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.

Do mast cells affect villous architecture? Facts and conjectures.

In adult life, the architecture of the intestinal villus is maintained by a complex series of epithelial-stromal interactions that involve different types of fixed and mobile cells located in the intestinal mucosa. Mast cells (MC) are normal constituents of the small bowel mucosa where they reside in the villous and pericryptal lamina propria as well as within the columnar epithelial cell layer. Besides being involved in numerous immune and inflammatory reactions in the context of both innate and acquired host defence, MC are known to exert important non-immunological functions like wound repair, extracellular matrix remodelling, angiogenesis and neurotrophism as well as modulation of fibroblast, epithelial cell and smooth muscle cell activity. These pleiotropic functions put MC in a central, strategic position to organize tissue defence, restore tissue damage and maintain tissue homeostasis. This review summarizes the most recent advances concerning the functional anatomy of the crypt-villus unit and discusses the way intestinal MC might become part of the instructive circuits that ultimately lead to the maintenance of a proper villous shape.

Low mast cell density in the human duodenal mucosa from chronic inflammatory duodenal bowel disorders is associated with defective villous architecture.

BACKGROUND: Mast cells (MC) have recently been implicated in the processes of tissue homeostasis, remodeling and repair. DESIGN: In this study, the total and tryptase-reactive mast cell populations were quantified in the duodenal mucosa of 27 subjects suffering from chronic inflammatory bowel disorders. Mast cell density was both related to the general villous architecture (normal or defective) and to the microvascular density in the duodenal mucosa. RESULTS: Total mast cell and tryptase-positive mast cell subpopulation densities were found to be significantly reduced in the samples with defective villous architecture in comparison with those exhibiting a normal villous profile. In these last samples, a relevant proportion of mucosal mast cells exhibited ultrastructural features of secretory activity, in particular piecemeal degranulation. Finally, no correlation was established between microvascular density and tryptase activity, as it has been previously demonstrated in other pathological conditions. CONCLUSIONS: Overall, these findings indicate a significant correlation between mast cell density and the duodenal mucosal architecture.

Evidence that human cardiac myocytes divide after myocardial infarction.

BACKGROUND: The scarring of the heart that results from myocardial infarction has been interpreted as evidence that the heart is composed of myocytes that are unable to divide. However, recent observations have provided evidence of proliferation of myocytes in the adult heart. Therefore, we studied the extent of mitosis among myocytes after myocardial infarction in humans. METHODS: Samples from the border of the infarct and from areas of the myocardium distant from the infarct were obtained from 13 patients who had died 4 to 12 days after infarction. Ten normal hearts were used as controls. Myocytes that had entered the cell cycle in preparation for cell division were measured by labeling of the nuclear antigen Ki-67, which is associated with cell division. The fraction of myocyte nuclei that were undergoing mitosis was determined, and the mitotic index (the ratio of the number of nuclei undergoing mitosis to the number not undergoing mitosis) was calculated. The presence of mitotic spindles, contractile rings, karyokinesis, and cytokinesis was also recorded. RESULTS: In the infarcted hearts, Ki-67 expression was detected in 4 percent of myocyte nuclei in the regions adjacent to the infarcts and in 1 percent of those in regions distant from the infarcts. The reentry of myocytes into the cell cycle resulted in mitotic indexes of 0.08 percent and 0.03 percent, respectively, in the zones adjacent to and distant from the infarcts. Events characteristic of cell division--the formation of the mitotic spindles, the formation of contractile rings, karyokinesis, and cytokinesis--were identified; these features demonstrated that there was myocyte proliferation after myocardial infarction. CONCLUSIONS: Our results challenge the dogma that the adult heart is a postmitotic organ and indicate that the regeneration of myocytes may be a critical component of the increase in muscle mass of the myocardium.

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.

Myocyte death in the failing human heart is gender dependent.

Cardiovascular disease is delayed and less common in women than in men. Myocyte death occurs in heart failure, but only apoptosis has been documented; the role of myocyte necrosis is unknown. Therefore, we tested whether necrosis is as important as apoptosis and whether myocyte death is lower in women than in men with heart failure. Molecular probes were used to measure the magnitude of myocyte necrosis and apoptosis in 7 women and 12 men undergoing transplantation for cardiac failure. Myocyte necrosis was evaluated by detection of DNA damage with blunt end fragments, whereas apoptosis was assessed by the identification of double-strand DNA cleavage with single base or longer 3' overhangs. An identical analysis of these forms of cell death was performed in control myocardium. Heart failure showed levels of myocyte necrosis 7-fold greater than apoptosis in patients of both sexes. However, cell death was 2-fold higher in men than in women. Heart failure resulted in a 13-fold and 27-fold increase in necrosis in women and men, respectively. Apoptosis increased 35-fold in women and 85-fold in men. The differences in cell death between women and men were confirmed by the electrophoretic pattern of DNA diffusion and laddering of isolated myocytes. The lower degree of cell death in women was associated with a longer duration of the myopathy, a later onset of cardiac decompensation, and a longer interval between heart failure and transplantation. In conclusion, myocyte necrosis and apoptosis affect the decompensated human heart; each contributes to the evolution of cardiac failure. However, the female heart is protected, at least in part, from necrotic and apoptotic death signals.

Changes in DNA content of myocardial cells after cardiac explantation.

To evaluate the changes in DNA content of myocardial cells, image cytometric measurement was performed on a series of specimens obtained from 7 explanted hearts with respect to different fixation times after cardiac explantation. Prior to fixation, the tissue samples were stored at 4 degrees C or at room temperature. When the tissue samples were stored at room temperature, the integrated optical density decreased after 48 hours from cardiac explantation. Meanwhile, the coefficient of variation of integrated optical density and the sum of intermediate ploidies of myocyte nuclei increased. However, no significant changes were found when tissue samples were kept at 4 degrees C. This study indicates that no significant changes in DNA content are found within two days from cardiac explantation.

Image sampling in static telepathology for frozen section diagnosis.

BACKGROUND: A frozen section diagnostic service is often not directly available in small rural or mountain hospitals. In these cases, it could be possible to provide frozen section diagnosis through telepathology systems. Telepathology is based on two main methods: static and dynamic. The former is less expensive, but involves the crucial problem of image sampling. AIMS: To characterise the differences in image sampling for static telepathology when undertaken by pathologists with different experience. METHODS: As a test field, a previously studied telepathology method based on multimedia email was adopted. Using this method, three pathologists with different levels of experience sampled images from 155 routine frozen sections and sent them to a distant pathology institute, where diagnoses were made on digital images. After the telepathology diagnoses, the glass slides of both the frozen sections and the definitive sections were sent to the remote pathologists for review. RESULTS: Four of 155 transmissions were considered inadequate by the remote pathologist. In the remaining 151 cases, the telepathology diagnosis agreed with the gold standard in 146 (96.7%). There was no significant divergence between the three pathologists in their sampling of the images. Each case comprised five images on average, acquired in four minutes. The overall time for transmission was about 19 minutes. CONCLUSIONS: The results suggest that in routine frozen section diagnosis an inexperienced pathologist can sample images sufficiently well to permit remote diagnosis. However, as expected, the internet is too unreliable for such a time dependent task. An improvement in the system would involve integrated real time features, so that there could be interaction between the two pathologists.

Myocyte proliferation in end-stage cardiac failure in humans.

Introduced several decades ago, the dogma persists that cardiac myocytes are terminally differentiated cells and that division of muscle cells is impossible in the adult heart. More recently, nuclear mitotic divisions in myocytes occasionally were seen, but those observations were challenged on the assumption that the rate of cell proliferation was inconsequential for actual tissue regeneration. Moreover, mitoses were never detected in normal myocardium. However, the analysis of routine histologic preparations constituted the basis for the belief that myocytes were unable to reenter the cell cycle and divide, ignoring the limitations of these techniques. We report here by confocal microscopy that 14 myocytes per million were in mitosis in control human hearts. A nearly 10-fold increase in this parameter was measured in end-stage ischemic heart disease (152 myocytes per million) and in idiopathic dilated cardiomyopathy (131 myocytes per million). Because the left ventricle contains 5.8 x 10(9) myocytes, these mitotic indices imply that 81.2 x 10(3), 882 x 10(3), and 760 x 10(3) myocytes were in mitosis in the entire ventricular myocardium of control hearts and hearts affected by ischemic and idiopathic dilated cardiomyopathy, respectively. Additionally, mitosis lasts less than 1 hr, suggesting that large numbers of myocytes can be formed in the nonpathologic and pathologic heart with time. Evidence of cytokinesis in myocytes was obtained, providing unequivocal proof of myocyte proliferation.

Image selection in static telepathology through the Internet.

A telepathology study was carried out to examine the differences occurring when the images were selected by an experienced pathologist, a junior pathologist and a first-year resident. One hundred and fifty-five consecutive frozen-section pathology cases were collected and sent for consultation to a remote experienced pathologist using multimedia email. Local diagnoses (as reported in the files of the Institute, not from the image selector) and remote diagnoses (based on the images) were compared with those performed on paraffin-embedded sections. Acquisition time and number of selected images were recorded for each case and used to compare the different behaviour of the three local pathologists. Of the 155 cases sent by telepathology, four were considered insufficient for a diagnosis by the remote pathologist and thus the diagnosis was postponed. In the remaining 151 cases, the overall diagnostic agreement between remote and definitive diagnosis was 96.7%. The results indicate that in the routine diagnostic work of a frozen-section service, an inexperienced pathologist can select images which are sufficiently informative for a remote diagnosis, in a sufficiently short time.

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.

Telepathology using Internet multimedia electronic mail: remote consultation on gastrointestinal pathology.

The feasibility of using the Internet for remote pathology consultation was examined. We assessed the diagnostic agreement between two groups of pathologists who independently evaluated histopathological cases exchanged by Internet electronic mail. The exchange was between two different workstations using readily available software not specifically developed for telemedicine. Data and images from 76 cases were transmitted to four pathologists. An average of 4.5 images per case were transmitted at compression ratios of between 6:1 and 40:1, corresponding to 250 kByte of data per case. In two cases the remote pathologists could not make a diagnosis. Agreement was reached in 63 of the other 74 (kappa = 0.79). In 11 cases (15%) there was a misdiagnosis. However, the results are encouraging and suggest that Internet electronic mail can be used successfully for remote consultation in pathology.