Fibroblast growth patterns for diagnosis of alzheimer's disease

Abstract

Methods of diagnosing Alzheimer's disease are provided. At least five methods of diagnostic measurements are presented: Method 1: Integrated score; Method 2: Average aggregate area per number of aggregates; Method 3: Cell migration analysis; Method 4: Fractal analysis; Method 5: Lacunarity Analysis. In certain embodiments, a sample of a subject's skin provides a network of fibroblasts that is imaged and a fractal dimension of the image is calculated. The fractal dimension can be compared to an aged-matched control (non-Alzheimer's) database to determine if the subject has Alzheimer's disease. The network of fibroblasts may be cultured in a matrix, for example in a protein mixture.

1 - 94 . (canceled) 
     
     
         95 . A method comprising: (a) obtaining one or more cells from a human subject; (b) culturing said one or more cells for a time period; (c) obtaining an image of said cells at the conclusion of said time period; (d) determining a fractal dimension associated with a network of cells on said image; (e) comparing the determination of step (d) with an independently determined fractal dimension associated with known non-Alzheimer's disease cells. 
     
     
         96 . The method of  claim 95 , wherein if the fractal dimension calculated in step (d) is statistically significantly lower than the fractal dimension associated with known non-Alzheimer's Disease cells, the comparison is indicative of Alzheimer's Disease. 
     
     
         97 . The method of  claim 96 , wherein Alzheimer's Disease is confirmed using one or more diagnostic methods. 
     
     
         98 . The method of  claim 97 , wherein said one or more diagnostic methods are selected from the group consisting of methods comprising determining an integrated score, methods comprising calculating area per number of aggregates, methods comprising cell migration analysis, methods comprising fractal analysis and methods comprising lacunarity analysis. 
     
     
         99 . The method of  claim 95 , wherein the fractal dimension is calculated using a box counting procedure. 
     
     
         100 . The method of  claim 99 , wherein said box counting procedure comprises an edge detection procedure. 
     
     
         101 . The method of  claim 95 , wherein said subject is aged-matched with a control subject providing said known non-Alzheimer's disease cells. 
     
     
         102 . The method of  claim 95 , wherein said time period is about 24 hours. 
     
     
         103 . The method of  claim 95 , wherein said cell is cultured in a protein mixture. 
     
     
         104 . The method of  claim 103 , wherein the protein mixture comprises an extracellular matrix preparation comprising laminin, collagen, heparin sulfate proteoglycans, entactin/nidogen, and/or combinations thereof. 
     
     
         105 . The method of  claim 104 , wherein the protein mixture further comprises growth factor. 
     
     
         106 . The method of  claim 104 , wherein the extracellular matrix protein is extracted from a tumor. 
     
     
         107 . The method of  claim 106 , wherein the tumor is the EHS mouse sarcoma. 
     
     
         108 . A method comprising: (a) determining a fractal dimension of an image of a network of fibroblasts from a human subject; (b) determining a fractal dimension of an image of a network of fibroblasts from known non-Alzheimer's disease cells; (c) comparing the determinations of steps (a) and (b). 
     
     
         109 . The method of  claim 108 , wherein if the fractal dimension determined in step (a) is statistically significantly lower than the fractal dimension determined in step (b), the diagnosis is indicative of Alzheimer's Disease. 
     
     
         110 . The method of  claim 108 , wherein said subject is aged-matched with a control subject providing said known non-Alzheimer's Disease cells. 
     
     
         111 . A method of diagnosing Alzheimer's disease in a human subject, the method comprising: (a) calculating a fractal dimension of an image of a network of fibroblasts from said subject; (b) comparing the calculation of step (a) with an independently determined fractal dimension associated with known non-Alzheimer's disease cells; wherein if the fractal dimension calculated in step (a) is statistically significantly lower than the fractal dimension associated with known non-Alzheimer's disease cells, the diagnosis is positive for Alzheimer's Disease in said subject. 
     
     
         112 . The method of  claim 111 , wherein said subject is aged-matched with a control subject providing said known non-Alzheimer's disease cells. 
     
     
         113 . A method of diagnosing Alzheimer's disease in a human subject, the method comprising: (a) using a surgical blade to obtain a sample of said subject's peripheral skin fibroblasts; (b) using an incubator to incubate said sample for a time period; (c) using an imager to take an image of said sample at the conclusion of said time period; (d) using a computer to calculate a fractal dimension associated with a network of fibroblasts on said image; (e) comparing the calculation of step (d) with an independently determined fractal dimension associated with known non-Alzheimer's disease cells; wherein if the fractal dimension calculated in step (d) is statistically significantly lower than the fractal dimension associated with known non-Alzheimer's disease cells, the diagnosis is positive for Alzheimer's Disease in said subject. 
     
     
         114 . The method of  claim 113 , wherein said sample is incubated in a protein mixture. 
     
     
         115 . The method of  claim 114 , wherein said protein mixture comprises an extracellular matrix preparation comprising laminin, collagen, heparin sulfate proteoglycans, entactin/nidogen, and/or combinations thereof. 
     
     
         116 . The method of  claim 115 , wherein the protein mixture further comprises growth factor. 
     
     
         117 . The method of  claim 115 , wherein the extracellular matrix protein is extracted from a tumor. 
     
     
         118 . The method of  claim 117 , wherein the tumor is the EHS mouse sarcoma. 
     
     
         119 . A method of diagnosing Alzheimer's Disease in a human subject, the method comprising: (a) using a surgical blade to obtain a sample of said subject's peripheral skin fibroblasts; (b) using an incubator to incubate said sample for a time period; (c) using an imager to take an image of said sample at the conclusion of said time period; (d) using a computer to calculate a fractal dimension associated with a network of fibroblasts on said image; (e) using a computer to input the fractal dimension of step (d) into a database having fractal dimension data generated from non-Alzheimer's disease cells obtained from control subjects of various ages; (f) using a computer to diagnose said subject by comparing the calculated fractal dimension of step (d) with the data of said database. 
     
     
         120 . The method of  claim 119 , wherein said sample is incubated in a gelatinous protein mixture. 
     
     
         121 . The method of  claim 120 , wherein the gelatinous protein mixture comprises an extracellular matrix preparation comprising laminin, collagen, heparin sulfate proteoglycans, entactin/nidogen, and/or combinations thereof. 
     
     
         122 . The method of  claim 120 , wherein the gelatinous protein mixture further comprises growth factor. 
     
     
         123 . The method of  claim 121 , wherein the extracellular matrix protein is extracted from a tumor. 
     
     
         124 . The method of  claim 123 , wherein the tumor is the EHS mouse sarcoma. 
     
     
         125 . A computer readable medium having a database of fractal dimension data generated from non-Alzheimer's disease cells obtained from control subjects of various ages, said medium containing instructions to: (a) calculate a fractal dimension of an image; (b) compare said fractal dimension with said database of fractal dimension data; and (c) output a diagnosis based on the comparison of step (b). 
     
     
         126 . A method of diagnosing Alzheimer's Disease in a subject comprising the steps of: (a) obtaining one or more cells from said subject and growing said one or more cells in a tissue culture medium; (b) measuring the fractal dimension of said one or more cells over a time period; (c) plotting said fractal dimension as a function of time to obtain a fractal dimension curve; (d) comparing said fractal dimension curve to fractal dimension curves obtained from non-Alzheimer's Disease cells and non-Alzheimer's Disease Dementia (non-ADD) cells; and (e) diagnosing the presence or absence of Alzheimer's Disease in said subject. 
     
     
         127 . The method of  claim 126 , wherein said diagnosis is positive for Alzheimer's Disease in said subject if said fractal dimension curve measured from a cell or cells obtained from said subject is statistically significantly different from said fractal dimension curves obtained from said non-Alzheimer's Disease cells and said non-ADD cells. 
     
     
         128 . The method of  claim 127 , wherein said cell or cells obtained from said subject is a fibroblast cell. 
     
     
         129 . The method of  claim 126 , wherein said diagnosis is confirmed using one or more additional diagnostic methods. 
     
     
         130 . The method of  claim 129 , wherein said one or more additional diagnostic methods are selected from the group consisting of methods comprising determining an integrated score, methods comprising calculating area per number of aggregates, methods comprising cell migration analysis, methods comprising fractal analysis and methods comprising lacunarity analysis. 
     
     
         131 . A method of diagnosing Alzheimer's Disease in a subject comprising the steps of (a) obtaining one or more cells from said subject and growing said one or more cells in a tissue culture medium; (b) determining the number of migrating cells; (c) comparing the number of migrating cells to the number of migrating cells for non-Alzheimer's Disease cells; (d) diagnosing the presence or absence of Alzheimer's Disease in said subject. 
     
     
         132 . The method of  claim 131 , wherein the diagnosis is positive for AD if the number of migrating cells obtained from said subject is statistically significantly smaller than the number of migrating non-Alzheimer's Disease cells. 
     
     
         133 . The method of  claim 131 , wherein said cells are fibroblasts. 
     
     
         134 . The method of  claim 131 , wherein said diagnosis is confirmed using one or more additional diagnostic methods. 
     
     
         135 . The method of  claim 134 , wherein said one or more additional diagnostic methods are selected from the group consisting of methods comprising determining an integrated score, methods comprising calculating area per number of aggregates, methods comprising cell migration analysis, methods comprising fractal analysis and methods comprising lacunarity analysis. 
     
     
         136 . A method of diagnosing Alzheimer's Disease in a subject comprising the steps of (a) obtaining one or more cells from said subject and growing said one or more cells in a tissue culture medium; (b) determining the lacunarity of said cells; (c) comparing the lacunarity of said cells to the lacunarity of non-Alzheimer's Disease cells; (d) diagnosing the presence or absence of Alzheimer's Disease in said subject. 
     
     
         137 . The method of  claim 136 , wherein the diagnosis is positive for AD if the lacunarity of the cells taken from said subject is statistically significantly higher than the lacunarity of the non-Alzheimer's Disease cells. 
     
     
         138 . The method of  claim 136 , wherein said diagnosis is confirmed using one or more additional diagnostic methods. 
     
     
         139 . The method of  claim 138 , wherein said one or more additional diagnostic methods are selected from the group consisting of methods comprising determining an integrated score, methods comprising calculating area per number of aggregates, methods comprising cell migration analysis, methods comprising fractal analysis and methods comprising lacunarity analysis. 
     
     
         140 . The method of  claim 136 , wherein said cells are fibroblasts. 
     
     
         141 . A method of screening for a lead compound useful for the development of one or more drug candidates for the treatment or prevention of Alzheimer's disease comprising the steps of (a) growing one or more AD cells in a cell culture medium; (b) contacting said AD cells with a compound; (c) determining whether one or more characteristics of said AD cells is altered to resemble the characteristics of non-Alzheimer's Disease cells that have not been contacted with said compound. 
     
     
         142 . The method of  claim 141 , wherein said cells are fibroblasts. 
     
     
         142 . The method of  claim 141 , wherein said characteristic is fractal dimension. 
     
     
         143 . The method of  claim 141 , wherein said characteristic is an integrated score. 
     
     
         144 . The method of  claim 141 , wherein said characteristic is an average aggregate area per number of aggregates. 
     
     
         145 . The method of  claim 141 , wherein said characteristic is cell migration. 
     
     
         146 . The method of  claim 141 , wherein said characteristic is lacunarity. 
     
     
         147 . A method of distinguishing between the presence of Alzheimer's Disease and non-Alzheimer's Disease Dementia in a subject comprising: (a) obtaining one or more cells from a subject (b) measuring the fractal dimension of said one or more cells over a time period; (c) plotting said fractal dimension as a function of time to obtain a fractal dimension curve; (d) comparing said fractal dimension curve to fractal dimension curves obtained from known non-Alzheimer's Disease cells, known non-Alzheimer's Disease Dementia (non-ADD) cells and known AD cells; and (e) distinguishing between AD and non-ADD in said subject. 
     
     
         148 . The method of  claim 147 , wherein said cells are fibroblasts. 
     
     
         149 . A method of distinguishing between the presence of Alzheimer's Disease and non-Alzheimer's Disease Dementia in a subject comprising: (a) obtaining one or more cells from a subject (b) obtaining one or more cells from said subject and growing said one or more cells in a tissue culture medium; (c) determining the number of migrating cells; (d) comparing the number of migrating cells to the number of migrating cells for known non-Alzheimer's Disease cells, known AD cells and known non-ADD cells; (e) distinguishing between AD and non-ADD in said subject. 
     
     
         150 . The method of  claim 149 , wherein said cells are fibroblasts.