Ultrastructure of the tunica albuginea in congenital penile curvature.

PURPOSE: We investigated the ultrastructure of the tunica albuginea in individuals with congenital penile curvature to explain the pathology of this disease. MATERIALS AND METHODS: Included in our study were 15 patients 17 to 24 years old with congenital penile curvature. Study material consisted of samples of the tunica albuginea excised from the greater curvature of the corpus cavernosum during surgical correction. Control samples were obtained from the lesser curvature on the side opposite the study material during the same operation. The 2 types of tissue were analyzed using transmitter electron microscopy. RESULTS: Ultrastructural examination of the control material revealed numerous collagen fibers that were homogenous in size and organization on cross section. Periodic striation was typical in collagen that produced fibers. In the study group the tunica albuginea structure had a chaotic pattern of collagen fibers that formed bundles with disrupted 3-dimensional organization. Diameter of the fibers differed greatly on cross section. We observed periodic widening and fragmentation of collagen fibers with the complete disappearance of striation and transformation into electron dense, fibrous granulated material. Disrupted fibroblasts without cell membrane and cellular organelles between collagen fibers were also visible. There was elastin accumulation without any morphological differences in the control and study groups. CONCLUSIONS: Our results show that ultrastructural changes in the tunica albuginea may cause congenital penile curvature, possibly by altering mechanical properties.

Effect of the cytokine rhTNF-alpha on the population of mast cells in the growth of MethA fibrosarcoma--a TEM study.

The aim of the present study was the ultrastructural characteristics of mast cell (MC) involved in host antitumor responses induced by local (i.t.) administration of recombinant human tumor necrosis factor alpha (rhTNF-alpha) in the primary focus of methA fibrosarcoma. MC were involved in tumor interstitium remodeling. Numerous mitochondria, well-developed RER and Golgi apparatus, clusters of polyribosomes, considerable polymorphism of granules and differentiated lamellar structures which frequently presented myelinic forms were observed after rhTNF-alpha application. In the study numerous fibres of the fibrous tissue, richly vascularized, occurred in the peripheral and intermediate tumor zones. Cluster of MC and tumor cells were seen on the border of the necrotic foci. However, proteolytic enzymes released by MC cause interstitial lysis, ensuring the place for tumor growth, and are involved in angiogenesis. Thus, it is not clear whether MC contribute to the inhibition of tumor growth or have an adjunctive role in tumor progression.

The antitumor effect of intraperitoneal treatment with rhTNF-alpha muteins on Ehrlich ascites tumor growth.

The intraperitoneal (i.p.) treatment with recombinant human tumor necrosis factor-alpha (rhTNF-alpha) is one of the possible therapies for tumors that are confined to the abdominal cavity. Clinical trials aiming at the exploitation of the antitumor effects of rhTNF-alpha have been largely disappointing. In this model the activity of some rhTNF-alpha derivatives was studied. Ehrlich's ascites tumor (EAT) bearing Swiss albino male mice were treated i.p. three times a week with 10 micrograms/mice of rhTNF-alpha, mutein V or mutein VI for two weeks, starting on the 4th day after tumor inoculation. Control mice received PBS. The effect of the rhTNF-alpha derivatives on the course of EAT was evaluated basing on: total ascites volume (TAV); packed cell volume (PCV); total packed cell volume (TPCV); inhibitory growth rate (IGR); cellular population of EAT fluid; morphological EAT cell changes and mean survival time (MST). In the study mutein VI had only a slight effect on MST but significant on TAV- and TPCV-IGR (p < 0.001). In mice treated with rhTNF-alpha and mutein V the enhancement of MST (p < 0.01) was accompanied by TAV- and TPCV-IGR (p < 0.001). The number of EAT cells in ascites decreased after rhTNF-alpha and mutein V administration (p < 0.001). We conclude that treatment with high-dose of this modified molecule lacking the possibility of binding with p75R and not producing so intensified side effects is likely to find wider application in therapy and prevent the ascites growth just as rhTNF-alpha dosage.

Comparative studies on the ultrastructure of the rat lungs after intratumor treatment of Morris hepatoma with rhTNF-alpha and its muteins.

The aim of the present study was the comparative analysis of morphological changes found in the lungs of Buffalo rats in the course of Morris hepatoma 5123 after i.t. treatment with recombinant human TNF-alpha (rhTNF-alpha) and its muteins. Modification of the native TNF-alpha molecule and synthesis of mutagenized analogues can prevent undesirable symptoms observed in the case of therapeutic administration of rhTNF-alpha. TNF-alpha has been shown to interact with two distinct membrane receptors (TNF-R): p55R and p75R. Mutagenized mutein V binds selectively with p55R. Mutein VI fails to recognize either TNF-R. The cytokines were applied in a dose of 10 micrograms protein in a cycle of 8 days. The control group consisted of tumor-bearing animals which were given PBS. Ultrastructural examinations were based on transmission electron microscope (TEM). Mutein VI-receiving animals showed enhanced changes of cytotoxic nature. Severe damage to endothelial cells (necrosis inclusive) was observed. Blood vascular lumen showed accumulation of neutrophils and monocytes. Features of enhanced activity of endothelial cells were noted. Focally, within pulmonary alveoli conglomerates of fibrin and fragments of damaged cells were found, with erythrocytes, neutrophils and macrophages in their vicinity. The epithelium of pulmonary alveoli showed signs of considerable damage, including necrosis. The lumen of pulmonary capillaries in rhTNF-alpha-treated animals showed a predominance of eosinophils and monocytic cells. Features of endothelial stimulation were observed, although without a tendency to form microthrombi. Much less pronounced changes both in the lung capillary bed and in the alveolar epithelial cells were noted in the mutein V-given animals. Our findings confirm the possibility of peripheral activation of cells involved in the cytokine-induced antitumor response. Mutein V with the smallest effect on the lung tissue rebuilding seems to be a rhTNF-alpha derivative which can delimit the undesirable symptoms in the course of antitumor therapy reduced to i.t. injections.

AgNORs in duct epithelial lesions in chronic pancreatitis and in pancreas cancer cells.

BACKGROUND/AIMS: Argyrophilic nucleolar organizer regions (AgNORs) reflect the proliferative activity of cells. Since the majority of pancreatic cancers are ductal carcinomas, the aim of the study was to determine the AgNORs expression of potential pre-neoplastic ductal epithelial lesions in advanced chronic pancreatitis compared with pancreatic cancer cells. METHODOLOGY: Histological preparations obtained from 24 patients with chronic pancreatitis and 16 patients with pancreatic cancer were used to estimate the number of AgNORs per nucleus. Four types of AgNORs were distinguished and histograms with cell percentage of each type were performed for all forms of epithelial anomalies. RESULTS: In simple hyperplasia, squamous and mucous metaplasia the number of AgNORs ranged from 1.92 to 2.23; type I was predominant. In papillary hyperplasia, dysplasia and in situ carcinoma the number ranged from 2.98 to 3.34, with a predominance of type II-IV. In invasive carcinoma the number was 4.29 and 74% of cells were of type II-IV. CONCLUSIONS: Both counts of AgNORs and the percentage of type II-IV cells showed a gradual increase from simple hyperplasia through papillary hyperplasia and dysplasia to invasive carcinoma which in this respect differs significantly from all forms of the epithelial anomalies examined.

Epithelial anomalies in chronic pancreatitis as a risk factor of pancreatic cancer.

BACKGROUND/AIMS: The relationship between chronic pancreatitis and the development of pancreatic cancer is still a matter of dispute. Our aim was to determine the frequency of hyperplastic, metaplastic and dysplastic epithelial anomalies in the course of chronic pancreatitis and the potential steps in their development to malignancy. METHODOLOGY: The study was based on biopsy material of 70 patients with clinically diagnosed advanced chronic pancreatitis, who underwent partial or total pancreatectomy, as well as other operations. The patients were assigned to 2 groups: Group I (n = 41) with calcifying chronic pancreatitis; Group II (n = 29) with other forms of the disease. Histological sections were stained with hematoxylin-eosin, Mallory-azan, Gomori's silver method, and glycosaminoglycans (PAS and Alcian blue staining). Special interest was focused on the type and incidence of epithelial ductal and acinar cell anomalies, and on the degree of parenchymal scarring. RESULTS: Hyperplasia of the ductal epithelium was present in 31.4%, focal squamous metaplasia in 21.4%, mucous metaplasia in 11.1%, cellular dysplasia in 8.6%, dysplastic acinar cell nodules in 21.4%, and "tubular complexes" in 30.0% of all cases. The differences in the frequency of these changes, except for ductal epithelial hyperplasia, were not statistically significant in two comparable groups. Advanced pancreatic fibrosis was associated with epithelial anomalies in 65.7% of all cases. CONCLUSIONS: From the morphological point of view, the adequate prerequisites for the consideration of advanced forms of chronic pancreatitis, independent of type, as a risk factor of pancreatic cancer exist, necessitating the surgical removal of pathological lesions.

Regression of Morris hepatoma in response to intralesional treatment with tumor necrosis factor muteins.

We examined the antitumor effects of human recombinant tumor necrosis factor alpha (rhTNF-alpha) and its muteins with the N-terminal amino acid sequence altered by point mutations against transplantable Morris hepatoma 5123 in rats. In vivo studies showed antiproliferative activity of the drugs in the dose range tested. For in vivo studies rhTNF-alpha and muteins were administered intratumorly (i.t.). The preparations were given at a dose of 10 microg/rat, once daily for eight days. Although the therapy was significantly effective in inhibiting tumor growth, complete growth inhibition could not be achieved. Nevertheless, there was a significant increase in survival time of tumor-bearing rats.

Protective effect of human recombinant tumour necrosis factor-alpha against lung metastases of the Morris hepatoma in rats.

The effect of intratumour (i.t.) administration of human recombinant tumour necrosis factor-alpha (hrecTNF-alpha) on Morris 5123 hepatoma spontaneous lung metastases was studied. Tumour was implanted in the skeletal muscles of the right hind limb of Buffalo rats. Two weeks after the implantation treatment with cytokine started. The cytokine was injected i.t. in a single dose of 10 micrograms in 4- and 8-day cycles. In control animals PBS was administered, respectively. Then all rats were necropsied and the extent of lung metastases was determined. Data were expressed as volume of lung metastases. In this study we observed significant antimetastatic effects of hrecTNF-alpha (p < 0.05) determined by reduced volume of lung metastases in treated animals.

Alveolar epithelial cells after intratumour administration of HREC TNF-alpha mutein VI.

The aim of this study was to explore the effect of intratumour mutein VI-Hrec TNF-alpha administration upon the ultrastructural changes within the pulmonary tissue, with special attention paid to type II alveolar epithelial cells. The experiment was carried out on Buffalo rats with implantable Morris hepatoma series 5123 in the skeletal muscles of the limb. Mutein VI-Hrec TNF-alpha was administered in a dose of 10 micrograms once a day in a cycle of eight days. Control animals were given saline (PBS). Ultrastructural changes within the pulmonary tissue were evaluated in the electron transmission microscope (TEM), with special attention paid to alveolar epithelial cells. In the animals receiving hrec TNF-alpha mutein VI, damage to the alveolar epithelial cells was found. In the later period (14 days after the mutein treatment), repairing processes were observed, accompanied by intensified fibrotic processes in the interalveolar septal interstitium, with the subsequent pulmonary tissue rebuilding. The study confirmed the possibility of a peripheral action of hrec TNF-alpha mutein VI after its administration to the experimental Morris hepatoma and found the alveolar epithelial cells to be a key element of the pulmonary tissue subjected to the cytokine effect.

Quantitative and morphological changes in BAL-isolated cells after the administration of TNF-alpha into Morris hepatoma.

The aim of this study was to explore the effect of intratumor TNF-alpha administration upon the composition and adherence degree of cells isolated from the lungs through multiple bronchoalveolar lavages (BAL). Ultrastructural evaluation of BAL-isolated cells was performed in the scanning electron microscope (SEM). The experiment used Buffalo rats. A suspension of 3 x 10(6) cells of Morris hepatoma (5123 series) was injected to the right hind leg of the animals. After fourteen days, TNF-alpha was administered into the tumor in a dose of 1.5 x 10(4) U in 0.5 ml PBS solution. The animals of group I were given 4 doses of TNF-alpha and group II-8 doses of TNF-alpha every 24 hours. Control groups consisted of rats with injected Morris hepatoma which were given PBS solution instead of TNF-alpha (group III A, B) and animals without the hepatoma, given intramuscullary 4 or 8 TNF-alpha, respectively (groups IV A, B). No statistically significant differences were noticed between groups I and II with regard to the number of macrophages and neutrophils isolated from the rat lungs compared with control group IV. However, such differences were observed compared with group III. In group II and IV B, an increase in the adherence of isolated cells was found compared with group III, as well as arise in the number of macrophages with the largest diameters. We found a correlation between the increase in cell adherence and ultrastructural changes (in SEM) suggesting an increased activity of BAL-isolated cells.

The ethanol effect on lung metastases development in experimental Morris 5123 hepatoma.

The experiment was carried out on 45 male Wistar rats which were divided into 3 groups: group I-15 rats which were given water to drink during the experiment, group II-15 rats obtaining 10% ethanol solution during the experiment and group III-15 rats obtaining 20% ethanol solution during the experiment. All animals were injected 0.3 ml suspension of hepatoma Morris 5123 cells directly to the liver on the 14th day of the experiment. After the 9 weeks of the experiment the animals from all groups were narcotized, decapitated and the lungs were taken into the morphometric, histological and ultrastructural examinations. They showed that ethanol has a stimulating effect on formation and development of hepatoma Morris 5123 in rat lungs. The increase of number and extensiveness of metastases as well as the increase of mean metastases focuses volume, and the increase of lung weight in animals which obtained ethanol are the exponent of the influence. On the basis of ultrastructural examinations, it can be noted that promoting activity of ethanol can be connected: a) with the increase of neoplastic cells activity, b) with changes appearing in pulmonic epithelium and endothelium of vessel enabling neoplastic cells to adhere to basal membrane as well as their crossing the vessel wall.

Ultrastructural evaluation of microcirculatory vessels in rheumatoid synovial membrane.

Since morphological lesions in microcirculatory vessels are often difficult to be found in the light microscope, the electron microscope investigations were performed on the synovial membrane biopsy specimens from 70 patients with rheumatoid arthritis (RA). Most common lesions referred to venules, capillaries and arterioles were swelling and proliferation of endothelial cells, adherence of lymphocytes, monocytes and neutrofiles to the endothelium, their margination and diapedesis. Also destructive changes in the endothelial cells, basement membrane thickening due to their multiplication and microthrombi were observed. Platelet aggregates, fibrin, fragments of desintegrated cells and deposits of granulofibrillary substance corresponding to fibrinoid necrosis were frequently seen. In 7 patients, lesions of this kind were found only in electronograms. It can be assumed that the evaluation of ultrastructural changes in the microcirculatory vessels may be of great significance as a complementary diagnostic examination in future determination of RA progression and further prognostication.

Microenvironment of Morris hepatoma 5123 metastases to the lungs--ultrastructural analysis of intratumour injections of rhTNF-alpha.

We analysed the effect of rhTNF-alpha on the development of Morris hepatoma 5123 spontaneous metastases to the lungs of Buffalo rats. After intratumour administration of the cytokine the lungs were found to contain scarce metastatic foci. In precapillaries and capillaries neoplastic cells were single or in small clusters, without contact with endothelial cells. Endothelial cells showed high pinocytic activity and frequently tightly closed vascular lumen. The stroma of interalveolar septa and the capillary bed contained multiple eosinophils. Moreover, eosinophils, histiocytes and monocytes mixed with abundant collagen fibres were found on the margin of single small metastatic foci and among necrotic neoplastic cells.

Tumor necrosis factor-alpha induces emphysema-like pulmonary tissue rebuilding. Changes in type II alveolar epithelial cells.

The effect of repeated doses of TNF-alpha on the histological picture of the pulmonary tissue was analyzed in the present study. Special attention was paid to the lung rebuilding processes. TNF-alpha was applied intraperitoneally for two weeks in a dose of 10 micrograms/0.5 ml PBS/24h. Morphological analysis of the pulmonary tissue was performed after 1 and 28 days following the last TNF-alpha dose. The study revealed focal pulmonary tissue rebuilding with emphysema-like changes twenty eight days following termination of TNF-alpha administration. The rebuilding processes included interalveolar septal atrophy, collagen accumulation and damage-repair changes in type II alveolar epithelial cells. It has been demonstrated that apart from the protease-antiprotease hypothesis of the lung emphysema, the inflammatory-repair hypothesis should be considered. Both hypotheses are complementary to each other and interpret the emphysema-like changes as complications of various pathological conditions of the pulmonary tissue.

Type II alveolar epithelial cells and free alveolar cells after intratumor TNF-alpha administration.

The experiment used Morris hepatoma 5123 series growing in muscles of the Buffalo rats. A suspension of 3 x 10(6) neoplastic cells was injected into the right hind leg of the animals. After fourteen days, TNF-alpha was administered into the tumour in a dose of 1.5 x 10(4) U/24 hours in 0.5 ml PBS solution. The group I animals were injected for 4 days and group II for 8 days. Control groups consisted of rats with injected Morris hepatoma which were given PBS solution instead of TNF-alpha (group III A and B) and animals without the hepatoma, given 4 or 8 TNF-alpha, respectively (groups IV A and B). In the present study, we have explored the effect of intratumor TNF-alpha administration on the composition of cells isolated from the lungs through multiple bronchoalveolar lavages (BAL). Ultrastructural evaluation of the pulmonary tissue was done using a transmission electron microscope (TEM), with special attention paid to type II alveolar epithelial cells and free alveolar cells. Examinations in TEM in groups I, II and IV (A and B) found, in the lumen of alveoli, an increase in the number of alveolar macrophages (AM) with morphological features of intensified activity and AM with numerous secondary lysosomes containing material of phospholipid structure. Also, numerous type II alveolar epithelial cells with emptied lamellar bodies were observed. The above mentioned changes were especially marked after eightfold TNF-alpha administration. In groups I, II and IV (A and B), compared with group III, a significant increase was found in the total number of cells isolated by BAL as well as in the number of cells with positive reaction in staining according to Beckstead's method. It may indicate that the changes in the parameters mentioned above are related to TNF-alpha action. The results obtained indicate the possibility of systemic effect of TNF-alpha after its administration into the experimental Morris hepatoma.

The effect of local hrec TNF-alpha administration upon the spontaneous lung metastases in rats with Morris 5123 hepatoma.

The experiment used Morris hepatoma 5123 series growing in muscles of the right hind limb of Buffalo rats. The group I animals were given intratumor 4 doses of TNF-alpha and group II-8 doses of TNF-alpha (10 micrograms/day). Control groups (III and IV) consisted of rats with injected Morris hepatoma, which were given PBS solution instead of TNF-alpha. A decrease in the volume of neoplastic metastases was observed in groups I and II, compared with groups III and IV. At the same time an increase was found in the volume of metastatic tumors in group II (8 x TNF-alpha), compared with group I (4 x TNF-alpha). Histological and ultrastructural analysis of the pulmonary tissue revealed intensified fibrotic reactions and inflammatory infiltrations around the metastatic tumors. The change were much more enhanced in group II, which might affect the results of neoplastic metastatic volume measurements. We concluded that multiple human recombinant TNF-alpha, hrec TNF-alpha, local injections inhibited dissemination of tumor cells and prolonged the survival time of rats up to the 76th day of the follow-up.