OBJECTIVETo explore the formation of pre-metastatic niche in the mouse lung and to study the underlying molecular mechanisms whereby primary breast carcinoma-derived factors mediate recruitment of bone marrow-derived cells (BMDCs) and affect the formation of pre-metastatic lung environment before the arrival of tumor cells.

METHODSMammary carcinoma 4T1 cells were inoculated into the mammary gland to construct mouse model of breast cancer. Confocal microscopy was used to detect the recruitment of BMDCs in the pre-metastatic lungs. The expression of factors in the mouse sera and 4T1 cell culture media was assayed using RayBio Custom mouse cytokine antibody array kit. The mice were injected daily with recombinant VEGF for 7 consecutive days to observe the effect of VEGF on BMDCs recruitment in the mouse lung.

RESULTSNo BMDCs were observed in the lungs of control and 4T1-tumor-bearing mice on day 0. On day 7 and 14, clusters of BMDCs observed in the lungs of 4T1-tumor-bearing mice were 8.7±2.2/objective field and 48.8±3.2/objective field, respectively, significantly higher than those in the control mice (1.1±0.8/objective field and 3.1±1.7/objective field) (P<0.05 for both). Confocal microscopic observation found that metastatic breast cancer cells preferentially facilitate BMDCs recruitment sites in the pre-metastatic mouse lungs. The levels of VEGF, GM-CSF, and IL-6 in the serum of 4T1-tumor-bearing mice were significantly increased compared with those in the control group (P<0.05 for all). However, VEGF was detected only in the culture media of 4T1 cells. The amount of BMDCs in the mouse lung tissue was (22.8±3.6)/objective field in the VEGF group and (3.1±0.4)/objective field in the control group (P<0.05). There were 36.8±5.4 metastatic foci in the lung tissue of VEGF group and 12.6±2.2 in the control group (P<0.05).

CONCLUSIONSThe results of this study demonstrate that primary breast cancer cells can alter the lung microenvironment during the pre-metastatic phase and induce the formation of pre-metastatic niche. Primary tumor cell-derived VEGF may be a crucial factor responsible for the formation of pre-metastatic niche.

Animals ; Bone Marrow Cells ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Disease Models, Animal ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; blood ; Humans ; Interleukin-6 ; blood ; Lung ; pathology ; Lung Neoplasms ; secondary ; Mice ; Recombinant Proteins ; administration & dosage ; Time Factors ; Tumor Microenvironment ; Vascular Endothelial Growth Factor A ; administration & dosage ; physiology ; secretion

The intestinal immune system maintains oral tolerance to harmless antigens or nutrients. One mechanism of oral tolerance is mediated by regulatory T cell (Treg)s, of which differentiation is regulated by a subset of dendritic cell (DC)s, primarily CD103+ DCs. The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, plays an important role in regulating immunity. The intestines are exposed to various AhR ligands, including endogenous metabolites and phytochemicals. It was previously reported that AhR activation induced tolerogenic DCs in mice or in cultures of bone marrow-derived DCs. However, given the variety of tolerogenic DCs, which type of tolerogenic DCs is regulated by AhR remains unknown. In this study, we found that AhR ligand 3,3'-diindolylmethane (DIM) inhibited the development of CD103+ DCs from mouse bone marrow cells stimulated with Flt3L and GM-CSF. DIM interfered with phosphorylation of STAT3 and STAT5 inhibiting the expression of genes, including Id2, E2-2, IDO-1, and Aldh1a2, which are associated with DC differentiation and functions. Finally, DIM suppressed the ability of CD103+ DCs to induce Foxp3+ Tregs.
Animals ; Bone Marrow Cells ; Dendritic Cells* ; Granulocyte-Macrophage Colony-Stimulating Factor ; Immune System ; Intestines ; Ligands ; Mice ; Phosphorylation* ; Phytochemicals ; Receptors, Aryl Hydrocarbon ; Transcription Factors

BACKGROUND AND OBJECTIVES: Glucocorticoids are a potent anti-inflammatory agent. The au-thors conducted this study to investigate the effect of Omnaris(R) on suppression of inflammation induction and mucin gene expression in nasal polyp epithelial cells. SUBJECTS AND METHOD: Primary nasal polyp epithelial cells were stimulated by 5 ug/mL of streptococcal enterotoxin B (SEB) and lipopolysaccharide (LPS). To determine the effects of Omnaris(R), cells were pretreated with 200, 100, 10, 1 ng/mL of Omnaris(R). The anti-inflammatory effect of epithelial cells were confirmed by measuring interleukin (IL)-6, IL-8, and granulocyte-macrophage colony stimulating factor (GM-CSF), and mucin gene expressions were determined by real time PCR for MUC4, MUC5AC, MUC5B, MUC8. RESULTS: SEB and LPS enhanced the production of IL-6, IL-8, and GM-CSF from nasal polyp epithelial cells. The increased cytokine levels were significantly suppressed by Omnaris(R) at 100 and 10 ng/mL. The expressions of MUC4, MUC5AC, MUC5B, MUC8 mRNA, and MUC4 mRNA were increased by SEB and LPS, respectively. The increased expression of these mucin genes were significantly suppressed by 100, 10, and 1 ng/mL of Omnaris(R). CONCLUSION: Omnaris(R) significantly suppressed the production of chemical mediators and mucin gene expression, which indicated that Omnaris(R) is effective in improving and treating inflammatory diseases in the nasal cavity.
Colony-Stimulating Factors ; Enterotoxins ; Epithelial Cells* ; Gene Expression* ; Glucocorticoids ; Granulocyte-Macrophage Colony-Stimulating Factor ; Inflammation ; Interleukin-6 ; Interleukin-8 ; Interleukins ; Mucins* ; Nasal Cavity ; Nasal Polyps* ; Real-Time Polymerase Chain Reaction ; RNA, Messenger

BACKGROUND AND OBJECTIVES: The nasal epithelium is the first barrier encountered by airborne allergens and is an active participant in airway inflammation. The aim of this study was to determine the activation mechanism of nasal epithelial cells with Alternaria and the effect of rhinovirus on the Alternaria induced activation of nasal epithelial cells. MATERIALS AND METHODS: Cultured epithelial cells were stimulated by Alternaria with or without rhinovirus-16 (RV-16) infection. Release of interleukin (IL)-6, IL-8, and granulocyte macrophage colony-stimulating factor (GM-CSF) into culture supernatants were measured to determine the activation of epithelial cells. Nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) of the epithelial cells were analyzed using western blot analysis. Intracellular NF-kappaB and AP-1 activity were evaluated by enzyme-linked immunosorbent assay. To determine the epithelial cell activation mechanism, cytokine production was inhibited with NF-kB, AP-1, and mitogen activated protein kinase (MAPK) inhibitors. RESULTS: Exposure of epithelial cells to Alternaria enhanced the production of cytokines. Intracellular NF-kB expression and activity were significantly increased by Alternaria, but not by RV-16. AP-1 expression and activity were not influenced by Alternaria. Increased IL-6 production was significantly inhibited by transcription factor inhibitors. However, IL-8 and GM-CSF production were not inhibited by these transcription factor inhibitors. CONCLUSIONS: Our in-vitro results demonstrate that Alternaria activates nasal polyp epithelial cells via NF-kB pathway and that NF-kB, AP-1, and MAPK are involved in the production of IL-6.
Allergens ; Alternaria ; Blotting, Western ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; Epithelial Cells ; Granulocyte-Macrophage Colony-Stimulating Factor ; Granulocytes ; Inflammation ; Interleukin-6 ; Interleukin-8 ; Interleukins ; Macrophage Colony-Stimulating Factor ; Nasal Mucosa ; Nasal Polyps ; NF-kappa B ; Protein Kinases ; Rhinovirus ; Transcription Factor AP-1 ; Transcription Factors

Pulmonary alveolar proteinosis (PAP) is a rare condition that is treated using whole lung lavage. A recent study suggested that granulocyte-macrophage colony stimulating factor (GM-CSF) plays roles in both the pathogenesis and treatment of PAP. We present a 69-year-old man with PAP who deteriorated despite bilateral whole lung lavage; that said, his symptoms, chest X-ray findings, and pulmonary function test improved after GM-CSF inhalation therapy over 12 months. GM-CSF therapy is an effective treatment modality for PAP.
Aged ; Bronchoalveolar Lavage ; Colony-Stimulating Factors ; Granulocyte-Macrophage Colony-Stimulating Factor ; Humans ; Inhalation ; Lung ; Pulmonary Alveolar Proteinosis ; Respiratory Function Tests ; Respiratory Therapy ; Thorax

Idiopathic pulmonary alveolar proteinosis (PAP) is a rare disorder characterized by surfactant component accumulation in the alveolar space. Idiopathic PAP has recently been recognized as a autoimmune disease of impaired alveolar macrophage function caused by autoantibodies against granulocyte-macrophage colony-stimulating factor (GM-CSF). While whole lung lavage has been the standard treatment, not every patient shows a complete response. Subcutaneous injection or inhalation of GM-CSF is another promising treatment option for PAP. A 45-year-old patient visited our hospital for dyspnea, he was diagnosed as PAP and underwent whole lung lavage. Eighteen months later, the patient had not achieved complete remission in despite of initial response. After then he was administered with GM-CSF (5 microgram/kg/day, subcutaneous injection) for fivetimes a week during 2 months. Nine months later, the abnormal shadows in high-resolution computed tomography (HRCT) decreased and the patient fully recovered in forced vital capacity. After 60 months, the HRCT scan showed complete remission of PAP.
Autoantibodies ; Autoimmune Diseases ; Bronchoalveolar Lavage ; Colony-Stimulating Factors ; Dyspnea ; Granulocyte-Macrophage Colony-Stimulating Factor ; Humans ; Inhalation ; Injections, Subcutaneous ; Lung ; Macrophages, Alveolar ; Middle Aged ; Pulmonary Alveolar Proteinosis ; Vital Capacity

PURPOSE: Gastric cancer has the highest incidence rate among cancers in Asia. The advanced type of signet ring cell carcinoma has poor prognosis compared to other types of gastric cancer. The immuno-gene therapy with cytokine-based tumor vaccines has not yet been investigated for gastric cancer. The granulocyte macrophage colony-stimulating factor (GM-CSF)-based tumor vaccine has been demonstrated as the most potent stimulator for specific and long-lasting systemic tumor immunity. MATERIALS AND METHODS: In the present study, KATO III cells, the human signet ring cell gastric carcinoma cell line, were genetically modified by the transduction with the human GM-CSF cDNA or the modified hGM-CSF in replication-deficient retroviruses. The genomic integrations and mRNA expressions of the transgenes were determined by Southern and Northern blot analyses. RESULTS: Wild type (wt) or modified hGM-CSF was integrated into the genome of KATO III cells. The modified hGM-CSF mRNA was more stable than that of wt. The KATO III cells with the modified hGM-CSF produced higher level of hGM-CSF (12.4-19 ng/10(6)cells/48hrs) than that with wt hGM-CSF, when determined by enzyme-linked immunosorbent assay (ELISA). The secreted recombinant hGM-CSF could support the proliferation of the GM-CSF-dependent cell line, indicating that the hGM-CSF secreted by the transduced KATO III cells has biological activities. Irradiated, transduced KATO III cells continued to secret hGM-CSF without proliferation. CONCLUSION: Our results suggest that GM-CSF secreting KATO III cells could be tested for the treatment of gastric cancer as an allogeneic tumor vaccine as a part of immunotherapeutic treatment.
Base Sequence ; Blotting, Northern ; Blotting, Southern ; Cell Line, Tumor ; Enzyme-Linked Immunosorbent Assay ; Granulocyte Macrophage Colony-Stimulating Factors, ; Humans ; Mutagenesis ; RNA, Messenger/genetics/metabolism ; Recombinant Proteins/metabolism/*secretion ; Stomach Neoplasms/genetics/metabolism/pathology ; Transduction, Genetic

Mesenchymal stem cells (MSC) are multipotent in nature and believed to facilitate the engraftment of hematopoietic stem cells (HSC) when transplanted simultaneously in animal studies and even in human trials. In this study, we transfected culture-expanded MSC with granulocyte macrophage-colony stimulating factor (GMCSF) and stem cell factor (SCF) cytokine genes and then cotransplanted with mononuclear cells (MNC) to further promote HSC engraftment. MNC were harvested from cord blood and seeded in long-term culture for ex vivo MSC expansion. A total of 1 x 10(7) MNC plus MSC/microliter were introduced to the tail vein of nonobese diabetic/severe combined immunodeficiency mice. After 6-8 weeks later, homing and engraftment of human cells were determined by flow cytometry and fluorescence in situ hybridization studies. The total nucleated cell count and the engraftment of CD45+/CD34+ cells and XX or XY positive human cells were significantly increased in cotransplanted mice and even higher with the cytokine gene-transfected MSC (GM-CSF>SCF, p<0.05) than in transplantation of MNC alone. These results suggest that MSC transfected with hematopoietic growth factor genes are capable of enhancing the hematopoietic engraftment. Delivering genes involved in homing and cell adhesions, CXCR4 or VLA, would further increase the efficiency of stem cell transplantation in the future.
Transfection/*methods ; Stem Cell Factor/genetics/*metabolism ; Mice, SCID ; Mice ; Mesenchymal Stem Cells/*metabolism ; Mesenchymal Stem Cell Transplantation/*methods ; Hematopoietic Stem Cell Transplantation/*methods ; Granulocyte Macrophage Colony-Stimulating Factors, Recombinant/*metabolism ; Graft Survival/*immunology ; Genetic Enhancement/methods ; Animals

PURPOSE: To evaluate the effects of autologous tumor vaccine alone or in combination with dendritic cell vaccines, as a method of stimulating antigen-presenting cells in patients with a locoregionally confined renal cell carcinoma (RCC) or metastatic disease. MATERIALS AND METHODS: Twenty-seven patients with RCC pathological stages II to IV were treated with autologous tumor cell vaccine, either with or without dendritic cell vaccine. Interleukin 2 (IL-2) based immunotherapy was also applied to the patients with metastatic disease. Immunomagnetic beads were used to isolate CD14+ monocytes from patient or donor in dendritic cell preparations. IL-4 and granulocyte-macrophage colony stimulating factor (GM-CSF) were used for maturation of dendritic cells. Flow cytometry evaluations were performed for dendritic cell maturation and changes in the immunological profiles following our treatment. RESULTS: Both the isolation of CD14+ monocyte, using Immunomagnetic beads, and the maturation of dendritic cells, using IL-4 and GM-CSF stimulation, were effective. Tumor immunological profiles showed increased CD3 and CD56 populations after treatment. Side effects related with vaccine were minimal and tolerable. Patients were stratified by the purpose for the vaccination; 8 patients for post-nephrectomy adjuvant therapy and 19 for adjuvant immunotherapy of a metastatic disease. All 8 patients in the former showed a disease free state, while only one of the 19 in the latter group remained in complete remission, while 6 showed short-term responses. CONCLISIONS: Autologous RCC vaccine, combined with or without dendritic cell vaccine, might be effective in the suppression of tumor recurrence in locoregionally confined RCC, although a longer follow-up will be required. These vaccines should be further developed to reach their therapeutic purpose in metastatic RCC.
Antigen-Presenting Cells ; Carcinoma, Renal Cell* ; Colony-Stimulating Factors ; Dendritic Cells* ; Flow Cytometry ; Follow-Up Studies ; Granulocyte-Macrophage Colony-Stimulating Factor ; Humans ; Immunotherapy ; Interleukin-2 ; Interleukin-4 ; Monocytes ; Recurrence ; Tissue Donors ; Vaccination ; Vaccines*

BACKGROUND AND OBJECTIVES: Airway epithelial cells contribute to the pathogenesis of air disease by their interaction with inhalant pathogenic extracts. Airborne fungi interact with nasal epithelial cell and enhance the production of inflammatory cytokines. Glucocorticosteroids (GCs) have been used therapeutically for nasal polyps and allergic disease with potent anti-inflammatory effects. The purpose of this study was to investigate the inhibitory effect of GCs on fungi induced nasal epithelial cell activation. MATERIALS AND METHODS: The epithelial cells of nasal polyps were obtained from patients and stimulated with Alternaria. To evaluate the anti-inflammatory effects of GCs, Alternaria was pretreated with GCs (triamcinolone, dexamethasone, and budesonide) and cultured with epithelial cells. Interleukin-8 (IL-8) and granulocyte-macrophage colony stimulating factor (GM-CSF) were measured to determine the activation of epithelial cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) test for protease-activated receptors (PARs) mRNA expression in nasal epithelial cells were performed. RESULTS: Alternaria enhanced the production of IL-8 and GM-CSF from nasal epithelial cells. GCs inhibited the activation of nasal epithelial cells, but the PAR2 and PAR3 mRNA expression were not suppressed by GCs. CONCLUSION: These data suggest that GCs inhibit the production of chemical mediators by Alternaria, but anti-inflammatory effect of GCs are not associated with PARs.
Adrenal Cortex Hormones* ; Alternaria ; Colony-Stimulating Factors ; Cytokines ; Dexamethasone ; Epithelial Cells* ; Fungi ; Granulocyte-Macrophage Colony-Stimulating Factor ; Humans ; Interleukin-8 ; Nasal Polyps* ; Receptors, Proteinase-Activated ; RNA, Messenger