ABSTRACT
Alveolar bone is a mechanosensitive tissue that provides structural support for teeth. Alveolar bone loss is common with aging, menopause, tooth loss, and periodontitis and can lead to additional tooth loss, reduced denture fixation, and challenges in placing dental implants. The current studies suggest that sclerostin and DKK1, which are established osteocyte-derived inhibitors of bone formation, contribute to alveolar bone loss associated with estrogen ablation and edentulism in rats. Estrogen-deficient ovariectomized rats showed significant mandibular bone loss that was reversed by systemic administration of sclerostin antibody (SAB) alone and in combination with DKK1 antibody (DAB). Osteocytes in the dentate and edentulous rat maxilla expressed Sost (sclerostin) and Dkk1 (DKK1) mRNA, and molar extraction appeared to acutely increase DKK1 expression. In a chronic rat maxillary molar extraction model, systemic SAB administration augmented the volume and height of atrophic alveolar ridges, effects that were enhanced by coadministering DAB. SAB and SAB+DAB also fully reversed bone loss that developed in the opposing mandible as a result of hypo-occlusion. In both treatment studies, alveolar bone augmentation with SAB or SAB+DAB was accompanied by increased bone mass in the postcranial skeleton. Jaw bone biomechanics showed that intact sclerostin-deficient mice exhibited stronger and denser mandibles as compared with wild-type controls. These studies show that sclerostin inhibition, with and without DKK1 coinhibition, augmented alveolar bone volume and architecture in rats with alveolar bone loss. These noninvasive approaches may have utility for the conservative augmentation of alveolar bone.
Subject(s)
Alveolar Bone Loss/drug therapy , Alveolar Ridge Augmentation/methods , Bone Morphogenetic Proteins/pharmacology , Intercellular Signaling Peptides and Proteins/pharmacology , Absorptiometry, Photon , Alveolar Bone Loss/metabolism , Animals , Bone Morphogenetic Proteins/metabolism , Female , Genetic Markers , In Situ Hybridization , Intercellular Signaling Peptides and Proteins/metabolism , Male , Mice, Knockout , Ovariectomy , Phenotype , Rats , Rats, Sprague-Dawley , Tooth Extraction , X-Ray MicrotomographyABSTRACT
Whereas much is known regarding the musculoskeletal responses to full unloading, little is known about the physiological effects and response to pharmacological agents in partial unloading (e.g. Moon and Mars) environments. To address this, we used a previously developed ground-based model of partial weight-bearing (PWB) that allows chronic exposure to reduced weight-bearing in mice to determine the effects of murine sclerostin antibody (SclAbII) on bone microstructure and strength across different levels of mechanical unloading. We hypothesize that treatment with SclAbII would improve bone mass, microarchitecture and strength in all loading conditions, but that there would be a greater skeletal response in the normally loaded mice than in partially unloaded mice suggesting the importance of combined countermeasures for exploration-class long duration spaceflight missions. Eleven-week-old female mice were assigned to one of four loading groups: normal weight-bearing controls (CON) or weight-bearing at 20% (PWB20), 40% (PWB40) or 70% (PWB70) of normal. Mice in each group received either SclAbII (25mg/kg) or vehicle (VEH) via twice weekly subcutaneous injection for 3 weeks. In partially-unloaded VEH-treated groups, leg BMD decreased -5 to -10% in a load-dependent manner. SclAbII treatment completely inhibited bone deterioration due to PWB, with bone properties in SclAbII-treated groups being equal to or greater than those of CON, VEH-treated mice. SclAbII treatment increased leg BMD from +14 to +18% in the PWB groups and 30 ± 3% in CON (p< 0.0001 for all). Trabecular bone volume, assessed by µCT at the distal femur, was lower in all partially unloaded VEH-treated groups vs. CON-VEH (p< 0.05), and was 2-3 fold higher in SclAbII-treated groups (p< 0.001). Midshaft femoral strength was also significantly higher in SclAbII vs. VEH-groups in all-loading conditions. These results suggest that greater weight bearing leads to greater benefits of SclAbII on bone mass, particularly in the trabecular compartment. Altogether, these results demonstrate the efficacy of sclerostin antibody therapy in preventing astronaut bone loss during terrestrial solar system exploration.
Subject(s)
Antibodies, Monoclonal/administration & dosage , Bone Density/drug effects , Glycoproteins/antagonists & inhibitors , Muscle Weakness/drug therapy , Weight-Bearing/physiology , Adaptor Proteins, Signal Transducing , Animals , Biomechanical Phenomena , Female , Glycoproteins/immunology , Hindlimb Suspension , Intercellular Signaling Peptides and Proteins , Mice , Mice, Inbred C57BLABSTRACT
Inhibition of the receptor activator of NF-κB ligand (RANKL) is a novel therapeutic option in the treatment of osteoporosis and related diseases. The aim of this study was to evaluate bone metabolism and structure in pigs after RANKL inhibition. 12 growing pigs were assigned to 2 groups with 6 animals each. The OPG group received recombinant human OPG-Fc (5 mg/kg IV) at day 0, the control group was given 0.9% NaCl solution. Serum levels of OPG-Fc, calcium (Ca), phosphorus (P), and bone turnover markers were evaluated every 5 days, and pigs were euthanized on day 20. Serum OPG-Fc concentration peaked at day 5 and coincided with significantly decreased Ca, P, and bone turnover markers. By day 15, measureable OPG-Fc serum levels could only be detected in 2/6 animals. With OPG-Fc clearance starting at day 10, serum Ca and P concentrations were not different between the 2 groups. TRACP5b, P1CP, and BAP levels significantly decreased by 40-70% relative to vehicle controls in the OPG-Fc group between days 5 and 10, indicating that pharmacologic concentration of OPG-Fc led to systemic concomitant inhibition of bone formation and resorption in young growing pigs. Dual X-ray absorptiometry data derived from the proximal femur did not differ between the 2 groups. µCT analysis of selected bone sites demonstrated an OPG-Fc-induced improvement of specific bone architectural indices and bone mineralization.
Subject(s)
Bone Resorption , Bone and Bones/metabolism , Calcium/blood , Immunoglobulin Fc Fragments/pharmacology , Osteoprotegerin/pharmacology , Absorptiometry, Photon , Alkaline Phosphatase/metabolism , Animals , Biomarkers/metabolism , Bone and Bones/diagnostic imaging , Female , Humans , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/metabolism , Male , Osteoprotegerin/genetics , Osteoprotegerin/metabolism , Phosphorus/blood , RANK Ligand/antagonists & inhibitors , RANK Ligand/metabolism , SwineABSTRACT
Receptor activator for nuclear factor-kappa B ligand (RANKL) is an essential mediator of osteoclastogenesis. We hypothesized that administration of soluble RANKL to mice would result in high turnover and deleterious effects on both cortical and trabecular bone. For 10 days, 10-week-old C57BL/6J female mice (n = 12/group) were given twice-daily subcutaneous injections of human recombinant RANKL (0.4 or 2 mg/kg/day) or inert vehicle (VEH). Bone turnover was greatly accelerated by RANKL, as evidenced by the 49-84% greater levels of serum TRAP-5b (bone resorption marker) and 300-400% greater levels of serum alkaline phosphatase (bone formation marker). RANKL resulted in significantly greater endocortical bone erosion surface (79-83%) and periosteal bone formation rate (64-87%) vs. VEH. Microcomputed tomographic (microCT) analysis of the proximal tibia indicated a reduction in trabecular volume fraction (-84%) for both doses of RANKL. Cortical bone geometry and strength were also negatively influenced by RANKL. MicroCT analysis of the femoral diaphysis indicated significantly lower cortical bone volume (-10% to -13%) and greater cortical porosity (8-9%) relative to VEH. Biomechanical testing of the femur diaphysis revealed significantly lower maximum bending load (-19% to -25%) vs. VEH. Bone strength remained correlated with bone mass, independent of RANKL stimulation of bone turnover. These findings are consistent with the hypothesis that soluble RANKL could be an important etiologic factor in pathologic bone loss. RANKL also has potential utility as a model for studying the consequences of high bone turnover on bone quality and strength in animals.
Subject(s)
Bone Density/drug effects , Bone Remodeling/drug effects , Femur/drug effects , RANK Ligand/pharmacology , Tibia/drug effects , Acid Phosphatase/blood , Animals , Biomarkers/blood , Bone Density/physiology , Bone Remodeling/physiology , Compressive Strength/drug effects , Dose-Response Relationship, Drug , Female , Femur/diagnostic imaging , Femur/metabolism , Humans , Injections, Subcutaneous , Isoenzymes/blood , Mice , Mice, Inbred C57BL , Recombinant Proteins , Tartrate-Resistant Acid Phosphatase , Tibia/diagnostic imaging , Tibia/metabolism , Tomography, X-Ray ComputedABSTRACT
As anti-inflammatory treatments used in rheumatoid arthritis, such as glucocorticoids, often result in secondary detrimental effects on bone health, the objective of this study was to investigate the effects of oestrogen therapy (ET) on the development and activity of collagen-induced arthritis (CIA) in rats, with a focus on assessment of chondroprotective effects using biomarkers of type II collagen degradation. Forty female Lewis rats were allocated into four intervention groups: (i) control + vehicle; (ii) CIA + vehicle; (iii) CIA + ET; and (iv) CIA + prednisolone. During the 28-day intervention period we monitored body weight, time-point of disease onset, incidence of manifest disease and paw volume. Levels of the type II collagen degradation marker (CTX-II) were measured in serum. At euthanasia, hind paws were isolated, extracted for proteins and measured for the concentration of CTX-II. Matrix metalloproteinase (MMP) activity was evaluated using gelatinase zymography. Oestrogen treatment delayed the time-point of disease onset and reduced the incidence and degree of manifest immunoarthritis significantly, assessed by macroscopic evaluation of hind paw inflammation and paw volume. Measures of serum or tissue levels of CTX-II showed significantly reduced type II collagen degradation elicited by oestrogen treatment. In alignment, a decreased activity of MMP-2 and MMP-9 was found in the paw protein extracts. We have demonstrated that the anti-inflammatory effect of ET is linked to chondroprotective effects in an animal model of systemic immunoarthritis. As ET has positive rather than negative effects on bone health in contrast to prednisolone, these observations may be important for potential combination therapy.
Subject(s)
Arthritis, Experimental/prevention & control , Collagen Type II/metabolism , Estrogen Replacement Therapy , Animals , Anti-Inflammatory Agents/therapeutic use , Arthritis, Experimental/metabolism , Arthritis, Experimental/pathology , Biomarkers/metabolism , Body Weight/drug effects , Collagen Type I/metabolism , Disease Models, Animal , Female , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Peptides/metabolism , Prednisolone/therapeutic use , Rats , Rats, Inbred Lew , Treatment OutcomeABSTRACT
The antitumor efficiency of secondary lymphoid organ chemokine (SLC), a CC chemokine that chemoattracts both dendritic cells (DCs) and T lymphocytes,was evaluated in SV40 large T-antigen transgenic mice that develop bilateral multifocal pulmonary adenocarcinomas. Injection of recombinant SLC in the axillary lymph node region led to a marked reduction in tumor burden with extensive lymphocytic and DC infiltration of the tumors and enhanced survival. SLC injection led to significant increases in CD4 and CD8 lymphocytes as well as DC at the tumor sites, lymph nodes, and spleen. The cellular infiltrates were accompanied by the enhanced elaboration of Type 1 cytokines and the antiangiogenic chemokines IFN-gamma inducible protein 10, and monokine induced by IFN-gamma (MIG). In contrast, lymph node and tumor site production of the immunosuppressive cytokine transforming growth factor beta was decreased in response to SLC treatment. In vitro, after stimulation with irradiated autologous tumor, splenocytes from SLC-treated mice secreted significantly more IFN-gamma and granulocyte macrophage colony-stimulating factor, but reduced levels of interleukin 10. Significant reduction in tumor burden in a model in which tumors develop in an organ-specific manner provides a strong rationale for additional evaluation of SLC in regulation of tumor immunity and its use in lung cancer immunotherapy.
Subject(s)
Adenocarcinoma, Bronchiolo-Alveolar/drug therapy , Angiogenesis Inhibitors/pharmacology , Chemokines, CC/pharmacology , Lung Neoplasms/drug therapy , Adenocarcinoma, Bronchiolo-Alveolar/blood supply , Adenocarcinoma, Bronchiolo-Alveolar/genetics , Adenocarcinoma, Bronchiolo-Alveolar/immunology , Angiogenesis Inhibitors/metabolism , Animals , Antigens, Polyomavirus Transforming/genetics , Antigens, Polyomavirus Transforming/immunology , Chemokine CCL21 , Chemokines/metabolism , Cytokines/metabolism , Dendritic Cells/drug effects , Dendritic Cells/immunology , Disease Models, Animal , Endothelial Growth Factors/metabolism , Lung Neoplasms/blood supply , Lung Neoplasms/genetics , Lung Neoplasms/immunology , Lymph Nodes/cytology , Lymph Nodes/immunology , Lymphokines/metabolism , Mice , Mice, Transgenic , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/immunology , Neovascularization, Pathologic/metabolism , Recombinant Proteins/pharmacology , Spleen/cytology , Spleen/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Transforming Growth Factor beta/metabolism , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
Inducible costimulator (ICOS) and B7-related protein-1 (B7RP-1) constitute a receptor-ligand pair involved in T cell costimulation. In this study, the stimulatory effects of B7RP-1 on cellular and humoral immune responses were investigated giving mice a construct with the extracellular domain of murine B7RP-1 fused with human IgG1 Fc (B7RP-1-Fc). B7RP-1-Fc stimulated contact hypersensitivity (CH) given near either the time of sensitization or challenge with oxazolone. When given near challenge time, B7RP-1-Fc stimulated CH more than a construct containing the extracellular domain of murine B7.2 and Fc (B7.2-Fc). B7RP-1-Fc increased the number of cells in lymph nodes draining the skin sensitized with oxazolone, especially activated T cells. B7RP-1-Fc also increased the ability of the cells in these lymph nodes to induce CH when transfused into naive mice. B7RP-1-Fc stimulated the production of anti-keyhole limpet hemocyanin (KLH) Ab, increasing anti-KLH IgG, IgG2a, and IgE, whereas B7.2-Fc did not affect this production. B7RP-1-Fc also increased the number of cells in lymph nodes draining the skin immunized with KLH and their production of IFN-gamma, IL-4, and IL-10 in response to KLH. Finally, B7RP-1-Fc increased the presence of eosinophils in the bronchoalveolar lavage and lungs of mice sensitized and challenged with OVA so to mount an asthmatic reaction. B7RP-1-Fc stimulates both cellular and humoral immune responses in vivo by increasing number and function of T and B cells reacting to Ag exposure.
Subject(s)
Adjuvants, Immunologic/administration & dosage , B-Lymphocytes/immunology , B7-1 Antigen/immunology , Immunoconjugates , Immunoglobulin G/biosynthesis , T-Lymphocytes/immunology , Abatacept , Administration, Cutaneous , Animals , Antigens, CD/administration & dosage , Antigens, Differentiation/administration & dosage , Asthma/immunology , B7-1 Antigen/administration & dosage , B7-2 Antigen , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , CTLA-4 Antigen , Dermatitis, Contact/immunology , Dermatitis, Contact/pathology , Drug Administration Schedule , Drug Combinations , Female , Hemocyanins/administration & dosage , Hemocyanins/immunology , Humans , Immunoglobulin Fc Fragments/administration & dosage , Inducible T-Cell Co-Stimulator Ligand , Injections, Intraperitoneal , Lymph Nodes/immunology , Lymph Nodes/pathology , Lymphocyte Count , Membrane Glycoproteins/administration & dosage , Mice , Mice, Inbred BALB C , Oxazolone/administration & dosage , Oxazolone/immunology , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/immunology , Skin/immunology , Skin/pathology , Spleen/cytology , Spleen/immunologyABSTRACT
We and others recently reported tumor necrosis factor (TNF) and apoptosis ligand-related leukocyte-expressed ligand 1 (TALL-1) as a novel member of the TNF ligand family that is functionally involved in B cell proliferation. Transgenic mice overexpressing TALL-1 have severe B cell hyperplasia and lupus-like autoimmune disease. Here, we describe expression cloning of a cell surface receptor for TALL-1 from a human Burkitt's lymphoma RAJI cell library. The cloned receptor is identical to the previously reported TNF receptor (TNFR) homologue transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI). Murine TACI was subsequently isolated from the mouse B lymphoma A20 cells. Human and murine TACI share 54% identity overall. Human TACI exhibits high binding affinities to both human and murine TALL-1. Soluble TACI extracellular domain protein specifically blocks TALL-1-mediated B cell proliferation without affecting CD40- or lipopolysaccharide-mediated B cell proliferation in vitro. In addition, when injected into mice, soluble TACI inhibits antibody production to both T cell-dependent and -independent antigens. By yeast two-hybrid screening of a B cell library with TACI intracellular domain, we identified that, like many other TNFR family members, TACI intracellular domain interacts with TNFR-associated factor (TRAF)2, 5, and 6. Correspondingly, TACI activation in a B cell line results in nuclear factor kappaB and c-Jun NH(2)-terminal kinase activation. The identification and characterization of the receptor for TALL-1 provides useful information for the development of a treatment for B cell-mediated autoimmune diseases such as systemic lupus erythematosus.
Subject(s)
B-Lymphocytes/immunology , Membrane Proteins/physiology , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor/immunology , Tumor Necrosis Factor-alpha/physiology , Amino Acid Sequence , Animals , B-Cell Activating Factor , Burkitt Lymphoma , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Gene Library , Humans , Ligands , Lymphocyte Activation , Lymphoma, B-Cell , Mice , Molecular Sequence Data , Receptors, Tumor Necrosis Factor/chemistry , Sequence Alignment , Sequence Homology, Amino Acid , Transmembrane Activator and CAML Interactor Protein , Tumor Cells, CulturedABSTRACT
In this study, we show that Delta-9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, suppresses host immune reactivity against lung cancer. In two different weakly immunogenic murine lung cancer models, intermittent administration of THC (5 mg/kg, four times/wk i.p. for 4 wk) led to accelerated growth of tumor implants compared with treatment with diluent alone. In contrast to our findings in immunocompetent mice, THC did not affect tumor growth in tumor-bearing SCID mice. The immune inhibitory cytokines, IL-10 and TGF-beta, were augmented, while IFN-gamma was down-regulated at both the tumor site and in the spleens of THC-treated mice. Administration of either anti-IL-10- or anti-TGF-beta-neutralizing Abs prevented the THC-induced enhancement in tumor growth. Both APC and T cells from THC-treated mice showed limited capacities to generate alloreactivity. Furthermore, lymphocytes from THC-treated mice transferred the effect to normal mice, resulting in accelerated tumor growth similar to that seen in the THC-treated mice. THC decreased tumor immunogenicity, as indicated by the limited capacity for tumor-immunized, THC-treated mice to withstand tumor rechallenge. In vivo administration of a specific antagonist of the CB2 cannabinoid receptor also blocked the effects of THC. Our findings suggest the THC promotes tumor growth by inhibiting antitumor immunity by a CB2 receptor-mediated, cytokine-dependent pathway.
Subject(s)
Carcinoma, Lewis Lung/immunology , Cytokines/physiology , Dronabinol/pharmacology , Immunosuppressive Agents/pharmacology , Receptors, Drug/physiology , Adoptive Transfer , Animals , Antibodies, Monoclonal/administration & dosage , Antigen-Presenting Cells/immunology , Carcinoma, Lewis Lung/pathology , Carcinoma, Lewis Lung/prevention & control , Cell Division/drug effects , Cell Division/immunology , Dronabinol/antagonists & inhibitors , Dronabinol/metabolism , Growth Inhibitors/administration & dosage , Immunity, Innate/drug effects , Immunosuppressive Agents/antagonists & inhibitors , Immunosuppressive Agents/metabolism , Injections, Intraperitoneal , Interleukin-10/immunology , Lymphocyte Culture Test, Mixed , Lymphocyte Subsets/drug effects , Lymphocyte Subsets/immunology , Lymphocyte Subsets/metabolism , Lymphocyte Subsets/transplantation , Lymphocyte Transfusion , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, SCID , Neoplasm Transplantation , Receptors, Cannabinoid , Severe Combined Immunodeficiency/immunology , T-Lymphocytes/immunology , Transforming Growth Factor beta/immunology , Tumor Cells, CulturedABSTRACT
Secondary lymphoid tissue chemokine (SLC, also referred to as Exodus 2 or 6Ckine) is a recently identified high endothelial-derived CC chemokine. The ability of SLC to chemoattract both Th1 lymphocytes and dendritic cells formed the rationale to evaluate this chemokine in cancer immunotherapy. Intratumoral injection of recombinant SLC evidenced potent antitumor responses and led to complete tumor eradication in 40% of treated mice. SLC-mediated antitumor responses were lymphocyte dependent as evidenced by the fact that this therapy did not alter tumor growth in SCID mice. Studies performed in CD4 and CD8 knockout mice also revealed a requirement for both CD4 and CD8 lymphocyte subsets for SLC-mediated tumor regression. In immunocompetent mice, intratumoral SLC injection led to a significant increase in CD4 and CD8 T lymphocytes and dendritic cells, infiltrating both the tumor and the draining lymph nodes. These cell infiltrates were accompanied by the enhanced elaboration of Th1 cytokines and chemokines monokine induced by IFN-gamma and IFN-gamma-inducible protein 10 but a concomitant decrease in immunosuppressive cytokines at the tumor site. In response to irradiated autologous tumor, splenic and lymph node-derived cells from SLC-treated tumor-bearing mice secreted significantly more IFN-gamma, GM-CSF, and IL-12 and reduced levels of IL-10 than did diluent-treated tumor-bearing mice. After stimulation with irradiated autologous tumor, lymph node-derived lymphocytes from SLC-treated tumor-bearing mice demonstrated enhanced cytolytic capacity, suggesting the generation of systemic immune responses. These findings provide a strong rationale for further evaluation of SLC in tumor immunity and its use in cancer immunotherapy.
Subject(s)
Carcinoma, Lewis Lung/immunology , Chemokines, CC/immunology , Chemokines, CC/therapeutic use , Lung Neoplasms/immunology , T-Lymphocyte Subsets/immunology , Animals , Carcinoma, Lewis Lung/metabolism , Carcinoma, Lewis Lung/therapy , Chemokine CCL21 , Chemokine CXCL10 , Chemokine CXCL9 , Chemokines, CC/administration & dosage , Chemokines, CXC/biosynthesis , Cytokines/biosynthesis , Dendritic Cells/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/biosynthesis , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Injections, Intralesional , Interferon-gamma/biosynthesis , Interferon-gamma/metabolism , Interleukin-12/biosynthesis , Lung Neoplasms/metabolism , Lung Neoplasms/therapy , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, SCID , T-Lymphocyte Subsets/metabolism , Tumor Cells, CulturedABSTRACT
In two murine lung cancer models adenoviral interleukin 7-transduced dendritic cells (DC-AdIL-7) were administered intratumorally, resulting in complete tumor regression. Intratumoral DC-AdIL-7 therapy was as effective as DCs pulsed with specific tumor peptide antigens. Comparison with other intratumoral therapies including recombinant IL-7, AdIL-7 vector alone, unmodified DCs, IL-7-transduced fibroblasts, or DCs pulsed with tumor lysates revealed DC-AdIL-7 therapy to be superior in achieving antitumor responses and augmenting immunogenicity. Mice with complete tumor eradication as a result of either DC-AdIL-7 or AdIL-7 therapy were rechallenged with parental tumor cells 30 days or more after complete tumor eradication. All the DC-AdIL-7-treated mice completely rejected a secondary rechallenge, whereas the AdIL-7-treated mice had sustained antitumor effects in only 20-25% of the mice. DC-AdIL-7 therapy was more effective than AdIL-7 in achieving systemic antitumor responses and enhancing immunogenicity. After complete tumor eradication, those mice treated with DC-AdIL-7 evidenced significantly greater release of splenocyte GM-CSF and IFN-gamma than did controls or AdIL-7-treated mice. After intratumoral injection, gene-modified DCs trafficked from the tumor to lymph node sites and spleen. DCs were detected in nodal tissues for up to 7 days after intratumoral injection. We report that intratumoral DC-AdIL-7 leads to significant systemic immune responses and potent antitumor effects in murine lung cancer models.
Subject(s)
Adenoviridae/genetics , Dendritic Cells/immunology , Interleukin-7/genetics , Lung Neoplasms/therapy , Animals , Female , Genetic Therapy , Immunotherapy , Injections, Intralesional , Interleukin-7/administration & dosage , Lung Neoplasms/immunology , Lymph Nodes/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Neoplasm Transplantation , Remission Induction , Spleen/immunologyABSTRACT
We report that the tumor neurosis factor homolog APRIL (a proliferation-inducing ligand) stimulates in vitro proliferation of primary B and T cells and increases spleen weight due to accumulation of B cells in vivo. APRIL functions via binding to BCMA (B cell maturation antigen) and TACI (transmembrane activator and CAML-interactor) and competes with TALL-I (also called BLyS or BAFF) for receptor binding. Soluble BCMA and TACI specifically prevent binding of APRIL and block APRIL-stimulated proliferation of primary B cells. BCMA-Fc also inhibits production of antibodies against keyhole limpet hemocyanin and Pneumovax in mice, indicating that APRIL and/or TALL-I signaling via BCMA and/or TACI are required for generation of humoral immunity. Thus, APRIL-TALL-I and BCMA-TACI form a two ligands-two receptors pathway involved in stimulation of B and T cell function.
Subject(s)
Membrane Proteins/immunology , Neuropeptides/immunology , Nuclear Proteins/immunology , Receptors, Tumor Necrosis Factor/immunology , Tumor Necrosis Factor-alpha/immunology , Animals , Antibody Formation/immunology , Antigens/immunology , B-Cell Activating Factor , B-Cell Maturation Antigen , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Binding, Competitive , Cell Count , Female , Hemocyanins/immunology , Humans , Lymphocyte Activation/immunology , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Neuropeptides/antagonists & inhibitors , Neuropeptides/metabolism , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism , Organ Size/physiology , Receptors, Tumor Necrosis Factor/metabolism , Recombinant Fusion Proteins/immunology , Spleen/anatomy & histology , Spleen/cytology , Spleen/immunology , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Transmembrane Activator and CAML Interactor Protein , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Cyclooxygenase-2 (COX-2), the enzyme at the rate-limiting step of prostanoid production, has been found to be overexpressed in human lung cancer. To evaluate lung tumor COX-2 modulation of antitumor immunity, we studied the antitumor effect of specific genetic or pharmacological inhibition of COX-2 in a murine Lewis lung carcinoma (3LL) model. Inhibition of COX-2 led to marked lymphocytic infiltration of the tumor and reduced tumor growth. Treatment of mice with anti-PGE2 mAb replicated the growth reduction seen in tumor-bearing mice treated with COX-2 inhibitors. COX-2 inhibition was accompanied by a significant decrement in IL-10 and a concomitant restoration of IL-12 production by APCs. Because the COX-2 metabolite PGE2 is a potent inducer of IL-10, it was hypothesized that COX-2 inhibition led to antitumor responses by down-regulating production of this potent immunosuppressive cytokine. In support of this concept, transfer of IL-10 transgenic T lymphocytes that overexpress IL-10 under control of the IL-2 promoter reversed the COX-2 inhibitor-induced antitumor response. We conclude that abrogation of COX-2 expression promotes antitumor reactivity by restoring the balance of IL-10 and IL-12 in vivo.
Subject(s)
Carcinoma, Lewis Lung/enzymology , Carcinoma, Lewis Lung/immunology , Interleukin-10/biosynthesis , Interleukin-12/biosynthesis , Isoenzymes/pharmacology , Prostaglandin-Endoperoxide Synthases/pharmacology , Adjuvants, Immunologic/physiology , Animals , Carcinoma, Lewis Lung/metabolism , Carcinoma, Lewis Lung/prevention & control , Cyclooxygenase 2 , Dinoprostone/antagonists & inhibitors , Dinoprostone/biosynthesis , Dinoprostone/physiology , Down-Regulation/immunology , Enzyme Induction/immunology , Female , Interleukin-12/antagonists & inhibitors , Isoenzymes/biosynthesis , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Neoplasm Transplantation , Prostaglandin-Endoperoxide Synthases/biosynthesis , Spleen/cytology , Spleen/metabolism , Tumor Cells, CulturedABSTRACT
We have found previously that human lung cancers potently induce T lymphocyte IL-10 production in vitro. To assess the impact of enhanced T cell-derived IL-10 on antitumor immunity in vivo, we utilized transgenic mice expressing IL-10 under the control of the IL-2 promoter. We have shown previously that Lewis lung carcinoma cells (3LL) have more aggressive growth potential in IL-10 transgenic mice compared with control littermates. In this study, we show that transfer of T cells from IL-10 transgenic mice to control littermates transferred the IL-10 immunosuppressive effect and led to enhanced 3LL tumor growth. In addition to changes in T cell-mediated immunity, professional APC from IL-10 transgenic mice were found to have significantly suppressed capacity to induce MHC alloreactivity, CTL responses, and IL-12 production. Tumor Ag-pulsed dendritic cells from IL-10 transgenic mice also failed to generate antitumor reactivity. These results suggest that increased levels of T cell-derived IL-10 severely impair antitumor immunity in vivo, due to defects in both T cell and APC function.
Subject(s)
Antigen-Presenting Cells/immunology , Carcinoma, Lewis Lung/immunology , Carcinoma, Lewis Lung/pathology , Immunosuppressive Agents/pharmacology , Interleukin-10/physiology , T-Lymphocytes/immunology , Adoptive Transfer , Animals , Antigen-Presenting Cells/metabolism , Carcinoma, Lewis Lung/etiology , Carcinoma, Lewis Lung/genetics , Cell Division/immunology , Cytokines/biosynthesis , Cytokines/deficiency , Cytotoxicity, Immunologic/genetics , Dendritic Cells/immunology , Female , Humans , Interleukin-10/biosynthesis , Interleukin-10/genetics , Interleukin-12/biosynthesis , Interleukin-12/deficiency , Interleukin-12/genetics , Lymphocyte Culture Test, Mixed , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Transgenic , T-Lymphocytes/metabolism , T-Lymphocytes/transplantation , T-Lymphocytes, Cytotoxic/immunology , Th1 Cells/metabolism , Tumor Cells, CulturedABSTRACT
The overall T cell response to a multideterminant antigen consists of the sum of responses to a limited number of different determinants on the protein. Antigen-presenting cells (APCs) are crucial in delimiting the determinants on the protein to which a response will be mounted. This influence is apparent at two levels. First, the determinants that are generated and displayed by APCs in the thymus are pivotal in shaping the T cell repertoire that will be available for responding to antigen determinants in the periphery. Second, antigen processing affects the selection of determinants that become displayed by the various peripheral APC populations that are involved in inducing and promoting a T cell response. We have studied the effect of the display hierarchy on tolerance induction to individual determinants in transgenic mice expressing different serum levels of hen egg lysozyme. We have also analysed aspects of the processing machinery that contribute to shaping the hierarchy of determinant display on MHC class II molecules: proteolysis and reduction of disulfide bonds.
Subject(s)
Antigen-Presenting Cells/immunology , Epitopes , Immune Tolerance , T-Lymphocytes/immunology , Amino Acids, Diamino/immunology , Animals , Antigen Presentation , Disulfides/metabolism , Mice , Peptide Fragments/immunologyABSTRACT
Tumor-derived prostaglandin E2 (PGE2) modifies cytokine balance and inhibits host immunity. We hypothesized that a high level of PGE2 production by lung tumor cells is dependent on tumor cyclooxygenase (COX)-2 expression. We found that PGE2 production by A549 non-small cell lung cancer (NSCLC) cells was elevated up to 50-fold in response to interleukin (IL)-1beta. Reversal of IL-1beta-induced PGE2 production in A549 cells was achieved by specific pharmacological or antisense oligonucleotide inhibition of COX-2 activity or expression. In contrast, specific COX-1 inhibition was not effective. Consistent with these findings, IL-1beta induced COX-2 mRNA expression and protein production in A549 cells. Specific inhibition of COX-2 abrogated the capacity of IL-1beta-stimulated A549 cells to induce IL-10 in lymphocytes and macrophages. Furthermore, specific inhibition of A549 COX-2 reversed the tumor-derived PGE2-dependent inhibition of macrophage IL-12 production when whole blood was cultured in tumor supernatants. Our results indicate that lung tumor-derived PGE2 plays a pivotal role in promoting lymphocyte and macrophage IL-10 induction while simultaneously inhibiting macrophage IL-12 production. Immunohistochemistry of human NSCLC tissues obtained from lung cancer resection specimens revealed cytoplasmic staining for COX-2 within tumor cells. This is the first description of functional COX-2 expression by NSCLC cells and the definition of a pathway whereby tumor COX-2 expression and a high level of PGE2 production mediate profound alteration in cytokine balance in the lung cancer microenvironment.
Subject(s)
Carcinoma, Non-Small-Cell Lung/enzymology , Interleukin-10/metabolism , Interleukin-12/metabolism , Interleukin-1/pharmacology , Isoenzymes/metabolism , Lung Neoplasms/enzymology , Lymphocytes/metabolism , Macrophages/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Adenocarcinoma/enzymology , Carcinoma, Squamous Cell/enzymology , Cyclooxygenase 2 , Down-Regulation , Gene Expression Regulation, Neoplastic/drug effects , Humans , Immunologic Techniques , Isoenzymes/genetics , Lymphocytes/enzymology , Macrophages/enzymology , Membrane Proteins , Oligonucleotides, Antisense/pharmacology , Prostaglandin-Endoperoxide Synthases/genetics , Tumor Cells, Cultured , Up-RegulationABSTRACT
Lung cancer, the leading cause of cancer death in the United States, is resistant to most currently available therapies. To evaluate a multicomponent gene therapy approach that replaces tumor-bearing host immune deficits, we genetically modified Line 1 (L1C2), a weakly immunogenic alveolar cell carcinoma cell line. L1C2 was transduced ex vivo with a retroviral construct that contained two components: a cytokine gene (granulocyte-macrophage colony-stimulating factor) and a drug sensitivity gene (herpes simplex virus thymidine kinase). The third component of this therapy, in vitro-activated syngeneic bone marrow-derived dendritic cells, was included to augment antigen presentation. The addition of ganciclovir (GCV) caused the lysis of transduced tumor cells, resulting in the release of potential tumor antigens. Ex vivo-transduced tumor cells regressed in vivo following GCV therapy but were not effective in the treatment of established parental tumors. To treat established tumors, dendritic cells were administered in combination with transduced tumor cells and GCV. A total of 50% of these mice rejected the 5-day-old established tumors and were immune to rechallenge with parental L1C2 cells. Thus, this multicomponent gene therapy system leads to both the regression of established tumors and enhanced immunogenicity in this weakly immunogenic murine lung cancer model.
Subject(s)
Adenocarcinoma, Bronchiolo-Alveolar/therapy , Dendritic Cells/immunology , Dendritic Cells/metabolism , Genetic Therapy , Granulocyte-Macrophage Colony-Stimulating Factor/therapeutic use , Lung Neoplasms/therapy , Simplexvirus/enzymology , Thymidine Kinase/therapeutic use , Adenocarcinoma, Bronchiolo-Alveolar/immunology , Animals , Female , Ganciclovir/therapeutic use , Genetic Vectors , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Interferon-gamma/biosynthesis , Lung Neoplasms/immunology , Lymphocytes/immunology , Mice , Mice, Inbred BALB C , Mice, SCID , Neoplasm Transplantation , Simplexvirus/genetics , Thymidine Kinase/metabolism , Transduction, Genetic , Tumor Cells, CulturedABSTRACT
Multiple antitumor modalities may be necessary to overcome lung tumor-mediated immunosuppression and effectively treat non-small cell lung cancer (NSCLC). To evaluate a multimodality gene therapy approach for control of local tumor growth, a weakly immunogenic murine alveolar cell carcinoma, L1C2, was transduced with either the interleukin-7/hygromycin-herpes simplex thymidine kinase (IL-7/HyHSVtk) internal ribosome entry site (IRES) retroviral vector or a vector containing the HyHSVtk, but not the IL-7 gene. Of the many cytokines available for gene transfer, IL-7 was chosen for these studies because it both stimulates CTL responses and down-regulates tumor production of the immunosuppressive peptide TGF-beta. Following selection in hygromycin, IL-7 transduction was confirmed by ELISA. Clones produced 1.25 to 10 ng of IL-7/ml/10(6) cells per 24 h. In vitro, genetically modified tumor cells were significantly more sensitive to ganciclovir (GCV) than unmodified parental tumor cells. The in vivo growth of ex vivo modified L1C2 cells was evaluated. There was a dose-response relationship between the amount of IL-7 secreted in vitro and the growth of genetically modified murine tumor in vivo. Transduced tumor cells regressed in mice following GCV therapy. Although ex vivo gene modification of tumor cells led to complete resolution of the tumor following implantation in vivo, IL-7 and HSVtk gene modified tumor cells were not effective in treating established parental tumors. However when 5 x 10(5) bone marrow-derived, in vitro activated dendritic cells (DC) were administered in combination with transduced tumor and GCV, 5 day old established tumors were eradicated in 80% of mice. These studies suggest that multicomponent vaccines may facilitate improved host responses by replacing host immune deficits and thus could have a role in adjuvant therapy and local control of NSCLC.
Subject(s)
Carcinoma, Non-Small-Cell Lung/therapy , Genetic Therapy/methods , Interleukin-7/genetics , Lung Neoplasms/therapy , Transfection/methods , Vaccines, DNA , Animals , Antimetabolites/therapeutic use , Carcinoma, Non-Small-Cell Lung/immunology , Dendritic Cells/immunology , Ganciclovir/therapeutic use , Lung Neoplasms/immunology , Mice , Mice, Inbred BALB C , Simplexvirus/enzymology , Thymidine Kinase/genetics , Tumor Cells, CulturedABSTRACT
The effects of low doses of environmental ionizing radiation on reproductive functions of seven generations of CC57W/Mv mice from Chernobyl experimental population has been investigated. A decrease in the number of litters per female during reproductive period, the number of newborn animals per litter, and an increase in the postnatal mortality during the first month of life were shown. These effects are due to the stable disorders of parental reproductive system and the high level of in-vivo preimplantation deaths in F1--F7 generations of CC57W/Mv mice from the Chernobyl experimental populations.
Subject(s)
Mice, Inbred C57BL/physiology , Power Plants , Radioactive Hazard Release , Reproduction/radiation effects , Animals , Dose-Response Relationship, Radiation , Female , Fetal Death/etiology , Genome , Litter Size/radiation effects , Male , Mice , Mice, Inbred C57BL/genetics , Time Factors , UkraineABSTRACT
Frequency of reversions to wild type varies for thi and its derivatives from 10(-2) to 10(-5). A broad spectrum of mutations arises in thi alleles and thi+ revertants. Among them there are r, w, Bd and new mutations causing scalloped wings with thickened veins in all the autosomes. In the offspring of a Cy/thi 1 male a fly is found carrying a thi 1 allele in the Cy chromosome; this is not caused by crossing-over, but probably by a transposition. Mutations in the thi, Bd strains and their derivatives are supposed to be caused by small insertion. The nature of these insertions are under discussion.