Stimulation of islet cell proliferation enhances pancreatic ductal carcinogenesis in the hamster model.

Previous studies have shown that some N-nitrosobis (2-oxopropyl)amine (BOP)-induced ductal/ductular pancreatic cancers in the hamster model develop within islets and that streptozotocin (SZ) pretreatment that caused islet degeneration and atrophy inhibits pancreatic cancer induction. Hence, it appears that in this model islets play a significant role in exocrine pancreatic carcinogenesis. To examine whether stimulation of islet cell proliferation (nesidioblastosis) enhances pancreatic exocrine cancer development, we tested the effect of the pancreatic carcinogen BOP in hamsters after induction of nesidioblastosis by cellophane wrapping. Before wrapping, hamsters were treated with SZ to inhibit pancreatic tumor induction in the unwrapped pancreatic tissues. Control groups with a wrapped pancreas did not receive SZ. Six weeks after SZ treatment, all hamsters were treated with BOP (10 mg/kg body weight) weekly for 10 weeks and the experiment was terminated 38 weeks after the last BOP treatment. Many animals recovered from their diabetes at the time when BOP was injected and many more after BOP treatment. Only nine hamsters remained diabetic until the end of the experiment. Both SZ-treated and control groups developed proliferative and malignant pancreatic ductal-type lesions primarily in the wrapped area (47%) but less frequently in the larger segments of the pancreas, including the splenic lobe (34%), gastric lobe (13%), and duodenal lobe (6%). Only a few lesions developed in the unwrapped pancreatic region of nine diabetic hamsters with atrophic islets, whereas seven of these hamsters had tumors in the wrapped area. Histologically, most tumors appeared to originate from islets, many invasive carcinomas had foci of islets, and some tumor cells showed reactivity with anti-insulin. The results show that, in the BOP hamster model, islets are the site of formation of the major fraction of exocrine pancreatic cancer and that induction of nesidioblastosis enhances pancreatic carcinogenesis.     

Modification of pancreatic carcinogenesis in the hamster model. 3. Inhibitory effect of alloxan.

Alloxan, when given intravenously at a dose of 60 mg/kg body weight 2 hours prior to subcutaneous injection of the potent pancreatic carcinogen N-nitrosobis (2-oxopropyl) amine (BOP), inhibited the induction of hyperplastic and neoplastic pancreatic lesions in a statistically significant fashion (P less than 0.01). The number of lesions per animal affected was markedly less in these animals, compared with BOP-treated control animals. BOP administration 2 weeks after alloxan treatment, at which time pancreatic islet cell regeneration is considered completed, did not alter either the incidence or number of lesions. The results support our view that the pancreatic islet cells are the primary source of BOP metabolism. The concomitant inhibition of gallbladder tumors, but not of common duct neoplasms, in hamsters receiving BOP 2 hours after alloxan could indicate that alloxan's inhibitory effects on BOP carcinogenesis are not restricted to the pancreas.     

Modification of pancreatic carcinogenesis in the hamster model. 7. Inhibitory effect of bethanechol chloride

Experiments were designed to investigate in the hamster model the effect on pancreatic carcinogenesis of bethanechol chloride (BC), which is known to increase pancreatic protein synthesis in rats. Hamsters received a single (15 mg/kg body weight) dose of BC either before, simultaneously with, or after a single dose of N-nitrosobis(2-oxopropyl)amine (BOP; 20 mg/kg body weight). A second group was treated daily with BC (7.5 mg/kg body weight) for 24 weeks, following BOP. The control groups consisted of animals treated with BOP only, with BC only, and with solvent only. The surviving hamsters were killed 46 weeks after BOP treatment. BC, whether given before, simultaneously with, or after BOP, significantly reduced the incidence of pancreatic ductal/ductular carcinomas. The multiplicity, size, and latency of carcinomas were also affected by BC. A more pronounced inhibition of cancer induction occurred in the group treated daily with BC after BOP. The possible mechanisms involved in the inhibitory action of BC on pancreatic carcinogenesis are discussed.     

An immunohistochemical, histological, and electron-microscopic study of the human periodontal ligament during orthodontic treatment

The periodontal ligament lies between the hard tissues of alveolar bone and cementum of teeth and serves to anchor the tooth to the alveolus and functions as a cushion between these hard tissues to migrate occlusal force during mastication. This tissue is always exposed to mechanical stress during mastication. When occlusal forces exceed the adaptive capacity of the periodontal ligament, the periodontal ligament tissue will be injured and then occlusal trauma will occur. The different modifications of periodontal ligament during load deformation can be monitored by analysis of the expression of different collagen types and fibronectin, with immunohistochemical techniques, and by morphological study of ligament, with light- and transmission electron-microscopic techniques. The use of continued and light orthodontic force generates a pressure of ligament with ejection of parodontal fluid externally and partial closing of vessels. On these basis we performed a study in order to evaluate periodontal ligament collagen types I and IV and the fibronectin modifications induced by application of a precalibrated orthodontic strength. We integrated these results, with light and transmission electron-microscopic observations, in order to evaluate the morphological modifications of periodontal tissue. Our observations showed that the type I collagen immunofluorescence staining is increased in the pressure side; in the tension side, it shows prior to treatment an increase, and after 72 h of treatment, a diminution of the staining pattern. Type IV collagen staining is reduced in both sides, but increased gradually after 7 days from treatment; finally, fibronectin staining pattern is gradually increased in the pressure side and reduced in the tension side. In light and transmission electron-microscopic observations it is possible to show a reduction of vessels at 72 h from treatment, and an increase of vessels after 7 days from treatment. The Malassez's epithelial residues are decreased at 72 h, while they are increased after 7 days from treatment. The modifications of immunofluorescence staining patterns of tested proteins revealed angiogenesis and reparative processes, and a thickening of fibrillar matrix as a defensive reply to mechanical stress. The modification of normal staining patterns of tested protein in our observations, could be determined by variation of scaffold geometry of periodontal ligament. The reduced movements of contraction and relaxation of periodontal ligament, due to orthodontic treatment, provoke a loss of mechanical stresses transmitted over ligament surface. Mechanical signals, therefore, could be integrated with other environmental signals and transduced into biochemical signals through force-dependent changes in scaffold geometry. Physical forces of gravity, hemodynamic stresses, and movement play a critical role in tissues, since the cells use tensegrity architecture for their structural organization.     

Modification of pancreatic carcinogenesis in the hamster model. 2. The effect of partial pancreatectomy.

The effect of partial pancreatectomy (PP) on the pancreatic carcinogenicity of N-nitrosobis (2-oxopropyl)amine (BOP) was investigated in Syrian golden hamsters by subcutaneous injection of a single dose of BOP (20 mg/kg, body weight) given 30 minutes after (Group 1), 1 week after (Group 2), or 1 week before 70% PP (Group 3). Additional groups consisted of animals with PP alone (Group 4), sham operation (laparotomy) followed 30 minutes later by BOP treatment (Group 5), and BOP treatment only (Group 6). The experiment was terminated 46 weeks after BOP administration in each group. The pancreas and extrahepatic bile ducts, including the common duct and gallbladder, were examined histologically. Tumor patterns were compared in hamsters with PP and in the corresponding segments of the pancreas in BOP-treated control groups. The pancreatic cancer incidence was highest (31%) in Group 2 and lowest in Group 1 (3%), a difference that was statistically significant (P less than 0.01). Also, a statistically highly significant larger number of tumors occurred in Group 2, compared with group 1, 3, or 5 (P less than 0.0005). In a comparison of the number of carcinomas per tumor-bearing hamster, there were greater numbers of carcinomas in Group 2 (2.6 carcinomas) than in Groups 1, 3, 5, and 6 (1.0, 1.0, 1.3, and 2.6 tumors, respectively). Moreover, pancreatic tumors in Group 2 hamsters were larger (average diameter, 10 mm) than in Group 1 (4 mm), Group 3 (3.5 mm), Group 5 (4 mm), and Group 6 (average, 9mm). The incidence of extrapancreatic tumors did not vary among the PP groups but was equally lower than those in BOP-treated control groups. The data indicated BOP carcinogenesis was inhibited by surgery (regardless of whether PP was per formed) when the carcinogen was given 30 minutes after the surgery but was significantly enhanced when BOP was administered 1 week after PP. The possible reasons for these conflicting results are discussed. Morphologically all tumors were of ductular, ductal, and mixed ductular-insular patterns and most developed at the resected margins, where proliferation of islets, ducts, and ductules, but not of acinar cells, occurred. The results confirm our view that the ductal and ductular cells are the progenitor cells for BOP-induced pancreatic tumors in hamsters.     

Cellular differentiation of epithelioid sarcoma. An electron-microscopic, enzyme-histochemical, and immunohistochemical study.

For the purpose of clarifying cellular differentiation of epithelioid sarcoma, studies based on various methods were performed. Enzyme histochemical studies showed that epithelioid sarcoma tumor cells have characteristics intermediate between epithelial cells and the large plump cells of synovial sarcoma-incomplete epithelial differentiation. For alkaline phosphatase and adenosine triphosphatase particularly, positive cells and negative cells coexisted, as in the large plump cells of synovial sarcoma. Immunohistochemical studies for alpha 1-antitrypsin, alpha 1-antichymotrypsin, vimentin, and keratin also showed that epithelioid sarcoma tumor cells are very similar to the large plump cells of synovial sarcoma and have incomplete epithelial differentiation. For example, the examinations of serial sections and double staining methods revealed that keratin-positive cells are always vimentin-positive in epithelioid sarcoma and in the monophasic area of synovial sarcoma. Electron-microscopically, bundles of intermediate filaments and filopodia toward the intercellular lumen were observed, as in the monophasic area of synovial sarcoma. The results of enzyme-histochemical and immunohistochemical studies of non-neoplastic synovial lining cells, performed here for the first time, are also discussed.     

Modification of pancreatic carcinogenesis in the hamster model. 6. The effect of ductal ligation and excision

The effect of ligation and excision of the pancreatic duct in pancreatic carcinogenesis was examined in the hamster model. Animals were treated with a single dose (20 mg/kg body weight) of N-nitrosobis(2-oxopropyl)amine (BOP) either immediately (Group 1) or on Days 1 (Group 2), 3 (Group 3) or 7 (Group 4) after ligation and excision of the duct of the splenic lobe. Group 5 received BOP shortly after laparoscopy, and Group 6 consisted of BOP-treated controls. All hamsters were killed 46 weeks after BOP treatment. The results showed that despite advanced atrophy of the splenic lobe distal to the excised duct in Groups 1-4, some hamsters in Groups 2, 3, and 4 showed hyperplasia, dysplasia, and increased mitotic activities of ductal and ductular cells. However, carcinomas in the duct-excised atrophic lobe were found only in Groups 1-3. These data indicate that BOP carcinogenesis is mediated through blood circulation, and that cancer development is not inhibited in the duct-excised lobe for up to 3 days after surgery. However, in the entire pancreas, a significant reduction in tumor incidence was seen when the carcinogen was given immediately, or to a lesser extent, 1 day after surgery, regardless of whether or not excision was made. On the contrary, BOP, when given 3 and 7 days after duct excision, enhanced tumor development in the nonexcised (intact) pancreas, compared with other test groups and with BOP controls. Both inhibition and enhancement seemed due to a proportional decrease and increase, respectively, of BOP-responsive cells throughout the intact pancreas.     

Experimental pancreatic carcinogenesis. I. Morphogenesis of pancreatic adenocarcinoma in the Syrian golden hamster induced by N-nitroso-bis(2-hydroxypropyl)amine.

In serial sacrifice experiment, outbred male Syrian golden hamsters were treated once weekly for life with subcutaneous injections of N-nitroso-bis(2-hydroxypropyl) amine (DIPN). The pancreas was examined by high resolution light (1-micro sections) and transmission electron microscopy. Early nonspecific changes in all pancreatic epithelial cellular elements were followed by a progressive proliferation of intra- and interlobular duct cells, with the development of multicentric foci of cystic and papillary cystic adenomas, intraductal carcinomas, and invasive ductal neoplasms. These observations were consistent with multistage morphogenesis of pancreatic adenocarcinoma of ductal origin.     

An immunohistochemical and in situ hybridization study of carbamyl phosphate synthetase I in altered liver cells during carcinogenesis

In order to study the changes of phenotype and gene expression of carbamyl phosphate synthetase I (CPS 1) in preneoplastic altered liver cells in situ, serial cryostat sections of rat liver initiated by diethylnitrosamine (DENO) and promoted by phenobarbital (PB) were hybridized with 35S-GPS 1 cDNA probe by in situ hybridization, and immunohistochemical demonstration (PAP) of CPS 1 were made simultaneously. The results showed that abnormal expression of CPS 1 in the altered foci and nodules increased much more rapidly than that in normal ones, and consequently they might be more susceptable to develop into tumor.     

Isolated pancreatic islets of the rat: An immunohistochemical and morphometric study

Although there is a recent increase in the use of the isolated pancreatic islets of the rat in the transplantation and functional studies, there has been no detailed quantitative assessment on the size and cellular constituents of islets after the isolation procedure. The present work was undertaken to study the size classes of the isolated islets and the morphometry of their cellular populations. Islets of the rat pancreas were isolated by using the intraductal collagenase digestion technique, the most commonly used procedure for the isolation of pancreatic islets. Different endocrine cells of the isolated islets were stained by immunoperoxidase staining techniques. The distribution of the cellular constituents of the isolated islets was similar to that of the intact islets of the normal pancreas; A, D, and PP cells were peripherally arranged around the centrally located B cells. However, morphometric quantitative study showed that the percent volume and percent number of A, D, and PP cells of the isolated islets were lower than those of the corresponding intact ones. Further, the mean true diameter of the isolated islets was lower than that of the intact ones. These data indicate loss of islet cells during the process of isolation. Most of the lost cells were from the periphery of islets. This may provide an explanation for the incomplete metabolic control and recurrence of hyperglycemia encountered after isolated islet transplantation in the treatment of diabetes mellitus. It seems that further refinements of the isolation techniques are necessary to obtain islet tissue with total cellular integrity, before a complete success in transplantation could be achieved. © 1993 Wiley-Liss, Inc.     

Isomyosin expression pattern during formation of the tubular chicken heart: a three-dimensional immunohistochemical analysis

Three-dimensional (3-D) distribution of atrial and ventricular isomyosins is analysed immunohistochemically during the formation of the tubular chicken heart (stage 7 to 12 [H/H]) using antibodies specific for adult chicken atrial and ventricular myosin heavy chains, respectively. This analysis revealed that both types of isomyosins can be first detected at stage 8 (H/H, possessing four pairs of somites), i.e., when the heart primordium still exists as two separate cardiogenic plates. The ventricular type of isomyosin is initially expressed in those areas of cardiogenic plates in the vicinity of the anterior intestinal portal. The atrial type of isomyosin is initially expressed in zones caudal and lateral to the areas of ventricular isomyosin expression. Medial to the atrial isomyosin-expressing areas, cardiogenic plate areas exist that initially lack myosin expression. Those parts of the cardiogenic plates that fuse in front of the anterior intestinal portal, thereby forming the heart tube, are characterized by the expression of both isomyosins; however, the caudolateral parts of the heart primordium maintain their single atrial isomyosin expression during further development. Cardiac contractions are therefore first observed at stage 10 (H/H, possessing ten pairs of somites) in myocardium that coexpresses both isomyosins.     

Early lesions of pancreatic ductal carcinoma in the hamster model.

Syrian golden hamsters were treated weekly with 10 mg/kg body weight N-nitrosobis (2-oxopropyl) amine for life (Group 1) or 6 weeks and were sacrificed at biweekly intervals from 2 weeks (Group 1) and 8 weeks (Group 2) after initiation of the experiment. The pancreas was examined in step sections, and the sequential alterations noted for each interval were recorded. Lesions were found in intrapancreatic and extrapancreatic ducts. Equivalent alterations consisting of hyperplasia, metaplasia, atypia, and lesions characteristic of carcinoma in situ developed ubiquitously and simultaneously in pancreatic ducts of different sizes and in ductules, but not in acinar cells. Among the most significant findings were intrainsular ductular formations, their proliferation, and sequential malignant alteration comparable to the involved preexisting ductules. Differences between the two experimental groups were of a quantitative rather than qualitative nature. The incidence and multiplicity of neoplastic lesions at each interval according to group, sex, and anatomic locations of adenocarcinomas are outlined. Predilected areas for some lesions were found. Results indicate a common origin of all induced tumors from a pluripotent cell populating the pancreatic ductal system.     

Nephrotoxicity of ferric nitrilotriacetate. An electron-microscopic and metabolic study.

Repeated intraperitoneal injections of ferric nitrilotriacetate (Fe-NTA) induce nephrotoxic features such as proximal tubular necrosis and renal failure, an unexpected phenomenon for a ferric compound. The mechanism of Fe-NTA toxicity was investigated by electron microscopy and respiration studies of renal cortical mitochondria in rats. Four hours after a single intraperitoneal injection of Fe-NTA, 5 mg iron/kg body wt, loss of microvilli, increased number of cytoplasmic vacuoles, electron-dense cytoplasmic deposits, mitochondrial swelling, karyorrhexis, and rupture of cytoplasmic membrane were observed in proximal tubular epithelia. At 24 hours, an increased number of cells had become necrotic. Polarographic studies of mitochondria from renal cortex 4 hours after Fe-NTA treatment showed a significant decrease in State 3 respiration and DNP-uncoupled respiration, whereas little change was observed in State 4 respiration and ADP/O.     

Histogenesis of clear cell sarcoma of tendons and aponeuroses. An electron-microscopic, biochemical, enzyme histochemical, and immunohistochemical study.

For the purpose of clarifying the histogenesis of clear cell sarcoma of tendons and aponeuroses (CCS) as well as the problem of whether or not CCS is a heterogeneous group of neoplasms, studies based on various methods were performed. Analysis of glycosaminoglycans gave the same results for amelanotic CCS and synovial sarcoma, and the DOPA reaction gave the same negative results for amelanotic CCS and synovial sarcoma. However, the DOPA reaction was also negative in an amelanotic recurrent tumor of a melanotic CCS, and electron-microscopic studies revealed a close resemblance between amelanotic CCS and melanotic CCS. Further, enzyme histochemical studies showed definite differences between synovial sarcoma and amelanotic CCS but gave identical results for amelanotic and melanotic CCS. Immunohistochemical studies revealed the presence of S-100 protein in all CCS cases, both amelanotic and melanotic. These results indicate that CCS is not a heterogeneous group of neoplasms, and that both amelanotic and melanotic CCS are of neural crest origin.     

Microglandular adenosis of the breast. An immunohistochemical comparison with tubular carcinoma.

Microglandular adenosis (MA) of the breast is a benign, disorganized proliferation of glands lined by a single layer of cells. As such, differential diagnosis between MA and tubular carcinoma may be challenging in selected cases. A panel of antibodies was applied to 10 cases of MA and 10 of tubular carcinoma to investigate the potential benefit of immunohistochemistry in the separation of these lesions and the possible role of myoepithelial cells in MA. The luminal cells in nine cases of MA were surrounded by a cuff of muscle-specific actin-reactive cells, which also coexpressed cytokeratin and vimentin. The immunophenotype of these cells is characteristic of myoepithelial differentiation, which was heretofore thought to be lacking in MA. This finding demonstrates that myoepithelial cells are indeed present in MA subjacent to luminal epithelial cells; moreover, it distinghuishes MA from tubular carcinoma, all examples of which were actin negative in this analysis. In addition, circumferential type IV collagen deposition was observed around constituent glands of MA in nine cases but was lacking in all tubular carcinomas. Other markers included in this evaluation (S100 protein, gross cystic disease fluid protein 15, carcinoembryonic antigen, estrogen receptor protein) were of no differential diagnostic value.     

An immunohistochemical study on the pancreatic endocrine cells of the three-toed sloth, Bradypus variegatus.

The cellular composition and relative frequency of the occurrence of pancreatic endocrine cells were studied immunohistochemically in a primitive eutherian and arboreal folivore, the three-toed sloth, since previous histochemical and ultrastructural studies on the endocrine pancreas of the sloth have detected only a single islet cell type, the A cell. In the sloth pancreas, four types of endocrine cells immunoreactive for glucagon, insulin, somatostatin and serotonin (5-hydroxytryptamine) were found as reported in the pancreas of human and common experimental mammals, but pancreatic polypeptide-immunoreactive cells were not detected by either avian- or bovine-pancreatic polypeptide antiserum. The endocrine cells were distributed mainly in the islets and partly also in the exocrine tissue including the pancreatic ducts. Larger or smaller clusters consisting of glucagon- and insulin-immunoreactive cells were also found frequently in the interlobular connective tissue. In the islets, glucagon- and insulin-immunoreactive cells were the most prominent cell type, while somatostatin- and serotonin-immunoreactive cells were sparse. The most striking feature in the sloth pancreas is the high frequency of glucagon-immunoreactive cells, because these cells are by far less in number than insulin-immunoreactive cells in the islets of human and common experimental mammals. This appears to be an intriguing characteristic of the sloth pancreas in a possible relation to the animal's unique metabolic system and the phylogenetical position.     

Myospherulosis. An electron-microscopic study of a human case.

An electronmicroscopic study of a human case of myospherulosis, which involved the nose and maxillary sinus, is reported. Large parent bodies limited by a thick electron-dense wall, enclosing variable numbers of spherules, were seen in cystic tissue spaces or adjacent connective tissue. Free spherules and shrunken degenerative forms were present as well. Similarity of these bodies to sporangium-like structures is suggested. The etiology of the disease remains unsettled.     

Mechanism of pain and cytoskeletal properties in angioleiomyomas: An immunohistochemical study

Angioleiomyoma is a solitary subcutaneous tumor characterized by pain in about half of patients with this tumor, and the pathogenesis of this pain has been a cause of much debate. To clarify the mechanism of pain and cytoskeletal property of tumor cells, 50 angioleiomyomas were studied clinicopathologically and immunohistochemically. The tumors occurred preferentially on the extremities, particularly the lower leg (46%), and the female to male ratio was 1.9:1. They were classified into three histological subtypes: (i) solid (30 cases); (ii) venous (15 cases); and (iii) cavernous (five cases). The pain and/or tenderness were present in 26 out of 49 patients (52%), in which small nerve fibers immunoreactive for S-100 protein and PGP9.5 were identified within the capsule of 20 tumors (77%) and the tumor stroma of 18 (69%), irrespective of the histological subtypes. In 24 patients where the pain was absent or unknown, nerves were observed within the capsule of 19 tumors (79%) and tumor parenchyma of 10 (42%). Many cells in all 50 tumors were positive for a-smooth muscle actin, and a relatively large number of cells in many tumors were positive for vimentin, desmin and collagen type IV. Also, cytokeratin (CAM5.2) reactivity was scattered in a few cells of four tumors. From these findings, the peculiar pain of angioleiomyomas could be mediated by the nerve fibers especially located within the tumor parenchyma. Although the expression of intermediate filaments in angioleiomyomas was heterogeneous, the overall cytoskeletal features were of smooth muscle cell differentiation.