Pineal gland function is required for colon antipreneoplastic effects of physical exercise in rats.

Light-at-night exposure enhances the risk of cancer. Colon cancer is among the most dangerous tumors affecting humankind. Physical exercise has shown positive effects against colon cancer. Here, we investigated whether pineal gland modulates antipreneoplastic effects of physical exercise in the colon. Surgical and non-surgical pineal impairments were performed to clarify the relationship between the pineal gland activity and manifestation of colonic preneoplastic lesions. Next, a progressive swimming training was applied in rats exposed or not to either non-surgical pineal impairment or carcinogen treatment for 10 weeks. Both surgical and non-surgical pineal impairments increased the development of colon preneoplasia. It was further found that impairing the pineal gland function, higher rates of DNA damage were induced in colonic epithelial and enteric glial cells. Physical exercise acted positively against preneoplasia, whereas impairing the pineal function with constant light exposure disrupts its positive effects on the development of preneoplastic lesions in the colon. This was yet related to increased DNA damage in glial cells and enteric neuronal activation aside from serum melatonin levels. Our findings suggest that protective effects of physical exercise against colon cancer are dependent on the pineal gland activity.

Frequency and Duration Modulate Anticarcinogenic Effects of a Physical Training in the Colon.

Physical exercise has proven protective against colon carcinogenesis. We sought to clarify whether the frequency and duration of physical training were key factors for its anticarcinogenic effects on the colon. Either sedentary or physically trained male Wistar rats (n=82) were either exposed or not to the carcinogen dimethylhidrazine (DMH). The first protocol investigated whether swimming for 60 min in different frequencies modulates antipreneoplastic effects of physical training. Another protocol then explored whether the duration for training 5 times a week impacts on the development of colon preneoplastic lesions. After 8 weeks, serum and colon samples were collected and analyzed afterwards. Swimming once a week for 60 min did not promote those anticarcinogenic effects found in rats trained 5 times weekly. Such weekly sustained physical training not only decreased the development of colon preneoplastic, but also epithelial proliferation, and subepithelial cyclooxygenase 2 (COX-2) expression. Interestingly, a 5 time per week training for less than 60 min was not as protective against colon carcinogenesis as swimming for 90 min. This 90 min training indeed reduced serum cholesterol and triglycerides levels, as well as colonic lipid peroxidation in carcinogen-exposed rats. Our collective data suggest anticarcinogenic effects of physical exercises are potentially promoted when training 5 times a week for at least 60 min.

Effects of the levonorgestrel-releasing intrauterine system on cell proliferation, Fas expression and steroid receptors in endometriosis lesions and normal endometrium.

BACKGROUND: The objectives of this study were: (i) to evaluate the effects of the levonorgestrel-releasing intrauterine system (LNG-IUS) on both proliferation and apoptosis markers and hormone receptors of the eutopic and ectopic endometrium of women experiencing pain related to endometriosis and (ii) to compare the results with those obtained with GnRH agonist (GnRHa) injections. METHODS: Pre- and post-treatment endometrium and endometriosis specimens were obtained from 22 women experiencing pain related to endometriosis who were treated with LNG-IUS (n = 11) or GnRHa (n = 11) for 6 months. Changes in the expression of proliferating cell nuclear antigen, Fas, progesterone receptor (PRA) and estrogen receptor alpha (ER-alpha) were analyzed by immunohistochemistry. RESULTS: The cell proliferation index was significantly reduced in the epithelium and stroma of both the eutopic and the ectopic endometrium after treatment with the LNG-IUS and GnRHa. Only LNG-IUS users showed an increased H-score for Fas in the epithelium of the eutopic and ectopic endometrium (P < 0.05). Expression of ER-alpha and PRA by the glandular epithelium was lower in the eutopic endometrium after both treatments, but this reduction was noted in the ectopic endometrium only after LNG-IUS treatments (P < 0.05). No difference was detected between groups for any of the markers. CONCLUSIONS: LNG-IUS reduced both cell proliferation and the expression of PRA and ER-alpha and increased Fas expression in the eutopic and ectopic endometrium of patients with endometriosis. Some of these actions were not observed with GnRHa.

Hypolipemic and antioxidant activities from Tamarindus indica L. pulp fruit extract in hypercholesterolemic hamsters.

Dietary modifications may significantly reduce cardiovascular disease (CVD) risk factors, including cholesterol and atherosclerosis. The present study addressed the effects of the crude extract from the pulp fruit of Tamarindus indica L. on lipid serum levels and early atherosclerotic lesions in hypercholesterolemic hamsters in vivo, and the extract's antioxidant action, in vitro. Animals were fed on either chow or atherogenic diet during 10 weeks and concomitantly received either water or T. indica L. extract for drinking. Treatment of hypercholesterolemic hamsters with the T. indica pulp fruit extract (5%) led to a decrease in the levels of serum total cholesterol (50%), non-HDL cholesterol (73%) and triglyceride (60%), and to an increase of high-density lipoprotein (HDL) cholesterol levels (61%). In vitro, the extract presented radical scavenging ability, as assessed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radicals assays, and led to decreased lipid peroxidation in serum, as assessed by the thiobarbituric acid reactive substances (TBARS) assay. In vivo, the extract improved the efficiency of the antioxidant defense system, as assessed by the superoxide dismutase, catalase and glutathione peroxidase activities. Together these results indicate the potential of tamarind extracts in diminishing the risk of atherosclerosis development in humans.

Trypanosoma cruzi infection and/or administration of the nonsteroidal anti-inflammatory nimesulide increase the number of colonic crypts overexpressing metallothioneins in rat colon carcinogenesis.

Trypanosoma cruzi infection and nonsteroidal anti-inflammatory drugs inhibit colorectal carcinogenesis by mechanisms not completely known and metallothionein proteins (MTs) may be involved in this process. Sixty-six male Wistar rats weighing 90 to 120 g were randomly divided into seven groups (GI to GVII). GI, GII and GIII animals were subcutaneously infected with 200,000 trypomastigote forms of the Y strain of T. cruzi. After 8 weeks, GI, GII, GIV, and GVI were injected with one weekly subcutaneous dose of 12 mg/kg dimethylhydrazine for 4 weeks. In sequence, GI, GIV and GV were treated with nimesulide (10 mg/kg per dose, five times per week for 8 weeks). Groups I, III, IV, and VI had 12 animals, and each of the other groups had 6 animals. All the animals were euthanized 8 weeks after the last dimethylhydrazine injection. The colons were fixed and processed for MT immunohistochemistry. The index of MT-overexpressing colonic crypts (MTEC) was estimated as the percentage of MT-stained crypts in relation to the total number of crypts scored. Five hundred crypts per animal were scored. Data were analyzed by the Kruskal-Wallis test followed by the Dunn test. There was an increase in MTEC index in the groups either infected with T. cruzi or treated with nimesulide or both infected and treated when compared to control (401, 809, and 1011%, respectively). We suggest that the increased formation of MTEC may be related to the protection against carcinogenesis provided both by T. cruzi infection and nimesulide.

Mouse ear spleen grafts: a model for intrasplenic immunization with minute amounts of antigen.

The production of monoclonal antibodies to protein antigens which can only be obtained in tiny amounts has been a major task, since classical in vivo immunization procedures are not always efficient. In order to circumvent this problem, two methods have been developed: (1) in vitro immunization, in which the immunogen is presented directly to spleen cell cultures; (2) intrasplenic immunization, a technique in which the immunogen is deposited in the spleen tissue. The latter approach requires less laboratory work and the risk of contamination, often a problem with in vitro cultures (Nilsson and Larsson, Immunol. Today (1990) 11, 10), is greatly reduced. Here, we describe a novel method of grafting neonatal spleens in the pinna of the mouse ear. Histological and functional studies show that these spleen grafts have white and red pulp and contain normal percentages of functional T and B cells. The results indicate that this procedure is extremely efficient in priming mice for a secondary humoral immune response, since very small amounts of soluble antigen (ovalbumin) were required. The data are discussed in terms of the advantages of this new technique over current procedures for intrasplenic immunization.

Origins and morphogenesis of colorectal neoplasms.

Gastrointestinal stem cells are considered pivotal in colonic carcinogenesis. There is evidence to suggest that early microadenomas in the colon are polyclonal in origin. Adenomas, once initiated, enlarge by the process of crypt fission. It is also the main mechanism by which neoplastic clones spread through the colorectal epithelium. Both concepts are important for our understanding of the early events in colonic carcinogenesis.

Topical application of benzalkonium chloride to the stomach serosa increases gastric emptying time, acid secretion, serum gastrin and size of the mucosa.

AIM: In the present study we evaluated the effects of gastric myenteric denervation using benzalkonium chloride (BAC) on the time for gastric emptying, as well as gastric secretion, and mucosal epithelial cell size and population in rats. METHODS AND RESULTS: Wistar rats were treated with topical serosal application of BAC to the stomach. Control animals received saline. Ninety days after surgery, gastric emptying time, gastric acid secretion and serum gastrin levels were studied. Next, the animals were sacrificed and the stomachs were removed, fixed in formalin and histologically processed for histomorphometry of the height, area and volume of the glandular portion, and volume and population of mucous, chief, parietal, G- and labelled cells. BAC animals showed a significant delay in gastric emptying and an increase in gastric acid secretion and serum gastrin levels. These animals also presented a significant reduction of myenteric neuron number, hypertrophy of parietal and chief cells, hyperplasia of G cells and an increase in the gastric mucosa area. CONCLUSION: The absence of the myenteric plexus seems to protect the stomach from the hyperplastic effects of hypergastrinemia. Gastric food stasis may act as a factor triggering morphological and functional alterations of the gastric epithelium. Although gastric food stasis is a common finding in medical practice, its physiopathological consequences are poorly understood and have not been frequently discussed in the literature.

Trends of accuracy of clinical diagnoses of the basic cause of death in a university hospital.

AIMS: To determine the agreement between clinical and necropsy diagnoses of the basic cause of death, and to compare the results with those obtained in a previous study carried out at the same university hospital. METHODS: In total, 4828 necropsies, performed between 1990 and 1995 in the University Hospital of the Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil, were reviewed. Examinations were concluded at the macroscopic part of the necropsy in nearly 35% of the cases. Statistical analysis was carried out using the kappa coefficient comparing the clinical diagnosis and the diagnosis obtained after necropsy. The jackknife method was used to identify comparable kappa values for the comparison of the two periods. RESULTS: Compared with the 1978-80 period, a significant increase in diagnostic agreement was seen for the group submitted to complete necropsy, whereas no similar increase was detected when only the macroscopic step was analysed. CONCLUSIONS: There was a discrete tendency to an improved correlation between clinical and postmortem data stated by full necropsy analysis. The findings show that microscopic analysis remains important to confirm the cause of death in many cases. Diagnostic discrepancies remained high, and therefore complete necropsy continues to be an essential instrument for the assessment of clinical diagnosis.

No reduction with ageing of the number of myenteric neurons in benzalkonium chloride treated rats.

The number of myenteric neurons may be reduced by topical serosal application of benzalkonium chloride (BAC). We studied the effects of ageing in the population of neurons that survive after the application of BAC. Ten treated and ten control animals were killed at intervals of 2, 6, 12 and 18 months after the surgery. We performed myenteric neurons counting in serially cut histological preparations of the descending colon. The control animals revealed a continuous loss of myenteric neurons number with increasing of age. Interestingly, contrary to control animals, the BAC-treated rats presented no neuron loss with ageing at any experimental time. The reasons for their survival with ageing could be related to a neuroplasticity phenomenon.

Does the correlation between EBNA-1 and p63 expression in breast carcinomas provide a clue to tumorigenesis in Epstein-Barr virus-related breast malignancies?

Several investigators have identified Epstein-Barr virus (EBV) particles in breast carcinomas, a fact that supports a role for EBV in mammary tumorigenesis. The possible mechanism involved in this process is not clear. The present study was carried out in an attempt to determine whether there is a relationship between latent infection with EBV and p53 and p63 expression in breast carcinomas. Immunohistochemistry developed with 3.3-diaminobenzidine tetrahydrochloride was performed in 85 formalin-fixed paraffin-embedded breast carcinomas using anti-EBV EBNA-1, anti-p63, anti-p53, anti-estrogen receptor (ER) and anti-progesterone receptor (PR) antibodies. The cases were selected to represent each of the various histologic types: intraductal carcinoma (N=12), grade I invasive ductal carcinoma (N=15), grade II invasive ductal carcinoma (N=15), grade III invasive ductal carcinoma (N=15), tubular carcinoma (N=8), lobular carcinoma (N=10), and medullary carcinoma (N=10). The ductal breast carcinomas were graded I, II and III based on the Scarff-Bloom and Richardson grading system modified by Elston and Ellis. One slide containing at least 1000 neoplastic cells was examined in each case. ER, PR, p63, p53 and EBNA-1 were positive in 60, 40, 11.8, 21.2 and 37.6% of carcinomas, respectively. There was a correlation between EBNA-1 and p63 expression (P<0.001), but not between EBNA-1 and p53 (P=0.10). These data suggest a possible role for p63 in the mammary tumorigenesis associated with Epstein-Barr virus infection.

Profiles of Asian American students with LD at initial referral, assessment, and placement in special education.

Alhough Asian American students are underrepresented in special education, recent studies have highlighted concerns about the cultural and linguistic appropriateness of the identification and placement process. This study examined the characteristics of Asian American elementary students with learning disabilities (LD) in a school district in the Southwest that was selected because it served the largest number of Asian American students with LD in the state. Due to the small numbers of students served, the sample included all Asian American students with LD in Grades K to 5 (N = 26). Variables of interest included students' demographic characteristics, factors associated with referral, assessment practices and student profiles, and instructional recommendations for special education services. The results suggest that the experiences of Asian American students with LD are similar to those of Mexican American students with LD reported in earlier studies. These findings also reflect the challenges faced by educators in providing appropriate educational services for language minority students.

The inflammatory stimulus of a natural latex biomembrane improves healing in mice.

The aim of the present study was to compare healing obtained with biomembranes with the natural healing process (sham) using biochemical and immunohistological assays. C57BL/6 mice were divided into 4 groups of 15 mice each and received different subcutaneous implants: natural latex biomembrane (NLB), denatured latex (DL), expanded polytetrafluorethylene (ePTFE), or sham. On the 2nd, 7th, and 14th days post-treatment, 5 mice per group were sacrificed and biopsied for the following measurements: oxidative stress based on malondialdehyde (MDA), myeloperoxidase (MPO) and hydrogen peroxide by the method of ferrous oxidation-xylenol orange (FOX), as well as glutathione and total proteins; histological evaluation to enumerate inflammatory cells, fibroblasts, blood vessels, and collagen, and immunohistochemical staining for inducible nitric oxide synthase, interleukin-1ß, vascular endothelial growth factor (VEGF), and transforming growth factor-ß1 (TGF-ß1). On day 2 post-treatment, NLB stimulated a dense inflammatory infiltrate mainly consisting of polymorphonuclear cells, as indicated by increased MPO (P < 0.05), but oxidative stress due to MDA was not observed until the 7th day (P < 0.05). The number of blood vessels was greater in NLB (P < 0.05) and DL (P < 0.05) mice compared to sham animals on day 14. NLB induced fibroplasia by day 14 (P < 0.05) with low expression of TGF-ß1 and collagenesis. Thus, NLB significantly induced the inflammatory phase of healing mediated by oxidative stress, which appeared to influence the subsequent phases such as angiogenesis (with low expression of VEGF) and fibroplasia (independent of TGF-ß1) without influencing collagenesis.

Intrinsic denervation of the colon is associated with a decrease of some colonic preneoplastic markers in rats treated with a chemical carcinogen.

Denervation of the colon is protective against the colon cancer; however, the mechanisms involved are unknown. We tested the hypothesis that the denervated colonic mucosa could be less responsive to the action of the chemical carcinogen dimethylhydrazine (DMH). Three groups of 32 male Wistar rats were treated as follows: group 1 (G1) had the colon denervated with 0.3 mL 1.5 mM benzyldimethyltetradecylammonium (benzalkonium chloride, BAC); G2 received a single ip injection of 125 mg/kg DMH; G3 was treated with BAC + the same dose and route of DMH. A control group (Sham, N = 32) did not receive any treatment. Each group was subdivided into four groups according to the sacrifice time (1, 2, 6, and 12 weeks after DMH). Crypt fission index, ss-catenin accumulated crypts, aberrant crypt foci, and cell proliferation were evaluated and analyzed by ANOVA and the Student t-test. G3 animals presented a small number of aberrant crypt foci and low crypt fission index compared to G2 animals after 2 and 12 weeks, respectively. From the second week on, the index of ss-catenin crypt in G3 animals increased slower than in G2 animals. From the 12th week on, G2 animals presented a significant increase in cell proliferation when compared to the other groups. Colonic denervation plays an anticarcinogenic role from early stages of colon cancer development. This finding can be of importance for the study of the role of the enteric nervous system in the carcinogenic process.

The inflammatory stimulus of a natural latex biomembrane improves healing in mice

The aim of the present study was to compare healing obtained with biomembranes with the natural healing process (sham) using biochemical and immunohistological assays. C57BL/6 mice were divided into 4 groups of 15 mice each and received different subcutaneous implants: natural latex biomembrane (NLB), denatured latex (DL), expanded polytetrafluorethylene (ePTFE), or sham. On the 2nd, 7th, and 14th days post-treatment, 5 mice per group were sacrificed and biopsied for the following measurements: oxidative stress based on malondialdehyde (MDA), myeloperoxidase (MPO) and hydrogen peroxide by the method of ferrous oxidation-xylenol orange (FOX), as well as glutathione and total proteins; histological evaluation to enumerate inflammatory cells, fibroblasts, blood vessels, and collagen, and immunohistochemical staining for inducible nitric oxide synthase, interleukin-1β, vascular endothelial growth factor (VEGF), and transforming growth factor-β1 (TGF-β1). On day 2 post-treatment, NLB stimulated a dense inflammatory infiltrate mainly consisting of polymorphonuclear cells, as indicated by increased MPO (P < 0.05), but oxidative stress due to MDA was not observed until the 7th day (P < 0.05). The number of blood vessels was greater in NLB (P < 0.05) and DL (P < 0.05) mice compared to sham animals on day 14. NLB induced fibroplasia by day 14 (P < 0.05) with low expression of TGF-β1 and collagenesis. Thus, NLB significantly induced the inflammatory phase of healing mediated by oxidative stress, which appeared to influence the subsequent phases such as angiogenesis (with low expression of VEGF) and fibroplasia (independent of TGF-β1) without influencing collagenesis.

p63 correlates with both BRCA1 and cytokeratin 5 in invasive breast carcinomas: further evidence for the pathogenesis of the basal phenotype of breast cancer.

AIMS: To study the expression of p63, cytokeratin (CK) 5 and CK8/18 in invasive ductal carcinomas and their relationship with BRCA1 and other pathological and immunohistochemical features of clinical significance. METHODS AND RESULTS: Immunohistochemistry with the antibodies p63, CK5, CK8/18, BRCA1, oestrogen receptor, progesterone receptor, p53, c-erbB-2 and Ki67 was performed in 102 formalin-fixed paraffin-embedded samples of invasive ductal carcinomas. The CK5+ cases were submitted to a double-immunolabelling study with p63. There was a strong relationship between CK5 and p63 expression and both markers were associated with hormonal receptor-negative high-grade carcinomas with high proliferative rate. Furthermore, there was coexpression of CK5 and p63 in neoplastic cells, indicating that p63, like CK5, is a marker of the basal phenotype of breast cancer. There was a strong relationship between reduced expression of BRCA1 with both p63 and CK5 expression as well as an inverse correlation between p63 and CK8/18 expression, suggesting that loss of p63 expression is required for the transition between a basal to a luminal phenotype of breast carcinoma. CONCLUSIONS: Since p63 is thought to be a marker of stem cells and may act as an oncogene, our data support the idea that BRCA1 acts as stem cell regulator.

Endocrine cells in the denervated intestine.

This study deals with the effects of myenteric denervation of the proximal jejunum on endocrine cell population of the crypt-villus unit, 5 months after treatment with benzalkonium chloride (BAC). Male Wistar albino rats weighing on average 100 g were allocated to two groups: the BAC group - the proximal jejunal serosa was treated with 2 mM BAC for 30 min, and the control group - treated with saline solution (0,9% NaCl). There was a significant reduction in neurone number in the jejunal myenteric plexus of the BAC group and the endocrine cell population (serotoninergic and argyrophilic cells) was significantly increased in this intestine segment. In conclusion, the present findings provide further evidence that the myenteric denervation induced by BAC may lead to the development of a local imbalance of the neurotransmitters, with a consequent induction of enteroendocrine cell (argyrophilic and serotoninergic cells) hyperplasia in the crypt and villus.

The clonal origin and clonal evolution of epithelial tumours.

While the origin of tumours, whether from one cell or many, has been a source of fascination for experimental oncologists for some time, in recent years there has been a veritable explosion of information about the clonal architecture of tumours and their antecedents, stimulated, in the main, by the ready accessibility of new molecular techniques. While most of these new results have apparently confirmed the monoclonal origin of human epithelial (and other) tumours, there are a significant number of studies in which this conclusion just cannot be made. Moreover, analysis of many articles show that the potential impact of such considerations as patch size and clonal evolution on determinations of clonality have largely been ignored, with the result that a number of these studies are confounded. However, the clonal architecture of preneoplastic lesions provide some interesting insights --many lesions which might have been hitherto regarded as hyperplasias are apparently clonal in derivation. If this is indeed true, it calls into some question our hopeful corollary that a monoclonal origin presages a neoplastic habitus. Finally, it is clear, for many reasons, that methods of analysis which involve the disaggregation of tissues, albeit microdissected, are far from ideal and we should be putting more effort into techniques where the clonal architecture of normal tissues, preneoplastic and preinvasive lesions and their derivative tumours can be directly visualized in situ.

Experimental megaileum.

Typical megaileum occurred in young male Wistar rats three months after ileum myenteric plexus denervation. An average of 58.4% denervation of the Auerbach plexus was obtained by serosal application of benzalkonium chloride (0.2% v/v). Denervation was assessed by ganglion cell counts in an 8 nm ring-shaped histological sectfrom the midportion of the treated segment. A morphometric study showed that the increased thickness of the megaileum wall was due to muscle hypertrophy and mucosal hyperplasia. The potential usefulness of this model of megaileum is emphasized.

The relationship between megacolon and carcinoma of the colon: an experimental approach.

'Carcinoma of the colon does not occur in cases of megacolon' is an axiom held by Brazilian physicians working in endemic areas for Chagas' disease. The objective of the present study was to test this axiom experimentally by submitting rats with experimental megacolon to a carcinogen which causes carcinoma of the colon. Eighty young male Wistar rats received serosal application of either saline (0.9% NaCl) or 2 mM benzalkonium chloride (BAC) to the distal colon. Ten months later randomly chosen saline and BAC rats were injected weekly with dimethylhydrazine (DMH) for 20 weeks. Non-DMH-treated rats from both original groups were maintained, for a total of four experimental groups. Three months after the injections all surviving rats were killed. At autopsy the presence of absence of carcinomas along the colon was recorded. The induction of megacolon was evaluated by morphometry of the wall from the distal colon and myenteric denervation was assessed by neuron counts. An increase of at least 2-fold in distal colon wall thickness confirmed the induction of megacolon in BAC-treated rats. Neuronal counts from BAC and control rats not treated with DMH showed an average denervation of 63%. The number of distal colon carcinomas in BAC+DMH-treated rats was significantly lower than that in DMH-treated rats. These findings appear to contradict the traditional concept of carcinogenesis of the colon. The clinical axiom was reproduced experimentally.