Avaliação da cinética do epitélio corneano com marcadores de proliferação celular / Evaluation of the corneal epithelium kinetics using cell proliferation markers

OBJETIVO: Avaliar a cinética celular do epitélio corneano de coelhas em três situações: controle, hiperproliferação e hipoproliferação celular, com a utilização dos marcadores de proliferação celular BrdU, Ki-67/MIB-1 e AgNOR. MÉTODOS: Foram utilizadas quinze coelhas albinas que tiveram seus olhos aleatoriamente divididos em 3 grupos (A, B e C). O grupo A incluiu olhos que foram submetidos à instilação de tampão fosfato (total de 10 olhos); o grupo B, instilação de tampão fosfato após a remoção de uma área central do epitélio corneano de 10 mm (total de 10 olhos) e o grupo C, instilação de 5-fluoruracil em superfície ocular íntegra (total de 10 olhos). RESULTADOS: Os resultados da média e desvio-padrão do número de células marcadas pela BrdU nos grupos A, B e C foram, respectivamente, de 7,17 ± 0,74; 35,00 ± 3,01 e 0,22 ± 0,1 células marcadas por 100 células basais. As diferenças entre os grupos foram estatisticamente significantes. A média e o desvio padrão do número de células marcadas utilizando o Ki-67 foram de 7,55 ± 1,22 no grupo A; 35,55 ± 3,84 no grupo B e 0,34 ± 0,14 no grupo C. As diferenças entre os grupos foram estatisticamente significantes. A média e o desvio-padrão da medida da área das NORs no grupo A foram de 1,92 ± 0,24, no grupo B foram de 3,61 ± 0,27 e no grupo C foram de 1,71 ± 0,26. CONCLUSÕES: Os marcadores BrdU, Ki-67 e AgNOR apresentaram uma correlação positiva e estatisticamente significante nas situações de proliferação celular avaliadas (controle, hiperproliferação e hipoproliferação); o emprego do AgNOR não permitiu identificar diferenças na proliferação celular nas situações controle e hipoproliferação e houve maior concordância de resultados entre a BrdU e o Ki-67 nas três situações de proliferação celular.

PURPOSE: In order to maintain its clear and uniform structure, the corneal epithelium needs constant equilibrium between production (division) and desquamation of its epithelial cells. The author aimed to evaluate the cell kinetics of corneal epithelium of rabbits in three situations (control, hypoproliferation and hyperproliferation) using BrdU, Ki-67/MIB-1 and AgNORs proliferation markers. METHODS: Fifteen white female rabbits had their eyes randomly divided into three groups (A, B and. C). Group A included eyes submitted to phosphate buffer saline instillation (total 10 eyes); group B, instillation of phosphate buffer saline after removing a 10 mm central area of the corneal epithelium (total 10 eyes) and group C, instillation of 5-fluorouracil in normal epithelium (total 10 eyes). RESULTS: The results of the mean number and standard deviation of the marked cells using BrdU in groups A, B e C were, respectively, 7.17 ± 0.74; 35.00 ± 3.01 e 0.22 ± 0.1 marked cells per 100 basal cells. Differences among groups were statistically significant. The mean number and standard deviation of the labelled cells using Ki-67 were 7.55 ± 1.22 in group A; 35.55 ± 3.84 in group B and 0.34 ± 0.14 in group C. Differences among groups were statistically significant. The mean area and standard deviation of NORs in group A were 1.92 ± 0.24, in group B, 3.61 ± 0.27 and in group C, 1.71 ± 0.26. CONCLUSIONS: The markers BrdU, Ki-67 and AgNOR showed a positive correlation with statistical significance among the cellular proliferation situations studied (control, hypoproliferation and hyperproliferation); the AgNOR did not show statistically significant differences among the control and hypoproliferation situations and there was more agreement in the results among markers BrdU and Ki-67 in three cell proliferation situations.

GC-rich heterochromatin in silver stained nucleolar organizer regions (NORs) fluoresces with Chromomycin A3 (CMA3) staining in three species of teleostean fishes (Pisces).

In a bid to ascertain the molecular architecture of the silver positive regions (NORs) in chromosomes of three species of fish, namely, Hemibagrus menoda (Hamilton), Sperata seenghala (Sykes) (Fam: Bagridae) and Mastacembelus armatus (Lacep6de) (Fam: Mastacembelidae), an additional staining methodology using a fluorochrome dye (Chromomycin A3) was deployed along with the AgNO3 technique. The nucleolar organizing regions (NORs) were located terminally at the shorter arms (Tp) of one pair of submetacentric chromosomes (No.3) in H. menoda (2n=58), at the longer arms (Tq) of one pair of submetacentric chromosomes (No.5) in S. seenghala (2n=50) and at the shorter arm (Tp) of one pair of homologous submetacentric chromosomes (No.6) in M. armatus (2n=48). Staining with Chromomycin A3 produced bright fluorescing zones in GC-rich heterochromatin of Ag-positive NORs. The results indicate a more general trend of existence of an overlapping region between NOR and GC-rich fluorescing zones, the active sites of rRNA genes (rDNA) in this primitive group of vertebrates although exceptions to this situation has been reported in a couple of extant fish species earlier. More data utilizing such combined methodologies are warranted to understand the structural organization of fish chromosomes more precisely.

Cell adhesion molecule E-cadherin and cellular proliferation marker AgNOR in bladder lesions--usefulness and limitations.

The objective of this study on bladder lesions was to assess the relationship of (a) the proliferative activity measured by AgNOR counts, and (b) the loss of cell adhesion leading to acquisition of invasive properties as assessed by E-Cadherin expression. Paraffin embedded tissue biopsies from normal urothelium and malignant urothelial lesions were randomly selected from our surgical pathology files. AgNORs were analysed by the silver staining method and E_cadherin expression by immunohistochemistry. An increase in M&P AgNOR counts was observed from normal through benign to malignant tumors. A corresponding decrease in E Cadherin expression was noticed from normal through different grades of transitional cell carcinoma (TCC). A correlation was found between a high P AgNOR and decreased E_cadherin expression with stromal and muscle invasive tumors. It is deduced that when low grade tumors show a high P AgNOR and loss of E_cadherin expression, it is a significant indicator of the possible aggressive behavior in terms of stromal or muscle invasion and progression to a higher grade in future.

Cytology. A valuable tool in prognostication of carcinoma breast.

Sixty consecutive cases of carcinoma breast diagnosed on fine needle aspiration cytology (FNAC) between July 1993 to June 1994 were studied prospectively. Nuclear grading was done on all cytologic smears, histologic grading was possible in only 22 cases. A good correlation was seen between nuclear grading on cytology and histology. Silver staining for nucleolar organizer region, and immunocytochemical staining for Ki-67 and C-erbB-2 could be done with ease on cytologic smears, providing necessary additional information on which to base the theraputic decisions.

Application of the AgNOR method to distinguish pseudoepitheliomatous hyperplasia from squamous cell carcinoma.

The AgNOR stain was performed on seventeen cases of pseudoepitheliomatous hyperplasia of the oral cavity and genital tract, seventeen cases of squamous cell carcinomas of the same regions, and nineteen cases of squamous cell carcinoma of the cervix, to determine whether the stain could help to distinguish pseudoepitheliomatous hyperplasia from squamous cell carcinoma. No constant relationship of the AgNOR score to the grade of the lesion could be determined. Follow up of some of the cases was possible. Here, too, it was seen that the AgNOR score could not reliably predict which cases of pseudoepitheliomatous hyperplasia would progress to squamous cell carcinoma, and which cases of squamous cell carcinoma would suffer a relapse.

Malignant inverted papilloma of the urinary bladder: the histopathological aspect of malignant potential of inverted papilloma

To investigate the histopathological characteristics of inverted papillomas of the urinary bladder, including the possibility of malignant transformation, we studied the indicators of cellular proliferation activity in 7 inverted papillomas of the bladder including two cases of malignant inverted papilloma of the bladder. PCNA expression rates in two cases of malignant inverted papilloma were higher than in benign inverted papillomas. Mean numbers of AgNORs per nucleus in malignant inverted papillomas were much more than in benign inverted papillomas. The c-erbB-2 oncoprotein was expressed only in malignant inverted papillomas. These results suggest that PCNA expression rate, mean number of AgNORs per nucleus and c-erB-2 oncoprotein expression may be merited as good indicators to detect the inverted papilloma with more proliferative and aggressive lesions, and with the potential of malignant transformation.

Correlation between proliferating index and prognostic factors in papillary cystic tumors of the pancreas

Fifteen cases of papillary cystic tumor of the pancreas (PCTP) were studied (14 female patients, one male patient; mean age: 23.5 years). Most tumors developed in the head of the pancreas as a well circumscribed large mass. The tumor had a mean diameter of 6.7 cm(range; 2 to 15 cm). Histopathologically abundant delicate papillary fragments, monomorphic tumor cells and degenerative changes of the solid area of the tumor were characteristic. All but two cases had completely circumscribed capsules. Two cases had duodenal invasion; one of all cases had cul de sac metastasis. Compared with 12 non-aggressive tumors, the aggressive cases had larger tumor size (more than 9 cm) with a thicker capsule (more than 2 mm). In studies to investigate the prognostic index using nucleolar organizing region (NOR), proliferating cell nuclear antigen (PCNA) and flow cytometry as well as nuclear grade and mitotic index, we could not find the useful parameter to detect the malignant potential of PCTP. In the flow cytometric analysis of cellular DNA contents, two invasive cases and the only one case of the male patient among the non-aggressive group were aneuploid. In conclusion, although it is hard to predict the prognosis by microscopic findings only, those with a thick capsule and aneuploidy tend to be related to malignant potential.