ABSTRACT
BACKGROUND: Macrophage migration inhibitory factor (MIF) has been proposed as a link between inflammation and tumorigenesis. Despite its potentially broad influence in tumour biology and prevalent expression, the value of MIF as a therapeutic target in cancer remains unclear. We sought to validate MIF in tumour models by achieving a complete inhibition of its expression in tumour cells and in the tumour stroma. METHODS: We used MIF shRNA-transduced B16-F10 melanoma cells implanted in wild-type and MIF-/- C57Bl6 mice to investigate the effect of loss of MIF on tumour growth. Cytokine detection and immunohistochemistry (IHC) were used to evaluate tumours ex vivo. RESULTS: Macrophage migration inhibitory factor shRNA inhibited expression of MIF protein by B16-F10 melanoma cells in vitro and in vivo. In vitro, the loss of MIF in this cell line resulted in a decreased response to hypoxia as indicated by reduced expression of VEGF. In vivo the growth of B16-F10 tumours was inhibited by an average of 47% in the MIF-/- mice compared with wild-type but was unaffected by loss of MIF expression by the tumour cells. Immunohistochemistry analysis revealed that microvessel density was decreased in tumours implanted in the MIF-/- mice. Profiling of serum cytokines showed a decrease in pro-angiogenic cytokines in MIF-/- mice. CONCLUSION: We report that the absence of MIF in the host resulted in slower tumour growth, which was associated with reduced vascularity. While the major contribution of MIF appeared to be in the regulation of angiogenesis, tumour cell-derived MIF played a negligible role in this process.
Subject(s)
Macrophage Migration-Inhibitory Factors/biosynthesis , Melanoma, Experimental/blood supply , Animals , Cell Line, Tumor , Disease Models, Animal , Female , HCT116 Cells , HT29 Cells , Humans , Immunohistochemistry , Macrophage Migration-Inhibitory Factors/genetics , Male , Melanoma, Experimental/genetics , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , Stromal Cells/metabolism , Stromal Cells/pathology , Transduction, GeneticABSTRACT
Bovine leukemia virus (BLV)-induced persistent lymphocytosis is characterized by a polyclonal expansion of CD5+ B lymphocytes. To examine the role of the cytokine microenvironment in this virus-induced B-lymphocyte expansion, the expression of interleukin-2 (IL-2), IL-4, IL-10, and gamma interferon (IFN-gamma) mRNA, was measured in stimulated peripheral blood mononuclear cells from persistently lymphocytotic BLV-infected cows, nonlymphocytotic BLV-infected cows, and uninfected cows. IL-2 and IL-10 mRNA expression and IL-2 functional activity were significantly increased when peripheral blood mononuclear cells from persistently lymphocytotic cows were stimulated with concanavalin A (ConA). Additionally, during persistent lymphocytosis, peak IL-2 and IL-10 mRNA expression was delayed, and elevated expression was prolonged. To determine the potential biologic importance of increased IL-2 and IL-10 expression, the response of isolated B lymphocytes from persistently lymphocytotic cows to human recombinant cytokines and to cytokine-containing supernatants from isolated T lymphocytes was examined. While recombinant human IL-10 (rhIL-10) did not consistently induce detectable changes, rhIL-2 increased viral protein (p24) and IL-2 receptor expression in isolated B lymphocytes from persistently lymphocytotic cows. Additionally, rhIL-2 and supernatant from ConA-stimulated T lymphocytes enhanced B-lymphocyte proliferation. The stimulatory activity of the T-lymphocyte supernatant could be completely inhibited with a polyclonal anti-rhIL-2 antibody. Finally, polyclonal anti-rhIL-2 antibody, as well as anti-BLV antibody, inhibited spontaneous proliferation of peripheral blood mononuclear cells from persistently lymphocytotic cows, demonstrating that the spontaneous lymphoproliferation characteristic of BLV-induced persistent lymphocytosis is IL-2 dependent and antigen dependent. Collectively, these findings strongly suggest that increased T-lymphocyte expression of IL-2 in BLV-infected cows contributes to development and/or maintenance of persistent B lymphocytosis.
Subject(s)
B-Lymphocytes/cytology , Interleukin-2/metabolism , Leukemia Virus, Bovine/physiology , Lymphocytosis , T-Lymphocytes/metabolism , Animals , Antigens, Viral/metabolism , Cattle , Cell Division , Cells, Cultured , Concanavalin A/pharmacology , Culture Media, Conditioned , Gene Expression , Humans , Interleukin-10/genetics , Interleukin-2/genetics , Interleukin-2/pharmacology , Kinetics , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Mitogens/pharmacology , RNA, Messenger , Receptors, Interleukin-2/biosynthesis , Recombinant Proteins/pharmacology , Viral Core Proteins/biosynthesisSubject(s)
Abortion, Veterinary/virology , Bluetongue virus/isolation & purification , Dog Diseases/virology , Pregnancy Complications, Infectious/veterinary , Vaccines, Attenuated/adverse effects , Viral Vaccines/adverse effects , Abortion, Veterinary/pathology , Animals , Dog Diseases/pathology , Dogs , Female , Pregnancy , Pregnancy Complications, Infectious/mortality , Pregnancy Complications, Infectious/virology , Vaccines, Attenuated/administration & dosage , Viral Vaccines/administration & dosageABSTRACT
An antigen capture enzyme-linked immunosorbent assay was developed by using monoclonal antibodies to conserved epitopes on the Anaplasma marginale MSP1a surface protein. The assay sensitivity was 1.1 (+/- 0.5)% parasitized erythrocytes, and all infected cattle were detected prior to development of 2.0%-parasitized erythrocytes. Positive tests preceded the onset of anemia by a mean of 2 days. The assay was specific for anaplasmosis, as demonstrated by nonreactivity with another common hemoparasitic pathogens.
