Possible involvement of Pseudomonas fluorescens and Bacillaceae in structural modifications of Tuber borchii fruit bodies.

Previous studies on Tuber borchii fruit bodies in early maturation stages suggested a role of bacteria in sporocarp structural modifications. In order to verify this hypothesis, in the present study we investigated by means of microbial and ultrastructural approaches, the bacterial population of T. borchii sporocarps from intermediate maturation phases to advanced decomposition stages, paying particular attention to chitinolytic and cellulolytic bacteria and to their relationships with ascii and ascospores. We found that Pseudomonas fluorescens and spore-forming Bacillaceae, both able to degrade cellulose and chitin, are present inside the sporocarps in all maturation stages investigated. Moreover, rod-shaped bacteria seem able to erode ascus walls and colonize the interior of ascii containing mature spores. These results suggest a possible role of these bacteria in the process of ascus opening. Moreover, the presence of P. fluorescens and Bacillaceae on isolated mature spores after decontamination suggests an intimate association between these bacteria and the ascospores.

Disassembly of nuclear bodies during arousal from hibernation: an in vitro study.

In previous studies we demonstrated that during hibernation cell nuclei contain structural constituents usually absent in euthermia. The rapid disappearance of such nuclear bodies upon arousal makes very difficult the in vivo investigation of the disassembly process, which could clarify their functions in nuclear metabolism in the hibernator. In the present study we subjected liver samples taken from hibernating edible dormice ( Glis glis) to different in vitro experimental conditions: at 4 degrees C, to preserve the hypothermic state of the hibernating organism; at 37 degrees C, to simulate the drastic increase in body temperature occurring during arousal; at 37 degrees C, in culture medium containing 10(-5) M delta opioid D-Ala2- D-Leu5 enkephalin, which mimics the activity of the hibernation induction trigger in hibernators. Electron microscopic analysis of hepatocyte nuclei at increasing incubation times revealed the subsequent steps of disassembly of coiled bodies, amorphous bodies and fibro-granular material, the unusual structural constituents accumulating during hibernation in these nuclei. We demonstrated that: (1) a temperature of 37 degrees C induces the disappearance of all nuclear bodies typical of hibernation in a few minutes; (2) both low temperature and hibernation-triggering opioid are able to slow down, although to different extents, the process of disassembly of nuclear bodies; (3) the fibro-granular material rapidly disappears during the early phases of incubation; while (4) coiled bodies and amorphous bodies progressively disassemble as fibrous material. Our results support previous hypotheses based on in vivo observations about a possible role for coiled bodies, amorphous bodies and fibro-granular material as storage/assembly sites of molecules needed for the rapid and massive resumption of transcriptional and post-transcriptional activities upon arousal and suggest a strict correlation between the dynamics and metabolic rate of nuclear bodies.

Prostate-specific antigen synthesis and secretion by human placenta: a physiological kallikrein source during pregnancy.

Prostate-specific antigen (PSA), a kallikrein-like serine protease until recently thought to be prostate specific, has been demonstrated in various nonprostatic tissues and body fluids. PSA has been also found in human endometrium and amniotic fluids, even if the significance of this novel expression is unclear. In this study, we have demonstrated by multiple techniques that human placental tissue, obtained at delivery from normal full-term pregnancies, synthesizes and secretes PSA. RT-PCR showed the presence of PSA messenger ribonucleic acid; biochemical, chromatographic, and immunological studies revealed the expression of both free and complexed PSA forms; immunoelectron microscopy indicated the syncytiotrophoblast as the site of PSA synthesis and secretion. Moreover, in vitro experiments demonstrated that PSA production and secretion are up-regulated by 17beta-estradiol, a pregnancy-related steroid hormone. These results suggest that human placenta is a source of the PSA present in amniotic fluid and maternal serum during pregnancy.

Ultrastructural, morphometrical and immunocytochemical analyses of the exocrine pancreas in a hibernating dormouse.

Pancreatic acinar cells of euthermic, hibernating and arousing individuals of the hazel dormouse Muscardinus avellanarius (Gliridae) have been observed at the electron-microscopic level and analysed by means of ultrastructural morphometry and immunocytochemistry in order to investigate possible fine structural changes of cellular components during periods of strikingly different degrees of metabolic activity. During hibernation, the cisternae of the rough endoplasmic reticulum (RER) flatten assuming a parallel pattern, the Golgi apparatus is extremely reduced and the mitochondria contain many electron-dense particles. The cell nuclei appear irregularly shaped, with deep indentations containing small zymogen granules. They also contain abundant coiled bodies and unusual constituents, such as amorphous bodies and dense granular bodies. Large numbers of zymogen granules occur in all animals. However, the acinar lumina are open and filled with zymogen only in euthermic animals, whereas, in hibernating and arousing individuals, they appear to be closed. Morphometrical analyses indicate that, in pancreatic acinar cells, nuclei and zymogen granules significantly decrease in size from euthermia to hibernation, probably reflecting a drastic decrease of metabolic activities, mainly protein synthesis and processing. In all the studied animals, immunocytochemistry with specific antibodies has revealed an increasing gradient in alpha-amylase content along the RER-Golgi-zymogen granule pathway, reflecting the protein concentration along the secretory pathway. Moreover, during deep hibernation, significantly larger amounts of alpha-amylase accumulate in RER and zymogen granules in comparison to the other seasonal phases analysed. Upon arousal, all cytoplasmic and nuclear constituents restore their euthermic aspect and all morphometrical and immunocytochemical parameters exhibit the euthermic values, thereby indicating a rapid resumption of metabolic activities.

Ultrastructural characterization and biochemical profile of human gross cystic breast disease.

Human gross cystic breast disease is a benign condition affecting about 7-10% of adult women occurring with the highest incidence in the premenopausal decade. Although breast cysts do not represent a preneoplastic condition per se, several studies indicate an increased breast cancer risk in women affected by this pathology. In this report we study 115 breast cystic fluid samples obtained by needle-aspiration from women with gross cystic breast disease. The samples were analysed biochemically and the cells contained therein were observed at the electron microscope. According to their biochemical profiles, the cysts were subdivided into three types: Type I, showing a Na/K ratio < 0.5 and a typical protein content; Type II, showing a Na/K ratio >10 and a protein content quite similar to plasma; Type III, showing a Na/K ratio between 1 and 7 and an intermediate protein content. The electron microscopic examination demonstrated that Type I cystic fluid cells exhibit morphological features typical of actively synthesising and secreting cells, while the characteristics of Type II cells indicate a low metabolic activity. Type III cells have characteristics typical of both Type I and Type II cells, thereby confirming the intermediate nature of this cyst type. We hypothesise that these cyst types could represent different developmental stages of a structural evolution pathway, during which the biosynthetically active 'apocrine stage' would be the key step to cell neoplastic transformation.

Quantification of prostate-specific antigen immunoreactivity in human breast cyst fluids.

The frequency of gross cystic breast disease in premenopausal women and its possible association with increased breast cancer risk emphasises the importance of investigations relating to breast cyst fluid composition. In order to contribute to a better analysis of this medium, we have measured the presence of prostate-specific antigen immuno-reactivity in sixty-four human breast cyst fluids. Data analyses show that 35% of samples presented a level of this antigen < 0.05 micrograms/L, whereas 42 out of 64 cysts show a significant increase in the mean value of metabolically active apocrine cysts when compared to flattened cysts (p < 0.01). We report the first evidence that breast epithelium of gross cysts produces, secretes, and accumulates large amounts of prostate-specific antigen, a glycoprotein produced by prostatic tissue but recently detected in breast tumours, normal tissues, and during pregnancy. The production and intracystic accumulation of this serine protease in biosynthetically active apocrine type cyst can play a feasible role in the natural history gross cystic breast disease as well as in the mechanism of cyst formation, enlargement, and transformation.

Tissue polypeptide antigen immunostaining in fine needle aspirates from solid breast nodules.

The purpose of this study was to determine the role of Tissue Polypeptide Antigen (TPA) immunostaining in the evaluation of aspiration smears from solid breast nodules and to discuss its possible clinical applications. Seventy-one cytologic specimens were studied; 52 were carcinomas, 15 fibroadenomas and 4 fibrocystic dysplasia. TPA was visualized with the peroxidase-antiperoxidase method, using 3-3' diaminobenzidine as chromogen and hematoxylin as counterstaining. The incidence of TPA-positive aspiration smears was 88.5% in the carcinomas and 26.7% in the fibroadenomas. The four cases of fibrocystic dysplasia showed variable behaviour. The intensity of the TPA staining varied from - to and was most frequently high in the carcinomas. The results of this research show a good correlation between a diagnosis of carcinoma and TPA positivity in fine needle aspirates of solid breast nodules.

Establishment and characterization of two human small cell lung carcinoma cell lines.

Two human cellular lines of cancerous small lung cells obtained from biopsies specimens from two patients were characterized in vitro according to cellular morphology, growth modality, caryotype and antigenic profile. In vivo studies were carried out by inoculating the cells into nude mice of both sexes and various ages by different routes in order to study tumorigenicity and metastatic capacity and to identify a biological marker of the malignancy. Results obtained to date suggest that the two cellular lines have different biological properties similar to the classic and the variant form found in literature. A biological marker of the malignancy seems to be the antigenic profile of the cells.

Urothelial toxicity following conditioning therapy in bone marrow transplantation and bladder cancer: morphologic and morphometric comparison by exfoliative urinary cytology.

Since cyclophosphamide and busulphan used for therapy of bone marrow transplantation (BMT) can cause urothelial cell changes similar to those found in bladder cancer, comparative morphologic and morphometric urinary cytologic research was carried out, examining 812 urine samples taken from 121 patients undergoing BMT and 60 urine samples from 20 patients with bladder cancer. The morphological results showed some differences in the characteristics of the urinary sediment in urothelial toxicity caused by conditioning therapy in BMT and in bladder cancer; among these were background, cellularity, leukocytes, urothelial cell arrangement, cell shape and size, vacuolization, mitosis, and nucleoli. A comparative morphometric study was also carried out, showing differences regarding cell area, nuclear area and perimeter, and N/C ratio, especially between well-differentiated bladder cancer and urothelial toxicity.

Circulating immune complexes in human breast cyst fluids: relationship with intracystic immunoglobulin and electrolyte levels.

Circulating immune complexes, the major classes of immunoglobulins and electrolyte concentrations were measured in sixty-two breast cyst fluids aspirated in women affected by gross cystic breast disease. Two main classes of cysts were defined according to the Na/K ratio. Appreciable levels of immunoglobulins were found in almost all samples examined; 66% of breast cyst fluids showed increased levels of immune complexes. A highly significant linear correlation between increased values of immune complexes and immunoglobulin M (p less than 0.001) was found in apocrine cysts, characterized by Na/K ratio less than 3. However, a significant inverse linear correlation was found between positive values of immune complexes and lowered levels of immunoglobulins A (p less than 0.001) and G (p less than 0.001) in epithelial cysts with Na/K ratio greater than 3. These data suggest and confirm that the menstrual cycle can also influence or modulate the metabolic activity of human breast cells as a part of the secretory immune system. The relationship between immune complexes and immunoglobulins and electrolyte profiles may provide further knowledge about the immunological features of breast cyst fluid and suggest the possible alteration of immune-response in cystic breast lesions associated with increased cancer risk.

Urothelial cell changes due to busulfan and cyclophosphamide treatment in bone marrow transplantation.

Since the administration of cyclophosphamide and busulfan can cause hemorrhagic cystitis and changes in urothelial cells, an investigation was carried out to see whether patients undergoing bone marrow transplantation (BMT) who were treated with these drugs showed such urothelial changes and whether exfoliative urinary cytology can contribute to the early diagnosis and monitoring of such changes. Morphologic and morphometric analyses were performed on cytocentrifuged, Papanicolaou-stained preparations of 700 samples from 107 patients. Various degrees of urothelial cell changes were found in 30.8% of the cases. These changes consisted mainly of a considerable increase in the size of the nucleus and of a cytoplasm that was often bizarrely shaped. Even the structures of the nucleus and the cytoplasm changed. The results of this study showed that exfoliative urinary cytology permits an early diagnosis and monitoring of urothelial cell changes related to the administration of busulfan and cyclophosphamide in connection with BMT.

Ferritin and transferrin levels in human breast cyst fluids: relationship with intracystic electrolyte concentrations.

The concentrations of sodium and potassium and the content of ferritin and transferrin, proteins considered as potential markers for identifying cells undergoing divisional activity, were measured in fluid from 30 human breast cysts. On the basis of the relative electrolyte concentrations, two main classes of cysts were defined. When the cyst fluids were subdivided according to their Na+/K+ ratio, a significant difference was found between menstruating vs. menopausal patients. The relationship between the two major iron-binding proteins and the Na+/K+ ratio may indicate the expression of a local higher biosynthetic activity in apocrine cysts associated with higher cancer risk.

[Serum levels of Cd, Pb, Cu and Zn in cigarette smokers]. / Livelli sierici di Cd, Pb, Cu e Zn nei fumatori di sigarette.

In order to show the accumulation of Cd, Pb, Cu and Zn in smokers, levels of these metals in serum were determined in 108 subjects: 32 non-smokers, 37 average cigarette smokers and 39 heavy cigarette smokers. The analysis was carried out by potentiometric stripping analysis (PSA) with the Tecator "Striptec System". Backward oxidation time of the amalgamated metals, by means of electrolysis, in a thin "film" of mercury in an electrode, gives their concentration measure. Our data showed an increase in average values of Cd and Pb in the serum of heavy smokers compared with average and non-smokers. Instead, as regards Cu and Zn, no differences were found in the two groups of smokers compared with the non-smokers used as controls. Results obtained of the Cd and Pb levels in serum are compatible with the presence of these metals in cigarette tobacco and inhaling them could contribute to disease connected with their accumulation in the human organism. For these subjects the increase in Cd values is of particular importance as it could predispose pulmonary emphysema.

[Histochemical demonstration of CEA in pathology of the colorectum]. / Dimostrazione immunoistochimica del CEA nelle patologie del colon retto.

The evaluation of CEA serum levels and CEA tissue staining is one of the most common parameters used to help in the diagnosis and prognosis of colon-rectum disease. To this aim, an immunohistochemical demonstration of the CEA was carried out with the ABC staining system in sections of normal and diseased colon. 24 patients with polyps of the large intestine, 17 with rectocolitis, 22 with colon-rectum cancer and 11 normal subjects were studied. Results were positive in 9, 1% of the normal mucosa, 37, 5% of the polyps, 41, 2% of the rectocolitis and 100% of the carcinomas. However, the CEA in the positive reactions showed different degrees of staining and different cellular localization. From our results it would appear that the intracellular localization of this marker is connected with the degree of atypia in the histological sections. Immunohistochemical localization of the CEA could therefore be useful, above all in assessing the prognosis of colon-rectum disease.

Levels of human chorionic gonadotropin in breast cyst fluid.

The results of dosage of human chorionic gonadotropin (HCG) in 75 breast cyst fluids taken from 61 patients suffering from breast gross cystic disease (BGCD), 13 of which had multiple mono and/or bilateral cysts are discussed. The corresponding sera were also examined. Assays were carried out using the ELISA method. For comparison, HCG levels were also determined in 21 breast cyst fluids using RIA method. In 66.7% of the breast cyst fluids examined the hormone levels were higher than normal serum values. Corresponding sera showed HCG to be within the range of normal levels. In 5 patients the multiple and/or bilateral cysts showed widely differing hormone levels. The importance of these observations is discussed.

Lactate dehydrogenase, isoenzyme patterns and cation levels in human breast gross cyst fluid.

In order to investigate the diagnostic and/or prognostic value of total activity and isoenzymatic patterns of lactate dehydrogenase, 24 human breast gross cystic fluids were studied. A comparison of total lactate dehydrogenase activity to the serum level revealed an increased activity in about 63% of the cases examined; moreover, a significant increase in the slow-moving lactate dehydrogenase 4 and 5 isoenzymes was observed in some cyst fluids. The levels of Na+ and K+ concentrations were also analyzed and two classes of cysts were identified: one presenting Na+ and K+ levels similar to those found in extracellular compartment; the other with high K+ and low Na+ levels, characteristic of an intracellular fluid. This latter pattern could indicate an active metabolism of the epithelial cells lining the cysts. This breast cyst fluid also showed increased levels of lactate dehydrogenase 4 and 5 isoenzymes. The correlation between an increased activity of lactate dehydrogenase 4 and 5 isoenzymes and high K+ and low Na+ levels could be the expression of a high biosynthetic activity and of an anaerobic metabolism in some cysts, suggesting the evolution of the breast gross cyst lesion to malignancy. The importance of these observations is discussed.

[Carcinoembryonic antigen in a review of cases in the literature]. / L'antigene carcinoembrionario (CEA) nella revisione della casistica della letteratura.

Literature analysis of CEA serum levels tested in 21,147 patients, with non neoplastic and neoplastic disease of various organs, are reported. The sensitivity, the specificity, the positive and negative prognostic trend and the effective value of test in different pathologic situations are evaluated on the basis of organ localization and embryological origin.