Co-localization of tryptase and cathepsin-G in mast cells in cutaneous mastocytosis.

Mastocytosis is a heterogeneous disease characterized by an abnormal growth and/or accumulation of clonal mast cells (MC) in one or more organs. The most frequent site of organ involvement is the skin. The aim of this study was to investigate the immunoreactivity to tryptase and to cathepsin-G of MC from human cutaneous mastocytosis and to compare their number in normal skin and cutaneous mastocytosis. Immunohistochemistry and dual immunofluorescence using anti-tryptase and anti-capthepsin-G antibodies was performed on biopsy specimens from 20 cases diagnosed as cutaneous mastocytosis. Tryptase-positive MC was more numerous as compared to cathepsin-G positive MC. Dual immunofluorescence for tryptase and cathepsin-G demonstrated a colocalization of tryptase and cathepsin-G in skin MC secretory granules. Morphometric evaluation of MC number demostrated that the number of both tryptase- and cathepsin-G-positive MC was significantly higher in cutaneous mastocytosis as compared to normal skin and that in both conditions the number of tryptase-positive MC was significantly higher as compared to the number of cathepsin-G-positive MC. In conclusion, in this study, for the first time we have demonstrated the presence of MC with immunoreactivity to cathepsin-G in human cutaneous mastocytosis, as well as the co-localization of tryptase and cathepsin-G in MC secretory granules.

Angiogenesis and mast cells in human breast cancer sentinel lymph nodes with and without micrometastases.

AIMS: An increasing number of mast cells have been reported in angiogenesis associated with solid and haematopoietic tumours. Data concerning the number of mast cells in neoplastic lymph nodes and their relationship with microvessel density are controversial. The aim was to correlate the extent of angiogenesis with the number of mast cells reactive with tryptase in biopsy specimens of sentinel lymph nodes with and without micrometastases obtained from patients with breast cancer. METHODS AND RESULTS: Specimens from sentinel lymph nodes obtained from 80 patients (40 with and 40 without micrometastases) were investigated immunohistochemically by using anti-CD31 and anti-tryptase antibodies. Angiogenesis, measured as microvessel counts, increased in parallel with the number of tryptase-positive mast cells and their values were significantly higher in lymph nodes with micrometastases compared with those without. CONCLUSIONS: Tryptase-positive mast cells may contribute, at least in part, to angiogenesis occurring in sentinel lymph nodes with micrometastases from patients with breast cancer.

Nuclear size of myocardial cells in end-stage cardiomyopathies.

OBJECTIVE: To determine the alteration of nuclear size in myocardial cells and the relationship between nuclear size and DNA ploidy classes in normal and cardiomyopathic human hearts. STUDY DESIGN: The study group consisted of 46 hearts obtained at biopsy. These patients had undergone cardiac transplantation for intractable congestive heart failure (18 cases with ischemic cardiomyopathy and 28 cases with idiopathic dilated cardiomyopathy). Another 10 hearts were collected at autopsy and used as control hearts according to preautopsy, autopsy and histology criteria. One hundred fibroblasts and 200 myocytes were evaluated in each ventricle. The nuclear area and DNA content were estimated using image cytometry. RESULTS: End-stage ischemic and dilated cardiomyopathies were characterized by an increase in nuclear size of both the myocyte and nonmyocyte population. The nuclear area of interstitial cells increased about 30% in cardiomyopathic hearts. Augmentation of average nuclear area of myocytes was 1.2-fold in the ischemic group and about 1.5-fold in the dilated group as compared with the control group. Also, a tendency was found for the coefficient of variation of average nuclear area to decrease in the interstitial cell population and increased in the myocyte population in cardiomyopathic situations. Furthermore, the nuclear area of myocytes enlarged as augmentation of nuclear DNA content. The relative nuclear areas of myocytes can be presented as: 2c:4c:8c:16c :32c:64c = 1:1.65:2.75:4.60:7.25:9.18. CONCLUSION: The increase in nuclear size follows either one of two different processes: the first does not involve an increase in DNA content, whereas the second is concomitant with an incremental increase in DNA content. In the first instance, the enlargement of nuclear size is limited. In the second, augmentation of nuclear size can become very impressive. In end-stage ischemic and dilated cardiomyopathies, the nuclear growth of myocytes and interstitial cells may be due to different mechanisms. Enlargement of the nuclear area of myocytes represents a complex process, including simple nuclear hypertrophy, polyploidization and multinucleation. The main pattern of nuclear growth of interstitial cells is nuclear hypertrophy without an increase in DNA content.

DNA content, apoptosis and mitosis in transplanted human hearts.

Aim of the study: To analyze the changes in DNA content, the percentage of apoptosis and the nuclear mitotic frequency of myocytes in transplanted human hearts. Methods: Twenty-three transplanted hearts were obtained from 22 patients. The mean interval between transplantation and death was 649 days (ranging from 13 to 2558 days). Ten control hearts were selected from individuals whose death was not due to primary heart disease. Tissue samples were obtained from the mid section of the lateral wall of left and right ventricles. DNA content was evaluated on isolated myocardial cells using image cytometry. In situ detection of apoptosis was performed by the terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) technique. Mitotic figures were examined by staining the nuclear DNA with YOYO-1 iodide. Myocytes were distinguished from stromal cells by using antibodies reacting with a-sarcomeric actin. Results: Comparing with control hearts, the myocytic changes after cardiac transplantation are characterized by: 1) a decrease in mononucleated myocytes and an increase in binucleated and multinucleated myocytes; 2) a decrease in diploid myocytic nuclei and a distinct augmentation of intermediate ploidies; 3) an increase in myocytic nuclei in DNA ploidies higher than 4c; 4) a marked augmentation of percentage of apoptotic myocytes and 5) an increased frequency of nuclear mitosis of myocytes; this fact appears as a declining phenomenon after six months of cardiac transplantation. Conclusion: After cardiac transplantation the DNA content of myocytes shows two completely different aspects: 1) a distinct increase in subdiploidy and intermediate ploidies related to myocyte injury induced by apoptosis and necrosis; 2) an increase in multinucleation, polyploidization and mitotic proliferation. Both myocyte growth and myocyte injury alter the function of the allograft and contribute to adaptation or failure of the graft. Furthermore, a relevant difference of age between the recipient and the donor may lead to a more marked myocyte damage and a lower myocyte growth. This tendency provides an evidence that age matching could be an important aspect in selecting the donor for the recipient.

Proliferating cell nuclear antigen (PCNA), DNA synthesis and mitosis in myocytes following cardiac transplantation in man.

Cardiac transplantation is characterized by rejection, myocyte loss, interstitial and replacement fibrosis, and loading abnormalities. These modifications contribute to enhance mural and muscle cell stress, activating reactive growth processes in myocytes and interstitial cells. However, it is unknown whether cell cycle related gene product, such as PCNA, and DNA synthesis are stimulated under these conditions. Therefore, 62 endomyocardial biopsies obtained from 17 patients who underwent cardiac transplantation were examined for the immunocytochemical detection of PCNA protein in myocyte and non-myocyte nuclei. In addition, tissue samples were labeled in vitro with bromodeoxyuridine (BrdU) to document ongoing DNA synthesis. The presence of mitotic images in myocytes and non myocytes were also examined. Biopsies were collected from 1-768 days after surgery. Histologic examination of tissue sections documented that PCNA labeling involved nearly 30% of myocyte nuclei in all patients. Similar percentages of PCNA labeling were detected in interstitial cells, lymphocytes and mononuclear infiltrates. DNA synthesis in myocytes and connective tissue cells was observed in nine and 14 subjects, respectively. BrdU positive lymphocytes and mononuclear infiltrates in 13 cases. Three mitotic figures in myocyte nuclei were identified. PCNA, BrdU labeling and mitosis were not detected in eight myocardial samples obtained from control hearts. These results suggest that the evolution of the transplanted heart involves the expression of a gene which is implicated in DNA replication. The presence of ongoing DNA synthesis and mitosis support the notion that proliferation of myocytes and non muscle cells may be a component of ventricular remodeling after cardiac transplantation.

DNA Content in End-Stage Heart Failure.

AIM OF THE STUDY: Heart failure is the final clinical presentation of a variety of cardiovascular diseases, such as coronary artery disease, hypertensive, toxic, and inflammatory heart disease. However, the cellular mechanisms responsible for the progressive deterioration of myocardial function observed in heart failure remain unclear and may result from cell death (programmed or not) and from an increase in number of nuclei and in the degree of their ploidy. METHODS: We examined thirty-eight explanted hearts obtained during transplantation for DNA content in the myocytic population. All thirty-eight patients had severe chronic heart failure: 23 had idiopathic dilated cardiomyopathy, and 15 had ischemic cardiomyopathy. Ten hearts of people whose death was not due to primary heart disease or as a consequence of major risk factors of coronary artery disease, including hypertension, diabetes, obesity, or severe atherosclerosis, were used as controls. DNA content in the myocytic population was evaluated using Image Cytometry. RESULTS: The DNA content per nucleus and per myocyte in cardiomyopathic hearts are characterized by: a) a decrease of the diploid DNA content of myocytic nuclei; b) an increase of DNA ploidies higher than 4c; c) a decrease in mononucleated myocytes; d) an increase in binucleated and multinucleated myocytes. The changes are more prominent in dilated cardiomyopathy. e) The total ploidy index, used to calculate the total DNA content, is related to heart weight and ventricular weight. CONCLUSIONS: Ischemic and dilated cardiomyopathies result in reduction of ventricular mass-to-chamber volume ratio and in discrete foci of myocyte cell death, leading to an elevation in systolic and diastolic stress on the remaining viable cells. Therefore mechanical stimuli generated by global and local loading abnormalities associated with end-stage failure may contribute to activate genes implicated in cell proliferation. Observations in this investigation are consistent with recent results documenting that in the presence of overload conditions the myocytes may retain their capacity to proliferate throughout life and this growth reserve mechanism may become operative in response to severe myocardial dysfuntion and overt failure. Polyploidization and multinucleation are prominent phenomena in the end-stage of ischemic and dilated cardiomyopathy in humans.

Computer-assisted morphometric analysis of the heart.

OBJECTIVE: To assess the applicability of the image analyzer for morphometric study of cardiomyopathies. STUDY DESIGN: A computer-assisted methodology for the morphometric study of ischemic cardiomyopathy, dilated cardiomyopathy and right ventricular dysplasia. RESULTS: In each heart, 200 nuclear lengths and myocyte diameters, 160 fields for nuclear density, 420 fields for interstitial fibrosis and 350 fields for replacement fibrosis were evaluated. In ischemic cardiomyopathy, the multiple foci of replacement fibrosis of the myocardium, in addition to interstitial fibrosis, appear to be the major cause of ventricular remodelling. In dilated cardiomyopathy the major pathologic processes are myocyte cell death and segmental, replacement and interstitial fibrosis. In right ventricular dysplasia the heart develops congestive failure due to a conspicuous increase in the volume of the right ventricle without an appreciable loss of the number of myocytes. CONCLUSION: The traditional morphometric methods based on test grids for the study of pathologic hearts can be enhanced using an image analyzer. In this way one can study a large area of the myocardium and collect a very large number of data. This may be the method of choice for analyses of pathologic human hearts.

Microscopic thymoma and myasthenia gravis.

A rare case of microscopically sized thymoma is described in a 56 year old man suffering from myasthenia gravis. Histological examination of the surgically removed thymus showed the presence of several epithelial thymoma-like islands. As controls, 100 thymuses obtained from consecutive necropsies were sampled: 4% of these cases showed epithelial islands. This case is further proof that "microscopic thymoma" is a true pathological entity and suggests that every thymus removed from myasthenic patients in which there is no macroscopic evidence of thymoma should be examined microscopically on serial sections.

Structure analysis of breast lesions using neighborhood graphs.

The College of American Pathologists divides proliferative lesions of the breast into three categories, but the diagnostic criteria for classification are not easy to apply. Therefore, many morphologic, immunohistochemical and morphometric features were studied in order to clarify this subdivision; however, the reported results are not very satisfactory. We also attempted to identify and differentiate the architectural features useful for the diagnosis of proliferative breast lesions. The morphologic characteristics of proliferative lesions of the breast were considered amenable to a graph theoretical approach. From a set of images of mammary ducts acquired from a hematoxylin-eosin-stained section, a hierarchy of graphs was built. The main graphs representing the structure are: (1) neighborhood graph, (2) planar graph, derived from (1) and (3) a dual graph. A simplified prototype of a system for structure analysis of the lesions was implemented and tested. Forty duct images classified by two expert pathologists were acquired and subjected to the system. The data obtained were analyzed, and some significant graph features validating our approach to the structure representation of the lesions were discovered. This approach can also be applied to other, similar classification problems and may help in understanding the biologic significance of proliferative lesions of the breast.

Right ventricular dysplasia: Right and left ventricular involvement morphometrically evaluated.

Right ventricular dysplasia (RVD) is a cardiac anomaly characterized by replacement of variable amounts of right ventricular myocardium by adipose tissue. This condition is believed to be a selective disorder involving extensively the right ventricle, but there are occasional reports of concomitant "minor" abnormalities of the left ventricle. The object of this report concerns a patient who died after heart transplantation because of an unsuspected RVD of the donor heart. We present a morphometric study of the heart in order to evaluate the distribution of the fat on both ventricles and to understand the structural basis of the heart failure. The results show that a large portion of the right ventricle is replaced by fat with a quite homogeneous distribution; the left ventricle is also largely replaced by adipose tissue that is primarily localized at the apex and decreases from the apex to the basis. The remodeling of the heart is attributable to a conspicuous increase in volume of the right ventricle associated with a normal number of myocytes that are longer than normal. For these reasons, according to Starling's law, the heart develops congestive failure.