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A New Image Analysis Method Based on Morphometric and Fractal Parameters for Rapid Evaluation of In Situ Mammalian Mast Cell Status

  • Piper Wedman (a1), Ahmed Aladhami (a1), Mary Beste (a1), Morgan K. Edwards (a1), Alena Chumanevich (a1), John W. Fuseler (a1) and Carole A. Oskeritzian (a1)...


Apart from their effector functions in allergic disorders, tissue-resident mast cells (MC) are gaining recognition as initiators of inflammatory events through their distinctive ability to secrete many bioactive molecules harbored in cytoplasmic granules. Activation triggers mediator release through a regulated exocytosis named degranulation. MC activation is still substantiated by measuring systemic levels of MC-restricted mediators. However, identifying the anatomical location of MC activation is valuable for disease diagnosis. We designed a computer-assisted morphometric method based on image analysis of methylene blue (MB)-stained normal mouse skin tissue sections that quantitates actual in situ MC activation status. We reasoned MC cytoplasm could be viewed as an object featuring unique relative mass values based on activation status. Integrated optical density and area (A) ratios were significantly different between intact and degranulated MC (p<0.001). The examination of fractal characteristics is of translational diagnostic/prognostic value in cancer and readily applied to quantify cytoskeleton morphology and vasculature. Fractal dimension (D), a measure of their comparative space filling capacity and structural density, also differed significantly between intact and degranulated MC (p<0.001). Morphometric analysis provides a reliable and reproducible method for in situ quantification of MC activation status.


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Akin, C., Valent, P. & Metcalfe, D.D. (2010). Mast cell activation syndrome: Proposed diagnostic criteria. J Allergy Clin Immunol 126, 10991104.
Anderson, J.C., Babb, A.L. & Hlastala, M.P. (2005). A fractal analysis of the radial distribution of bronchial capillaries around large airways. J Appl Physiol 98, 850855.
Aon, M.A., Roussel, M.R., Cortassa, S., O’Rourke, B., Murray, D.B., Beckmann, M. & Lloyd, D. (2008). The scale-free dynamics of eukaryotic cells. PLoS One 3, e3624.
Arock, M., Schneider, E., Boissan, M., Tricottet, V. & Dy, M. (2002). Differentiation of human basophils: An overview of recent advances and pending questions. J Leuko Biol 71, 557564.
Brown, J.H., Gupta, V.K., Li, B.-L., Milne, B.T., Restrepo, C. & West, G.B. (2002). The fractal nature of nature: Power laws, ecological complexity and biodiversity. Phil Trans R Soc Lond B 357, 619626.
Da Silva, E.Z.M., Jamur, M.C. & Oliver, C. (2014). Mast cell function: A new vision of an old cell. J Histochem Cytochem 62, 698738.
Di Ieva, A. (2012). Fractal analysis of microvascular networks in malignant brain tumors. Clin Neuropathol 31, 342351.
Di Ieva, A., Grizzi, F., Ceva-Grimaldi, G., Russo, C., Gaetani, P., Aimar, E., Levi, D., Pisano, P., Tancioni, F., Nicola, G., Tschabitscher, M., Dioguardi, N. & Baena, R.R. (2007). Fractal dimension as a quantitator of the microvasculature of normal and adenomatous pituitary tissue. J Anat 211, 673680.
Dioguardi, N., Grizzi, F., Franceschini, B., Bossi, P. & Russo, C. (2006). Liver fibrosis and tissue architectural change measurement using fractal-rectified metrics and Hurst’s exponent. World J Gastroenterol 12, 21872194.
Dioguardi, N., Grizzi, F., Fiamengo, B. & Russo, C. (2008). Metrically measuring liver biopsy: a chronic hepatitis B and C computer-aided morphologic description. World J Gastroenterol 14, 73357344.
Doubal, F.N., MacGillivray, T.J., Patton, N., Dhillon, B., Dennis, M.S. & Wardlaw, J.M. (2010). Fractal analysis of retinal vessels suggests that a distinct vasculopathy causes lacunar stroke. Neurology 74, 11021107.
Ferro, D.P., Falconi, M.A., Adam, R.L., Ortega, M.M., Lima, C.P., De Souza, C.A., Lorand-Metze, I. & Metze, K. (2011). Fractal characteristics of May-Grünwald-Giemsa stained chromatin are independent prognostic factors for survival in multiple myeloma. PLoS One 6, e20706.
Fuseler, J.W., Bedenbaugh, A., Yekkala, K. & Baudino, T.A. (2010). Fractal and image analysis of the microvasculature in normal intestinal submucosa and intestinal polyps in Apc(Min/+) mice. Microsc Microanal 16, 7379.
Fuseler, J.W., Merrill, D.M., Rogers, J.A., Grisham, M.B. & Wolf, R.E. (2006). Analysis and quantitation of NF-kappaB nuclear translocation in tumor necrosis factor alpha (TNF-alpha) activated vascular endothelial cells. Microsc Microanal 12, 269276.
Fuseler, J.W., Millette, C.F., Davis, J.M. & Carver, W. (2007). Fractal and image analysis of morphological changes in the actin cytoskeleton of neonatal cardiac fibroblasts in response to mechanical stretch. Microsc Microanal 13, 133143.
Fuseler, J.W. & Valarmathi, M.T. (2012). Modulation of the migration and differentiation potential of adult bone marrow stromal stem cells by nitric oxide. Biomaterials 33, 10321043.
Galli, S.J., Borregaard, N. & Wynn, T.A. (2011). Phenotypic and functional plasticity of cells of innate immunity: Macrophages, mast cells and neutrophils. Nat Immunol 12, 10351044.
Galli, S.J., Grimbaldeston, M. & Tsai, M. (2008). Immunomodulatory mast cells: Negative, as well as positive, regulators of immunity. Nat Rev Immunol 8, 478486.
Galli, S.J. & Tsai, M. (2012). IgE and mast cells in allergic disease. Nat Immunol 18, 693704.
Gerber, P.A., Buhren, B.A., Schrumpf, H., Homey, B., Zlotnik, A. & Hevezi, P. (2014). The top skin-associated genes: A comparative analysis of human and mouse skin transcriptomes. Biol Chem 395, 577591.
Giannou, A.D., Marazioti, A., Spella, M., Kanellakis, N.I., Apostolopoulou, H., Psallidas, I., Prijovich, Z.M., Vreka, M., Zazara, D.E., Lilis, I., Papaleonidopoulos, V., Kairi, C.A., Patmanidi, A.L., Giopanou, I., Spiropoulou, N., Harokopos, V., Aidinis, V., Spyratos, D., Teliousi, S., Papadaki, H., Taraviras, S., Snyder, L.A., Eickelberg, O., Kardamakis, D., Iwakura, Y., Feyerabend, T.B., Rodewald, H.R., Kalomenidis, I., Blackwell, T.S., Agalioti, T. & Stathopoulos, G.T. (2015). Mast cells mediate malignant pleural effusion formation. J Clin Invest 125, 23172334.
Grizzi, F. & Dioguardi, N. (1999). A fractal scoring system for quantifying active collagen synthesis during chronic liver disease. Int J Chaos Theo Appl 21, 262266.
Grizzi, F., Russo, C., Colombo, P., Franceschini, B., Frezza, E.E., Cobos, E. & Chiriva-Internati, M. (2005). Quantitative evaluation and modelling of two-dimensional neovascular network complexity: The surface fractal dimension. BMC Cancer 5, 1423.
Hamilton, M.J., Hornick, J.L., Akin, C., Castells, M.C. & Greenberger, N.J. (2011). Mast cell activation syndrome: A newly recognized disorder with systemic clinical manifestations. J Allergy Clin Immunol 128, 147152.
Hart, P.H., Grimbaldeston, M.A., Swift, G.J., Jaksic, A., Noonan, F.P. & Finlay-Jones, J.J. (1998). Dermal mast cells determine susceptibility to ultraviolet B-induced systemic suppression of contact hypersensitivity responses in mice. J Exp Med 187, 20452053.
Hart, P.H., Grimbaldeston, M.A., Hosszu, E.K., Swift, G.J., Noonan, F.P. & Finlay-Jones, J.J. (1999). Age-related changes in dermal mast cell prevalence in BALB/c mice: Functional importance and correlation with dermal mast cell expression of Kit. Immunology 98, 352356.
Jelinek, H.F., Ristanovic, D. & Milosevic, N.T. (2011). The morphology and classification of α ganglion cells in the rat retinae: A fractal analysis study. J Neurosci Methods 201, 281287.
Kalesnikoff, J. & Galli, S.J. (2008). New developments in mast cell biology. Nat Immunol 9, 12151223.
Lagunoff, D. (1974). Analysis of dye binding sites in mast cell granules. Biochemistry 19, 39823986.
Mandelbrot, B.B. (1982). The Fractal Geometry of Nature, 1st ed. New York: W. H. Freeman and Company.
Marshall, J.S. (2004). Mast-cell responses to pathogens. Nat Rev Immunol 4, 787799.
Manera, M., Dezfuli, B.S., Borreca, C. & Giari, L. (2014). The use of fractal dimension and lacunarity in the characterization of mast cell degranulation in rainbow trout (Onchorhynchus mykiss). J Microsc 256, 8289.
Mcnally, J.G. & Mazza, D. (2010). Fractal geometry in the nucleus. EMBO J 29, 23.
Moon, T.C., Befus, A.D. & Kulka, M. (2014). Mast cell mediators: Their differential release and the secretory pathways involved. Front Immunol 5, 118.
Moledina, S., de Bruyn, A., Schievano, S., Owens, C.M., Young, C., Haworth, S.G., Taylor, A.M., Schulze-Neick, I. & Muthurangu, V. (2011). Fractal branching quantifies vascular changes and predicts survival in pulmonary hypertension: a proof of principle study. Heart 97, 12451249.
Nezadal, M., Zemeskal, O. & Buchnicek, M. (2001). The box-counting: Critical study, 4th Conference on Prediction, Synergetic and More, The Faculty of Management, Institute of Information Technologies, Faculty of Technology, Tomas Bata University in Zlin, HarFA software, October 25–26, p. 18.
Oldford, S.A. & Marshall, J.S. (2015). Mast cells as targets for immunotherapy of solid tumors. Mol Immunol 63, 113124.
Oskeritzian, C.A. (2015). Mast cell plasticity and sphingosine-1-phosphate in immunity, inflammation and cancer. Mol Immunol 63, 104112.
Oskeritzian, C.A., Alvarez, S.E., Hait, N.C., Price, M.M., Milstien, S. & Spiegel, S. (2008). Distinct roles of sphingosine kinases 1 and 2 in human mast cell functions. Blood 111, 41934200.
Oskeritzian, C.A., Hait, N.C., Wedman, P., Chumanevich, A., Kolawole, E.M., Price, M.M., Falanga, Y.T., Harikumar, K.B., Ryan, J.J., Milstien, S., Sabbadini, R. & Speigel, S. (2015). The sphingosine-1-phosphate/sphingosine-1-phosphate receptor 2 axis regulates early airway T-cell infiltration in murine mast cell-dependent acute allergic responses. J Allergy Clin Immunol 135, 10081018.
Oskeritzian, C.A., Price, M.M., Ryan, J.J., Hait, N.C., Kapitonov, D., Falanga, Y., Morales, J.K., Milstien, S. & Spiegel, S. (2010). Essential role of sphingosine-1-phosphate receptor 2 in human mast cell activation, anaphylaxis, and pulmonary edema. J Exp Med 207, 465474.
Qian, A.R., Li, D., Han, J., Gao, X., Di, S.M., Zhang, W., Hu, L.F. & Shang, P. (2012). Fractal dimension as a measure of altered actin cytoskeleton in MC3T3-E1 cells under simulated microgravity using 3-D/2-D clinostats. IEEE Trans Biomed Eng 59, 13741380.
Rafail, S., Kourtzelis, I., Foukas, P.G., Markiewski, M.M., Deangelis, R.A., Guariento, M., Ricklin, D., Grice, E.A. & Lambris, J.D. (2015). Complement deficiency promotes cutaneous wound healing in mice. J Immunol 194, 12851291.
Reber, L.L., Marichal, T. & Galli, S.J. (2012). New models for analyzing mast cell functions in vivo . Trends Immunol 33, 613625.
Ribatti, D. (2013). Mast cells and macrophages exert beneficial and detrimental effects on tumor progression and angiogenesis. Immunol Lett 152, 8388.
Rogers, J.A. & Fuseler, J.W. (2007). Regulation of NF-κB activation and nuclear translocation by exogenous nitric oxide (NO) donors in TNF-α activated vascular endothelial cells. Nitric Oxide 16, 379391.
Schwartz, L.B., Metcalfe, D.D., Miller, J.S., Earl, H. & Sullivan, T. (1987). Tryptase levels as an indicator of mast-cell activation in systemic anaphylaxis and mastocytosis. N Engl J Med 316, 16221626.
Sedivy, R., Thurner, S., Budinsky, A.C., Kostler, W.J. & Zielinski, C.C. (2002). Short-term rhythmic proliferation of human breast cancer cell lines: Surface effects and fractal growth patterns. J Pathol 197, 163169.
Smith, T.G., Lange, G.D. & Marks, W.B. (1996). Fractal methods and results in cellular morphology-dimensions, lacunarity and multifractals. J Neurosci Methods 39, 123136.
Streba, L., Forţofoiu, M.C., Popa, C., Ciobanu, D., Gruia, C.L., Mogoanta, S.Ş. & Streba, C.T. (2015). A pilot study on the role of fractal analysis in the microscopic evaluation of colorectal cancers. Rom J Morphol Embryol 56, 191196.
Thamrin, C., Stern, G. & Frey, U. (2010). Fractals for physicians. Paediatr Respir Rev 11, 123131.
Theoharides, T.C., Alysandratos, K.-D., Angelidou, A., Delivanis, D.-A., Sismanopoulos, N., Zhang, B., Asadi, S., Vasiadi, M., Weng, Z., Miniati, A. & Kalogeromitros, D. (2012). Mast cells and inflammation. Biochim Biophys Acta 1822, 2133.
Valent, P., Horny, H.-P., Triggiani, M. & Arock, M. (2011). Clinical and laboratory parameters of mast cell activation as basis for the formulation of diagnostic criteria. Int Arch Allergy Immunol 156, 119127.
Voehringer, D. (2013). Protective and pathological roles of mast cells and basophils. Nat Rev Immunol 13, 362375.
Walter, R.J. Jr. & Berns, M.W. (1986). Digital image processing and analysis. In Video Microscopy, Inoue S. (Ed.), pp. 327392. New York and London: Plenum Press.
Wernersson, S. & Pejler, G. (2014). Mast cell secretory granules: Armed for battle. Nat Rev Immunol 14, 478494.
Wick, N., Thurner, S., Paiha, K., Sedivy, R., Vietor, I. & Huber, L.A. (2003). Quantitative measurement of cell migration using time-lapse videomicroscopy and non-linear system analysis. Histochem Cell Bio 119, 1520.
Wolters, P.J., Mallen-St. Clair, J., Lewis, C.C., Villalta, S.A., Baluk, P., Erle, D.J. & Caughey, G.H. (2005). Tissue-selective mast cell reconstitution and differential lung gene expression in mast cell-deficient Kit W-sh /Kit W-sh sash mice. Clin Exp Allergy 35, 8288.
Zhang, L., Liu, J.Z., Dean, D., Sahgal, V. & Yue, G.H. (2006). A three-dimensional fractal analysis method for quantifying white matter structure in human brain. J Neurosci Methods 150, 242253.
Zouien, F.A., Kurdi, M., Booz, G.W. & Fuseler, J.W. (2014). Applying fractal dimension and image analysis to quantify fibrotic collagen deposition and organization in the normal and hypertensive heart. Microsc Microanal 20, 11341144.
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