AgNOR Method Research Articles

TRANSMISSIBLE VENEREAL TUMOR: CELL PROLIFERATION (AGNOR) AND RESPONSE TO CHEMOTHERAPY CORRELATED WITH CYTOMORPHOLOGICAL CLASSIFICATION

Transmissible venereal tumor (TVT) is a contagious neoplasm of round cells that occurs naturally and that affects mostly the genitalia. The TVT has been classified according to the predominant cell type as follows: linfocytoid, plasmacytoid and mixed. Various degrees of aggressiveness with wide range of biological behavior have been described based on this cell morphology. The present study aimed to evaluate the rate of cell proliferation, by cytochemical AgNOR method, and response to chemotherapy correlated with the cytomorphological classification of TVT. The results of the 22 cases showed that TVT was identified more frequently in female, adults and mixed-breed dogs. Plasmocytoid-classified tumors required a greater number of vincristine sulfate sessions, have higher cell proliferation and were the most resistant to chemotherapy, requiring additional treatment with doxorubicin. Based on these results, we can infer that the plasmacytoid pattern might have a more aggressive behavior compared to other cell types.

Possible correlation between Lactobacillus paracasei X12 intake and tumor characteristics in the rat model of colorectal cancer

Introduction: There are many different therapeutic approaches to control colorectal cancer (CRC) which is recognized as one of the deadliest diseases in the world today. One of the most recent of which is the probiotic interventions to change the gut microbiome. Probiotics can be related to the control of gastrointestinal cancers in a variety of ways. Therefore, the present study aimed to investigate the relationship between supplement use with macroscopic and physiological changes of tumor in 1,2-dimethylhydrazine (DMH) -induced rats. Methods: The male Wistar rats were divided into three groups. Lactobacillus paracasei X12 was administrated (40 weeks) to the DMH-induced rats. DMH injection (30 mg/kg BW) was used for 12 weeks to induce CRC. The "AgNOR method" was applied for the evaluation of cell proliferation. Real-time polymerase chain reaction (PCR) was used to measure the gene expression of apoptotic markers. Results: The findings of this study indicated that L. paracasei X12 intake prevented weight loss caused by CRC (P=001), and probiotic consumption could significantly prevent tumor growth. Additionally, a significant correlation was observed between apoptosis markers and weight of animals, and a strong negative correlation (P=000) between apoptosis parameters and tumor characteristics (incidence, volume, and multiplicity of adenomas). A close association between cell proliferation and tumor characteristics was illuminated as well (P<001). Conclusion: This study revealed a strong correlation between tumor incidence and growth and probiotic intake in CRC. Moreover, it could be believed that cancer prevention is a far more essential and cost-effective way than its cure.

Cytoprotective effect of non-opioid leu-enkephalin analogue in primary culture of pulmonary fibroblasts in oxidative stress

Aim. Analysis of the cytoprotective effect of non-opioid leu-enkephalin analogue (Phe-D-Ala-Gly-Phe-Leu-Arg) in the primary culture of pulmonary fibroblasts in conditions of oxidative stress.
 Methods. Pulmonary fibroblasts were incubated with the peptide of non-opioid leu-enkephalin analogue (Phe-D-Ala-Gly-Phe-Leu-Arg) in the concentration 0.1 μM for 6 hours. To simulate oxidative stress, 60 μM H2O2 was added to the culture medium for 2 hours. Experimental series included (1) «control»; (2) «non-opiate leu-enkephalin analogue» (the peptide was added to the culture medium 44 hours after the final passage); (3) «oxidative stress» (H2O2 was added to the culture medium 48 hours after the final passage); (4) «non-opiate leu-enkephalin analogue + oxidative stress» (the peptide and H2O2 were added to the culture medium 44 and 48 hours respectively after the final passage). In order to evaluate the generation of superoxide anion by pulmonary fibroblasts, the method of lucigenin-dependent chemiluminescence was used. Computer morphometry of the nucleo-nucleolar apparatus of fibroblasts stained with silver nitrate by the AgNOR method was used to assess the cell state: the area of fibroblast nuclei, the total nucleoli area in the nucleus, and the number of nucleoli in the nucleus were measured. These parameters correlate with the activity of anabolic processes in the cells.
 Results. The effect of H2O2 on the primary culture of pulmonary fibroblasts caused an increase of superoxide anion generation by the fibroblasts, reduction of fibroblast nuclei size, decrease of nucleoli amount and size. Pre-incubation of pulmonary fibroblasts with a non-opioid leu-enkephalin analogue reduced the H2O2-induced generation of superoxide anion, corrected changes in the nucleo-nucleolar apparatus of fibroblasts caused by oxidative stress. In our previous studies, similar effect in the same model was shown for non-selective μ/δ-opioid receptor agonist peptide sedatin (dermorphin analogue). The mechanism of cytoprotective effect of non-opioid leu-enkephalin analogue may include the affinity of this peptide to nociceptin receptors (NOR receptors) that requires further studies.
 Conclusion. The results of the study indicate a direct cytoprotective effect of the peptide Phe-D-Ala-Gly-Phe-Leu-Arg (non-opioid leu-enkephalin analogue) in oxidative stress.

Drought leads to reproductive quiescence in smooth‐billed anis: Phenotypic evidence for opportunistic breeding and reproductive readiness

Previous studies have suggested that the smooth-billed ani (Crotophaga ani, Linnaeus, 1758) breeds opportunistically following unpredictable rainfall in drought areas. To obtain proof of this phenomenon, the present study described and compared reproductive morphology and cell proliferation in the gonads of free-living smooth-billed anis during a wet season (April to June 2012) and the following dry season (July to September 2012) in a semiarid area using light and electron microscopy (transmission and scanning) and the AgNOR method. The morphological findings indicated distinct levels of reproductive activity related to seasonal changes. Morphological and morphometric analyses of the gonads confirmed intense gametogenic activity during the wet season, whereas gonadal involution occurred after rainfall ceased. The sizes of the testes and ovaries were significantly reduced compared to those in the wet season. The volumetric fraction of the seminiferous tubules in the testis decreased considerably, and no preovulatory follicles were detected in the ovary in the dry season. Moreover, the AgNOR count in the gonads revealed a significant decline in cell recruitment for gametogenesis after rainfall ceased. The histological findings indicated partial gonadal activation throughout the dry season. The analysis of the seminiferous epithelium confirmed the early testicular recrudescence phase, and sporadic postovulatory follicles indicated random ovulation during this time. The excurrent ducts and the oviduct also underwent remarkable involution in the dry season. Taken together, these findings confirm opportunistic breeding by smooth-billed anis in a semiarid habitat and suggest that gonadal recrudescence has been established as a reproductive strategy to cope with unexpected precipitation events.

Use of different fixation times and application of two immunohistochemical methods for detection of KIT and Ki67 proteins in canine cutaneous mast cell tumors

ABSTRACT: Due to the high prevalence of mast cell tumors (MCTs) in the diagnostic routine, several factors, especially prognostic, have been sought to determine the biological behavior of these neoplasms. Immunohistochemistry (IHC) is one of the main tools utilized to biologically differentiate more aggressive tumors from less aggressive ones. However, some immunostainings are influenced by formalin fixation, interfering with the results. This is both a retrospective and prospective study of MCTs diagnosed in laboratory routine. A total of 25 samples, without knowledge about fixation time, were analyzed in the retrospective study, whereas 12 samples, with known fixation times, were assessed in the prospective study. Two histologic grading systems (Patnaik and Kiupel), special staining of toluidine blue, and IHC for KIT and Ki67 proteins were applied in both studies. Additionally, two amplification systems (biotinylated and non-biotinylated) for Ki67 protein and counting of the argyrophilic nucleolar organizing regions (AgNOR method) were tested in the prospective study. In the retrospective study, greater agreement between the evaluating pathologists was observed when the Kiupel system was used. IHC staining for KIT protein was effective in both studies, regardless of fixation time. IHC staining for Ki67 protein was highly sensitive to formaldehyde, and staining failure was observed in 56% of the cases in the retrospective study. In the prospective study, samples fixed for longer than 24 hours showed a reduction in the number of stained cells (altering the determination of the cell growth fraction) or showed absence of IHC staining in both amplification systems. The use of the AgNOR method to evaluate the rate of cell proliferation may be an alternative when the fixation time of the neoplasm is unknown or longer than 24 hours.

Detection of telomeric sequences and ribosomal RNA genes in holokinetic chromosomes of five jumping plant-lice species: First data on the superfamily Psylloidea (Hemiptera: Sternorrhyncha)

Fluorescence in situ hybridization (FISH) is a technique used to determine the chromosomal position of DNA and RNA probes. The present study contributes to knowledge on jumping plant-lice genomes by using FISH with 18S rDNA and telomeric (TTAGG)n probes on meiotic chromosomes of Psylla alni (2n = 24 + X), Cacopsylla mali (2n = 22 + neo-XY and 20 + neo-X1X2Y), C. sorbi (2n = 20 + neo-XY), Baeopelma foersteri (2n = 14 + X), and Rhinocola aceris (2n = 10 + X). This is the first study that has used FISH on the hemipteran superfamily Psylloidea. We found that the chromosomes of all studied species contain the insect-type telomere motif, (TTAGG)n. In C. mali and C. sorbi, the neo-sex chromosomes originating from autosome-sex chromosome fusions showed no interstitially located clusters of TTAGG repeats, suggesting their loss or inactivation. Similarly, no interstitial (TTAGG)n clusters were detected in an extremely large autosome pair of B. foersteri that most likely originated from a fusion of at least five ancestral chromosome pairs. Clusters of 18S rDNA were detected on the fused and second largest autosome pairs of B. foersteri and on one of the large autosome pairs of the remaining species. In C. mali and B. foersteri, the rDNA clusters were shown to coincide with the NORs as detected by the AgNOR method. Finally, we speculate, based on the obtained FISH markers, on the mechanisms of karyotype evolution of psylloid species differing in chromosome numbers and sex chromosome systems.

Cytogenetics of two hylid frogs from Brazilian Cerrado.

Cytogenetic data can be useful for taxonomic and phylogenetic studies, as well as to provide information about chromosome evolution. Therefore, it may help design conservation priorities for some threatened species, such as anurans. Herein, we describe the karyotypes of Scinax constrictus and Ololygon centralis, native endemic species from the Brazilian Cerrado. Chromosome preparations for both species were stained with Giemsa for morphological analyses and then impregnated by the Ag-NOR method for localization of the nucleolar organizer region (NOR). Both species had 24 chromosomes, as confirmed by meiotic analyses, which showed 12 bivalents. Chromosome morphologies presented the same pattern for Scinax and Ololygon compared to species already karyotyped in both genera. The NOR was interstitially located in the long arm of pair 7 in S. constrictus, whereas in O. centralis it was found near the centromere in the long arm of pair 1, thus diverging from what is commonly found for other Ololygon species. Therefore, we provide the first description of the karyotype of O. centralis and the first report of the localization of the NOR for the karyotype of both species. Our study increases the cytogenetic knowledge in species of the genera Scinax and Ololygon, and provide support for further studies on the taxonomy, ecology, and evolution of hylid anurans.

EFEK KO-KEMOTERAPI FRAKSI ETIL ASETAT AKAR PASAK BUMI DAN DOXORUBICIN TERHADAP PROLIFERASI DAN EKPRESI BAX JARINGAN PAYUDARA TIKUS SD

The use of co-chemotherapy agent was needed since there were some toxicities on the normal tissues caused by the use of doxorubicin. The root of Eurycoma longifolia Jack, a part of plant, has a potential activity as co-chemotherapy. This study aimed to determine the effect of co-chemotherapy of ethyl acetate fraction of E. longifolia Jack roots and doxorubicin against cell proliferation activity by AgNOR method and determine expression of Bax protein of breast tissue in rats induced by DMBA. The rats were divided into five groups: I (baseline), II (DMBA 20 mg/kgBW), III (DMBA, doxorubicin 1.12 mg/kg), IV (DMBA, fraction of 100 mg/kg), and V (DMBA, doxorubicin, fractions). All the rats were sacrificed at week 16. Their breast tissue was evacuated. Cell proliferation and expression was observed using AgNOR method and immunohistochemistry, respectively. Results showed the percentage of p-AgNOR obtained by the healthy group, DMBA group, DMBA+Doxorubicin group, DMBA+ethyl acetate fraction of E. longifolia Jack root group, and DMBA+Doxorubicin+Ethyl acetate fraction of E. longifolia group were 0±0%, 14.67±2.11%, 1.83±1.21%, 6.83±2.03%, and 4.08±0.95%, respectively. The percentage of Bax expression obtained by the healthy group, DMBA group, DMBA+Doxorubicin group, DMBA+ethyl acetate fraction of E. longifolia Jack root group, and DMBA+Doxorubicin+Ethyl acetate fraction of E. longifolia group were 68.82±1.52%, 26.86±3.25%, 44.49±2.06%, 80.92±3.27%, and 78.70±4.87%, respectively. Based on the results, it was concluded that co-chemotherapy agent of ethyl acetate fraction of E. longifolia Jack roots and Doxorubicin could inhibit proliferation and trigger apoptosis through Bax expression in breast tissue of rats induced by DMBA.

How Useful is the AgNOR Staining Method for the Diagnosis of Salivary Gland Tumors?

Objectives: The purpose of this study was to determine the numbers and mean areas of the AgNOR dots in the 3 most common salivary gland tumors. Materials and Methods: One hundred and seventy paraffin blocks histologically diagnosed as pleomorphic adenoma, adenoid cystic carcinoma, and mucoepidermoid carcinoma were selected from the archives of the pathology department of Loghman hospital, Tehran, Iran and then cut and stained using the standard AgNOR method. The number and area of AgNOR dots were evaluated. Chi-square and Pearson correlation (2-tailed) tests were used for statistical analysis. The significance level was P < 0.05. Results: The chi-square test showed significant differences between tumor types and the numbers of AgNORs (P = 0.000) and between tumor types and areas of AgNORs (P = 0.000). Conclusions: The AgNOR technique can be used to diagnose salivary gland tumors.

English

Infection of the gastric mucosa by Helicobacter spp. is an important cause of gastric diseases and neoplasms in humans and animals. The objective of this study was to evaluate the presence of Helicobacter spp. in the livers of dogs with nonspecific histological changes. The association between Helicobacter spp. infection, hepatocyte proliferation and E-cadherin expression was also evaluated. Liver samples from 39 dogs were subjected to polymerase chain reaction (PCR) to identify Helicobacter spp. Hepatocyte proliferation was evaluated by the AgNOR method. The Helicobacter genus was detected in seven (17.9%) animals that exhibited liver histological changes. Twenty-six animals were PCR negative and exhibited microscopical changes, while six animals demonstrated no histological changes and were PCR negative. Infected animals showed a significant increase in liver lesional score and the area of NORs compared to non-infected dogs. E-cadherin expression showed no significant difference among groups. The results suggest that Helicobacter spp. and hepatocytes injury in the livers of dogs could contribute to increased cell proliferation, thereby influencing hepatocarcinogenesis. Key words: Dog, helicobacter, liver, AgNOR, immunohistochemistry, E-cadherin.

Quantitative analysis of agnor counts of buccal mucosal cells of chewers and non chewers of gutkha: A comparative cytologic study.

The present study was taken up to evaluate the AgNOR counts in the buccal mucosa cells of gutkha chewers and compare that with the sex-matched controls. In all, 100 gutkha chewers and 50 sex-matched non-chewers (controls) were chosen. None of the patients in both groups had any clinical oral lesions or systemic diseases. After rinsing with 0.9% sodium chloride, cytologic smears were prepared and stained using the AgNOR method and observed in immersion oil at 1000 × magnification. Finally, 50 cells were selected at random; AgNOR dots were counted and their mean was recorded. The student t-test was used for analysis of data. Comparison between mean AgNOR counts of gutkha chewers (2.68 ± 0.23) and non-chewers (2.01 ± 0.14) was found to be statistically significant. Cytology associated with AgNOR staining can effectively detect the early molecular changes within buccal mucosa cells of oral mucosa.

Evaluation of cellular proliferative activity in patients with oral lichen planus and hepatitis C through AgNOR method.

The objective of this study was to evaluate the cellular proliferative potential of oral lichen planus (OLP) lesions from patients without hepatitis C virus (HCV) by means of AgNOR method, as well as the cellular proliferative potential of the normal oral mucosa from patients with HCV, treated or untreated by interferon and ribavirin. A cross-sectional study was developed to investigate four groups: 10 HCV+ patients without clinical signs of OLP who had never been treated for HCV infection - Group 1; 10 HCV+ patients that were under interferon and ribavirin treatment - Group 2; 15 patients with reticular OLP lesions histopathologically confirmed, without HCV - Group 3; and 15 blood donors without HCV infection and no clinical signs of OLP GROUP 4 Control Group. The cytological material of all groups was collected by the liquid-based cytology technique. Then, the sedimented material from each patient was filled with the Nucleolar Organizer Regions impregnation by silver method (AgNOR). The count of NORs was performed on 100 epithelial cell nuclei per patient using the Image Tool(tm) software. The Tukey HSD test was used to compare the median value of NORs among the groups and showed that the oral mucosa of HCV+ patients previously treated with anti-HCV drugs (GROUP 2), presented a higher average number of NORs in relation to others (p<0.05). The anti-HCV treatment may be related to increased cell proliferation of oral mucosa, indicating a possible relationship between OLP and HCV+ patients treated with interferon and ribavirin.

Interstitial Telomeric Sequences (ITS) and major rDNA mapping reveal insights into the karyotypical evolution of Neotropical leaf frogs species (Phyllomedusa, Hylidae, Anura).

BackgroundThe combination of classical cytogenetics with molecular techniques represents a powerful approach for the comparative analysis of the genome, providing data for the systematic identification of chromosomal homologies among species and insights into patterns of chromosomal evolution within phylogenetically related groups. Here, we present cytogenetic data on four species of Neotropical treefrogs of the genus Phyllomedusa (P. vaillantii, P. tarsius, P. distincta, and P. bahiana), collected in Brazil and Ecuador, with the aim of contributing to the understanding of the chromosomal diversification of this genus.ResultsWith the exception of P. tarsius, which presented three telocentric pairs, all the species analyzed had conservative karyotypic features. Heterochromatic patterns in the genomes of these species revealed by C-banding and fluorochrome staining indicated the presence of a large number of non-centromeric blocks. Using the Ag-NOR method and FISH with an rDNA 28S probe, we detected NOR in the pericentromeric region of the short arm of pair 7 in P. vaillantii, pair 1 in P. tarsius, chromosomes 1 and 9 in P. distincta, and in chromosome 9 in P. bahiana, in addition to the presence of NOR in one homologue of chromosome pair 10 in some individuals of this species. As expected, the telomeric probe detected the terminal regions of the chromosomes of these four species, although it also detected Interstitial Telomeric Sequences (ITS) in some chromosomes of the P. vaillantii, P. distincta and P. bahiana karyotypes.ConclusionA number of conservative chromosomal structures permitted the recognition of karyotypic homologies. The data indicate that the presence of a NOR-bearing chromosome in pair 9 is the plesiomorphic condition in the P. burmeisteri group. The interspecific and intraspecific variation in the number and location of rDNA sites reflects the rapid rate of evolution of this character in Phyllomedusa. The ITS detected in this study does not appear to be a remnant of structural chromosome rearrangements. Telomeric repeats were frequently found in association with heterochromatin regions, primarily in the centromeres, which suggests that (TTAGGG)n repeats might be an important component of this heterochromatin. We propose that the ITSs originated independently during the chromosomal evolution of these species and may provide important insights into the role of these repeats in vertebrate karyotype diversification.

Antiproliferative Activity of Black Seed (Nigella sativa) on 7,12-dimethylbenz-[a]antracene (DMBA) Induced Mice Lung Cell

Lung cancer is the leading cause of death in the world. Nigella sativa is a plant that has an anticancer activity. Previous research showed that the chloroform extract of N. sativahave cytotoxic effect on T47D cells. This study aimed to observe the effect of cloroform extract of N. sativaseed (NSS) on mice lung cell after initiation of 7,12-dimethylbenz [a] anthracene. Sprague Dawleystrain female rats were divided intofive groups. Each group consisted of 12 rats. The experiment consisted of five mice groups, corn oil as a solvent control group, the DMBA dose 20 mg/kgBW p.o. twice a week during five weeks, DMBA+NSS dose 250 mg/kgBW, DMBA+NSS dose 500 mg/kgBW, and DMBA+NSS dose 750 mg/kgBW. Extract which was dissolved into corn oil was administered daily by the oral route 1 week before and during the DMBA induction. At the end of the study, the experimental mice were sacrified and colon organs were collected and then stained with Haematoxylin and Eosin (H&E) and AgNOR method. H&E staining showed there was a decrease damage inthe treatment group compare with DMBA group. In AgNOR staining result showed mAgNOR value in DMBA group was 1.47±0.558, in extract group (250, 500 and 750mg/kgBB) was 0.44±0.172, 1.38±0.140 and 1.25±0.164 respectively and in corn oil group was 0.65±0.050. The results showed that N. sativareduced the damage of colon cells and inhibit colon cell proliferation on mice induced DMBA. This study indicated that N. sativacan be developed into a chemopreventive agent for lung cancer.

Antiproliferative Activity of Ethanolic Extract of Ciplukan Herbs (Physalis angulata L.) on 7,12-Dimethylbenz[A]Nthracene-Induced Rat Mammary Carcinogenesis

Physalis angulata L. is an annual herb widely used as popular medicine for the treatment of cancer. Physalis angulata L. ethanolic extract (PEE) has been demonstrated to have strong cytotoxic activity against breast cancer, inhibited cancer cell’s proliferation and induced cell cycle arrest. The aim of our study is to investigate the effect of PEE as a cancer chemopreventive agent on 7,12-dimethylbenz[a]nthracene (DMBA)-induced rats mammary. The antiproliferative activity was characterized by monitoring the histopatology representation and expression of cell proliferation on DMBA-induced mammary rats that were treated with PEE against control groups. The histopatology representation were analyzed by Haematoksilin Eosin (HE) staining method, while proliferative activity was detected by AgNOR method. The HE staining results showed significant differences in cells morphology of treatment groups compared to the control groups. Thus results suggest that PEE was able to repair morphology of cells undergoing carcinogenesis. AgNOR method showed decreasing occurrence of black dots between treatment and control groups. Thus, we conclude that PEE has an antiproliferative activity on DMBA-induced rat mammary. Therefore, the ethanolic extract of Physalis angulata herbs is a potential chemopreventive agent on cancer. Further study on its molecular mechanism needs to be explored.Keywords: Physalis angulata, breast cancer, 7,12-dimethylbenz[a]nthracene, carcinogenesis, antiproliferative

Oral epithelial atypical changes in apparently healthy oral mucosa exposed to smoking, alcohol, peppers and hot meals, using the AgNOR and Papanicolaou staining techniques

To evaluate cytological atypical changes in apparently healthy oral mucosa exposed to smoking, alcohol, hot meals, and peppers using the AgNOR and Papanicolaou methods. A total of 180 individuals were evaluated, of which 60 were smokers, 34 were alcohol users, 52 were habitual peppers and hot meal (exposed) consumers, 24 were non-exposed, and 10 were patients with Oral Squamous Cell Carcinoma (OSCC), as an internal control. Cytological materials were obtained by brushing of buccal mucosa, on the border of the tongue and on the floor of the mouth, and participants underwent the Papanicolaou test for cytological changes and AgNOR staining for evaluation of the mean number of AgNOR dots per nucleus. SPSS program was used to perform the Pearson chi-square test. The 95% confidence level, Odds Ratio (OR), and the 95% Confidence Intervals (CI) were used. The features of cytological atypia were verified among 10 individuals, including 5 smokers, 2 alcohol users, 2 hot meals and peppers consumers, and one non-exposed. For atypia among tobacco smokers, the adjusted Odds Ratio (OR) and the 95% CI were found to be 2 (0.246-16.24). Increased keratinization was detected among 27 (45%) of the smokers (P < 0.0001), 17 (32.7%) of the pepper and hot meals consumers (P < 0.005), 4 (11.8%) of the alcohol consumers, and among 2 (3.7%) of the non-exposed group. Statistical analyses revealed a greater mean number of AgNORs per nucleus in smokers (3.68) followed by (2.82) alcohol consumers, compared to the habitual peppers and hot meal consumers (2.28) and the non-exposed group (2.00). What's more, 80% of the smears with cytological atypia were identified with 6 +/- 2 AgNOR mean count. The increase of the variables suggests that the evaluation of epithelial atypical changes in individuals exposed to smoking and alcohol carcinogens may be a useful screening tool. While hot meals and peppers did not seem to be a risk for oral mucosal proliferation, they increased the potency of keratinization and infection.

Proliferação celular nos linfomas caninos

O estudo dos linfomas caninos além de abordar a classificação morfológica e imunofenotípica necessita ser ampliado para que se tenha a avaliação da cinética celular. Esta verificação só pode ser feita com segurança quando são avaliados os parâmetros que indicam a taxa de proliferação celular. No homem, estes fatores têm influência no prognóstico e no tratamento dos limfomas. Os 40 linfomas utilizados neste trabalho foram classificados de acordo com a classificação de Kiel e a imunofenotipagem utilizou CD3 (linfócitos T) e CD79a (linfócitos B). A proliferação celular foi avaliada pelos métodos de contagem dos AgNORs e Ki-67 (MIB-1) De acordo com a classificação de Kiel os linfomas de alto grau foram os mais freqüentes, a imunofenotipagem mostrou a mesma freqüência de linfomas T e B. Quando avaliada a proliferação celular houve diferença significativa entre os linfomas de alto e baixo grau tanto pelo método do AgNOR como do KI-67.(MIB-1), porém não foram estatisticamente entre os imunofenótipos Entre as neoplasias de alto grau de malignidade a média de número de NORs por núcleo de célula neoplásica foi de 1,37 ± 0,32, e nos casos de baixo grau de 0,98 ± 0,36, de acordo com o KI-67 a média do percentual de células positivas foi de 43,19% ± 19,01, e 14,09% ± 11,74 nos casos de alto e baixo grau, respectivamente.

Unusual primitive heteromorphic ZZ/ZW sex chromosomes in Proceratophrys boiei (Anura, Cycloramphidae, Alsodinae), with description of C-Band interpopulational polymorphism

We performed cytogenetic analyses on specimens from three population samples of Proceratophrys boiei from southeastern and northeastern Brazil. We stained chromosomes of mitotic and meiotic cells with Giemsa, C-banding and Ag-NOR methods. All specimens of P. boiei presented a karyotype with a full chromosome complement of 2n=22, metacentric and submetacentric. We observed the secondary constriction within the short arm of pair 8, which was in the same position of the nucleolus organizer region (NOR). NOR heteromorphism was observed within two specimens from the municipality of Mata de São João (northeastern Bahia State). The C-banding evidenced an unusual heterochromatic pattern in the genome of P. boiei. In the southern most population samples (São Paulo State), we observed large blocks of heterochromatin in the centromeric regions of all chromosomes, whereas the northernmost samples (Bahia State) presented a small amount of constitutive heterochromatin. We suppose that this geographic variation in heterochromatin quantities could be due to heterochromatinization of some chromosome regions in the genome of the São Paulo samples. Furthermore, females from São Paulo presented, within chromosome pair 1 from C-banded karyotypes, one homologous chromosome almost heterochromatic, whereas males had heterochromatin restricted to the centromeric region. This unusual heterochromatic arrangement led us to assume that P. boiei owns a ZZ/ZW type of sexual determination system. This finding is very important, as this is the first record of ZZ/ZW sex chromosomes within Cycloramphidae. We believe that the cytogenetic differences found between southeastern and northeastern Brazilian population samples of P. boiei strongly supports the existence of a species complex under the name P. boiei, and the requirement of taxonomic and systematic reviews by morphological, bioacoustical, molecular, and cytogenetic data could define this taxonomic issue in the future.

Proliferative activity of thyroid hürthle cell tumors using PCNA, Ki-67 and AgNOR methods

Aim: We have undertaken an attempt to compare the application efficacy of the proliferative activity markers in differential diagnosis of thyroid Hürthle cell tumors using the PCNA and Ki-67 labeling and AgNOR visualization techniques.

Quantity of AgNORs in gastric endocrine carcinoid tumours as a potential prognostic tool.

In order to assess if the quantity of silver-stained nucleolar organizer region (AgNOR) proteins represents a prognostic tool in gastric carcinoids, a standardised AgNOR analysis was performed on 24 samples collected from the pathology archives of the Universities of Messina and Parma; the samples were taken at surgery from 11 males and 13 females (mean age 55 yrs, age range 28-77 yrs); 13 cases were defined as Type I, 1 case as Type II and 10 cases as Type III; 16 cases showed a diameter <1 cm, 8 >1 cm. Only 6 tumours were deeply invasive, breaking through the muscularis propria or the subserosa. The proliferative status of carcinoids performed by Ki67 protein antibodies was available in 20/24 cases. The quantification of AgNORs was performed according to the guidelines of the Committee on AgNOR Quantification and the mean area (microm2) of AgNORs per nucleus (NORA) was determined by means of image analyser and specific software programs. The relationship between NORA values and Ki67 data was investigated by Spearman correlation test. The mean NORA value of all 24 gastric carcinoids was 1.279 microm2 (SD 0.404); values ranged from 0.734 to 2.142 microm2. A significantly higher (p < 0.001) mean NORA value (1.736 microm2; SD 0.283) was found in tumours larger than 1 cm, in comparison to the smaller neoplasms (1.051 microm2; SD 0.214); moreover, cases showing deep wall invasion exhibited a mean NORA value of 1.765 microm2 (SD 0.276), significantly higher (p < 0.001) than those with superficial growth (1.118 microm2; SD 0.296). Finally, a similar highly significant difference was seen between type III carcinoids (1.615 microm2; SD 0.375) and type I-II (1.040 microm2; SD 0.208). A linear relationship between Ki67 and corresponding NORA values was obtained by the Spearman correlation test (p = 0.001). No other significant correlations were found between mean NORA values and other clinico-pathological parameters. The AgNOR method seems to be an additional tool potentially able to predict the prognosis of this kind of endocrine tumour, facilitating the identification of fast-growing tumours and being able to directly correlate with the size, deep invasion of gastric wall and tumour type, generally considered as the best prognostic indicators.