Estudio comparativo de tres modelos de melanomas murinos Harding-Passey, B16 y B 16F 10 / Comparative study of three murine melanoma models: Harding-Passey, B16 and B16F10

Planteamiento: Estudiamos las características clinicopatológicas, ultraestructurales y el comportamiento biológico de tres líneas de melanomas murinos: Harding-Passey. B16 y B16F10. Material y métodos: Se han utilizado 80 ratones C57B1/6J, a los que se inyectó una suspensión de 10° células tumorales por vía subcutánea a nivel inguinal. Se procesaron muestras de los tumores para el estudio microscópico óptico y electrónico. Se realizó el estudio estadístico. Resultados: Las tres líneas originaban tumores fácilmente trasplantables. De los tres modelos, el Harding-Passey supone el tumor de crecimiento más lento, presentando los menores pesos medios, el menor índice mitótico y una supervivencia mayor de los animales huéspedes. El B16 muestra un crecimiento intermedio con pesos tumorales de casi el doble que el anterior, un índice mitótico ligeramente superior y una supervivencia de los ratones ligeramente inferior. El B16F10 muestra la mayor capacidad de crecimiento, siendo los pesos medios de los tumores seis y cuatro veces mayores, respectivamente, que los de Harding-Passey y B16, con un índice mitótico más alto y una supervivencia de los animales inferior a la mitad de los anteriores. Conclusiones: Las tres líneas estudiadas constituyen modelos experimentales idóneos para el estudio del melanoma. Los tumores muestran un comportamiento biológico distinto, con una capacidad proliferativa variable, siendo el B16F10 el de mayor crecimiento. La baja supervivencia de los animales hace que sea un modelo ideal para estudios cortos. Todos mostraban alteraciones en la ultraestructura de los melanosomas (AU)

Fate of human retinal pigment epithelial cells seeded onto layers of human Bruch's membrane.

PURPOSE: To determine the fate of human retinal pigment epithelial (RPE) cells seeded onto different layers of human Bruch's membrane (BM). METHODS: Bruch's membrane explants were prepared from 16 human cadaver eyes (7 eyes age <50 years; 9 eyes >50 years) by removing native RPE cells with ammonium hydroxide to expose the RPE cell basal lamina (BL). The inner collagenous layer (ICL) and elastin layer (EL) were exposed by removing apical layers sequentially by mechanical and enzymatic means. Synchronized first passage human RPE cells (15,000 cells/(6-mm-diameter explant) were plated onto each layer of human BM. The RPE cell reattachment and apoptosis rates at 24 hours, proliferation rates and mitotic index 24 hours after growth stimulation, and the ability of RPE cells to repopulate the explant surface were determined on each layer. RESULTS: RPE cell reattachment was highest on BL but decreased on deeper layers of BM. The apoptosis rate of attached cells increased as deeper layers of BM were exposed. The proliferation rate and mitotic index of the grafted cells were higher on BL than on deeper layers. RPE cells plated onto BL repopulated the explant surface within 14 +/- 3 days, whereas cells plated onto the ICL and EL eventually died and never reached confluence. CONCLUSIONS: The fate of RPE cells seeded onto BM depends on the ultrastructural layer of BM available for reattachment. These findings suggest that the ability of transplanted RPE cells to repopulate bare BM will depend on the layer of BM available for RPE cell reattachment.

Successful long-term in vitro cultivation of Theileria annulata schizonts in media supplemented with homologous and heterologous sera.

The efficacy of medium RPMI-1640 supplemented with either foetal bovine, normal bovine, goat or sheep sera was compared for prolonged in vitro propagation of Theileria annulata (Hisar) schizonts. Medium RPMI-1640 supplemented with 20% foetal bovine serum (standard growth medium) resulted in optimum growth of T. annulata (Hisar) schizonts in vitro. Comparable viability and non-viability counts were observed in growth media supplemented with normal bovine or goat sera. However, viability counts in medium supplemented with sheep serum were significantly lower than that of the standard medium. Mitotic indices of cultures of T. annulata (Hisar) schizonts were directly related to the extent of cell growth and were lower in various growth media supplemented with normal bovine, goat or sheep sera than in that of the standard medium. The results suggested that normal bovine and goat sera could be successfully used in place of foetal bovine serum in the growth medium for long-term in vitro propagation of T. annulata schizonts. The study will help in reducing the cost of large-scale in vitro propagation of T. annulata aimed at mass production of the cell culture vaccine.

Ki-67 immunostaining of normal human epidermis: comparison with 3H-thymidine labelling and PCNA immunostaining.

BACKGROUND: The size of the germinative growth fraction (i.e. the number of actively proliferating germinative cells) of normal human epidermis is still a subject of debate. Ki-67 antigen and PCNA, an auxiliary protein of d-polymerase, are considered as markers of the growth fraction when used under optimal conditions. METHOD: In the present work, we have compared Ki-67 expression (detected with MIB1 antibody) with PCNA expression (detected with PC10 antibody) in biopsies of normal human epidermis fixed in neutral formalin and using antigen retrieval by microwave processing. To obtain additional information, such as the percentage of cells in S phase, biopsies were also incubated in 3H-thymidine before immunostaining. RESULTS: Before microwave treatment, 8% of the basal cells were positive for MIB1 antibody and 7.8% were positive for PC10 antibody. The 3H-thymidine labelling index was 2.8%. The proportion of MIB1-positive cells rose to 19% after antigen retrieval by microwave processing. In the same way, the 3H labelling index rose to 9%. In contrast, PC10 became positive in all epidermal nuclei. CONCLUSION: These results suggest that the growth fraction of the germinative cell population of normal human epidermis is not larger than 20% and is composed of cells with a short cell cycle time.

Regeneration of epithelial defects in corneas previously treated with excimer laser. A study of cell kinetics in the rat corneal epithelium.

PURPOSE: The present study was to investigate, using cell kinetic methods, whether previous excimer laser treatment affected healing of later corneal epithelial erosions. METHODS: The right eyes of rats underwent central excimer corneal stromal-epithelial ablations (ArF 193 nm, 228 pulses, diameter 3,5 mm, 17,3 mJ per pulse, depth about one third of corneal thickness) and were allowed to heal for 15 weeks. Then central circular epithelial abrasions (diameter 3 mm), using N-Heptanol and mechanical debridement, were made on both corneas. The rats were killed 1,2,4 and 6 days after the last treatment. The central stromal thickness, the epithelial cell density, the labelling index (LI) and the mitotic rate (MR) of the peripheral, the midperipheral and the central areas of the corneal epithelium were calculated for each timepoint. RESULTS: The central stromal thickness increased equally in the two groups the first day after making the erosion, normalising in both groups during the following days. The corneal epithelium was restored at the same rate in both groups. The cell number per microscopic visual field, the LI and the MR were very similar for the two groups at all timepoints. CONCLUSION: Previous excimer laser treatment does not seem to interfere with healing of later epithelial erosions, when studied with cell kinetic methods.

A measure of the mitotic index: studies of the abundance and half-life of p34cdc2 in cultured cells and normal and neoplastic tissues.

BACKGROUND: The cdc2 gene encodes a protein kinase, p34cdc2, that is essential for mitosis, and is present at high levels in dividing cells. Classical studies of the levels of this protein in dividing and resting cells used antibodies that cross-react with other members of the CDK family, in particular with CDK2. We have therefore re-examined the abundance of p34cdc2 in a variety of tissues and cell lines, using a highly specific, epitope-mapped monoclonal antibody that does not react with CDK2. RESULTS: We observed high levels of p34cdc2 in proliferating cells, especially those in neoplastic tissues. Cells that have withdrawn from the cell cycle have low or undetectable levels. At the end of mitosis, the level of p34cdc2 declines, with simple first-order kinetics, with a half-life which is never less than 6h and is more typically about 18h. The persistence of p34cdc2 after the last cell division is comparable to that of PCNA, a commonly used marker of proliferation. CONCLUSIONS: The immunochemical detection of p34cdc2 provides an accurate, reliable and meaningful measure of the proliferative activity of cells in tissues. We suggest that p34cdc2 should be considered as the most authentic molecular marker of the mitotic index.

Synovial sarcoma. Evaluation of prognosis with emphasis on the study of DNA ploidy and proliferation (PCNA and Ki-67) markers.

Controversy still exists regarding the validity of parameters commonly used in the evaluation of prognosis of patients with synovial sarcoma (SS). Forty-nine cases of previously untreated primary SS (23 females and 26 males, ranging in age from 7 to 81, with 31 tumors located in the lower extremity, 8 at the upper extremity and 10 at the trunchus), without regional lymph-node or distant metastases were studied. We investigated the relationship between (flow and image) DNA cytometry, proliferation activity, clinicopathologic parameters, and relapse-free and overall survival of the patients. The prognostic value of gender, age, duration of symptoms, location, compartmentalization, size, adequacy of surgical margins, residual tumor, adjuvant therapy, histologic subtype, extent of necrosis, glandular differentiation, calcification, and extent of hemangiopericytic areas, mitotic rate, amount of mast cells, blood vessel invasion, histologic (UICC and NCI) grades, DNA ploidy, percentage of cells in S and S+G2 phases, PCNA and Ki-67 labeling indices (LI), and TNM (UICC) stage of the tumors, were evaluated by univariate and multivariate (Cox hazard model) analyses. Short duration of symptoms (<12 months), biphasic SS, scarcity of mast cells (<10/10 HPF), high mitotic rate (> or =10/10 HPF), high histologic grade (grade 3), high PCNA-LI (> or =20%), high Ki-67-LI (> or =10%), DNA aneuploidy, and advanced TNM stage (stage III) were features associated with significantly shorter relapse-free and overall 5-year survival rates in the univariate analyses. Scarcity of mast cells, high mitotic rate, or high PCNA-LI were significant predictors of poor survival, in addition to TNM stage in the multivariate analyses. The amount of mast cells was inversely correlated with mitotic rate and PCNA-LI. Scarcity of mast cells, high mitotic rate, or high PCNA-LI are factors associated with poor prognosis, in addition to advanced TNM stage in patients with localized SS.

Depth of infiltration of primary gastric B-cell lymphomas--correlations with proliferation, apoptosis and expression of p53 and bcl-2.

Local extension has been shown to be a major prognostic factor in primary gastric B-cell lymphoma. In order to evaluate whether this parameter might be correlated with cell proliferation or its regulation, a retrospective study was performed. Fifty-three surgical specimens with primary gastric B-cell lymphomas were analysed concerning histological grading, depth of infiltration and Ann-Arbor stage. In addition, immunohistochemistry (p53[Do7], bcl-2[bcl-2-124], Ki-67 [MiB1]) and in situ end-labeling (apoptotic bodies) were applied. The depth of infiltration was significantly correlated with Ann-Arbor stage (p < 0.001) and histological grading (p < 0.002). Furthermore, the semiquantitatively evaluated expressions of Ki-67, apoptotic bodies and p53 revealed that tumours limited to the mucosa and submucosa had lower numbers of stained cells than lymphomas infiltrating the muscularis propria or beyond (p < 0.001 in all cases). Analysis of bcl-2 expression showed an inverse picture (p < 0.05). The importance of local spread of gastric lymphomas is underscored by our findings: in gastric lymphomas infiltrating the muscularis propria or beyond, powerful proliferative stimuli have been acquired, e.g. associated with p53 mutations, that are independent of the known mucosa-associated stimuli, Helicobacter pylori and auto-immunity.

[Analysis by computer simulation of cell kinetics of the liver]. / Analyse par simulation sur ordinateur de la cinétique cellulaire du foie.

A computer simulation model of cell kinetics is established to represent the variations of rat hepatocyte populations in several circumstances: normal growth, circadian rythms, response to partial hepatectomy under different conditions. This model differs from usual mathematical models-sets of equations--by the use of digital simulation techniques. Each cell in the population is represented by a set of variables in the computer memory. When experiments are simulated, the values of these variables are modified step by step according to the hypotheses we want to test. Counts and statistics derived at each step from the simulation are then compared to experimental values, in order to assess the validity of our hypotheses. This procedure enables us to establish a minimal set of conditions and outside causal effects necessary to mimic the behaviour of the liver under experimental situations similar to those we simulate. Such modelisation suggests further work on the mechanisms governing cell kinetics in the normal liver and during cancerisation.

The effect of a herbicide--sodium salt of 2,4-dichlorophenoxyacetic acid on guerin carcinoma.

The effect of sodium salt of 2,4-dichlorophenoxyacetic acid, being an active component of herbicide "PIELIK", upon the development of Guerin carcinoma implanted in male Wistar rats, was studied. 192 animals were divided in to 6 equal groups: I-animals which obtained physiological salt solution; II-rats exposed to the herbicide in postlactational period; III-animals with Guerin carcinoma, non exposed to the herbicide; IV- rats exposed to the herbicide in postlactational period+Guerin carcinoma; V-animals exposed to the herbicide from prenatal period to the end of an experiment, without Guerin carcinoma; VI-the same as in V group, but with Guerin carcinoma. The effect of the herbicide on tumor growth dynamism (diameters and mass), degree of tumour malignancy (metastases to lymph nodes), animals survival time and morfological changes in the primary tumour and in metastases was evaluated. Basing of the results obtained, it was stated that this herbicide accelerates the development of Guerin carcinoma and reduces the survival time in the rats exposed to it in the prenatal and postnatal period. However, it does not significantly influence the growth of the carcinoma in the rats exposed only in the postlactational period.

[Oncocytoma of the nose. A case report and review of the literature]. / Das Onkozytom der Nase. Ein Fallbericht und Ubersicht der Literatur.

Oncocytomas are rare epithelial tumors of the major and minor salivary glands. Reported is an oncocytoma of the lateral wall of the left nasal cavity in a 60-year-old man. Because of its locally invasive character, with rupture into the left nasal cavity and partial dedifferentiation with loss of typically oncocytic features, we classified the tumor as oncocytoma of low malignant potential.

Aspectos morfológicos, morfométricos e citoquímicos da parede duodenal de ratos com diferentes idades / Morphologic, morphometric and cytochemical aspects in the duodenal wall in rats with differents ages

Foram analisados as características morfológicas, morfométricas e citoquímicas ocorridas na parede duodenal de ratos com 3, 12 e 24 meses de idade. A conservaçao morfológica encontrou-se a existência de 2 padroes. O primeiro envolvendo animais de 3 a 12 meses e o segundo composto pelos animais de 24 meses. A morfometria evidenciou um aumento progressivo do número de células caliciformes, com envelhecimento do animal, e mais, o conteúdo de mucina destas células sofreu gradativa acidificaçao com decorrer da idade. Nao se observou alteraçao no índice mitótico nos 3 grupos de animais.

Proliferation indices as independent prognostic factors in papillary Ta-T1 transitional cell bladder tumours.

A cohort of 148 patients with papillary Ta-T1 transitional cell carcinomas (TCCs) was followed up for over 10 years and flow cytometric (DNA ploidy, S phase fraction) and morphometric variables (5 nuclear factors, volume corrected mitotic index) were related to prognosis during this period. Recurrence-free survival was significantly related to DNA ploidy, S phase fraction and M/V index. Progression in T-category was predicted by M/V index, S phase fraction, DNA ploidy and WHO grade. The same variables predicted progression in N- and M-categories. In a multivariate analysis only M/V index and S phase fraction were independent predictors of progression. Univariate analysis showed that M/V index, SPF, DNA ploidy and WHO grade predicted survival. In a multivariate survival analysis only M/V index and SPF were independent predictors. The results showed that proliferation indices had independent prognostic value in papillary Ta-T1 TCCs and the grading of these tumours could be based on the proliferation indices. Papillary Ta-T1 tumours with a M/V index value < or = 10/mm2 or SPF < or = 10% had a favourable prognosis whereas tumours with M/V index > 10/mm2 or SPF > 10% had a high malignant potential.

Cell kinetics of normal and healing rat corneal epithelium during organ culture.

Local epithelial proliferation in corneal, limbal, and conjunctival rat epithelium was investigated during organ culture of corneo-scleral shells, by relating the number of tritium labelled cells to the total number of cells (labelling index). The changes in labelling index after a mechanically induced central corneal abrasion were also studied. Local labelling indices were measured 2 h, 1, 2, 4 and 7 days after incubation. The average labelling index increased in un-abraded specimens 4-fold after 4 days of incubation, while the total number of cells only decreased 30-50%. In eyes with a central corneal abrasion the labelling index increased 10-fold in the surrounding epithelial cells after 1 day. The labelling index of the limbal and conjunctival epithelial cells increased to the same extent after 2 days of incubation. The finding of a reduced total number of cells and an increased cell proliferation in un-abraded eyes may be explained by increased loss of mature cells normally producing growth-inhibitory substances (chalones). Alternatively, the culture medium may contain an excess of growth-stimulatory substances. The results also suggest that the proliferative response spreads in a centrifugal direction during in vitro healing of a central corneal abrasion.

Okadaic acid inhibits a protein phosphatase activity involved in formation of the mitotic spindle of GH4 rat pituitary cells.

Okadaic acid, a selective inhibitor of serine/threonine protein phosphatases, was utilized to investigate the requirement for phosphatases in cell cycle progression of GH4 rat pituitary cells. Okadaic acid inhibited GH4 cell proliferation in a concentration-dependent manner with a half-maximal inhibition (IC50) of approximately 5 nM. Treatment of GH4 cells with 10 nM okadaic acid resulted in a 40-60% decrease in phosphatase activity and an increase in the proportion of phosphorylated retinoblastoma (RB) protein. Cell cycle analysis indicated that okadaic acid increased the percentage of cells in G2-M, decreased proportionally the percentage of cells in G1 phase, and had little effect on the percentage of cells in S-phase. The absence of a change in the proportion of S-phase cells indicates that G1-specific phosphatases responsible for dephosphorylation of RB protein were not inhibited by 10 mM okadaic acid. Mitotic index revealed that 10 nM okadaic acid decreased proliferation of GH4 cells specifically by slowing the progression through mitosis. Immunostaining with anti-tubulin demonstrated that 10 nM okadaic acid-treated mitotic cells contained mitotic spindles; however, the spindle apparatus in these cells frequently contained multiple poles. These results suggest that the organization of spindle microtubules during prometaphase requires a protein phosphatase that is sensitive to nanomolar concentrations of okadaic acid. Chromosomes in 10 nM okadaic acid-treated cells appear to be attached to spindle microtubules and the nuclear envelope is absent. The effects of okadaic acid on the spindle differ from those elicited by the calcium channel blocker, nimodipine, indicating that this okadaic acid sensitive phosphatase is not part of the calcium signalling events which participate in mitotic progression.

Efecto de la "microdosis" de Carnoy y de la proporcion KCI/Carnoy sobre el indice mitótico / Effects of Carnoy priming and the KCI/Carnoy ratio on the mititic index

Se estudiaron, en linfocitos de sangre periférica, los cambios en el Indice Mitótico (IM) producidos por la omisión o la adición de la "microdisis" (MD) de Carnoy (50ul), al inicio o al final de la incubación con KCI 75 mM. El efecto de diferentes proporciones y temperaturas de KGI y Carnoy también fue investigado. La prespuesta óptima, evidenciada por un Im de 9.8 +- 2.4 por ciento, se obtuvo del grupo en el que la MD, a 22oC, se agergó al inicio de la incubación con KGI a 37oC y la proporción KGI/Carnoy fue mayor de 2. El análisis conjunto de todos los resultados demostró que la omisión de la MD; que la MD añadida al final de la incubación; que la omisión del reposo entre las dos primeras fijaciones de Carnoy o éste adicionado a 4oC o el KGI a 22oC, produjeron IM bajos con valores comprendidos entre 0.6 +- 0.1 por ciento y 1.2 +- 0.6 por ciento (p <0.001 vs 9.8+- 2.4 por ciento).

Cell kinetics of melanocytes in common and dysplastic nevi and in primary and metastatic cutaneous melanoma.

The 3H-thymidine labelling (LI) and mitotic (MI) indexes were calculated in 29 cutaneous melanocytic lesions: 6 common nevi (CN), 11 dysplastic nevi, subclassified as nevi with architectural atypia (NAA = 4) and nevi with cyto-architectural atypia (NCAA = 7), 2 melanomas in situ (MIS), 4 invasive superficial spreading melanomas (IM) and 6 metastatic melanomas (MM). The LI mean values resulted to be: CN = 0.23%, NAA = 0.98%, NCAA = 1.79%, MIS = 5.75%, IM = 5.16%, MM = 3.80%. In CN, NAA, NCAA and MIS, these values were calculated at epidermal level; in IM and MM at dermal level. At dermal level, the LI mean values of CN, NAA and NCAA were: 0.20%, 0.20%, 0.23% respectively. The MI mean value was close to 0 in CN, NAA, NCAA, MIS; 0.18% in IM, 0.16% in MM. Confirming a low proliferative activity in CN and a high activity in melanomas (MIS, IM, MM), the results showed that dysplastic nevi (NAA, NCAA) had a proliferative activity intermediate between common nevi and melanomas. The lesions with melanocytic atypia (NCAA) resulted to have a higher proliferative activity than those without this histological feature (NAA).

Alterations in quantitative distribution of Na,K-ATPase activity along crypt-villus axis in animal model of malabsorption characterized by hyperproliferative crypt cytokinetics.

The distribution of sodium- and potassium-stimulated ATPase (Na,K-ATPase) along the crypt-villus axis and crypt cytokinetics were examined in an infective model of celiac disease produced by infection of the rat with the nematode Nippostrongylus brasiliensis. In controls, levels of enzyme activity remained stable during enterocyte migration to the villous apex. In the jejunum of infected rats, the structural lesion of villous atrophy and crypt hyperplasia, observed at day 10 of infection, was associated with a three-dimensional expansion of the crypts. Cell cycle time was shortened and this resulted in a markedly increased crypt cell production rate. Enterocytes emerged from the crypts at a faster rate, and this functional immaturity was paralleled by decreased Na,K-ATPase activity. Further decreases in enzyme levels were observed during enterocyte migration along the villi. This may reflect enterocyte damage or increased enzyme turnover. In the ileum of these animals, enterocyte maturation was prolonged and enzyme activity was increased at the level of the crypt villus junction with further increases noted during enterocyte transit. These changes in ileal Na,K-ATPase appear to be adaptive.