DETECTION OF CELL-PROLIFERATION IN TISSUE-SECTIONS

1. Cell proliferation is of interest since abnormal cell proliferation appears to be a precursor of tumorigenesis and also because the quantitative description of cell proliferation in tumors can be used to predict the biological behavior of a particular neoplasia.2. Them am several reliable methods of studying cell proliferation in tissues. One of the most important is the detection of the Ki67 defined antigen in frozen sections. The number of cells expressing Ki67 correlates with histological grades of tumors and can also be predictive of clinical outcome. The Ki67 can be localized in tissue sections using monoclonal antibodies in association with the immunoperoxidase technique.3. Proliferating cell nuclear antigen (PCNA) is a component of DNA polymerase-delta and is another important cell proliferation marker manifesting a striking increase in concentration during the S phase of the cell cycle. 19A2 and PC10 are two different monoclonal antibodies which can be employed to detect PCNA in paraffin-embedded tissues.4. Molecular biology has also been making a great contribution to the study of cell proliferation. The most recent innovation in tissue identification of proliferating cells is the use of in situ hybridization for the localization of histone H3 and/or H4 mRNA. H3 mRNA-positive cells appear to be present in basal cells of the skin and in crypt cells of the intestine which are sites with high proliferation rate.

The Archipelago of Fernando de Noronha: An Intriguing Malformed Toad Hotspot in South America

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Fiber fermentability effects on energy and macronutrient digestibility, fecal traits, postprandial metabolite responses, and colon histology of overweight cats

Considering the different potential benefits of divergent fiber ingredients, the effect of 3 fiber sources on energy and macronutrient digestibility, fermentation product formation, postprandial metabolite responses, and colon histology of overweight cats (Felis catus) fed kibble diets was compared. Twenty-four healthy adult cats were assigned in a complete randomized block design to 2 groups of 12 animals, and 3 animals from each group were fed 1 of 4 of the following kibble diets: control (CO; 11.5% dietary fiber), beet pulp (BP; 26% dietary fiber), wheat bran (WB; 24% dietary fiber), and sugarcane fiber (SF; 28% dietary fiber). Digestibility was measured by the total collection of feces. After 16 d of diet adaptation and an overnight period without food, blood glucose, cholesterol, and triglyceride postprandial responses were evaluated for 16 h after continued exposure to food. on d 20, colon biopsies of the cats were collected under general anesthesia. Fiber addition reduced food energy and nutrient digestibility. of all the fiber sources, SF had the least dietary fiber digestibility (P < 0.05), causing the largest reduction of dietary energy digestibility (P < 0.05). The greater fermentability of BP resulted in reduced fecal DM and pH, greater fecal production [g/(cat x d); as-is], and greater fecal concentration of acetate, propionate, and lactate (P < 0.05). For most fecal variables, WB was intermediate between BP and SF, and SF was similar to the control diet except for an increased fecal DM and firmer feces production for the SF diet (P < 0.05). Postprandial evaluations indicated reduced mean glucose concentration and area under the glucose curve in cats fed the SF diet (P < 0.05). Colon mucosa thickness, crypt area, lamina propria area, goblet cell area, crypt mean size, and crypt in bifurcation did not vary among the diets. According to the fiber solubility and fermentation rates, fiber sources can induce different physiological responses in cats, reduce energy digestibility, and favor glucose metabolism (SF), or improve gut health (BP).

Static electric fields interfere in the viability of cells exposed to ionising radiation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Selective Ablation of the Androgen Receptor in Mouse Sertoli Cells Affects Sertoli Cell Maturation, Barrier Formation and Cytoskeletal Development

The observation that mice with a selective ablation of the androgen receptor (AR) in Sertoli cells (SC) (SCARKO mice) display a complete block in meiosis supports the contention that SC play a pivotal role in the control of germ cell development by androgens. To delineate the physiological and molecular mechanism responsible for this control, we compared tubular development in pubertal SCARKO mice and littermate controls. Particular attention was paid to differences in SC maturation, SC barrier formation and cytoskeletal organization and to the molecular mediators potentially involved. Functional analysis of SC barrier development by hypertonic perfusion and lanthanum permeation techniques and immunohistochemical analysis of junction formation showed that SCARKO mice still attempt to produce a barrier separating basal and adluminal compartment but that barrier formation is delayed and defective. Defective barrier formation was accompanied by disturbances in SC nuclear maturation (immature shape, absence of prominent, tripartite nucleoli) and SC polarization (aberrant positioning of SC nuclei and cytoskeletal elements such as vimentin). Quantitative RT-PCR was used to study the transcript levels of genes potentially related to the described phenomena between day 8 and 35. Differences in the expression of SC genes known to play a role in junction formation could be shown from day 8 for Cldn11, from day 15 for Cldn3 and Espn, from day 20 for Cdh2 and Jam3 and from day 35 for ZO-1. Marked differences were also noted in the transcript levels of several genes that are also related to cell adhesion and cytoskeletal dynamics but that have not yet been studied in SC (Actn3, Ank3, Anxa9, Scin, Emb, Mpzl2). It is concluded that absence of a functional AR in SC impedes the remodeling of testicular tubules expected at the onset of spermatogenesis and interferes with the creation of the specific environment needed for germ cell development.

Histopathological changes induced by extracts from the tissue covering the stingers of Potamotrygon falkneri freshwater stingrays

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Skin and pulmonary models using coated bentonite particles for the study of the inflammation evoked by Paracoccidioides brasiliensis antigens in previously immunized mice

Bentonite particles coated with polysaccharide antigen or crude soluble antigen of Paracoccidioides brasiliensis were injected intradermally or intravenously in mice. In control animals that were not pre-immunized with P. brasiliensis antigens, coated and uncoated bentonite caused minimal and nonspecific inflammation around the cutaneous injection site or around the bentonite thrombi in small lung vessels after intravenous injection. However, in mice previously immunized with P. brasiliensis antigens, the coated bentonite particles boosted the humoral and cellular immune responses to P. brasiliensis and evoked intense inflammatory reactions. Twelve days after intradermal injection, the inflammatory reaction around the bentonite was rich in neutrophils, macrophages, lymphocytes and plasma cells associated with young granulation tissue. In intravenously injected mice, the pulmonary inflammation was maximal at day 2, and was characterized by a florid neutrophilic and macrophagic cellular infiltration around bentonite thrombi; in some foci, there was incipient organization to mature granuloma. However, in both models, there was no formation of epithelioid granulomata, demonstrating that in paracoccidioidomycosis cellular immunity alone, without the presence of intact micro-organisms, may not be enough for the development of this type of granuloma.

Performance and physiological parameters of broiler chickens subjected to fasting on the neonatal period

Broiler chicks aged 12 h after hatching were allotted according to a block design in a 7 x 2 factorial schedule of 14 treatments and four replications of 50 chicks each one. The main experimental factors were fasting for 0, 6, 12, 18, 24, 30, and 36 h after chick placement and sex. Independent of sex, fasting had a negative linear effect on weight and productivity of broilers at market age (42 d) without affecting feed conversion or mortality index. Groups subjected to 18 and 36 h of fasting after placement, corresponding to 30 and 48 h posthatching fasting, had lower biometrical values for small intestine (length, weight, and size; villus height; and crypt depth) than chicks fed immediately after placement. According to the Pearson test, BW of birds at 21 and 42 d were significantly correlated to BW at 7 d (r = 0.77) and 21 d (r = 0.45), respectively. Males performed better than females but had higher mortality rates. Fasting did not influence serum concentrations of corticosterone or sexual steroid hormones. Nevertheless, early signs of sexual dimorphism arose from the high estradiol (E2) concentration on female serum. Heterophil:lymphocyte ratio was not different among treatments, indicating that early fasting did not seem to be a stress factor 21 or 42 d after fasting. The results suggested a maximum fasting of 24 h after hatching in order to preserve broiler productivity at market age.

Desempenho e morfometria da mucosa de duodeno de frangos de corte alimentados com farelo de canola, durante o período inicial

A trial was carried to verify the effect of increasing levels (0, 10, 20, 30 and 40%) of canola meal in diets of broiler chickens on performance and mucous morphology, during the starting period (1 - 21 days). One thousand 1-d male chicks were assigned to a randomized experimental assay, with five treatments, four replicates and 50 birds per experimental unit. At the 1 st, 21 st and 41 st days, the birds and the rations were weighed. At the 21 st day, two birds of each experimental unit were sacrificed for collection of a duodenum segment to evaluate the intestinal morphometry. The results demonstrated that increasing levels of canola meal resulted in linear reduction on weight gain, weight and feed intake and worst feed conversion. During the growing period (21 to 41 days), when all birds received similar diet, average weight and weight gain decreased, as the canola meal levels increased, while feed conversion and feed intake were similar. Data regarding mucous morphology, submitted to the regression analysis, demonstrated that the crypt depth increased, as the canola meal levels increased. It is possible to include up to 20% of canola meal without damages on broiler performance and digestive tract.

Effects of crude extracts of Agaricus blazei on DNA damage and on rat liver carcinogenesis induced by diethylnitrosamine

The effects of crude extracts of the mushroom Agaricus blazei Murrill (Agaricaceae) on both DNA damage and placental form glutathione S-transferase (GST-P)-positive liver foci induced by diethylnitrosamine (DEN) were investigated. Six groups of adult male Wistar rats were used. For two weeks, animals of groups 3 to 6 were treated with three aqueous solutions of A. blazei (mean dry weight of solids being 1.2, 5.6, 11.5 and 11.5 mg/ml, respectively). After this period, groups 2 to 5 were given a single ip injection 200 mg/kg DEN and groups 1 and 6 were treated with 0.9% NaCl. All animals were subjected to 70% partial hepatectomy at week five and sacrificed 4, 24 and 48 h or 8 weeks after DEN or 0.9% NaCl treatments (10th week after the beginning of the experiment). The alkaline comet assay and GST-P-positive liver foci development were used to evaluate the influence of the mushroom extracts on liver cell DNA damage and on the initiation of liver carcinogenesis, respectively. Previous treatment with the highest concentration of A. blazei (11.5 mg/ml) significantly reduced DNA damage, indicating a protective effect against DEN-induced liver cytotoxicity/genotoxicity. However, the same dose of mushroom extract significantly increased the number of GST-P-positive liver foci.

Critical protective role for annexin 1 gene expression in the endotoxemic murine microcirculation

The inflammatory response is a protective process of the body to counteract xenobiotic penetration and injury, although in disease this response can become deregulated. There are endogenous biochemical pathways that operate in the host to keep inflammation under control. Here we demonstrate that the counter-regulator annexin 1 (AnxA1) is critical for controlling experimental endotoxemia. Lipopolysaccharide (LPS) markedly activated the AnxA1 gene in epithelial cells, neutrophils, and peritoneal, mesenteric, and alveolar macrophages-cell types known to function in experimental endotoxemia. Administration of LPS to AnxA1-deficient mice produced a toxic response characterized by organ injury and lethality within 48 hours, a phenotype rescued by exogenous application of low doses of the protein. In the absence of AnxA1, LPS generated a deregulated cellular and cytokine response with a marked degree of leukocyte adhesion in the microcirculation. Analysis of LPS receptor expression in AnxA1-null macrophages indicated an aberrant expression of Toll-like receptor 4. In conclusion, this study has detailed cellular and biochemical alterations associated with AnxA1 gene deletion and highlighted the impact of this protective circuit for the correct functioning of the homeostatic response to sublethal doses of LPS. Copyright © American Society for Investigative Pathology.

Tuberculose e o estudo molecular da sua epidemiologia

Systems that can distinguish epidemiologically-related Mycobacterium tuberculosis strains from unrelated ones are extremely valuable. Molecular biology techniques have allowed a great deal of information to be acquired about the infectious disease tuberculosis (TB) that was very hard or impossible to obtain by conventional epidemiology. A typing method based on bacterial DNA genome differences, known as RFLP (restriction fragment length polymorphism), is widely used to discriminate strains in the epidemiologic study of TB. However, RFLP is laborious and there is a tendency to replace it by other methods. Thus, other DNA sequences have been employed as epidemiological markers, as in Spoligotyping, a fast technique based on PCR followed by differential hybridization of amplified products. The polymorphism observed among different isolates is probably the product of strain-dependent recombination. MIRU (mycobacterial interspersed repetitive unit) typing is a reproducible and fast assay, involving the generation of genotypes based on the study of 12 loci containing VNTRs (variable-number tandem repeats) in strains of the M. tuberculosis complex. It compares strains from different geographic areas and allows the movement of individual lineages to be tracked, as in RFLP. This approach enables a greater number of isolates to be analyzed, leading to the identification of a larger number of foci of transmission within the population and thus to improved ways of slowing the progress of the disease.

Infantile epileptic encephalopathy with hypsarrhythmia (infantile spasms/west syndrome) and immunity

West syndrome is a severe epilepsy, occurring in infancy, that comprises epileptic seizures known as spasms, in clusters, and a unique EEG pattern, hypsarrhythmia, with psychomotor regression. Maturation of the brain is a crucial component. The onset is within the first year of life, before 12 months of age. Patients are classified as cryptogenic (10 to 20%), when there are no known or diagnosed previous cerebral insults, and symptomatic (80 to 90%), when associated with pre-existing cerebral damages. The time interval from a brain insult to infantile spasms onset ranged from 6 weeks to 11 months. West syndrome has a time-limited natural evolutive course, usually disappearing by 3 or 4 years of age. In 62% of patients, there are transitions to another age-related epileptic encephalopathies, the Lennox-Gastaut Syndrome and severe epilepsy with multiple independent foci. Spontaneous remission and remission after viral infections may occur. Therapy with ACTH and corticosteroids are the most effective. Reports about intravenous immunoglobulins action deserve attention. There is also immune dysfunction, characterized mainly by anergy, impaired cell-mediated immunity, presence of immature thymocytes in peripheral blood, functional impairment of T lymphocytes induced by plasma inhibitory factors, and altered levels of immunoglobulins. Changes in B lymphocytes frequencies and increased levels of activated B cells have been reported. Sensitized lymphocytes to brain extract were also described. Infectious diseases are frequent and may, sometimes, cause fatal outcomes. Increase of pro-inflamatory cytokines in serum and cerebrospinal fluid of epileptic patients were reported. Association with specific HLA antigens was described by several authors (HLA-DR7, HLA-A7, HLA-DRw52, and HLA-DR5). Auto-antibodies to brain antigens, of several natures (N-methyl-d-aspartate glutamate receptor, gangliosides, brain tissue extract, synaptic membrane, and others), were described in epileptic patients and in epileptic syndromes. Experimental epilepsy studies with anti-brain antibodies demonstrated that epileptiform discharges can be obtained, producing hyperexcitability leading to epilepsy. We speculate that in genetically prone individuals, previous cerebral lesions may sensitize immune system and trigger an autoimmune disease. Antibody to brain antigens may be responsible for impairment of T cell function, due to plasma inhibitory effect and also cause epilepsy in immature brains. © 2008 Bentham Science Publishers Ltd.

Evidence for the role of calcineurin in morphogenesis and calcium homeostasis during mycelium-to-yeast dimorphism of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a dimorphic fungus that causes paracoccidioidomycosis, the most prevalent human deep mycosis in Latin America. The dimorphic transition from mycelium to yeast (M-Y) is triggered by a temperature shift from 25°C to 37°C and is critical for pathogenicity. Intracellular Ca 2+ levels increased in hyphae immediately after temperature-induced dimorphism. The chelation of Ca 2+ with extracellular (EGTA) or intracellular (BAPTA) calcium chelators inhibited temperature-induced dimorphism, whereas the addition of extracellular Ca 2+ accelerated dimorphism. The calcineurin inhibitor cyclosporine A (CsA), but not tacrolimus (FK506), effectively decreased cell growth, halted the M-Y transition that is associated with virulence, and caused aberrant growth morphologies for all forms of P. brasiliensis. The difference between CsA and FK506 was ascribed by the higher levels of cyclophilins contrasted to FKBPs, the intracellular drug targets required for calcineurin suppression. Chronic exposure to CsA abolished intracellular Ca 2+ homeostasis and decreased mRNA transcription of the CCH1 gene for the plasma membrane Ca 2+ channel in yeast-form cells. CsA had no detectable effect on multidrug resistance efflux pumps, while the effect of FK506 on rhodamine excretion was not correlated with the transition to yeast form. In this study, we present evidence that Ca 2+/calmodulin-dependent phosphatase calcineurin controls hyphal and yeast morphology, M-Y dimorphism, growth, and Ca 2+ homeostasis in P. brasiliensis and that CsA is an effective chemical block for thermodimorphism in this organism. The effects of calcineurin inhibitors on P. brasiliensis reinforce the therapeutic potential of these drugs in a combinatory approach with antifungal drugs to treat endemic paracoccidioidomycosis. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Infliximabe no tratamento inicial da retocolite ulcerativa moderada e grave. Terapia top down: Relato preliminar

The first option for the treatment of UC is both: salicylates or corticoids. Recently, in late November of 2006, the Brazilian Ministry of Health has approved infliximab (Remicade c, Mantecorp, Brazil) to treat ulcerative colitis. We report the use of infliximab as a first option for the treatment of two patients with severe ulcerative colitis. Case report: Patient 1: AZF, 52 years-old, female, was first diagnosed with UC after history and clinical examination; colonoscopy showed pancolitis with positive biopsy (crypt microabscess). Her Mayo score was 10 (range: 0 to 12/asymptomatic to severe colitis). She received intra venous infusion of infliximab at a dose of 5mg/Kg of body weigh at week 0, 2, 6 and 14. Then, patient was given mesalazine 4.5 g/day for maintenance therapy. Clinical response was defined as a decreased from baseline in the total Mayo score of at least 3 points. At present, patient is asymptomatic with Mayo score of 3 one moth after the last dose of infliximab. Patient 2: MLA, 45 years-old, female was first diagnosed with bloody diarrhea; colonoscopy showed left colitis and the biopsy was positive for ulcerative colitis. Her Mayo score was 9. She was offered and accepted the step down treatment. She was given infliximab 5mg/Kg of body weight at week 0, 2, 6 and 14. After initial treatment with infliximab, she received mesalazine 4.2 g/day. At present, she is asymptomatic with Mayo score of 2 eighteen days after the last dose of infliximab. At our knowledge, this is the first Brazilian report of the use of infliximab as fist-line therapy in ulcerative colitis. Few days after the begging of the infusion, an impressive clinical and colonoscopy improvement was seen in these two patients. Recently, it has been reported the use of infliximab as first-line therapy in pediatric Crohn disease. Infliximab could be a good option in cases of moderate and severe UC to avoid the side effects of the use of high doses of corticoids in patients with moderate and severe UC. However, the question if step-down therapy in ulcerative colitis is better then conventional therapy with salicylates and corticoids needs to be answered by randomized trials.