![]() The information dimension (entropy) is the slope of the straight line. Log plot of a monocyte at rest (left) and in a diabetic patients (right). Transmission Electron Microscopy, 3,500 x.įigure 2. The pericellular membranes are drawn and extracted (right). Blood monocyte in a healthy subject (top, right) and in a type 2 diabetic patient (bottom). The method had been previously validated by measuring computer-generated shapes of known fractal dimension.įigure 1. The slope of the straight line (p<0.001) represented the entropy (information dimension, D1, Benoit v. 1/L for each image was drawn and the points were interpolated by a straight line (Figure 2). Briefly, each image was covered by a net of L square boxes (from 1 to 100 pixels) and the number of points in each box containing any part of the outline Nb(L) was counted. Fractal analysis was performed by using the box-counting method. Entropy evaluation of monocytic surfaceīy grey level threshold segmentation, single pixel outline of monocyte pericellular membrane was obtained (Jmicrovision v1.27 ImageJ) (Figure 1). Fifty monocytes for sample were grabbed at 3,500 x without any selection. Glutaraldehyde-fixed leukocytes were postfixed in osmium tetroxide (1%), dehydrated by acetone, embedded in Araldite, and stained with lead citrate and uranyl acetate. Leukocytes obtained by Ficoll-Hypaque from healthy subjects were stimulated with the calcium ionophore A23187, Sigma-Aldrich C7522 (2.5*10 -6 M) and with theoligopeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP, Sigma-Aldrich F3506) (10 -7 M) for 2 minutes. ![]() Monocytes obtained by Ficoll-Hypaque procedure were collected from type 2 diabetic patients (n=10) and from gender- and age-matched healthy controls (n=6). Monocytes from healthy subjects were also stimulated in vitro with the ionophore A23187 or with the oligopeptide FMLP. The aim of our study is to evaluate the information dimension (entropy, measure of geometrical complexity) of pericellular membrane of monocytes observed by transmission electron microscopy in type 2 diabetic patients and in control subjects. In the field of pathology, fractal geometry has been applied with remarkable success as a discrimant parameter in histopathology, also regarding the study of blood cells. Fractal geometry is a very useful tool for describing the irregular and complex shapes of many natural objects. Difficulty in wound healing, greater susceptibility to infections, macrovascular and microvascular diseases are complications commonly seen in type 2 diabetic patients and could carry an inflammatory background. These inflammatory components are important in the development of common diabetic complications. Inflammatory markers such as C-reactive protein (CRP), sialic acid, and diverse cytokines are predictors of the disease and they are found to be augmented in these patients. In effect, since ten years, a number of publications have shown that type 2 diabetic patients appear to be in a low grade inflammation status. Inflammation and activation of the innate immune system could be linked to type 2 diabetes pathogenesis and also to the development of common diabetic complications, mainly atherosclerosis. Monocytes, diabetes, fractals, entropy, morphometry Introduction Our approach was able to assess and quantitatively evaluate in diabetic patients morphological modifications of the monocyte linked to its activation, offering new parameters useful to follow the effects of therapeutic procedures. In effect, fractal analysis of stimulated in vitro monocytes (healthy subjects) showed a significant increase of entropy of pericellular membrane, compared with their controls (p<0.001). The mechanism underlying the observed increased complexity of pericellular membrane may be explained by the in vivo activation of the circulating monocyte in diabetes. Entropy of monocytes appeared statistically higher in diabetic patients, compared with sex- and age-matched controls (p<0.01). Box-counting method was then applied to determine the entropy of the monocytic surface. The cell contour was extracted, resized to a standard dimension and converted to a single pixel outline. Monocytes, obtained by Ficoll-Paque, were examined by transmission electron microscopy. To reproduce in vitro an activated status of the monocytes, monocytes from healthy volunteers were stimulated in vitro with the calcium ionophore A23187 or with the oligopeptide FMLP. Monocytes were collected from normal healthy volunteers (n=6) and from type 2 diabetic patients (n=10). Entropy (information dimension) of pericellular membrane of monocytes was evaluated in diabetic patients and in control subjects. Major evidence points towards a link between inflammation and type 2 diabetes, insulin resistance and diabetic complications, where monocytes are deeply involved. ![]()
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