytes and an increase in early granulocyte precursors accompanied by cell cycle acceleration was followed by a gradual increase in granulocytes originating from the immature populations. Upon infection, C/EBPß was upregulated at the protein level in all the granulopoietic subpopulations. The rapid increase in immature subpopulations #1 and #2 observed in C/EBPß knockout mice at 1 d postinfection was attenuated. Candidemia-induced cell cycle acceleration and proliferation of hematopoietic stem/progenitors were also impaired. Taken together, these data suggest that C/EBPß is involved in the efficient amplification of early granulocyte precursors during candidemia-induced emergency granulopoiesis.

Porphyromonas gingivalis induces myocarditis and/or myocardial infarction in mice and IL-17A is involved in pathogenesis of these diseases.

OBJECTIVES: Although an association between periodontitis and cardiovascular diseases has been suggested, the role of Porphyromonas gingivalis in cardiovascular diseases is not clear. In this study, we examined whether experimental bacteremia of P. gingivalis causes cardiovascular diseases and investigated the mechanism of pathogenesis of cardiovascular diseases induced by P. gingivalis. DESIGN: C57BL/6 mice were intravenously inoculated with 2.0 × 10(8)CFU of P. gingivalis A7436 strain. Mice were sacrificed at specified days and their hearts were collected. The collected organs were divided into two halves and used for histological evaluation and cytokine analysis. IL-17A(-/-), IFN-γ(-/-) and TNF-α(-/-) mice were also intravenously inoculated and the histological changes of hearts in mice were examined. RESULTS: Myocarditis and/or myocardial infarction were observed in mice injected with P. gingivalis. The levels of IL1-ß, IL-6, IL-17A, IL-18, TNF-α and IFN-γ mRNA increased significantly after P. gingivalis injection. In particular, high levels of IL-17A and IFN-γ mRNA expression were observed in hearts of mice after P. gingivalis injection in comparison with these levels before injection. Furthermore, the production of IL-17A was detected in hearts of wild-type mice after P. gingivalis injection. In wild-type, TNF-α(-/-) and IFN-γ(-/-) mice, moderate infiltration of neutrophils and monocytes was observed in hearts at 5 days after injection. In contrast, no inflammatory findings were observed in hearts of IL-17A(-/-) mice. CONCLUSION: We have demonstrated that an experimental bacteremia of P. gingivalis could induce myocarditis and/or myocardial infarction in mice, and IL-17A plays an important role in the pathogenesis of these diseases.

Interleukin-28B acts synergistically with cisplatin to suppress the growth of head and neck squamous cell carcinoma.

Interleukin-28B (IL-28B), also referred to as interferon-λ3, belongs to the type III interferon family. Earlier studies showed that IL-28B suppresses proliferation of some tumor cells in vitro. IL-28B gene transfection ex vivo also resulted in growth retardation of tumor cells in mice, through either direct antiproliferative action or induction of antitumor immunity. However, it has not been reported whether in vivo therapeutic administration of recombinant IL-28B can inhibit the growth of a pre-established tumor. Here, we found that repetitive subcutaneous administration of recombinant mouse IL-28B significantly induced tumor-specific cytotoxic T lymphocytes and augmented natural killer cytolytic activity, leading to moderate suppression of the growth of a murine head and neck squamous cell carcinoma (HNSCC) cell line that was completely resistant to the direct antiproliferative effect of IL-28B. Moreover, co-administration of recombinant mouse IL-28B and cisplatin (CDDP) more significantly inhibited in vivo growth of the tumor that had been established in syngenic mice and induced tumor-specific cytotoxic T lymphocytes. The CDDP treatment induced the expression of major histocompatibility complex class I and Fas molecules on the surface of HNSCC cells both in vitro and in vivo; this may be the mechanism underlying the synergistic tumor suppression activity of IL-28B and CDDP. Unlike type I interferon, IL-28B did not suppress growth of bone marrow cells in culture. Therefore, IL-28B may be useful as a tool for a novel multidisciplinary therapy against cancer, significantly potentiating innate and adaptive antitumor immune responses, especially when co-administrated with CDDP, which is currently the first choice chemotherapeutic agent against various tumors including HNSCCs.

Mitf silencing cooperates with IL-12 gene transfer to inhibit melanoma in mice.

Malignant melanoma is a malignant neoplasm originating from the melanocyte lineage. Microphthalmia-associated transcription factor (Mitf) is crucially involved in the melanin synthesis as well as proliferation and survival of melanocyte and melanoma. We previously showed that short interfering RNA (siRNA) that is specific for the Mitf gene (Mitf-siRNA) significantly inhibited growth of B16 melanoma after electro-transfected in vivo into preestablished tumor in mice. Here we assessed efficacy of electroporation-mediated co-transfection of Mitf-siRNA and IL-12 gene in the treatment of murine melanoma. As results, the tumor growth was more strongly inhibited by intratumor co-transfection with Mitf-siRNA and IL-12-encoding plasmid DNA than by transfection with either of the molecules alone. The co-transfection induced intratumor infiltration of CD4+ and CD8+ T cells, and hampered neoangiogenesis in the tumor. The findings suggest that the RNAi/cytokine gene combination therapy by means of electroporation may become a novel and efficacious therapeutic modality to treat neoplasms including melanoma.

MDR1a/1b gene silencing enhances drug sensitivity in rat fibroblast-like synoviocytes.

BACKGROUND: Drug resistance mediated by P-glycoprotein (P-gp) is one of the major reasons for the failure of rheumatoid arthritis (RA) therapy with disease modifying anti-rheumatic drugs and glucocorticoids. In the present study, we aimed to investigate the in vitro effectiveness of small interfering RNA (siRNA) to render rat fibroblast-like synoviocytes (FLS) susceptible to drugs. We also attempted the electroporation-mediated transfer of siRNA against multidrug resistance (MDR) genes into rat knee joints. METHODS: FLS were transfected with siRNAs corresponding to MDR1a and MDR1b genes. FLS were treated with dexamethasone (DEX) and lipopolysaccharide. The mRNA and protein levels of tumor necrosis factor-alpha, interleukin (IL)-6 and IL-1beta were measured. Both siRNAs were co-transduced into rat knee joints by an electroporation method and evaluated the target gene expressions in the synovium. RESULTS: Each siRNA could sequence-specifically reduce the target gene expression by over 70% and effectively suppressed P-gp expression and function in the FLS. Both gene expression and protein production of the inflammatory cytokines in the cells transfected with siRNA were reduced by a greater amount compared to in control cells. The in vivo electroporation-mediated transduction of siRNA could significantly inhibit the target gene expressions. CONCLUSIONS: MDR1a/1b gene silencing by siRNA could effectively inhibit P-gp in rat FLS, resulting in a significant enhancement of the anti-inflammatory effects of DEX. The in vivo siRNA transduction could successfully silence MDR gene expression in the rat synovium. These findings indicate that the siRNA targeting MDR gene could be a useful tool for treating refractory arthritis in RA.

N-acetylcysteine prevents nitric oxide-induced chondrocyte apoptosis and cartilage degeneration in an experimental model of osteoarthritis.

We investigated whether N-acetylcysteine (NAC), a precursor of glutathione, could protect rabbit articular chondrocytes against nitric oxide (NO)-induced apoptosis and could prevent cartilage destruction in an experimental model of osteoarthritis (OA) in rats. Isolated chondrocytes were treated with various concentrations of NAC (0-2 mM). Apoptosis was induced by 0.75 mM sodium nitroprusside (SNP) dehydrate, which produces NO. Cell viability was assessed by MTT assay, while apoptosis was evaluated by Hoechst 33342 and TUNEL staining. Intracellular reactive oxygen species (ROS) and glutathione levels were measured, and expression of p53 and caspase-3 were determined by Western blotting. To determine whether intraarticular injection of NAC prevents cartilage destruction in vivo, cartilage samples of an OA model were subjected to H&E, Safranin O, and TUNEL staining. NAC prevented NO-induced apoptosis, ROS overproduction, p53 up-regulation, and caspase-3 activation. The protective effects of NAC were significantly blocked by buthionine sulfoximine, a glutathione synthetase inhibitor, indicating that the apoptosis-preventing activity of NAC was mediated by glutathione. Using a rat model of experimentally induced OA, we found that NAC also significantly prevented cartilage destruction and chondrocyte apoptosis in vivo. These results indicate that NAC inhibits NO-induced apoptosis of chondrocytes through glutathione in vitro, and inhibits chondrocyte apoptosis and articular cartilage degeneration in vivo.

Asporin and transforming growth factor-beta gene expression in osteoblasts from subchondral bone and osteophytes in osteoarthritis.

BACKGROUND: To clarify the significance of subchondral bone and osteophytes in the pathology of osteoarthritis (OA), we investigated the expression of asporin (ASPN), transforming growth factor-beta1 (TGF-beta1), TGF-beta2, TGF-beta3, and runt-related transcription factor-2 (Runx2) genes involved in bone metabolism. METHODS: Osteoblasts were isolated from 19 patients diagnosed with knee OA and from 4 patients diagnosed with femoral neck fracture. Osteoblast expression of mRNA encoding ASPN, TGF-beta1, TGF-beta2, TGF-beta3, and Runx2 was analyzed using real-time RT-PCR. RESULTS: Expression of ASPN, TGF-beta1, and TGF-beta3 mRNA in the subchondral bone and osteophytes of OA patients increased compared with that of non-OA patients. The ratio of ASPN to TGF-beta1 mRNA in patients with severe cartilage damage was higher than that in patients with mild cartilage damage. CONCLUSIONS: The increased ratio of ASPN mRNA to TGF-beta1 mRNA in patients with severe relative to mild cartilage damage indicates that increased ASPN mRNA expression was significantly associated with the severity of cartilage degeneration. This finding suggests that ASPN may regulate TGF-beta1-mediated factors in the development of OA, which may provide clues as to the underlying pathology of OA.

Bone marrow angiotensin AT1 receptor regulates differentiation of monocyte lineage progenitors from hematopoietic stem cells.

BACKGROUND: The angiotensin II (Ang II) type 1 (AT(1)) receptor is expressed in bone marrow (BM) cells, whereas it remains poorly defined how Ang II regulates differentiation/proliferation of monocyte-lineage cells to exert proatherogenic actions. METHODS AND RESULTS: We generated BM chimeric apoE(-/-) mice repopulated with AT(1)-deficient (Agtr1(-/-)) or wild-type (Agtr1(+/+)) BM cells. The atherosclerotic development was significantly reduced in apoE(-/-)/BM-Agtr1(-/-) mice compared with apoE(-/-)/BM-Agtr1(+/+) mice, accompanied by decreased numbers of BM granulocyte/macrophage progenitors (GMP:c-Kit(+)Sca-1(-)Lin(-)CD34(+)CD16/32(+)) and peripheral blood monocytes. Macrophage-colony-stimulating factor (M-CSF)-induced differentiation from hematopoietic stem cells (HSCs:c-Kit(+)Sca-1(+)Lin(-)) to promonocytes (CD11b(high)Ly-6G(low)) was markedly reduced in HSCs from Agtr1(-/-) mice. The expression of M-CSF receptor c-Fms was decreased in HSCs/promonocytes from Agtr1(-/-) mice, accompanied by a marked inhibition in M-CSF-induced phosphorylation of PKC-delta and JAK2. c-Fms expression in HSCs/promonocytes was mainly regulated by TNF-alpha derived from BM CD45(-)CD34(-) stromal cells, and Ang II specifically regulated the TNF-alpha synthesis and release from BM stromal cells. CONCLUSIONS: Ang II regulates the expression of c-Fms in HSCs and monocyte-lineage cells through BM stromal cell-derived TNF-alpha to promote M-CSF-induced differentiation/proliferation of monocyte-lineage cells and contributes to the proatherogenic action.

Osteoblasts derived from osteophytes produce interleukin-6, interleukin-8, and matrix metalloproteinase-13 in osteoarthritis.

To clarify the significance of the osteophytes that appear during the progression of osteoarthritis (OA), we investigated the expression of inflammatory cytokines and proteases in osteoblasts from osteophytes. We also examined the influence of mechanical stress loading on osteoblasts on the expression of inflammatory cytokines and proteases. Osteoblasts were isolated from osteophytes in 19 patients diagnosed with knee OA and from subchondral bone in 4 patients diagnosed with femoral neck fracture. Messenger RNA expression and protein production of inflammatory cytokines and proteases were analyzed using real-time RT-PCR and ELISA, respectively. To examine the effects of mechanical loading, continuous hydrostatic pressure was applied to the osteoblasts. We determined the mRNA expression and protein production of IL-6, IL-8, and MMP-13, which are involved in the progression of OA, were increased in the osteophytes. Additionally, when OA pathological conditions were simulated by applying a nonphysiological mechanical stress load, the gene expression of IL-6 and IL-8 increased. Our results suggested that nonphysiological mechanical stress may induce the expression of biological factors in the osteophytes and is involved in OA progression. By controlling the expression of these genes in the osteophytes, the progression of cartilage degeneration in OA may be reduced, suggesting a new treatment strategy for OA.

Interleukin-27 activates natural killer cells and suppresses NK-resistant head and neck squamous cell carcinoma through inducing antibody-dependent cellular cytotoxicity.

Interleukin (IL)-27 is an IL-12 family cytokine playing a pivotal role in the induction of Th1 immune responses, although its action on natural killer (NK) cells has not been fully elucidated. Here, we show that IL-27 is capable of inducing phosphorylation of signal transducers and activators of transcription 1 and 3, as well as expression of T-bet and granzyme B in murine DX-5+ NK cells. IL-27 also enhances cytotoxic activity of NK cells both in vitro and in vivo, while the in vitro viability of NK cells is also improved by this cytokine. Therapeutic administration of the IL-27 gene drastically suppressed the growth of NK-unsusceptible SCCVII tumors that had been preestablished in syngenic mice, resulting in significant prolongation of the survival of the animals. This can likely be ascribed to the antibody-dependent cellular cytotoxicity machinery because IL-27 successfully induced tumor-specific IgG in the sera of the tumor-bearing mice, and supplementation of the sera enabled IL-27-activated NK cells to kill SCCVII cells in an Fcgamma receptor III-dependent manner. These findings strongly suggest that IL-27 may offer a powerful immunotherapeutic tool to eradicate head and neck squamous cell carcinoma and other poorly immunogenic neoplasms through activating NK cells and inducing tumor-specific immunoglobulin that may cooperatively elicit antibody-dependent cellular cytotoxicity activity.

Anxiolytic effect of aromatherapy massage in patients with breast cancer.

We examined how aromatherapy massage influenced psychologic and immunologic parameters in 12 breast cancer patients in an open semi-comparative trial. We compared the results 1 month before aromatherapy massage as a waiting control period with those during aromatherapy massage treatment and 1 month after the completion of aromatherapy sessions. The patients received a 30 min aromatherapy massage twice a week for 4 weeks (eight times in total). The results showed that anxiety was reduced in one 30 min aromatherapy massage in State-Trait Anxiety Inventory (STAI) test and also reduced in eight sequential aromatherapy massage sessions in the Hospital Anxiety and Depression Scale (HADS) test. Our results further suggested that aromatherapy massage ameliorated the immunologic state. Further investigations are required to confirm the anxiolytic effect of aromatherapy in breast cancer patients.

Comparison of anti-rheumatic effects of local RNAi-based therapy in collagen induced arthritis rats using various cytokine genes as molecular targets.

RNA interference (RNAi) provides a powerful means of sequence-specific gene silencing. Several studies show that RNAi may provide promising strategies to treat human diseases by suppressing disease responsible genes in vivo. In locomotor diseases, the progression of collagen-induced arthritis (CIA) is suppressed by tumor necrosis factor-alpha (TNF-alpha)-specific small interfering RNA (siRNA) delivered into the joint. The aim of this study, is to compare the effects of intraarticularly administered siRNAs targeting TNF-alpha, interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and receptor activator of NF-kappaB ligand (RANKL) on CIA in rats. We confirmed that the silencing effects of siRNA duplexes specific for rat TNF-alpha, IL-1beta, IL-6 and RANKL in vitro. Each siRNA was also delivered into the knee joint of CIA rats by the in vivo electroporation method 7, 10, 13 and 16 days after immunization with collagen. Local delivery of TNF-alpha or IL-1beta-specific siRNA ameliorated CIA in rats effectively at the gross morphological, radiographical and histological evaluations. Our results suggested that TNF-alpha and IL-1beta were the cytokines to be targeted in the joint for the treatment of rheumatoid arthritis. The in vivo siRNA transfection method may be useful for selection of target molecules to be silenced for treatment of joint diseases.

Enhanced expression of interleukin-6, matrix metalloproteinase-13, and receptor activator of NF-kappaB ligand in cells derived from osteoarthritic subchondral bone.

BACKGROUND: The aim of this study was to clarify the significance of subchondral bone in the pathology of osteoarthritis (OA) by investigating the expression of inflammatory cytokines, proteases, and receptor activator of NF-kappaB ligand (RANKL)/receptor activator of NF-kappaB (RANK)/osteoprotegerin (OPG) involved in cartilage degeneration. METHODS: Subchondral bone was obtained from 19 patients diagnosed with knee OA and 4 patients diagnosed with femoral neck fracture. Subchondral bone osteoblasts (SBOs) were isolated, and total RNA was extracted. Messenger RNA expression of inflammatory cytokines, proteases, and RANKL/RANK/OPG were analyzed using a real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Real-time RT-PCR showed that mRNA expressions of interleukin-6 (IL-6), matrix metalloproteinase-13 (MMP-13), and RANKL were significantly enhanced in OA SBOs compared to SBOs without OA. The expressions of these genes was greater in patients with severe cartilage damage than in those with mild cartilage damage. A high correlation between mRNA expression of IL-6 and that of MMP-13 was found in OA SBOs. CONCLUSION: The increases in IL-6, MMP-13, and RANKL expression in OA SBOs suggest that in subchondral bone OA progression involves abnormal osseous tissue remodeling, which induces mechanical property changes. Cartilage degeneration in OA may also be due, at least in part, to IL-6 and MMP-13 produced by SBOs. Comprehensive research on these pathological features may lead to the development of more effective therapies for OA by administration of molecules that affect bone remodeling and metabolism.

Levels of interleukin-18 are markedly increased in Helicobacter pylori-infected gastric mucosa among patients with specific IL18 genotypes.

BACKGROUND: The cellular immune response in gastric mucosa infected with Helicobacter pylori is proposed to be predominantly of the T helper cell type 1 type. METHODS: Interleukin (IL)-18, IL-12, and interferon (IFN)-gamma levels were measured in gastric mucosal biopsy specimens by reverse-transcription polymerase chain reaction (PCR) and by enzyme-linked immunosorbent assay; IL18 polymorphisms were determined by PCR. RESULTS: Biopsy specimens from 128 patients (56 with nonulcer dyspepsia, 28 with gastric ulcers, 28 with duodenal ulcers, and 16 with gastric cancer) were examined; 96 patients had H. pylori infection. IL-18 levels were markedly up-regulated in mucosa infected with H. pylori (P < .001), whereas IL-12 and IFN-gamma levels were independent of H. pylori status. IL-18 levels correlated with IFN-gamma levels only in infected patients (R = 0.31 to R = 0.51). IL-18 levels were the determining factor for monocyte infiltration in H. pylori-infected mucosa (P < .001). H. pylori-infected patients displaying IL18 -607C/C and -137G/G had higher IL-18 levels than did those with other genotypes and were more likely to experience treatment failure. CONCLUSION: H. pylori infection induces IL-18 in the gastric mucosa. H. pylori-infected patients with IL18 -607C/C and -137G/G have higher IL-18 levels, which causes severe gastric inflammation. IL18 genotype might be a marker for predicting the effects of eradication therapy.

Nanogel DDS enables sustained release of IL-12 for tumor immunotherapy.

For a valid cytokine immunotherapy of malignancies, a suitable delivery system that ensures slow-release of cytokines is required, because short half-life in vivo of the molecules ruins therapeutic efficacy while causing severe systemic toxic effects. We previously showed that the cholesterol-bearing pullulan (CHP)-based hydrogel nanoparticles, or nanogel, encapsulates, stabilizes and releases various molecules. Here we applied this nanogel to administration in vivo of interleukin-12 (IL-12). Recombinant murine IL-12 (rmIL-12) was successfully incorporated into CHP nanogel simply by incubated with CHP at room temperature. After subcutaneously injected into mice, the CHP/rmIL-12 complex led to a prolonged elevation in IL-12 concentration in the sera. Repetitive administrations of the CHP/rmIL-12, but not rmIL-12 alone, induced drastic growth retardation of preestablished subcutaneous fibrosarcoma without causing any serious toxic event. The present study proposes a novel therapeutic intervention technology, taking advantage of slow and sustained release of bioactive cytokines from the self-assembling biocompatible nanoparticles.