ABSTRACT
Acromegaly is a disease caused by the oversecretion of growth hormone. It is currently treated by intravenous injection with cyclic peptide drugs that activate somatostatin receptor subtype 2 (SSTR2). Here, novel nonpeptidic, small-molecule, and orally active SSTR2 agonists were identified from a hit compound (13). Pharmacophore studies enabled scaffold hopping to obtain a unique 3,4,5-trisubstituted pyridine motif. Further optimization conferred potent SSTR2 agonistic activity and metabolic stability. Several compounds were evaluated and these showed good oral pharmacokinetic profiles in rats, and one representative compound (25) showed highly potent inhibition of growth hormone secretion induced by growth hormone-releasing hormone in rats. Based on these results, 25 was identified as a promising lead for further optimization. A structure-activity relationship (SAR) study and the metabolic stability data for this compound are also described.
Subject(s)
Acromegaly , Acromegaly/drug therapy , Animals , Growth Hormone , Rats , Receptors, Somatostatin/agonists , Somatostatin , Structure-Activity RelationshipABSTRACT
Scaffold hopping from the amide group of lead compound ONO-7300243 (1) to a secondary alcohol successfully gave a novel chemotype lysophosphatidic acid receptor 1 (LPA1) antagonist 4. Wash-out experiments using rat isolated urethra showed that compound 4 possesses a tight binding feature to the LPA1 receptor. Further modification of two phenyl groups of 1 to pyrrole and an indane moiety afforded an optimized compound ONO-0300302 (19). Despite its high i.v. clearance, 19 inhibited significantly an LPA-induced increase of intraurethral pressure (IUP) in rat (3 mg/kg, p.o.) and dog (1 mg/kg, p.o.) over 12 h. Binding experiments with [3H]-ONO-0300302 suggest that the observed long duration action is because of the slow tight binding character of 19.
ABSTRACT
Lysophosphatidic acid (LPA) evokes various physiological responses through a series of G protein-coupled receptors known as LPA1-6. A high throughput screen against LPA1 gave compound 7a as a hit. The subsequent optimization of 7a led to ONO-7300243 (17a) as a novel, potent LPA1 antagonist, which showed good efficacy in vivo. The oral dosing of 17a at 30 mg/kg led to reduced intraurethral pressure in rats. Notably, this compound was equal in potency to the α1 adrenoceptor antagonist tamsulosin, which is used in clinical practice to treat dysuria with benign prostatic hyperplasia (BPH). In contrast to tamsulosin, compound 17a had no impact on the mean blood pressure at this dose. These results suggest that LPA1 antagonists could be used to treat BPH without affecting the blood pressure. Herein, we report the hit-to-lead optimization of a unique series of LPA1 antagonists and their in vivo efficacy.
ABSTRACT
We present a case of a 62-year-old man who underwent total hip arthroplasty for treatment of pathologic femoral neck fracture associated with adefovir dipivoxil-induced osteomalacia. He had a 13-month history of bone pain involving his shoulders, hips, and knee. He received adefovir dipivoxil for treatment of lamivudine-resistant hepatitis B virus infection for 5 years before the occurrence of femoral neck fracture. Orthopedic surgeons should be aware of osteomalacia and pathological hip fracture caused by drug-induced renal dysfunction, which results in Fanconi's syndrome. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1600344696739249.
Subject(s)
Adenine/analogs & derivatives , Antiviral Agents/adverse effects , Fanconi Syndrome/chemically induced , Femoral Neck Fractures/chemically induced , Hepatitis B, Chronic/drug therapy , Organophosphonates/adverse effects , Osteomalacia/chemically induced , Adenine/adverse effects , Arthroplasty, Replacement, Hip , Femoral Neck Fractures/diagnostic imaging , Femoral Neck Fractures/surgery , Hepatitis B, Chronic/diagnosis , Humans , Magnetic Resonance Imaging , Male , Middle Aged , Predictive Value of Tests , Radiography , Radiopharmaceuticals , Treatment Outcome , Whole Body ImagingABSTRACT
To identify new cost-effective prostaglandin D2 (DP) receptor antagonists, a series of novel 3-benzoylaminophenylacetic acids were synthesized and biologically evaluated. Among those tested, some representative compounds were found to be orally available. Receptor selectivity and rat PK profiles were also evaluated. The structure-activity relationship (SAR) study is presented.
Subject(s)
Benzamides/pharmacology , Drug Design , Phenylacetates/pharmacology , Receptors, Immunologic/antagonists & inhibitors , Receptors, Prostaglandin/antagonists & inhibitors , Animals , Benzamides/chemical synthesis , Benzamides/chemistry , Binding, Competitive/drug effects , CHO Cells , Cricetinae , Dose-Response Relationship, Drug , Male , Molecular Conformation , Phenylacetates/chemical synthesis , Phenylacetates/chemistry , Rats , Stereoisomerism , Structure-Activity Relationship , Tissue DistributionABSTRACT
A series of novel N-acylsulfonamide analogs were synthesized and evaluated for their binding affinity and antagonist activity for the EP3 receptor subtype. Representative compounds were also evaluated for their inhibitory effect on PGE(2)-induced uterine contraction in pregnant rats. Among those tested, a series of N-acylbenzenesulfonamide analogs were found to be more potent than the corresponding carboxylic acid analogs in both the in vitro and in vivo evaluations. The structure activity relationships (SAR) are also discussed.
Subject(s)
Receptors, Prostaglandin E/antagonists & inhibitors , Sulfonamides/pharmacology , Animals , Dinoprostone/pharmacology , Drug Discovery , Female , Pregnancy , Rats , Receptors, Prostaglandin E, EP3 Subtype , Structure-Activity Relationship , Sulfonamides/chemistry , Uterine Contraction/drug effectsABSTRACT
A series of 3-[2-{[(3-methyl-1-phenylbutyl)amino]carbonyl}-4-(phenoxymethyl)phenyl]propanoic acid analogs were synthesized and evaluated for their in vitro potency. In most cases, introduction of one or two substituents into the two phenyl moieties resulted in the tendency of an increase or retention of in vitro activities. Several compounds, which showed excellent subtype selectivity, were evaluated for their inhibitory effect against PGE(2)-induced uterine contraction in pregnant rats, which is thought to be mediated by the EP3 receptor subtype. The structure-activity relationships (SARs) are also discussed.
Subject(s)
Propionates/pharmacology , Receptors, Prostaglandin E/antagonists & inhibitors , Animals , CHO Cells , Cricetinae , Cricetulus , Female , Magnetic Resonance Spectroscopy , Mass Spectrometry , Pregnancy , Propionates/chemistry , Propionates/pharmacokinetics , Rats , Receptors, Prostaglandin E, EP3 Subtype , Uterine Contraction/drug effectsABSTRACT
Neutrophil recruitment is pivotal to the host defense against microbial infection, but it also contributes to the immunopathology of disease. We investigated the mechanism of neutrophil recruitment in human infectious disease by means of bioinformatic pathways analysis of the gene expression profiles in the skin lesions of leprosy. In erythema nodosum leprosum (ENL), which occurs in patients with lepromatous leprosy and is characterized by neutrophil infiltration in lesions, the most overrepresented biological functional group was cell movement, including E-selectin, which was coordinately regulated with interleukin 1beta (IL-1beta). In vitro activation of Toll-like receptor 2 (TLR2), up-regulated in ENL lesions, triggered induction of IL-1beta, which together with interferon gamma induced E-selectin expression on and neutrophil adhesion to endothelial cells. Thalidomide, an effective treatment for ENL, inhibited this neutrophil recruitment pathway. The gene expression profile of ENL lesions comprised an integrated pathway of TLR2 and Fc receptor activation, neutrophil migration, and inflammation, providing insight into mechanisms of neutrophil recruitment in human infectious disease.
Subject(s)
Leprosy/immunology , Neutrophil Infiltration/immunology , Cluster Analysis , E-Selectin/biosynthesis , E-Selectin/genetics , E-Selectin/immunology , Gene Expression Profiling , Humans , Inflammation/immunology , Interferon-gamma/biosynthesis , Interferon-gamma/genetics , Interferon-gamma/immunology , Interleukin-1beta/biosynthesis , Interleukin-1beta/genetics , Interleukin-1beta/immunology , Leprosy/genetics , Models, Biological , Mycobacterium lepraemurium/isolation & purification , Neutrophil Infiltration/drug effects , Neutrophil Infiltration/genetics , Oligonucleotide Array Sequence Analysis , Receptors, Fc/biosynthesis , Receptors, Fc/genetics , Receptors, Fc/immunology , Signal Transduction/drug effects , Skin/immunology , Skin/microbiology , Thalidomide/pharmacology , Toll-Like Receptor 2/biosynthesis , Toll-Like Receptor 2/genetics , Toll-Like Receptor 2/immunologyABSTRACT
A series of 3-(2-aminocarbonyl-4-phenoxymethylphenyl)propanoic acid analogs were synthesized and evaluated for their EP3 antagonist activity in the presence of additive serum albumin. Several compounds were biologically evaluated for their in vivo efficacy with respect to the PGE(2)-induced uterine contraction in pregnant rats as well as their pharmacokinetics. The discovery process of these potent and selective EP3 antagonists and their structure activity relationship are also presented.
Subject(s)
Receptors, Prostaglandin E/antagonists & inhibitors , Receptors, Prostaglandin E/metabolism , Uterine Contraction/drug effects , Animals , CHO Cells , Cattle , Cricetinae , Cricetulus , Female , Mice , Phenyl Ethers , Pregnancy , Propionates/chemistry , Rats , Receptors, Prostaglandin E, EP3 Subtype , Serum Albumin, Bovine/metabolismSubject(s)
Autoimmunity , Cell Differentiation/immunology , Dendritic Cells/immunology , Immunity, Innate/immunology , Monocytes/cytology , Monocytes/immunology , Receptors, IgG/immunology , T-Lymphocytes/immunology , Antigen-Antibody Complex/immunology , Autoimmune Diseases/immunology , Granulocyte-Macrophage Colony-Stimulating Factor , Humans , Immunoglobulin G/immunology , Japan , Lymphocyte Activation/immunology , Societies, MedicalABSTRACT
The formation of immune complexes results in activation of the innate immune system and subsequent induction of host inflammatory responses. In particular, the binding of IgG immune complexes to FcgammaR on monocytes triggers potent inflammatory responses leading to tissue injury in disease. We investigated whether activation of monocytes via FcgammaR induced cell differentiation, imparting specific inflammatory functions of the innate immune response. Human IgG alone induced monocytes to differentiate into cells with an immature dendritic cell (iDC) phenotype, including up-regulation of CD1b, CD80, CD86, and CD206. Differentiation into CD1b(+) iDC was dependent on activation via CD64 (FcgammaRI) and induction of GM-CSF. The human IgG-differentiated iDC were phenotypically different from GM-CSF-derived iDC at the same level of CD1b expression, with higher cell surface CD86, but lower MHC class II, CD32, CD206, and CD14. Finally, in comparison to GM-CSF-derived iDC, IgG-differentiated iDC were more efficient in activating T cells in both autologous and allogeneic mixed lymphocyte reactions but less efficient at presenting microbial Ag to T cells. Therefore, activation of FcgammaRI on monocytes triggers differentiation into specialized iDC with the capacity to expand autoreactive T cells that may contribute to the pathogenesis of immune complex-mediated tissue injury.
Subject(s)
Autoimmune Diseases/immunology , Cell Differentiation/immunology , Dendritic Cells/immunology , Dendritic Cells/metabolism , Monocytes/immunology , Receptors, IgG/blood , T-Lymphocyte Subsets/immunology , Antigens, CD1/biosynthesis , Antigens, CD1/genetics , Autoimmune Diseases/blood , Autoimmune Diseases/pathology , Cell Adhesion Molecules/metabolism , Cell Differentiation/genetics , Cells, Cultured , Dendritic Cells/pathology , Granulocyte-Macrophage Colony-Stimulating Factor/physiology , Humans , Immune Complex Diseases/blood , Immune Complex Diseases/immunology , Immune Complex Diseases/pathology , Inflammation Mediators/blood , Inflammation Mediators/physiology , Lectins, C-Type/metabolism , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Monocytes/cytology , Monocytes/metabolism , Receptors, Cell Surface/metabolism , Receptors, IgG/biosynthesis , Receptors, IgG/genetics , T-Lymphocyte Subsets/metabolism , T-Lymphocyte Subsets/pathologyABSTRACT
OBJECTIVE: Tissue hypoxia is closely associated with arthritis pathogenesis, and extracellular high mobility group box chromosomal protein 1 (HMGB-1) released from injured cells also has a role in arthritis development. This study was thus undertaken to investigate the hypothesis that extracellular HMGB-1 may be a coupling factor between hypoxia and inflammation in arthritis. METHODS: Concentrations of tumor necrosis factor alpha, interleukin-6, vascular endothelial growth factor, lactic acid, lactate dehydrogenase, and HMGB-1 were measured in synovial fluid (SF) samples from patients with inflammatory arthropathy (rheumatoid arthritis and pseudogout) and patients with noninflammatory arthropathy (osteoarthritis). The localization of tissue hypoxia and HMGB-1 was also examined in animal models of collagen-induced arthritis (CIA). In cell-based experiments, the effects of hypoxia on HMGB-1 release and its associated cellular events (i.e., protein distribution and cell viability) were studied. RESULTS: In SF samples from patients with HMGB-1-associated inflammatory arthropathy (i.e., samples with HMGB-1 levels >2 SD above the mean level in samples from patients with noninflammatory arthropathy), concentrations of HMGB-1 were significantly correlated with those of lactic acid, a marker of tissue hypoxia. In CIA models in which the pathologic phenotype could be attenuated by HMGB-1 neutralization, colocalization of HMGB-1 with tissue hypoxia in arthritis lesions was also observed. In cell-based experiments, hypoxia induced significantly increased levels of extracellular HMGB-1 by the cellular processes of secretion and/or apoptosis-associated release, which was much more prominent than the protein release in necrotic cell injury potentiated by oxidative stress. CONCLUSION: These findings indicate that tissue hypoxia and its resultant extracellular HMGB-1 might play an important role in the development of arthritis.
Subject(s)
Arthritis/metabolism , HMGB1 Protein/analysis , Hypoxia/metabolism , Inflammation/metabolism , Joints/metabolism , Synovial Fluid/chemistry , Adult , Aged , Aged, 80 and over , Animals , Arthritis/pathology , Arthritis, Experimental/chemically induced , Arthritis, Experimental/metabolism , Arthritis, Experimental/pathology , Blotting, Western , Cells, Cultured , Female , Fluorescent Antibody Technique , Humans , Hypoxia/pathology , Inflammation/pathology , Interleukin-1/analysis , L-Lactate Dehydrogenase/analysis , Lactic Acid/analysis , Male , Mice , Middle Aged , Rats , Reverse Transcriptase Polymerase Chain Reaction , Severity of Illness Index , Synovial Membrane/metabolism , Synovial Membrane/pathology , Tumor Necrosis Factor-alpha/analysis , Vascular Endothelial Growth Factor A/analysisABSTRACT
Loss or down-regulation of human leukocyte antigen (HLA) class I expression has been demonstrated in a variety of solid tumors. To date, such altered HLA expression has not been studied extensively in freshly isolated leukemic blasts. If it occurs, leukemic cells could escape T-cell surveillance as a consequence. Genotypes of nine leukemic cell lines were determined using a polymerase chain reaction for HLA classes I and II. Cells were also examined for HLA beta2-microglobulin, and allele-specific HLA protein expression using flow cytometry. Next, 44 samples of freshly isolated leukemic blasts from 43 patients with malignant hematological diseases were examined for allele-specific HLA expression using flow cytometry. Microsatellite analysis was performed to determine heterozygosity in the HLA region on chromosome 6. Genotype analysis for HLA class I together with microsatellite analysis demonstrated loss of HLA haplotype in HL-60 cells. No loss of HLA haplotype was observed in 44 samples of freshly isolated leukemic blasts. As reported previously, flow cytometric analysis rarely demonstrated loss or down-regulation of HLA expression at initial diagnosis (3/39; 7.7%); however, this was evident in two of five cases in relapse (40.0%), which contrasts with previous reports. In one patient with acute leukemia, HLA-A2 cell surface expression was present at initial diagnosis, lost at relapse, and completely restored after 48 h of culture in the presence of interferon-gamma. These results suggest loss of allele-specific HLA expression may be involved in the pathogenesis of relapse in patients with leukemia. The findings should be valuable in designing new strategies for clinical immunotherapy.
Subject(s)
Histocompatibility Antigens Class I/biosynthesis , Loss of Heterozygosity , Lymphoproliferative Disorders/genetics , Neoplasm Recurrence, Local/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Blast Crisis/genetics , Cell Line, Tumor , Down-Regulation , Female , Flow Cytometry , Gene Expression , Genotype , Haplotypes , Histocompatibility Antigens Class I/genetics , Humans , Male , Microsatellite Repeats , Middle Aged , Polymerase Chain ReactionABSTRACT
PURPOSE: NY-ESO-1 belongs to a class of cancer/testis antigens and has been shown to be immunogenic in cancer patients. We synthesized a complex of cholesterol-bearing hydrophobized pullulan and NY-ESO-1 protein (CHP/ESO) and investigated the in vitro stimulation of CD8 and CD4 T cells from peripheral blood mononuclear cells in healthy donors with autologous CHP/ESO-loaded dendritic cells as antigen-presenting cells. EXPERIMENTAL DESIGN: In vitro stimulation of CD8 or CD4 T cells was determined by IFNgamma ELISPOT assays against autologous EBV-B cells infected with vaccinia/NY-ESO-1 recombinant virus or wild-type vaccinia virus as targets and by ELISA measuring secreted IFNgamma. RESULTS: NY-ESO-1-specific CD8 and CD4 T cells were induced. In a donor expressing HLA-A2, CD8 T cells stimulated with CHP/ESO-loaded dendritic cells recognized naturally processed NY-ESO-1(157-165), an HLA-A2-binding CD8 T cell epitope. NY-ESO-1 CD4 T cells were Th1-type. We identified a new HLA-DR15-binding CD4 T cell epitope, NY-ESO-1(37-50). CONCLUSIONS: These findings indicate that CHP/ESO is a promising polyvalent cancer vaccine targeting NY-ESO-1.
Subject(s)
Antigens, Neoplasm/chemistry , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Dendritic Cells/immunology , Glucans/chemistry , Membrane Proteins/chemistry , Amino Acid Sequence , Antigen Presentation/immunology , Antigens, Neoplasm/immunology , Binding Sites/immunology , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Cholesterol/chemistry , Coculture Techniques , Dendritic Cells/chemistry , Dendritic Cells/cytology , Enzyme-Linked Immunosorbent Assay , Epitopes, T-Lymphocyte/immunology , Glucans/immunology , HLA-DR Antigens/immunology , HLA-DR Serological Subtypes , Humans , Hydrophobic and Hydrophilic Interactions , Interferon-gamma/biosynthesis , Membrane Proteins/immunology , Molecular Sequence DataABSTRACT
PURPOSE: XAGE-1 was originally identified by the search for PAGE/GAGE-related genes using expressed sequence tag database and was shown to exhibit characteristics of cancer/testis-like antigens. Four transcript variants XAGE-1a, XAGE-1b, XAGE-1c, and XAGE-1d have been identified thus far. We recently identified XAGE-1b as a dominant antigen recognized by sera from lung adenocarcinoma patients. We here investigated the mRNA expression of four XAGE-1 variants and XAGE-1 protein expression in non-small cell lung cancer (NSCLC). Humoral immune response to XAGE-1b was also evaluated in patients. EXPERIMENTAL DESIGN: Forty-nine NSCLC specimens were analyzed for the expression of four XAGE-1 transcript variants by conventional 30-cycle and real-time reverse transcription-PCR and XAGE-1 protein expression by immunohistochemistry. Sera from 74 patients were analyzed for XAGE-1b antibody production by ELISA and Western blot. RESULTS: XAGE-1b and XAGE-1d mRNA were detected in 15 and 6 of 49 lung cancer specimens, respectively. No XAGE-1a or XAGE-1c mRNA expression was observed. XAGE-1b mRNA expression was observed in 14 of 31 (45%) adenocarcinoma and 1 of 18 (6%) lung cancer with other histologic types. Immunohistochemical analysis using a XAGE-1 monoclonal antibody showed that 14 of 15 XAGE-1b mRNA-positive and 3 of 34 XAGE-1b mRNA-negative specimens expressed XAGE-1 protein. Seropositivity was observed in 5 of 56 patients with adenocarcinoma, whereas none of 18 patients with other histologic types produced XAGE-1b antibody. CONCLUSION: XAGE-1b is highly and strongly expressed in lung adenocarcinoma and immunogenic in patients, suggesting that XAGE-1b is a promising antigen for immunotherapy against lung adenocarcinoma.
Subject(s)
Antigens, Neoplasm/biosynthesis , Carcinoma, Non-Small-Cell Lung/metabolism , Lung Neoplasms/metabolism , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Antibodies, Monoclonal/chemistry , Antigens, Neoplasm/chemistry , Blotting, Western , DNA Primers/chemistry , DNA, Complementary/metabolism , Databases as Topic , Enzyme-Linked Immunosorbent Assay , Expressed Sequence Tags , Genetic Vectors , Humans , Immunohistochemistry , Immunotherapy/methods , Plasmids/metabolism , Protein Structure, Tertiary , RNA, Messenger/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Treatment OutcomeABSTRACT
Thrombomodulin (TM) is an endothelial anticoagulant cofactor that promotes thrombin-mediated formation of activated protein C (APC). We have found that the N-terminal lectin-like domain (D1) of TM has unique antiinflammatory properties. TM, via D1, binds high-mobility group-B1 DNA-binding protein (HMGB1), a factor closely associated with necrotic cell damage following its release from the nucleus, thereby preventing in vitro leukocyte activation, in vivo UV irradiation-induced cutaneous inflammation, and in vivo lipopolysaccharide-induced lethality. Our data also demonstrate antiinflammatory properties of a peptide spanning D1 of TM and suggest its therapeutic potential. These findings highlight a novel mechanism, i.e., sequestration of mediators, through which an endothelial cofactor, TM, suppresses inflammation quite distinctly from its anticoagulant cofactor activity, thereby preventing the interaction of these mediators with cell surface receptors on effector cells in the vasculature.
Subject(s)
HMGB1 Protein/metabolism , Inflammation/metabolism , Thrombomodulin/metabolism , Animals , COS Cells , Chlorocebus aethiops , Humans , Inflammation/pathology , Protein C/metabolism , Protein Structure, Tertiary , Skin/metabolism , Skin/pathology , Time FactorsABSTRACT
BACKGROUND: Thrombocytosis can accompany various cancers including lung cancer. This finding has recently been suggested to indicate poor prognosis. OBJECTIVES AND METHODS: We retrospectively examined the clinical records of 611 patients with lung cancer to investigate whether there is a correlation between thrombocytosis, other clinicopathologic factors, and survival. RESULTS: Ninety-eight of the patients (16%) manifested thrombocytosis at the time of their first evaluation at our hospital. Thrombocytosis and age (p = 0.0006) and thrombocytosis and performance status (p = 0.0002) are significantly correlated, but thrombocytosis is not related to gender, tumor histology, clinical stage, or serum lactate dehydrogenase concentrations. Survival is significantly shorter in patients with thrombocytosis: [median survival time (MST) 7.5 months; n = 98] than without thrombocytosis (MST 10.1 months; n = 513; p = 0.0029). Multivariate analysis of prognostic factors using the Cox proportional hazards model indicated that thrombocytosis had independent prognostic significance. CONCLUSION: Thrombocytosis at the first patient evaluation is an independent prognostic factor in lung cancer.
Subject(s)
Lung Neoplasms/diagnosis , Lung Neoplasms/epidemiology , Thrombocytosis/epidemiology , Adult , Age Distribution , Aged , Aged, 80 and over , Biomarkers/blood , Female , Humans , Japan/epidemiology , Lung Neoplasms/blood , Male , Middle Aged , Multivariate Analysis , Prognosis , Retrospective Studies , Survival AnalysisABSTRACT
A DNA vaccine for inducing a tumor immune response was investigated using a well-characterized murine model tumor antigen. We demonstrated that in vivo electroporation augmented the induction of IFNgamma enzyme-linked immunospot (ELISPOT) and cytotoxic T lymphocyte (CTL) generation against pRL1a peptide in BALB/c spleen cells upon immunization with RLakt plasmid. Immunization without in vivo electroporation resulted in only a marginal induction of IFNgamma ELISPOT and CTL generation. Furthermore, co-injection of GM-CSF and RLakt plasmids significantly enhanced the induction of IFNgamma ELISPOT and CTL generation compared to the injection of RLakt plasmid alone. Inhibition of RL male 1 tumor growth was observed by injecting BALB/c mice with GM-CSF and RLakt plasmids using in vivo electroporation, although no effect was observed against an established tumor using the same treatment. No growth inhibition was observed without in vivo electroporation. Immunization with either RLakt plasmid alone, or GM-CSF and pCIneo control plasmids using in vivo electroporation did not inhibit RL male 1 tumor growth.
Subject(s)
Antigens, Neoplasm/genetics , Cancer Vaccines , Electroporation , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Leukemia/drug therapy , Oligopeptides/genetics , T-Lymphocytes, Cytotoxic/immunology , Animals , Antigens, Neoplasm/immunology , Cell Line, Tumor , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Male , Mice , Oligopeptides/immunology , Plasmids , Vaccines, DNAABSTRACT
Using the recently developed ELISPOT cloning methodology, we obtained cDNA clone S35 coding for the Ag epitope recognized by a murine sarcoma Meth A-specific CTL clone AT-1. Analysis of truncated S35 constructs and overlapping peptides revealed that the peptide epitope was LGAEAIFRL. AT-1 CTL lysed peptide-pulsed CMS8 cells at a nanomolar concentration, and the peptide strongly stimulated IFN-gamma production in AT-1 CTL. Sequence homology indicated that the S35 was derived from a mouse homologue of human retinoic acid-regulated nuclear matrix-associated protein (ramp). The ramp gene consisted of 15 exons. The majority of the ramp mRNA was the transcript normally spliced between exons 14 and 15, but a minor population of mRNA with an extended exon 14 was also present in Meth A cells. The epitope was derived from the newly created open reading frame, which resulted from extension of exon 14 after splicing of the adjacent intronic sequence.
Subject(s)
Epitopes, T-Lymphocyte/biosynthesis , Epitopes, T-Lymphocyte/genetics , Exons/genetics , Methylcholanthrene , Sarcoma, Experimental/immunology , T-Lymphocytes, Cytotoxic/immunology , Amino Acid Sequence , Animals , Antigens, Neoplasm/biosynthesis , Antigens, Neoplasm/genetics , Antigens, Neoplasm/isolation & purification , Antigens, Neoplasm/metabolism , Base Sequence , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Carrier Proteins/isolation & purification , Carrier Proteins/metabolism , Clone Cells , Cloning, Molecular , Cytotoxicity Tests, Immunologic , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , DNA-Binding Proteins/isolation & purification , DNA-Binding Proteins/metabolism , Enzyme-Linked Immunosorbent Assay , Epitopes, T-Lymphocyte/isolation & purification , Epitopes, T-Lymphocyte/metabolism , Exons/immunology , Gene Library , Humans , Interferon-gamma/analysis , Mice , Mice, Inbred BALB C , Mice, Inbred DBA , Molecular Sequence Data , Sarcoma, Experimental/chemically induced , Transcription FactorsABSTRACT
Gingival metastasis is an extremely rare manifestation of lung cancer, and exhibits rapid growth with various clinical symptoms. Physicians must appropriately manage patients with lung cancer who develop gingival metastasis. Clinical records of patients with lung cancer treated at the Department of Internal Medicine II, Okayama University Hospital, between 1976 and 1998 were retrospectively reviewed. The medical literature was searched by Medline to identify reports of gingival metastasis from lung cancer. Three of 729 (0.41%) lung cancer patients developed gingival metastasis in our hospital between 1976 and 1998, and 9 additional cases of this type of metastasis were found in the literature. All were male, and median age was 57.5 years (range, 47 to 70). There were no clear correlations between development of gingival metastasis and either histologic type or location of the primary lesion. Chemotherapy or radiotherapy was effective for treatment of gingival metastasis, and the quality of life was improved. However, survival after development of gingival metastasis was very short, with median survival of only 4 months.