Platelet entropy is increased in familial hypercholesterolemia and in type 2 diabetic patients.

AIM: A plethora of abnormalities in platelet function have been described in diabetic patientsas well in familial hypercholesterolemia. This paper investigates the use of computerized fractal analysis for objective characterization of the entropy of the platelet surface of circulating platelets collected from healthy individuals, from type 2 diabetic patients and from familial hypercholesterolemic subjects, in order to search a structural biomarker to distinguish between them. METHODS: Circulating platelets were collected from 11 type 2 diabetic patients, from 6 familial hypercholesterolemic patients and 5 healthy subjects as platelet rich plasma. Platelet boundaries were observed by transmission electron microscopy and extracted by means of automatically image processing. The information dimension (entropy of the platelet contour) was automatically calculated. RESULTS: Platelet boundary observed by electron microscopy is fractal. Entropy of the platelet surface in the circulating platelets is significantly increased in the diabetic patients in comparison to healthy subjects (P<0.001), as well in familial hypercholesterolemic patients (P<0.01), with 100% correct classification in selected subjects. In vitro activated platelets from healthy subjects show an analogous increase of platelet entropy. CONCLUSION: Computerized shape analysis of circulating platelets observed by transmission electron microscopy provides accurate, quantitative, data to study platelet activation at morphological level in atherosclerosis-linked condition, as diabetes mellitus and familial hypercholesterolemia, able to distinguish the activated platelets of the patients from the platelets of healthy subjects. This method may be promising to follow the platelet activation in the circulating blood at morphological level in pathophysiological condition linked to platelet activation and after administration of drugs or other therapeutic procedures.

Nonlinear analysis of tRNAs nucleotide sequences by random walks: randomness and order in the primitive informational polymers.

In order to test the hypothesis that the nucleotide sequences of the primitive informational polymers might not be chosen randomly and in the attempt to compare among taxa, we propose a comparison of computer-generated random sequences with tRNAs nucleotide sequences present in the bacterial and archaeal genomes, being tRNAs molecules possible "fossils" of the time (billions years ago) in which life arose. Our approach is based on the analysis of sequences of tRNAs described as random walks and the distances from the origin evaluated by the use of nonlinear indexes (largest Lyapunov exponent, entropy, BDS statistic). Six different tRNAs of Bacteria and Archaea (ten Archaea and ten Bacteria, thermophilic and mesophilic ones; n = 120), and computer-generated random sequences (n = 50) were studied. Our data show that tRNAs present indices statistical lower than the ones of computer-generated random data (tRNAs own a more ordered sequence than random ones: Lyapunov, p < 0.01; entropy, p < 0.05; BDS, p < 0.01). The observed deviation from pure randomness should be arisen from some constraints like the secondary structure of this biologic macromolecule and/or from a "frozen" stochastic transition, or even from the possible peculiar origin of tRNA by replication of older proto-RNA. Comparing between taxa, in the species studied, Bacteria present BDS and Base ratio (G+C)/(A+T) indexes statistically lower than in Archaea, together which a 20% of entropy increase. The analysis of a greater number of tRNAs and species will permit to explain if this finding, showing a higher randomness in the bacterial tRNAs sequences, is linked to the different base ratio, to the different environments in which the microorganisms live or to an evolutionary effect.

Fractal analysis of circulating platelets in type 2 diabetic patients.

This paper investigates the use of computerized fractal analysis for objective characterization by means of transmission electron microscopy of the complexity of circulating platelets collected from healthy individuals and from type 2 diabetic patients, a pathologic condition in which platelet hyperreactivity has been described. Platelet boundaries were extracted by means of automatically image analysis. Local fractal dimension by box counting (measure of geometric complexity) was automatically calculated. The results showed that the platelet boundary observed by electron microscopy is fractal and that the shape of the circulating platelets is significantly more complex in the diabetic patients in comparison to healthy subjects (p <  0.01), with 100% correct classification. In vitro activated platelets from healthy subjects show an analogous increase of geometric complexity. Computerized fractal analysis of platelet shape by transmission electron microscopy can provide accurate, quantitative, data to study platelet activation in diabetes mellitus.

Microvascular abnormalities in Rett syndrome.

Rett syndrome (RTT) is a post-natal neurological disorder that represents the second most common cause for mental retardation. The presence of cold hands and feet, and blue, a feature frequently observed in these patients, is one of the non-neurological phenotypes that characterizes RTT, up to now not well explained. We have performed videocapillaroscopy in subjects affected by Rett syndrome. We have observed ramified and bushy capillaries, characteristic features of neoangiogenic capillaries, dilated capillaries and an irregular and chaotic microvascular pattern. To quantify these features and to evaluate the microvascular pattern complexity, we have performed a fractal analysis. Fractal dimension and Lempel-Ziv indexes resulted higher in Rett females than in age-matched healthy females (p < 0.001; p < 0.001). Our findings indicate the presence of previously unrecognized microvascular abnormalities in Rett syndrome.

Abnormal vascular network complexity: a new phenotypic marker in hereditary non-polyposis colorectal cancer syndrome.

BACKGROUND: Hereditary non-polyposis colorectal cancer (HNPCC) (Lynch cancer family syndrome I (LCFS1) and II (LCFS2)) is one of the most common hereditary cancer disorders. HNPCC results from dominantly inherited germline mutations in mismatch repair (MMR) genes, leading to genomic instability and cancer. No predictive physical signs of HNPCC are available to date. AIMS: Increased complexity in tumour associated vascular growth has been reported. Here, we tested the hypothesis that an increased vascular network complexity is a phenotypic marker for LCFS2. METHODS: Fourteen subjects from an LCFS2 kindred (gene carriers, n=5; non-carriers, n=9) and 30 controls were examined. Fractal dimension (D) at two scales (D (1-46), and D (1-15), tortuosity (minimum path dimension, Dmin), and relative Lempel-Ziev complexity (L-Z) of the vascular networks from the lower gingival and vestibular oral mucosa were measured. RESULTS: LCFS2 networks exhibited a significantly increased overall complexity at both larger (D (1-46): 1.82 (0.04) v 1.68 (0.08); p<0.0001) and smaller (D (1-15): 1.51 (0.11) v 1.20 (0.09); p<0.0001) scales, increased destructured randomness (L-Z: 0.77 (0.09) v 0.56 (0.03); p<0.0001), and decreased vessel tortuosity (DMIN: 1.02 (0.03) v 1.07 (0.04); p=0.0005) compared with control patterns. The vascular networks of LCFS2 gene carriers showed higher complexity at the smaller scale (D (1-15): 1.59 (0.12) v 1.47 (0.07); p=0.034), and higher destructured randomness (L-Z: 0.85 (0.11) v 0.73 (0.05); p=0.013) than those of non-carriers. CONCLUSIONS: Increased oral vascular network complexity is a previously unrecognised phenotypic marker for LCFS2, and is related to gene mutation carrier status.

Acute thymic involution in fetuses and neonates with chorioamnionitis.

Chorioamnionitis represents the leading cause of preterm birth and related pathologic conditions as well as of fetal death and frequently occurs in symptom-free mothers. Recent radiologic findings have indicated that thymus size is significantly reduced in preterm infants born to mothers with subclinical, histologically proven chorioamnionitis. However, an accurate morphologic description of the thymus gland in fetuses and neonates with chorioamnionitis is lacking, although it is known that infection and other stress processes may cause lymphocyte depletion in the thymuses of infants and older babies (acute stress involution). We describe morphologic modifications in the thymus of fetuses with histologically proven chorioamnionitis and newborn infants with chorioamnionitis and proven sepsis. The main findings included (1) decreased organ volume (ANOVA, P < .0024); (2) reduced corticomedullary ratio (P < 10(-6)); (3) significant changes in the relationship between thymic parenchyma and thymic interstitial tissue with resulting increased organ complexity (P = .03); (4) severe reduction of thymocytes; and (5) other degenerative processes such as monocyte/macrophage infiltration of Hassall's bodies. These results indicate that chorioamnionitis, with or without sepsis, is associated with significant morphologic modifications in the thymus. We wish to note that the described thymic pathology is only one aspect of the fetal systemic inflammatory response syndrome with which chorioamnionitis is associated.

World Health Organization (WHO) and the World Heart Federation (WHF) Pathobiological Determinants of Atherosclerosis in Youth (PBDAY) Study. Histomorphometric investigation of the aorta and coronary arteries in young people from different geographical locations.

BACKGROUND AND AIM: At the Morphometric Reference Center of the World Health Organization (WHO) and the World Heart Federation (WHF) for the Pathobiological Determinants of Atherosclerosis in Youth (PBDAY) Project, histomorphometric studies were carried out on aortic and coronary artery walls of 590 people ranging from 5 to 34 yr of age, who were of European, American, Asian and African origin (6 countries) and died of trauma. The aim was to evaluate the mean intimal and medial thickness of arterial tissues. METHODS AND RESULTS: Computerized operator-assisted morphometry with a semiautomatic image analyzer was carried out on 2,893 histological slides (prepared at the WHO Reference Center in Malmö, Sweden). Our data show that age-dependent trends for prevalence of fatty streaks and atherosclerotic plaques in arteries are different: prevalence of fatty streaks increases until a plateau is reached, while exponential growth characterizes atherosclerotic plaque prevalence. Age is statistically associated with an increase in mean intimal and medial thickness in all specimens. CONCLUSIONS: Mean intimal and medial thickness differs in arterial wall specimens of individuals from various geographic locations: persons from countries with high mortality rates from cardiovascular disease tend to have greater mean intimal thickness in youth. Men with hypertension and those who smoke have significantly greater combined means of intimal thickness than normotensives and non-smokers respectively. The also have significantly greater combined means of medial thickness than normotensive men. Histomorphometric data provide evidence of the development of atherosclerotic lesions at an early age and relate their progression to ethnic and lifestyle factors and to major coronary heart disease risk factors.

World Health Organization (WHO) and the World Heart Federation (WHF) Pathobiological Determinants of Atherosclerosis in Youth (PBDAY) Study. Lipid and raised lesion distribution in the right coronary artery of young people.

BACKGROUND AND AIM: As part of the WHO-WHF PBDAY Study, we examined the proximal segment of the right coronary artery (RCA) (n = 469) of 5-34 yr-old otherwise healthy trauma victims from 15 countries to establish the topographical relation of atherosclerotic lesions to age, sex and geographical location. METHODS AND RESULTS: Topography and extent of lesions were analyzed by image processing and probability-of-occurrence maps of lipid lesions (mostly fatty streaks) and raised lesions on the intimal surface of the RCA were produced. Extension of lesions varied considerably between the groups and between individuals in the same group. The prevalence of lipid lesions was 68% (319/469) compared with 100% in the aorta. The prevalence of raised lesions was 22% (102/469) compared with 7% and 26% in the thoracic and abdominal aorta. Females had more lipid lesions, whereas raised lesions prevailed in males. Lipid lesion extent increased with age in both sexes. CONCLUSIONS: High probability-of-occurrence areas for lipid and raised lesions prevailed in the proximal 5 cm of the intimal surface of the RCA (myocardial side) and were greatly overlapping. Regression analysis between lipid and raised lesion extent in the thoracic or abdominal aorta and the RCA showed no correlations between the lipid lesion extent, whereas significant correlations were found between the raised lesion extent in the RCA and the thoracic aorta only, showing that the two types of lesion behave differently in different anatomical locations.

Kidney vascular damage and cocaine.

Cardiovascular damage is common in young cocaine addicts, and similar atherosclerotic lesions seem likely in the kidneys. To confirm this hypothesis, we performed histological examination of 40 kidney autopsy specimens classified as "cocaine-related deaths"; as controls, kidney specimens of 40 road accident victims were examined. Semiquantitative analysis showed that the ratio of the number of glomeruli affected by hyalinosis to the total number of glomeruli was 0.09 +/- 0.13 in addicts and 0.005 +/- 0.01 in controls; the difference was highly significant. The degree of periglomerular fibrosis was significantly higher in cocaine addicts than in accident victims. The ratio of glomeruli to tubular casts was 0.15 +/- 0.17 in cocaine addicts and 0.17 +/- 0.18 in controls (not significant). The degree of interstitial cellular infiltration was significantly higher in addicts than controls. A monunuclear cell infiltrate was observed prevalently in the medullary region. Arteriolar sclerosis was significantly higher in addicts than controls. Medial thickening, luminal narrowing and vessel obstruction were absent in the control group. Quantitative morphometric analysis of arterial structure showed significantly greater lumen circumference, intima circumference, media circumference, intima area, media area, intima thickness and media thickness in cocaine addicts than in controls.

Correction of erythrocyte shape abnormalities in familial hypercholesterolemia after LDL-apheresis: does it influence cerebral hemodynamics?

It is well known that red blood cells incubated in low-density lipoprotein (LDL)-rich medium show shape abnormalities that revert to normal after reincubation in normal plasma. Patients with homozygous familial hypercholesterolemia (HFH) have an increased percentage of abnormally-shaped erythrocytes (mostly stomatocytes, knisocytes, and crenated cells) compared to normocholesterolemic controls: 7.73+/-0.96 versus 3.52+/-0.52 (mean+/-SEM; P = 0.001). To confirm the role of high LDL concentration in inducing red cell shape abnormalities we determined the percentage of abnormally shaped erythrocytes in seven HFH patients 1 day after the procedure of LDL-apheresis with a 40% cholesterol decrease. A reduction in kniscocytes, stomatocytes, and crenated cells was observed in the patients treated by LDL-apheresis (P < 0.01). To investigate the possible benefit of a reduction in erythrocyte shape abnormality on cerebral hemodynamics, cerebral flow velocity, as evaluated by transcranial Doppler, was evaluated concomitantly and found to be remarkably increased after apheresis (P < 0.01). No significant change in hematocrit, plasma viscosity, blood viscosity, mean pressure, or cardiac output was detected, 1 day after apheresis. An inverse correlation was demonstrated (r = 0.55; P = 0.04) between changes in the percentage of knisocytes+stomatocytes +crenated cells and percent changes in middle cerebral artery peak systolic velocity. The correction of erythrocyte shape abnormalities after LDL-apheresis might be related to dramatic changes in plasma phospholipid concentration and proportion occurring after this procedure in HFH patients. The reduction of erythrocyte shape abnormalities could contribute, together with other hemorheological factors, to the improvement of cerebral hemodynamics after LDL-apheresis.

Platelet Activation in Hypercholesterolemic Patients Submitted to Therapeutic Plasmapheresis. An Ultrastructural Study.

Therapeutic plasmapheresis has been recommended as the choice therapy in patients with familial hypercholesterolemia. Little is known about the effect of plasmapheresis on platelet behavior. By means of electron microscopy we have studied the platelet plasma membrane of 4 patients with familial hypercholesterolemia who were submitted to repeated plasmaphereses. After each procedure of plasmapheresis, at the 15th day, morphometrical studies revealed a statistically significant increase in the surface density of the Open Canalicular System, which is considered a marker of platelet activation. However, during 12 months of plasmapheresis, a significant mean decrease in the morphometric parameters was observed. On one hand, these results indicate the necessity to consider the blood hemocoagulatory state in the patient who has been submitted to the above mentioned treatment, since the variation of these parameters after the therapeutic procedure, in a short time, could be potentially harmful to the patient; on the other hand, these results indicate, for the first time, the ability of the procedure to improve platelet behavior after repeated treatments on long term basis.

Morphological aspects of endothelial cell-monocyte interactions in culture.

Monocytes obtained from the blood of healthy volunteers were added to cultures of bovine aortic endothelial cells (BAEC) at increasing time intervals (from 1 h to 24 h). Cultures of BAEC without the added blood elements were used as controls. The ultrastructural features of cellular interactions were studied by transmission electron microscopy. After 4 h, in the co-cultured endothelial cells focal cytoplasmic lesions appeared in the areas where activated monocytes were strictly in contact. Adhering monocytes frequently appeared apoptotic. After 24 h, pseudopod-like cytoplasmic projections originating from the endothelial cells gave rise to areas of close contact between the BAEC and the monocytes. These data reveal until now unknown endothelial cell taxis toward blood cells.

The proliferating cell marker monoclonal antibody Ki-67 recognizes specific antigens associated with the nuclear envelope of the early Drosophila embryo.

Immunofluorescence and immunoelectron microscopy indicated that the antibody raised against the nuclear antigen Ki-67 of mammalian cells recognized antigenic determinants of early Drosophila embryos, localized on the outside of the nuclear envelope. Hence, the nuclear envelope of Drosophila appears to share a similar epitope with the chromosome scaffold of mitotic mammalian cells. With the progression of mitosis the antigen persisted around the mitotic spindle region and was also found in the pole regions at metaphase and anaphase. The antibody also stained the equatorial regions of the spindles from anaphase to late telophase. The antibody may therefore be used as a biochemical marker of the nuclear envelope for studying nuclear membrane biogenesis and behavior during the mitotic divisions of the Drosophila embryo.

Distribution of lipid and raised lesions in aortas of young people of different geographic origins (WHO-ISFC PBDAY Study). World Health Organization-International Society and Federation of Cardiology. Pathobiological Determinants of Atherosclerosis in Youth.

At the Morphometric Reference Center of the World Health Organization-International Society and Federation of Cardiology PBDAY (Pathobiological Determinants of Atherosclerosis in Youth) project, we studied left hemiaortas of 5- through 34-year-old male and female healthy subjects who died of traumatic injury. The subjects were either of European, American, Asian, or African origin. Three hundred fifty-five thoracic and 343 abdominal left hemiaortas, stained and photographed at the Malmö, Sweden, World Health Organization Reference Center, were studied. Lipid and raised lesion extent was evaluated by using computerized techniques. Probability-of-occurrence maps of lipid and raised lesion distribution were obtained by image processing. Our data have shown that the distributions of atherosclerotic lesions in the aortic intimal surface, which were similar in the different ethnic groups, also prevailed in branching regions, where low-blood flow shear stress and turbulence occur. The areas involved by raised lesions and by lipid lesions only partially overlapped. Lipid lesion extent, which was different among the ethnic groups, continuously increased with age in males but not in females, in whom the increase ceased at an age range from 15 through 24 years. This suggests that ethnic and dietary factors influence the extent but not the distribution of atherosclerotic lesions in the human aorta. Probability-of-occurrence maps also provided evidence that not every fatty streak will develop into a raised lesion, or will not develop quickly.

Preliminary morphometric data on lipid lesion distribution in aortas of young people (WHO-ISFC PBDAY study).

As the Morphometric Reference Center of the WHO-ISFC PBDAY Project (Pathobiological Determinants of Atherosclerosis in Youth) we are studying lesion involvement of left hemiaortas of young people from different parts of the world. Gross specimens were stained and photographed at the Malmö reference Center (Dir. Prof. N.H. Sternby). Percentage of lipid lesions were evaluated and probability maps of lipid lesion distribution at the aortic surface were obtained by image processing. In all samples from Italy, Mexico and Sri Lanka, high probability of lipid lesion occurrence was found between the emergences of the intercostal arteries, next to the emergence of the celiac trunk, between the emergence of the superior and inferior lumbar arteries and between the emergence of the 1st and the 2nd mesenteric arteries. Our data reveal that a) lipid lesions are localized in low shear-stress regions or in turbuecent regions; b) our samples taken from groups of different geographical origin present the same lipid lesion distribution at the aortic surface.

Blood cell activation: new perspectives from ultrastructural morphometry.

The new computerized approaches for image analysis available for a few years permit, in an excellent and detailed way, monitoring cellular structural modifications during activation processes. Here, we presented morphometric data obtained on blood cells (platelets, monocytes, and PMN cells) after in vitro activation and under in vivo conditions related to atherosclerosis studied by means of electron microscopy. Area and perimeter form factors, nucleus to cytoplasma area ratio, and the surface density of platelet plasma membrane openings of SCCS presented significant differences compared with the respective significant differences compared with the respective controls. Ultrastructural markers of blood cell activation were revealed by the computerized morphometry applied on ultrastructural images.

Updating on in-vivo and in-vitro effects of heparin and other glycosaminoglycans (mesoglycan) on arterial endothelium: a morphometrical study.

Glycosaminoglycans, which include heparin, heparansulfate and dermatansulfate, are substances that exhibit many significant biological activities. In-vitro and in-vivo experiments for studying the effects of heparin and an association of heparan-like glycosaminoglycan and dermatansolfate (mesoglycan) on aortic arterial endothelium were performed. The studies were developed by means of computerized morphometric techniques. The in-vitro tests, performed on bovine aortic endothelial cells, have revealed an increase in survival rate, enhancement of cell density at confluence, and increase of nucleus/cytoplasm ratio, after "in-vitro" administration of heparin or mesoglycan. The in-vivo tests have revealed a minor development of aortic intimal lipid deposition in mesoglycan-treated hypercholesterolaemic rabbits. Our morphometrical results confirmed by statistical tests strongly support the data collected in the literature over many years on the protective effects of mesoglycan and heparin on endothelium.