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.

Cholestasis without cirrhosis alters regulatory liver gene expression and inhibits hepatic regeneration.

Partial hepatectomy (PH) initiates cellular signals for regeneration. Sequential expression of nuclear and cytosolic protooncogenes accompanies the restoration of normal liver function and architecture. Although cirrhosis is known to inhibit liver regeneration, the effects of noncirrhotic cholestasis on hepatocellular proliferation, differentiation, and regulatory gene expression are unknown. To examine this, 25 male Fisher rats underwent common bile duct ligation and division. A 47% +/- 5% PH was performed 10 days after common bile duct ligation and division when histologic analysis revealed cholestasis without cirrhosis. Despite early elevations of total hepatic DNA and RNA values, cholestatic livers demonstrated a significant threefold suppression of expected hepatocyte mitotic indexes 48 and 72 hours after PH, compared with livers after PH alone. Weight restoration in cholestatic livers was 11% +/- 5.2% compared with 40% +/- 4.3% in control livers (+/- SEM; p less than 0.001) 5 days after PH. Analysis of regenerating liver messenger RNA with complementary DNA probes revealed an abnormal, sustained elevation of K-ras expression in cholestatic livers through all time points. Cholestasis blunted but did not obliterate normal sequential elevations in H-ras found in control livers. The expression of c-myc was inhibited threefold with cholestasis 72 hours after PH. These results are the first indication that cholestasis alone inhibits hepatocyte proliferation and the expression of c-myc that normally precedes the first wave of mitosis. This implies that cholestasis without cirrhosis may alter programmed liver gene expression, inhibiting normal hepatic regeneration.

Mitotic activity and DNA synthesis of rat islet cells following partial pancreatectomy.

The regenerative potential of the adult rat pancreas following a 50% pancreatectomy was investigated. Blood glucose values and body weight were unchanged by the procedure. The mitotic index (MI) of alpha, beta, and exocrine cells was examined in formalin-fixed sections of the pancreas stained for hormones and 5-bromo-2'-deoxyuridine (BrDU) after treating the animals with 2.5 mg/kg colchicine and/or 50 mg/kg BrDU prior to sacrifice. The MI was unchanged within the control group. For pancreatectomized versus control animals, in beta cells after 5 days the MI was 0.84% versus 0.41% (p less than 0.05), while 7 days postoperatively it was 1.04% versus 0.57% (p less than 0.01); in alpha cells, the MI was 0.32% versus 0.17% (p less than 0.05) at 5 days and 0.47% versus 0.24% (p less than 0.05) at 7 days. The MI of exocrine cells rose transiently on day 3 postoperatively to 0.74% in the pancreatectomized group but remained low at 0.15% (p less than 0.01) in the control group. The mean ratio of BrDU + ve beta cells was 1.19% in the pancreatectomy group and 0.45% in the control group on day 5 postoperatively (p = 0.01). A highly significant (r = 0.97; p less than 0.001) correlation existed between BrDU + ve and mitotic cells. There was no difference between the two groups in the area occupied by insulin-positive cells in pancreatic sections, nor in the number of beta cells per islet.(ABSTRACT TRUNCATED AT 250 WORDS)

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).

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.

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.

[Apud cells in endometrial cancer]. / Apudotsity v rake éndometriia.

55 endometrial carcinomas containing apud cells are studied. Correlation between the endocrine-metabolic disturbances and mitotic index, on the one hand, and number of apud cells and their product on the other, is revealed. If the percentage of apud cells is above 20 of all parenchymal cells, low mitotic index in the tumour, marked production of serotonin, calcitonin and other hormones and high incidence of endocrine disturbances together with a relatively favourable prognosis are observed. Low number of apud cells associated with high mitotic index and high incidence of unfavourable outcomes while the hormone production and endocrine disturbances were rare or absent.

[Proliferative activity of pigmented nevus cells and malignant melanoma of human skin]. / Osobennosti proliferativnoi aktivnosti pigmentnykh kletok nevusov i zlokachestvennykh melanom kozhi cheloveka.

The findings in the study of special features of proliferative activity in 58 pigmented neoplasms of skin have revealed a pronounced heterogeneity of tumor cell populations, malignant melanomas based on the values of the mitotic index (MI) and label index (LI). It is shown that while evaluating tumor anaplasia, these parameters provide more information as compared to the morphological characteristics.

[The importance of prognostic factors in malignant melanoma of the skin]. / Zur Bedeutung der Prognosefaktoren des malignen Melanoms der Haut.

122 primary malignant melanomas of the skin were investigated to estimate the histologically and clinical criteria of melanoma for the prognostic evaluation of the disease. As result the tumor thickness (Breslow) in mm is the best prognostic value. The prognostic index allows an assay to determine the risk of metastases. On this basis it is possible to distinguish between melanomas with high and low risk of metastases. This is important as a criteria for the determination for the adjuvant treatment.

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.

[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.

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.

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.

Age-related changes in the cell kinetics of rat foot epidermis.

The durations of the cell cycle and its component phases have been determined for the basal layer of the epidermis of the skin from the upper surface of the hind foot of the rat using single pulse [3H]-thymidine labelling and the percent labelled mitosis (PLM) technique. Rats of three age groups were used, namely 7, 14 and 52 weeks. The duration of DNA synthesis (Ts) and the G2 plus M phase (TG2 + M) were comparable in 7-week and 52-week-old rats (P greater than 0.1). The major difference between 7-week and 52-week-old rats was in the duration of the G1 phase (TG1). In 7-week-old rats TG1 was 15.0 +/- 0.8 h and in 52-week-old rats TG1 was 31.2 +/- 3.5 h. A consequence of this variation was that the overall duration of the cell cycle was longer in 52-week-old rats (53.9 +/- 5.3 h) than in 7-week-old rats (30.1 +/- 1.3 h). Difficulties were found in fitting a simple curve to the PLM data for 14-week-old rats. This suggests that the proliferative cell population of the epidermis of rats of this age group may be heterogeneous. A satisfactory fit to the data was obtained using a computer model which assumed that the proliferative population of the epidermis of 14-week-old rats was a mixture of cells with cell cycle parameters the same as those of the 7-week and the 52-week-old rats. These two sub-populations of relatively slowly and rapidly proliferating cells were present in the ratio of 2:1.

Changes in beta-tubulin isoforms and their RNA level in synchronized tobacco cells.

Tobacco BY-2 cells were synchronized by an aphidicolin treatment, and their beta-tubulin isoforms and their mRNA were analyzed by Western, Northern and dot blottings. The relative ratio of the beta-tubulin isoforms changed with the progress of cell cycle stage. By Northern blot hybridization of poly(A)+RNAs with a cloned carrot beta-tubulin cDNA probe, a single band of about 1.6 kb was detected throughout the cell cycle. Dot blot hybridization showed that beta-tubulin mRNA existed in all stages in the cell cycle at a relatively constant level, though it accumulated slightly more than average at M phase and decreased during G1 phase.

[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.

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.

Analysis of cytosolic phosphoethanolamine and ethanolamine and their correlation with prognostic factors in breast cancer.

Availability of accurate prognostic factors is vital in making decisions on cancer therapy. We have measured the cytosolic contents of phosphoethanolamine and ethanolamine in tumor tissues of 53 breast cancer patients in an attempt to explore the possibility that these amines could be used as prognostic indicators. The levels of phosphoethanolamine and ethanolamine were determined by high-performance liquid chromatography. The ratios of the molar quantity of these amines or amino acids to that of alanine plus tyrosine, which eluted as a single peak, were used to analyze and compare the results among different tumor samples. The results indicated that the values for phosphoethanolamine or ethanolamine varied significantly more than the values for amino acids, such as glycine plus threonine or glutamine plus serine (these amino acids were eluted as single peaks, respectively). The values for phosphoethanolamine, ethanolamine, and phosphoethanolamine plus ethanolamine were analyzed in relation to several commonly used prognostic factors of breast disease. The results indicated that groups having higher mitotic indices had significantly higher values for phosphoethanolamine or phosphoethanolamine plus ethanolamine than the group having lower mitotic indices. As the stage of the disease increased, the values for phosphoethanolamine plus ethanolamine also seemed to become higher. No correlation, however, was observed between steroid hormone receptor positive and negative groups or between positive and negative groups with regard to involved axillary lymph nodes. The content of phosphoethanolamine or phosphoethanolamine plus ethanolamine in cytosol therefore seems to be correlated with some prognostic indicators.