ABSTRACT
BACKGROUND AND PURPOSE: The lack of an appropriate animal model has been a limitation in studying hemorrhage from arteriovenous malformations (AVMs) in the central nervous system. METHODS: Novel mouse central nervous system AVM models were generated by conditionally deleting the activin receptor-like kinase (Alk1; Acvrl1) gene with the SM22-Cre transgene. All mice developed AVMs in their brain and/or spinal cord, and >80% of them showed a paralysis or lethality phenotype due to internal hemorrhages during the first 10 to 15 weeks of life. The mice that survived this early lethal period, however, showed significantly reduced lethality rates even though they carried multiple AVMs. RESULTS: The age-dependent change in hemorrhage rates allowed us to identify molecular factors uniquely upregulated in the rupture-prone AVM lesions. CONCLUSIONS: Upregulation of angiopoietin 2 and a few inflammatory genes were identified in the hemorrhage-prone lesions, which may be comparable with human pathology. These models will be an exceptional tool to study pathophysiology of AVM hemorrhage.
Subject(s)
Activin Receptors/genetics , Aging/pathology , Intracranial Arteriovenous Malformations/genetics , Intracranial Arteriovenous Malformations/mortality , Microfilament Proteins/genetics , Models, Animal , Muscle Proteins/genetics , Aging/metabolism , Angiopoietin-2/metabolism , Animals , Intracranial Arteriovenous Malformations/complications , Intracranial Hemorrhages/epidemiology , Intracranial Hemorrhages/metabolism , Mice , Mice, Mutant Strains , Mice, Transgenic , Prevalence , Risk Factors , Up-RegulationABSTRACT
Folate biochemical pathway enzymes such as folylpolyglutamate synthetase (FPGS) are key elements in the folate pathway. The role of FPGS is to add glutamate residues to folates and antifolates, trapping them in the cell and increasing their affinity for subsequent enzymatic reactions. FPGS may also be an indicator of response to both clinically established and novel antifolate drugs such as pemetrexed; knowledge of their level of expression in tumors may enable their optimal use by identifying potentially responsive subgroups of patients. In spite of its key role in both nucleotide biosynthesis and possible role as a determinant of response in chemotherapy, monoclonal antibodies to FPGS suitable for immunohistochemical analysis of formalin fixed and paraffin embedded biopsy samples, or that can be used for Western blot analysis, are not commercially available. The aim of this study was to generate a monoclonal antibody that could be used to detect specific expression of FPGS in paraffin embedded tissues. A 228 amino acid region of the FPGS sequence was expressed as a recombinant fusion protein and used as an antigen to generate monoclonal antibodies. ELISA and Western blot studies identified specific reactivity of the NN3.2 antibody to the recombinant protein and a single 60 kDa protein in whole cell lysates from cell lines known to express FPGS. Immunohistochemical analysis of FPGS using hybridoma clone NN3.2 in a panel of normal tissues demonstrated wide expression including strong immunoreactivity in the brush border and crypts of colon, liver hepatocytes, and lymphoid cells. Analysis of a panel of malignant and benign tissues demonstrated wide expression with variable intensities of staining and patterns of cytoplasmic reactivity. Stronger staining was observed in malignant tissue compared with that of normal adjacent tissue, particularly in ovarian and colon adenocarcinoma cases. Our results show that clone NN3.2 is a sensitive tool for detection of FPGS in paraffin-embedded tissues.
Subject(s)
Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/metabolism , Paraffin Embedding , Peptide Synthases/metabolism , Blotting, Western , Cell Line, Tumor , DNA Primers/genetics , Enzyme-Linked Immunosorbent Assay , Humans , Immunohistochemistry/methods , Microvilli/metabolism , Peptide Synthases/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
Folate biochemical pathway components such as FR-alpha are determinants of response to novel antifolate drugs such as pemetrexed. Knowledge of their level of expression in tumors will enable their optimal use by identifying potentially responsive subgroups of patients. In spite of its importance in the diagnosis and treatment of cancer, monoclonal antibodies to FR-alpha suitable for immunohistochemical analysis of formalin-fixed and paraffin-embedded biopsy samples, or that can be used for Western blot analysis, are not available. The aim of this study was thus to generate a monoclonal antibody that could be used to detect specific expression of FR-alpha in paraffin-embedded tissues. A 189 amino acid region of the FR-alpha sequence was expressed as a recombinant fusion protein and used as antigen to generate monoclonal antibodies. Studies by ELISA and Western blot identified specific reactivity of the BN3.2 antibody to the recombinant protein and a single 40 kD protein in whole cell lysates from cell lines known to express FR-alpha. Immunohistochemical analysis of FR-alpha using hybridoma clone BN3.2 in a panel of normal tissues demonstrated expression limited to ovarian epithelia, placental trophoblasts, and proximal kidney tubules. Analysis of a panel of malignant and benign tissues demonstrated limited expression with variable intensities of staining and patterns of both membrane and cytoplasmic reactivity observed between cases. In the majority of malignant ovarian tumors, high intensity staining was observed, predominantly localized to the plasma membrane. Our results show that clone BN3.2 is a sensitive tool for detection of FR-alpha in paraffin-embedded tissues. This preliminary study also supports its use in immunohistochemical studies to determine the role of FR-alpha as a tumor prognostic marker and a possible therapeutic target.
Subject(s)
Antibodies, Monoclonal , Carrier Proteins/analysis , Carrier Proteins/immunology , Paraffin Embedding , Receptors, Cell Surface/analysis , Receptors, Cell Surface/immunology , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/chemistry , Cell Line , Female , Folate Receptors, GPI-Anchored , Humans , Mice , Mice, Inbred BALB C , Tumor Cells, CulturedABSTRACT
Recently, several antibodies have allowed the detection of estrogen receptor beta (ER-beta) in paraffin-embedded tissue; however, these attempts have failed to specifically identify the wild-type form and revealed technical difficulties such as the necessity for alterations to standard staining protocols and amplification detection systems. The aim of this study was to generate a monoclonal antibody that could provide enhanced sensitivity for detection of ER-beta in paraffin embedded tissues. A 130-amino acid region of the C-terminus of ER-beta was expressed as a fusion protein and used as an antigen to generate monoclonal antibodies. Immunohistochemical analysis of ER-beta using clone EMR02 in normal and inflamed tissues demonstrated nuclear staining. In benign and malignant tumors, variable intensities of staining and patterns of nuclear reactivity were observed between cases. Intense ER-beta positivity was also observed in tumor-infiltrating lymphocytes. Mapping studies by ELISA and Western blotting have identified specific reactivity of EMR02 to a 17-amino acid sequence of the full-length wild-type ER-beta protein (ERbetawt). Our results show that clone EMR02 is a sensitive tool for the detection of ERbetawt in paraffin-embedded tissues. This preliminary study also supports its use in immunohistochemical studies to determine the role of ERbetawt as a tumor prognostic marker and a possible therapeutic target.
