Hypothesis for a new method to measure the dynamic patterns of tissue injury.

This note illustrates a novel method to measure the dynamics of pathological states using primary biliary cirrhosis as a prototype disease manifesting a dynamic course. The basis of the new method is the evaluation of liver sections using quantitative metrical concepts able to provide scalars useful for statistical purposes while abandoning the qualitative or semi-quantitative commonly used categorizations. The metrical measurements are provided by our fully automated machine coined the Metrizer. The method includes physical determinators that describe the history of the pathologic event with the time-related changes occurring in the liver tissue sections. The vectorial sum of the areas covered by two irreversible determinators (cytokeratin 7 positive cells representing ductular regeneration and liver fibrosis) is then utilized to illustrate the empirical trajectory of PBC. This ultimately provides a theoretical trajectory that includes all possible disease changes over time. The proposed approach includes both a description of the tissue architecture and an objective computerised dynamic diagnosis. Major strengths of this method include the solid physics and mathematics rationale and the high reproducibility irrespective the operator expertise along with the rapidity warranted by the computerised approach.

Metrically measuring liver biopsy: a chronic hepatitis B and C computer-aided morphologic description.

AIM: To describe a quantitative analysis method for liver biopsy sections with a machine that we have named "Dioguardi Histological Metriser" which automatically measures the residual hepatocyte mass (including hepatocytes vacuolization), inflammation, fibrosis and the loss of liver tissue tectonics. METHODS: We analysed digitised images of liver biopsy sections taken from 398 patients. The analysis with Dioguardi Histological Metriser was validated by comparison with semi-quantitative scoring system. RESULTS: The method provides: (1) the metrical extension in two-dimensions (the plane) of the residual hepatocellular set, including the area of vacuoles pertinent to abnormal lipid accumulation; (2) the geometric measure of the inflammation basin, which distinguishes intra-basin space and extra-basin dispersed parenchymal leukocytes; (3) the magnitude of collagen islets, (which were considered truncated fractals and classified into three degrees of magnitude); and (4) the tectonic index that quantifies alterations (disorders) in the organization of liver tissue. Dioguardi Histological Metriser machine allows to work at a speed of 0.1 mm(2)/s, scanning a whole section in 6-8 min. CONCLUSION: The results are the first standardized metrical evaluation of the geometric properties of the parenchyma, inflammation, fibrosis, and alterations in liver tissue tectonics of the biopsy sections. The present study confirms that biopsies are still valuable, not only for diagnosing chronic hepatitis, but also for quantifying changes in the organization and order of liver tissue structure.

Fractal dimension as a quantitator of the microvasculature of normal and adenomatous pituitary tissue.

It is well known that angiogenesis is a complex process that accompanies neoplastic growth, but pituitary tumours are less vascularized than normal pituitary glands. Several analytical methods aimed at quantifying the vascular system in two-dimensional histological sections have been proposed, with very discordant results. In this study we investigated the non-Euclidean geometrical complexity of the two-dimensional microvasculature of normal pituitary glands and pituitary adenomas by quantifying the surface fractal dimension that measures its space-filling property. We found a statistical significant difference between the mean vascular surface fractal dimension estimated in normal versus adenomatous tissues (P = 0.01), normal versus secreting adenomatous tissues (P = 0.0003), and normal versus non-secreting adenomatous tissues (P = 0.047), whereas the difference between the secreting and non-secreting adenomatous tissues was not statistically significant. This study provides the first demonstration that fractal dimension is an objective and valid quantitator of the two-dimensional geometrical complexity of the pituitary gland microvascular network in physiological and pathological states. Further studies are needed to compare the vascular surface fractal dimension estimates in different subtypes of pituitary tumours and correlate them with clinical parameters in order to evaluate whether the distribution pattern of vascular growth is related to a particular state of the pituitary gland.

Geometry of human vascular system: is it an obstacle for quantifying antiangiogenic therapies?

It is now recognized that all human natural and diseased anatomic systems are characterized by irregular shapes and very complex behaviors. In geometrical terms, tumor vascularity (which is the result of a nonlinear dynamic process called angiogenesis) is an archetypal anatomic system that irregularly fills a 3-dimensional Euclidean space. This characteristic, together with the highly variable nature of vessel shapes and surfaces, leads to considerable spatial and temporal heterogeneity in the delivery of oxygen, nutrients, and drugs, and the removal of metabolites. Although these biologic features have been well established, the quantitative analysis of neovascularity in 2-dimensional histologic sections still fails to view its architecture as a non-Euclidean geometrical object, thus allowing errors in visual interpretation and discordant results concerning the same tumor from different laboratories. We discuss here the tumor-induced vascular system as a fractal object, and what changes this new way of observing may bring to the quantification of effective antiangiogenic therapies.

Vascular architecture: is it a helpful histopathological biomarker for hepatocellular carcinoma?

Hepatocellular carcinoma (HCC) remains one of the major public health problems throughout the world. Although originally associated with tumorigenic processes, liver angiogenesis has also been observed in the context of different liver inflammatory, fibrotic, and ischemic conditions. Here we investigate the fractal dimension as a quantitator of non-Euclidean two-dimensional vascular geometry in a series of paired specimens of primary HCC and surrounding non-tumoral tissue, and discuss why this parameter might provide additional information regarding cancer behavior. The application of fractal geometry to the measurement of liver vascularity and the availability of a computer-aided quantitative method can eliminate errors in visual interpretation, and make it possible to obtain closer-to-reality numerals that are compulsory for any measurement process.

Sampling variability of computer-aided fractal-corrected measures of liver fibrosis in needle biopsy specimens.

AIM: To assess the sampling variability of computer-aided, fractal-corrected measures of fibrosis in liver biopsies. METHODS: Samples were derived from six to eight different parts of livers removed from 12 patients with clinically and histologically proven cirrhosis undergoing orthotopic liver transplantation. Sirius red-stained sections with a thickness of 2 mum were digitized using a computer-aided image analysis system that automatically measures the surface of fibrosis, as well as its outline perimeter, fractal surface and outline dimensions, wrinkledness, and Hurst coefficient. RESULTS: We found a high degree of inter-sample variability in the measurements of the surface [coefficient of variation (CV) = 43% +/- 13%] and wrinkledness (CV = 28% +/- 9%) of fibrosis, but the inter-sample variability of Hurst's exponent was low (CV = 14% +/- 2%). CONCLUSION: This study suggests that Hurst's exponent might be used in clinical practice as the best histological estimate of fibrosis in the whole organ, and evidences the fact that biopsy sections, which are fundamental for the qualitative diagnosis of chronic hepatitis, play a key role in the quantitative estimate of architectural changes in liver tissue.

The complex functions of mast cells in chronic human liver diseases.

Mast cells (MCs) are multifunctional effector cells of the immune system. MCs were originally thought to be involved in IgE-associated immediate hypersensitivity and allergic disorders, but it is now known that they contain or elaborate an array of mediators with a multitude of effects on many other cells. A number of studies have found that MCs are involved in various liver diseases. Although still controversial, they seem to be involved in the liver's fibrotic response to chronic inflammation and parasitic infection. Hepatic fibrosis is the most frequent liver response to toxic, infectious, or metabolic agents. During the establishment of this pathological condition, there is an increase in the components of the basement membrane that leads to continuous basement membrane-like structures being raised within Disse's space and a decrease in the number of sinusoid endothelial fenestrae. This leads to a complex process called "sinusoidal capillarization." At the cellular level, liver fibrogenesis is initiated by hepatocyte necrosis, which induces the recruitment of a large number of inflammatory cells, including MCs, which can be considered the primary effectors of the process changing sinusoidal endothelial cells into capillary-type endothelial cells. We review the roles played by MCs in hepatic chronic diseases and describe a biopsy section of hepatic tissue taken from a patient with chronic C virus-related hepatitis showing diffuse sinusoidal capillarization and a high density of MCs. This observation has led us to hypothesize a relationship between these highly specialized cells and sinusoidal capillarization.

Liver fibrosis and tissue architectural change measurement using fractal-rectified metrics and Hurst's exponent.

AIM: To provide the accurate alternative metrical means of monitoring the effects of new antiviral drugs on the reversal of newly formed collagen. METHODS: Digitized histological biopsy sections taken from 209 patients with chronic C virus hepatitis with different grade of fibrosis or cirrhosis, were measured by means of a new, rapid, user-friendly, fully computer-aided method based on the international system meter rectified using fractal principles. RESULTS: The following were described: geometric perimeter, area and wrinkledness of fibrosis; the collation of the Knodell, Sheuer, Ishak and METAVIR scores with fractal-rectified metric measurements; the meaning of the physical composition of fibrosis in relation to the magnitude of collagen islets; the intra- and inter-biopsy sample variability of these parameters; the"staging" of biopsy sections indicating the pathway covered by fibrosis formation towards its maximum known value; the quantitative liver tissue architectural changes with the Hurst exponent. CONCLUSION: Our model provides the first metrical evaluations of the geometric properties of fibrosis and the quantitative architectural changes of the liver tissue. The representativeness of histological sections of the whole liver is also discussed in the light of the results obtained with the Hurst coefficient.

Sperm protein 17 is expressed in human nervous system tumours.

BACKGROUND: Human sperm protein 17 (Sp17) is a highly conserved protein that was originally isolated from a rabbit epididymal sperm membrane and testis membrane pellet. It has recently been included in the cancer/testis (CT) antigen family, and shown to be expressed in multiple myeloma and ovarian cancer. We investigated its immunolocalisation in specimens of nervous system (NS) malignancies, in order to establish its usefulness as a target for tumour-vaccine strategies. METHODS: The expression of Sp17 was assessed by means of a standardised immunohistochemical procedure [(mAb/antigen) MF1/Sp17] in formalin-fixed and paraffin embedded surgical specimens of NS malignancies, including 28 neuroectodermal primary tumours (6 astrocytomas, 16 glioblastoma multiforme, 5 oligodendrogliomas, and 1 ependymoma), 25 meningeal tumours, and five peripheral nerve sheath tumours (4 schwannomas, and 1 neurofibroma). RESULTS: A number of neuroectodermal (21%) and meningeal tumours (4%) were found heterogeneously immunopositive for Sp17. None of the peripheral nerve sheath tumours was immunopositive for Sp17. The expression pattern was heterogeneous in all of the positive samples, and did not correlate with the degree of malignancy. CONCLUSION: The frequency of expression and non-uniform cell distribution of Sp17 suggest that it cannot be used as a unique immunotherapeutic target in NS cancer. However, our results do show the immunolocalisation of Sp17 in a proportion of NS tumour cells, but not in their non-pathological counterparts. The emerging complex function of Sp17 makes further studies necessary to clarify the link between it and immunopositive cells.

Sperm protein 17 expression defines 2 subsets of primary esthesioneuroblastoma.

Esthesioneuroblastomas (ENBs) are rare malignant tumors of the nasal vault, the origin, diagnosis, and management of which are still subjects of discussion. That there is no related prognostic factor or generally recognized therapeutic regimen highlights the need for further analyses of its underlying biologic features and investigations of new marker proteins that allow more reliable clinical testing. We here show that sperm protein 17 (Sp17) is expressed in the ciliated cells of the normal olfactory epithelium and in a proportion of primary ENB lesions. We found an association between Sp17 expression and metastases at relapse (P = .035), chromogranin expression (P = .014), and a female sex prevalence. A statistically nonsignificant relation was found between Sp17 and S-100, synaptophysin, and neurofilament expression. No correlation was also found between Sp17 expression and the proliferative capacity of the lesion that was evaluated by Ki-67 immunohistochemistry. The results of this study show the usefulness of Sp17 as a means of discriminating 2 subsets of primary ENB lesions and seem to suggest the existence of 2 distinct cell pathways in their origin and development.

Sperm protein 17 is expressed in human somatic ciliated epithelia.

It was once believed that sperm protein 17 (Sp17) was expressed exclusively in the testis and that its sole function was to bind to the oocyte during fertilization. However, immunohistochemistry of the human respiratory airways and reproductive systems show that it is abundant in ciliated cells but not in human cells with stereocilia and microvilli. The high degree of sequence conservation throughout its N-terminal half, and the presence of an A-kinase anchoring protein (AKAP)-binding motif within this region, suggest that Sp17 plays a regulatory role in a PKA-independent AKAP complex in both male germinal and ciliated somatic cells.

Immunolocalization of sperm protein 17 in human testis and ejaculated spermatozoa.

Sperm protein 17 (Sp17) is a highly conserved mammalian protein whose primary function is still poorly understood. Immunohistochemistry (IHC) in the human testis reveals the presence of Sp17 in some spermatocytes and abundantly in spermatids. All spermatogonia, Sertoli cells, and Leydig cells appear to be immunonegative for Sp17, whereas some interstitial cells are immunopositive. IHC recognized two distinct populations (immunopositive or not for Sp17) in the ejaculated spermatozoa. Although it will be necessary to clarify why some ejaculated spermatozoa do not contain Sp17, its distribution suggests that this protein may be associated with some phases of germinal cell differentiation.

Mast cells and human hepatocellular carcinoma.

AIM: To investigate the density of mast cells (MCs) in human hepatocellular carcinoma (HCC), and to determine whether the MCs density has any correlations with histopathological grading, staging or some baseline patient characteristics. METHODS: Tissue sections of 22 primary HCCs were histochemically stained with toluidine blue, in order to be able to quantify the MCs in and around the neoplasm using a computer-assisted image analysis system. HCC was staged and graded by two independent pathologists. To identify the sinusoidal capillarisation of each specimen 3 ?m thick sections were histochemically stained with sirius red, and semi-quantitatively evaluated by two independent observers. The data were statistically analysed using Spearman's correlation and Student's t-test when appropriate. RESULTS: MCs density did not correlate with the age or sex of the patients, the serum alanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels, or the stage or grade of the HCC. No significant differences were found between the MCs density of the patients with and without hepatitis C virus infection, but they were significantly higher in the specimens showing marked sinusoidal capillarisation. CONCLUSION: The lack of any significant correlation between MCs density and the stage or grade of the neoplastic lesions suggests that there is no causal relationship between MCs recruitment and HCC. However, as capillarisation proceeds concurrently with arterial blood supply during hepatocarcinogenesis, MCs may be considered of primary importance in the transition from sinusoidal to capillary-type endothelial cells and the HCC growth.

The role of amino acids in corneal stromal healing: a method for evaluating cellular density and extracellular matrix distribution.

PURPOSE: To evaluate how amino acid supplementation influences the response of corneal stromal keratocytes and other components of the corneal stroma. METHODS: We compared two groups of patients undergoing cataract surgery. Each group included 20 eyes of 20 patients. Patients in Group 1 were treated with amino acids for 15 days before surgery, and patients in Group 2 did not receive amino acids (control). To evaluate differences and change in corneal stroma, we analyzed all of the operated corneas with confocal microscopy. The confocal images were then analyzed with a new method for measurement of corneal structure based on fractal dimension calculation. This led to a numerical value, D index (fractal surface dimension), an objective measurement of corneal stromal characteristics. This index is related to three factors: keratocyte density, stromal distribution pattern, and intercellular matrix structure. RESULTS: Patients in the preoperative amino acid group showed higher keratocyte density values than those in the control group. We observed differences in cell distribution patterns and intercellular matrix structures between groups. Amino acid-treated patients had a corneal stroma with a D index value 10% higher than control group patients. CONCLUSIONS: We hypothesized that preoperative oral supplements with amino acids may change the corneal stroma structure by increasing keratocyte density and modifying the intercellular matrix. A new method for the measurement of these changes was applied, which provides quantification of the stromal structure complexity with a single numerical value.

Fractal dimension rectified meter for quantification of liver fibrosis and other irregular microscopic objects.

OBJECTIVE: To introduce a new mathematical method based on the principles of fractal geometry analysis that permits more realistic quantification of some of the physical (morphologic) aspects of irregular bodies appearing under microscopy. STUDY DESIGN: The principles of the method were tested on microscopic images of irregular collagen deposition in liver tissue. The method uses an ad hoc rectified meter implemented in a computer-assisted planar image analysis system that has been adapted to give metric measures of irregular outlines and surfaces that can be used to produce an index capable of quantifying the typical wrinkledness of biologic objects. Prototypical example measures of liver fibrosis were made on biopsy specimens showing chronic hepatitis C virus-related disease. Measurements were also made of the microscopic images of the abnormal deposition of lipid droplets in hepatocytes, a case of amyloid deposition in an osteoarthromuscular structure and a cytologic specimen of human dendritic cells. RESULTS: The proposed computer-aided method permits rapid measurements of the image of a whole biopsy section digitized at high magnification. The snapshot measurement of liver fibrosis deposition offered by a biopsy pattern is a valid means of more rigorously identifying the staging of the process. CONCLUSION: This method can measure liver fibrosis during chronic liver disease as well as any other irregular biologic structure that cannot be correctly quantified using traditional Euclidean-based metric methodologies.

Mast cell density: a quantitative index of acute liver inflammation.

OBJECTIVE: To evaluate mast cell (MC) density, in liver tissues taken from young and aging rats treated with carbon tetrachloride (CCl4) or untreated, as a quantitative marker of acute liver inflammation and to investigate whether the density of MCs varied with the rats' age. STUDY DESIGN: Rats aged 2, 6, 12 and 19 months treated intraperitoneally with CCl4 were killed 2 and 24 hours after intoxication. Hepatocellular damage was established by measuring alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity. Four histologic sections of 12 specimens from each age group were stained with toluidine blue to identify the MCs, which were counted using a computer-assisted image analysis system. RESULTS: Histology showed hepatocellular necrosis with inflammatory infiltration both 2 and 24 hours after intoxication. Serum AST levels were high in the 6- and 12-month-old rats, whereas ALT levels were high in the those aged 2 and 19 months. Two and 24 hours after intoxication, MC density increased considerably in young rats but less so in rats aged 19 months. CONCLUSION: MC density can be a useful marker of acute liver inflammation. The greater density in young rats suggests that older rats have a reduced immune response or recruit fewer MCs.

Reduced collagenolytic activity of matrix metalloproteinases and development of liver fibrosis in the aging rat.

Although moderate fibrosis is a histological hallmark of the aging liver, the molecular mechanisms underlying this phenomenon are little known. Here, we provide a comprehensive description of hepatic collagen expression and metabolism during natural aging in rats. Interstitial collagen accumulated significantly in the oldest animals, mainly in the periportal area (P<0.05, 19- vs. 2-month-old rats). This was ascribed to COL-III protein deposition (P<0.05 vs. 2-month-old rats), rather than COL-I. Conversely, the transcription activity of COL-III gene decreased (P<0.05) during the considered lifespan (2-19-months), whereas COL-I and transforming growth fator-beta1 (TGF-beta1) mRNA content was substantially unchanged. In the aged rats, hepatic matrix metalloproteinases (MMP) activity, (both MMP-1 and MMP-2) dropped significantly (P<0.05), with a concomitant increase of the inactive tissue inhibitor of MMP (TIMP-1)/MMP-1 complex (P<0.05). MMP-2 and TIMP-1 levels were weakly affected. All together, these results suggest that during natural aging, (i) COL III is the protein that accumulates preferentially in the liver; (ii) liver fibrosclerosis is mainly explained by a reduced proteolytic activity of matrix MMP, in which TIMP-1 seems to be a major regulating factor.