A grading scheme for the assessment of proliferative lesions of the mouse prostate in the TRAMP model

To improve the precision and consistency of experimental results, we have developed a scoring system for proliferative epithelial lesions in the mouse prostate based on histological growth patterns observed in individual lobes. Severity of proliferative lesions was divided into 6 categories; the grade of the most advanced lesion was identified for each lobe and its distribution estimated semiquantitatively. A numerical score combining grade and distribution of the most advanced lesion in each lobe was assigned and termed the "distribution-adjusted lesion grade"; the mean of these scores was calculated for each treatment group. Using this grading scheme, we assessed lesion development in ad libitum-fed and 20%-diet-restricted groups of TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice that were started on study at 7 weeks of age and sacrificed when 11 and 20 weeks old. The anterior, dorsal, lateral, and ventral prostate lobes showed clear reductions in lesion severity in diet-restricted TRAMPS at 11 and 20 weeks. This method for scoring the epithelial pathology of the prostate in the TRAMP model with minimal to severe proliferative lesions utilizes the natural history of lesion development for assessing the effects of chemical and dietary interventions.     

Immunohistochemical analysis of the proliferative activity of neuroendocrine tumors from various organs

Small-cell neuroendocrine carcinomas (NECs) of the prostate are believed not to derive from benign orthotopic NE epithelial cells. Instead, an origin from a putative stem cell is actually the most favored concept. Whether this concept can also be applied to neuroendocrine tumors (NETs) of other organs, especially whether there are indications for well-differentiated NET-NEC sequence, is subject of the present study. A double-labeling technique for the proliferation marker MIB-1 and the NE markers chromogranin A (ChrA) and synaptophysin (SNP) was used for the immunohistochemical analysis of 45 well-differentiated NETs, 16 well-differentiated (low-grade) NECs, and 63 high-grade NECs of the esophagus, stomach, small intestine, appendix, colon, lung, prostate, and urinary bladder. The lowest proliferative activity was found in NETs (0.85% of tumor cells), and the highest activity was found in high-grade NECs (72.5%). The expression of ChrA was highest in NETs and lowest in high-grade NECs. None of the NETs and only sporadic cells in low-grade NECs showed double labeling (up to 0.05%). Up to 50% of the tumor cells in high-grade NECs were positive for MIB-1 and SNP. The percentage of double-labeled cells ranged between 0.9 and 39.6 (mean 9.7). No double-labeled cells were found in the normal epithelium adjacent to the tumors. Transitions from NET to NEC could not be observed. NETs and low-grade NECs differ in their proliferative activity from high-grade NECs, suggesting that they may arise from different precursor cell populations.     

Failure of dietary restriction to influence natural killer activity in old rats

Natural killer activity of Sprague-Dawley rats maintained on an ad libitum versus restricted diet was compared using an 18 hour 51Cr-release assay, against the K562 erythroleukemic line, Yac-1 lymphoma cells and SV40-3T3 cells. The results indicated that no enhancement of natural killer function was induced by dietary restriction of 10.5-month-old rats from weaning. Prolongation of the restricted diet into late life (24 months) similarly did not enhance basal natural killer activity over levels observed in the ad libitum controls. This suggests that the improved resistance to some tumours seen after prolonged dietary restriction depends on another defensive mechanism, reduced metabolic activity and/or a reduction of available nutrients at cancerous foci.     

Effects of preweaning and postweaning feed restriction on the development of polygenic obese mice

Polygenic obese (M16) and control (ICR) mice were raised in litters of 8 (N8) or preweaning restricted in litters of 14 (N14). Mice were fed either ad libitum or postweaning restricted by automatic feeders from 4 to 10 weeks of age. All mice were fed ad libitum from 10 to 16 weeks. Preweaning restriction (N14) resulted in mice at 4 weeks with smaller body weights, fat percent, subcutaneous fat depot and adipocyte diameter than N8 mice. Most of the effects of preweaning restriction were overcome by 10 weeks. Obese mice, at 10 and 16 weeks, were heavier and had higher fat percent and larger fat depots with more and larger adipocytes than control mice. Postweaning restriction resulted in mice at 10 weeks with smaller body weight but greater fat percent, especially in the control line. Postweaning restriction of obese mice resulted in a higher percent of fat at 16 weeks. The change in eating pattern, stress or other factors associated with postweaning restriction, as imposed in this study, resulted in an altered metabolism that distributed a greater proportion of energy to fat storage than in the ad libitum fed mice. Ultimate body size and adipose tissue characteristics were determined primarily by genotype, whereas most effects of nutritional restriction were temporary.     

Metastasizing neuroblastomas from taste buds in rats transgenic for the Simian virus 40 large T antigen under control of the probasin gene promoter.

During establishment of a prostate cancer model in rats transgenic for the Simian virus 40 large T antigen, under control of the probasin gene promoter, with protein expression specific to the prostate, tongue, and spinal cord, undifferentiated small round cell tumors were frequently observed. Extensive examination of tongues of the transgenic rats, despite a macroscopically normal appearance, revealed the tumors to have come from taste buds of the papilla circumvallata and papilla foliata. The lesions were positive for the SV40 T antigen, PGP9.5 (ubiquitin C-terminal hydrolase), and synaptophysin, neuron and neuroendocrine markers. Morphologically and immunohistochemically, the tumors were diagnosed as neuroblastomas, considering the neuroepithelial origin. Histologically identical tumor cells in the spinal cord and lung were observed only in the rats with deeply invading tongue tumors, suggesting that metastasis from the tongue tumors had occurred. Castration or supplementation with testosterone propionate did not alter tumor development, indicating the tumors to be androgen-independent. These results clearly show that taste buds can give rise to metastasizing neuroblastomas.     

The gross morphology of skeletal disease in adult male breeding turkeys

The incidence of skeletal disease has been studied in two groups of male turkeys which were reared and kept as breeding birds. One group was fed ad libitum throughout life whereas the other was reared under dietary restriction to give body weights between 50% and 80% of the ad libitum values, depending on age. Losses during rearing were higher in ad libitum fed birds with lameness the major single cause. The majority of birds were killed at 55 weeks of age. It was noted that before death, more than half of the ad libitum birds were lame and the remainder showed gait abnormalities. By contrast, gait and posture was judged to be normal in many of the birds kept under feed restriction and none was lame. Post mortem dissections revealed a much higher incidence of skeletal disease in birds fed ad libitum. Although these observations suggest that feed restriction may alleviate skeletal disease in male breeding turkeys, it is suggested that such a strategy is of no long term benefit in parent breeding stock.     

Failure of dietary restriction to enhance cytolytic and chemiluminescent activity of peritoneal exudate cells from ageing rats.

Investigations into the cytolytic activity of peritoneal macrophages from male Sprague-Dawley rats (activated in vivo with Corynebacterium parvum) utilised SV40-3T3 and L-929 target cells in a 72 h [3H]thymidine release assay. The dietary restricted rats under test were given a measured amount of food from weaning sufficient to maintain their body weights at approximately 50% of age-matched controls fed ad libitum. Cytolytic activity was similar in the two dietary groups at both 13 and 21 months of age, nor were chemiluminescent responses of peritoneal cells to latex or zymosan particles affected by the dietary restriction. No differences were seen in the effectiveness of zymosan particles opsonised with serum pooled from dietary restricted versus control rats in stimulating chemiluminescent responses by standard preparations of peritoneal cells, and concentrations of serum C3 and IgG appeared to be unchanged by the dietary regime.     

The effects of a prolonged undernutrition on serum lipids and fatty acid composition of reindeer calves during winter and spring

We examined the effects of undernutrition on lipid metabolism in reindeer (<1 year) during mid-winter and spring, with particular focus on the proportions of polyunsaturated fatty acids (PUFAs) in major serum lipids. The reindeer (n=8) were fed their winter feed, lichen, ad libitum for 5 weeks, followed by 40% restriction of energy for 8 weeks and refeeding to normal for 6 weeks. The concentrations of major serum lipids, cholesterol and phospholipids decreased significantly during the ad libitum period (by 50 and 44%, respectively). The proportion of major PUFA, linoleic acid in serum cholesteryl esters, decreased from 48.2 to 38.4% during the ad libitum period (P < 0.01), and to 29.2% during the restriction period (P < 0.001). The proportion of linoleic acid in phospholipids decreased from 27.9 to 15.6% during the ad libitum period (P < 0. 001), and to 13.0% during the restriction (P < 0.01). Also alpha-linolenic acid in the major lipids decreased significantly during the ad libitum and restriction periods. The decreases in the major lipids and linoleic acid were reversed during the refeeding. The control group (n=8) which was fed high-quality concentrates ad libitum gained weight most of the spring but showed similar although slower decreases in the major serum lipids and PUFAs than the lichen group. Our results indicate that feeding reindeer on lichen during winter leads to the retardation of growth and reductions in major serum lipids and their principal C18-PUFA proportions. The decreased proportions of the principal PUFAs most probably reflect their low dietary intake but may have been modified also by seasonal factors.     

肺腺癌神经内分泌分化的生物学特性

目的探讨肺腺癌伴神经内分泌(neuroendocrine NE)分化的生物学特性。方法采用免疫组化S-P法检测47例肺腺癌神经内分泌标记物的表达。结果肺腺癌组织中NE标记物神经元特异性烯醇化酶(NSE)和突触素(Syn)的阳性表达率分别为63.83%、44.68%,均显著高于正常组织(P<0.01)。肺腺癌伴NE分化与肺癌的淋巴结转移、分化程度无关。NSE的表达在死亡组显著高于生存组(P<0.05)。结论肺腺癌伴有NE分化与生存时间呈负相关,与淋巴结转移和分化程度无相关性。     

Hepatocellular carcinoma with an unusual neuroendocrine component

We report a rare case of hepatocellular carcinoma (HCC) with an unusual neuroendocrine component. During a follow-up study for chronic hepatitis C in a 71-year-old man, a nodular lesion showed rapid growth from 1 cm to 4 cm in diameter within 3 months. Histologically, the tumor was consistent with moderately differentiated HCC, but was intermingled with nests of small round cells with scarce cytoplasm, which resembled those found in small cell carcinoma. This population formed small solid nests among the trabecular structures. Immunohistochemically the small round cell component of the tumor was strongly positive for neuron-specific enolase (NSE), chromogranin A and synaptophysin, but hepatocyte paraffin-1 (HP-1) and alpha-fetoprotein (AFP) were negative. In contrast, HP-1 and AFP were positive, and NSE, chromogranin A and synaptophysin were negative in moderately differentiated HCC tissues. Electron microscopy revealed many intracytoplasmic neurosecretory granules in the small round cells. The labeling indexes of p53 and Ki-67 were significantly higher in the small round cell component than in the moderately differentiated HCC component. Overall, we conclude that this nodule was HCC with neuroendocrine differentiation.     

Pancreatic Carcinoma with Multilineage (Acinar, Neuroendocrine, and Ductal) Differentiation

The preponderance of pancreatic tumors is adenocarcinoma of the ductal type; carcinomas with multiple lineage differentiation are extremely rare. We report an unusual case of pancreatic carcinoma with combined acinar and neuroendocrine differentiation and minor ductal component with concurrent acinar-ductal metaplasia (ADM), an early lesion implicated in ductal carcinogenesis. The patient is a 56-year-old man with vague complaints of dull left upper quadrant pain with radiation across the mid-portion of his abdomen. A computer tomography scan revealed an irregular enlargement of the distal 3.2 cm of the pancreatic body. A distal pancreatectomy was then performed. Histologic examination revealed a pancreatic carcinoma with cellular features of eosinophilic granular cytoplasm and salt-pepper nuclei. The acinar differentiation of the carcinoma was confirmed by positivity on periodic acid-Schiff stain resistant to diastase digestion (dPAS), positivity for antitrypsin on immunohistochemistry (IHC), and presence of zymogen granules on electron microscopy (EM). The neuroendocrine differentiation was evident by positive synaptophysin and chromogranin stain on IHC and neuroendocrine granules on EM. The ductal component was only visible by PAS stain and immunostains for CEA and CK19A and accompanied by a number of the acinar-ductal metaplasia lesions adjacent to the main tumor. Thus, the histological, histochemical, immunohistochemical and electron-microscopic evidence all suggested that the pancreatic carcinoma underwent trilineage differentiation.     

Carcinoids of the gastrointestinal tract: importance of determining differentiation and proliferation markers

The group of 35 carcinoid tumours obtained from 34 patients was reviewed according to recent histopathological criteria. Consequently, evaluation of the Grimelius staining and immunohistochemical detection of chromogranin A (CgA), Leu-7 (CD-57), synaptophysin, neuron-specific enolase (NSE), (beta-III tubulin, Ki-67 and proliferating cell nuclear antigen (PCNA) was performed. The majority of tumours (29, i.e. 83%) were classified as typical carcinoids composed predominantly of mixed solid and trabecular or solid and tubular growth patterns. Six tumours (17%) revealed more prominent cytological abnormalities corresponding with the diagnosis of atypical carcinoid. The majority of tumours (31, i.e. 93.9%) showed granular cytoplasmic positivity in Grimelius staining and diffuse cytoplasmic positivity of NSE (34, i.e. 97.1%). All of the 32 stained tumour samples showed positive immunoreactivity for synaptophysin. A high percentage of tumours (32, i.e. 91.4%) revealed also a positive reaction with antibody TU-20 detecting (beta-III tubulin, a marker of an early stage of neuronal differentiation. Thirty-four tumours (97.1%) showed granular cytoplasmic positivity for both markers of neuroendocrine granules (CgA and Leu-7). One tumour (2.9%) was positive only for Leu-7. Tumour cells revealed predominantly low proliferative activity evaluated by PCNA and Ki-67 immunodetection. Higher degree of proliferation was observed especially in atypical carcinoids.     

The effect of dietary restriction on myc protooncogene expression in mice: a preliminary study

The effect of chronic dietary restriction on the expression of the c-myc protooncogene was determined in the livers of a hybrid mouse strain (C57Bl6 x C3H F1 hybrid) at three time points during a 24-h period: 1 h after lights on (1 HALO), 5 h prior to feeding (12 HALO), and 2 h after feeding (19 HALO). In addition, in whole animals studies, changes in core body temperature were monitored. In mice which had been subjected to a chronic diet restriction (60% of the intake of ad libitum controls), c-myc expression was significantly reduced at 1 HALO and 19 HALO compared to corresponding ad libitum animals. In addition, significant differences in c-myc expression were found between time points, in both the ad libitum and restricted groups, suggesting that myc protooncogene expression in the liver may be regulated in a circadian fashion. C-myc expression may correlate with body temperature, suggesting a possible association with metabolic output.     

Effect of VMH lesion on sucrose-Fed nociceptive responses

An initial analgesia followed by hyperalgesia to phasic noxious stimuli occurs after ingestion of sucrose ad libitum. However, the mechanism underlying hyperalgesia is not known. The present study was designed to explore the role of VMH in the mediation of the hyperalgesic effect of sucrose ingestion. Adult male albino rats received sucrose solution (20% p.o.) in addition to laboratory food pellets and tap water ad libitum. Their behavioural responses to various phasic and tonic noxious stimuli were recorded after 6, 12 and 48 h during pre and post-sucrose fed states in both the control and VMH lesion groups of rats. Sucrose feeding to control rats significantly reduced the tail flick latency (TFL) and threshold of vocalization during stimulus (SV) and after discharge (VA) indicating hyperalgesia, while the threshold of tail flick remained unaffected. The average pain rating during the formalin test (tonic pain) decreased significantly indicating analgesia. VMH lesion decreased the latency (mean +/- SD) for tail flick (11.26 +/- 4.65 from 15.61 +/- 5.12 s), threshold (median) for tail flick (0.04 from 0.08 mA), vocalization during stimulus (0.05 from 0.1 mA) and vocalization after discharge (0.15 from 0.2 mA), while the tonic pain rating increased, thereby suggesting a hyperalgesic state. However, sucrose feeding to lesioned rats neither potentiated nor attenuated their hyperalgesia. The results suggest that sucrose feeding for 6-48 h ad libitum produces hyperalgesia to phasic noxious and analgesia to tonic noxious stimuli, while VMH lesion produces hyperalgesia to both phasic and tonic noxious stimuli. Secondly, sucrose ingestion by VMH lesion rats does not affect their responses to pain, suggesting the possible role of VMH in the mediation of sucrose-fed nociceptive responses.     

Effect of dietary restriction and aging on polyploidy in rat liver

Liver polyploidy levels were compared as a function of age and diet in male Fischer 344 rats between 1 and 24 months of age. Dietary restriction was imposed on one group by reducing their food intake to 60% of ad libitum food intake. Histological sections of the livers of animals at each age and diet were examined. Diploid, tetraploid and octaploid nuclei were observed, and their size and frequency established. There were no differences in the diameter or volume of these size classes as a function of age or diet. An age-related decline in the percentage of diploid nuclei, coupled with an increase in the percentage of tetraploid and octaploid nuclei was observed in both groups. The major difference between the two groups was that the adult level of liver polyploidy was attained more slowly in the animals on dietary restriction as compared to the ad libitum fed controls. Polyploid cell formation in the liver is under the control of growth hormone, thyroid hormone and thymus, all of which might be influenced by dietary restriction.     

An electron microscopic examination of age-related changes in the rat liver. The influence of diet

The influence of age and diet on the ultrastructure of hepatocytes is reported. The following dietary manipulations were investigated: Group 1, fed ad libitum a diet containing 21% protein; Group 2, fed a similar diet but restricted to 60% of the intake of Group 1 from 6 weeks of age onwards; Group 3, restricted from 6 weeks to 6 months of age and thereafter fed ad libitum; Group 4, restriction started at 6 months of age; Group 5, fed ad libitum a diet containing 12.6% protein. In all groups the size of hepatocytes was found not to increase during adult life. The size of hepatocytes in Groups 2 and 4 was the same as or larger than that of the other groups; thus food restriction resulted in a decreased number of hepatocytes. Changes in the structure of some organelles and the accumulation of lipofuscin granules occurred with advancing age and the extent of these age-related changes was less in Groups 2 and 4 than in the other groups. These morphologic findings in conjunction with our previously reported metabolic findings provide a new view of the action of food restriction on the aging process.     

Relationship of dietary intake to the development of glomerulosclerosis in obese Zucker rats

Obese Zucker rats are hyperphagic and develop premature glomerulosclerosis. The purpose of this study was to find out the effects of restriction of dietary intake on this glomerulosclerosis. The obese and lean male Zucker rats were fed with restricted amounts of balanced diet for periods of 40 and 50 weeks, sacrificed, and the body weight, the light and ultrastructural alterations of glomeruli, and the serum levels of blood urea nitrogen, triglyceride, and cholesterol were examined. Obese and lean male rats of identical ages were fed ad libitum with the diet and studied similarly. The dietary restriction significantly lessened the development of the glomerulosclerosis in obese rats, while those on the nonrestricted diet manifested an advanced glomerulosclerosis. The dietary restriction, however, did not normalize the obesity nor correct the elevated serum lipid level to the range of lean control rats. The spontaneous glomerular lesions of the obese rats were characterized by segmental mesangial expansion, disappearance of podocytes and endothelia, and obliteration of capillary lumina. The lean rats maintained essentially normal renal morphology. A similar study on the renal morphology done in female Zucker rats also revealed a preventive effect of dietary restriction on the development of glomerulosclerosis. In conclusion, there is a strong association between the glomerulosclerosis and the hyperphagia of the obese Zucker rats, both in males and females, and the emergence of this lesion is preventable to a significant degree.     

The Immunophenotype of Pituitary Adenomas

Although the production of pituitary hormones by adenohypophysial tumors has been studied extensively, an examination of the immunophenotype of pituitary adenomas using a broad spectrum of antibodies has not been previously investigated. We studied 23 pituitary adenomas using a large panel of antibodies to determine if these tumors exhibited a common immunophenotype. Various neuroendocrine markers, synaptophysin, neuron-specific enolase (NSE), and the intermediate filament protein, low-mol-wt keratin were expressed in most examples. There was, however, differential expression of chromogranin A in that few prolactin (PRL) and adenocorticotrophic hormone (ACTH) adenomas stained positively, whereas all other adenoma subtypes were reactive. The ACTH adenomas had a unique profile with positive staining for galanin, neurophysin, vasopressin, and ubiquitin. These results indicate that (1) pituitary adenomas do not express a single “generic” immunophenotype; (2) synaptophysin is the most reliable and best broad spectrum marker for pituitary adenomas; (3) the neuroendocrine granule marker chromogranin A is useful in the identification of null cell adenoma, a tumor that usually does not stain for anterior pituitary tumors; and (4) among pituitary tumors, ACTH adenomas have a unique immunoprofile.