Search

Factors regulating the expression of the angiotensin II subtype 1 (AT1) receptor during fetal life have not been investigated previously. The present study was designed 1) to characterize the ontogeny of AT1 receptor gene expression in the kidney of fetal and newborn sheep and 2) to determine the influence of both glucocorticoids and renal nerves in modulating AT1 gene expression during fetal life and during the transition from fetal to newborn life. We first isolated and cloned a PCR product that has 98 and 94% homology with the cDNA encoding the bovine and pig AT1 receptors, respectively, and 99 and 98% homology with the corresponding deduced protein sequences. Probing with this cDNA, we demonstrated that renal AT1 mRNA expression did not change significantly during the last trimester of gestation in fetal sheep or immediately after birth but decreased significantly 10 d after birth. We also demonstrated that renal denervation in the fetus had no effect on renal AT1 gene expression in 24-h-old newborn lambs. On the other hand, we observed in 130-d twin fetuses that a continuous intraperitoneal infusion (1 mL/h) of cortisol (3 mg/h or 6.2 mumol/h) for 48 h in one of the twins increased the fetal plasma cortisol concentration from 32.0 +/- 7.1 to 1126 +/- 231 nmol/L and produced a significant decrease (p < 0.005) in renal AT1 gene expression compared with the control twin receiving an intraperitoneal infusion of 0.9% NaCl. In summary, this study demonstrates that renal AT1 gene expression is elevated during fetal life and decreases after birth. It is also shown that glucocorticoids, but not renal nerves, contribute to the regulation of renal AT1 gene expression during development.

A prognostic value of DNA content in cervix carcinoma for local recurrence following radiotherapy is suggested by both retro- and prospective studies. The purpose of this study was to confirm these data. Flow cytometry and computerized image cytometry were used to measure DNA content of tumor cells retrospectively in paraffin-embedded archive material from 98 patients with carcinoma of the uterine cervix FIGO stage IB-III. All patients were treated with combined megavoltage irradiation and brachytherapy. Minimum follow-up has been 5.1 years. Pelvic recurrence was seen in 10, 26, and 54% of patients with stages IB II, and III, respectively (P = 0.003). DNA content, expressed as DNA index (DI), was not correlated with the FIGO stage (P = 0.25), histopathologic grading (P = 0.26), or age (P = 0.63) at diagnosis in this series. Using univariate analysis pelvic disease-free survival (PDFS) was best predicted by the clinical stage (P = 0.004) and by an age greater than 55 years (P = 0.04) before treatment. PDFS tended to be better for patients with aneuploid tumors (DI > 1.2), but this difference was not statistically significant (P = 0.17). Using Cox's regression model for multivariate analysis, only stage was independently correlated to PDFS (P = 0.009). After stratification for stage, ploidy level was significantly correlated with PDFS in stage II patients (P = 0.04). Overall results suggest aneuploid tumors to be more radiosensitive than diploid tumors in this series.

Ninety-one cases of primary breast cancers and their nodal metastases were examined with DNA flow cytometry. No differences were found between the stemline distributions in the primary tumors and nodal metastases. At both sites stemlines clustered around a DNA index of 1.0 (33-40% of cases) and 1.8. The mean S-phase fractions were 7.9 in primary tumors versus 5.6% in nodal metastases (p = 0.02); this difference was also observed if the analysis was restricted to cases with DNA aneuploidy at both sites (10.2 versus 7.6%, p = 0.04). Our results indicate that axillary nodal ploidy and proliferation reflect primary tumor characteristics rather than displaying changes associated with selection during the lymphatic metastatic process. Lymph nodes may have a suppressive effect on the proliferation of tumor cells.

To assess the reliability of DNA estimation using image cytometry, deparaffinized lymph nodes from 70 patients with Hodgkin's disease were examined and the results obtained were compared with those from flow cytometry. Image analysis without discriminating between the various cell types, as found in Hodgkin's disease, revealed no separate aneuploid peak. Selecting on morphologically defined nuclear types DNA aneuploidy was detected in 20% of the cases (14/70). The aneuploid populations were limited to the population of nuclei defined as Reed-Sternberg (RS)-like or medium-sized lymphocytes. Benign lymph nodes DNA aneuploidy was not found in any of the controls. Comparison of DNA histograms obtained by image and flow cytometry showed aneuploid peaks using image cytometry in 4 of 30 diploid and 10 of 40 aneuploid flow histograms. In conclusion, image analysis using the CAS 200 system as compared to flow cytometry is more time-consuming and less sensitive to assess ploidy status, although it may provide extra information in some selected cases. Evidence is obtained that DNA aneuploidy in Hodgkin's disease is preferentially expressed by cells with the RS/H-like and medium-sized lymphocyte morphology.

Paraffin-embedded lymph nodes from patients with Hodgkin's disease were examined for flow cytometric DNA content. In order to increase the sensitivity of the assay we tried to enrich for the neoplastic cells by bivariate analysis using a polyclonal anti-nucleolar antibody (AN-AB) and the forward scatter (FSC). DNA aneuploidy was found to be present in 67 of all 137 cases (49%), in 24 cases only demonstrable by dual-parameter analysis. The DNA index varied from 0.69 to 1.89 with a total of 22 hypo-diploid cases. The number of aneuploid nuclei exceeded the expected frequency of Reed-Sternberg (RS) and Hodgkin (H) cells in most of the analysed specimens. In conclusion, flow cytometry in Hodgkin's disease appears to give useful information regarding the ploidy status and evidence has been provided that the malignant cell population in Hodgkin's disease is not limited to the classical RS/H cells.

A physical map of 4q35 was constructed through radiation hybrid analysis of 134 clones generated from the cell line HHW416, a chromosome 4-only human-hamster somatic cell hybrid. This subtelomeric region contains the as-yet-unidentified gene responsible for facioscapulohumeral muscular dystrophy. The most likely order of 15 loci within 4q35 was determined. The loci ordered on this radiation hybrid map include both genes and polymorphic loci, as well as monomorphic loci which cannot be placed on a genetic linkage map. The physical distance spanning these loci was estimated to be approximately 4.5 Mb, by using a kilobase/centiray conversion factor derived from 4p16.3 marker analysis through the same set of radiation hybrids. The comparison of this physical map to establish genetic maps suggests that this region is smaller than initially estimated and that recombination rates are increased near the telomere.

We evaluated breast cancer specimens from 241 patients of a controlled clinical trial by means of DNA flow cytometry. We report the correlations between DNA index (DNI) and fraction of cells in S-phase (SPF) and other prognostic parameters. Both univariately and in a Cox model, the predictive power of these factors is evaluated after a follow-up of more than 10 years. There are strong correlations between DNI and SPF (P = 0.0001) and between flow cytometry parameters and clinical and histopathological factors such as axillary lymph node involvement, tumour size and histological grade. In univariate analysis DNI fails to provide prognostic information, whereas SPF turns out to be able to differentiate between patients at high and low risk for relapse and death (P = 0.002). In the multivariate Cox model, too, SPF is an important prognostic parameter with respect to patient survival (relative risk: +86%), only surpassed by nodal involvement. DNI, however, turns out to be an independent predictor of relapse free survival and distant recurrence free survival. By combination of DNI and SPF, patients can be divided into three prognostic subgroups. We conclude that data from DNA flow cytometry can be of great importance for the decision on the level of aggressiveness of adjuvant therapy for an individual patient and therefore may help to avoid overtreatment and toxicity.

To determine the effects of dietary ethanol or fibre on 2(3)-tert-butyl-4-hydroxyanisole (BHA)-induced alterations in cell kinetics in gastro-intestinal tract tissues, groups of six male Wistar rats were fed diets containing 0% (control) or 1.5% BHA for 2 wk. One group fed 1.5% BHA and one pair-fed control group received 10% ethanol in the drinking-water; two similarly fed groups received drinking-water only. Another group fed 1.5% BHA and a pair-fed control group received a diet supplemented with 20% cellulose; two similar groups received no fibre supplementation. Cell kinetics in the forestomach, glandular stomach and oesophageal tissue were determined, after 14 days, by bivariate 5-bromo-deoxyuridine/DNA analysis using immunocytochemistry and flow cytometry. In the fibre experiment, colorectal tissue was also examined. In both experiments the labelling indices in all the gastro-intestinal tract tissues were significantly altered in the BHA-fed groups compared with the corresponding control groups. In the ethanol experiment no statistically significant difference in the labelling indices was observed in the forestomach or glandular stomach between the two control groups or between the two BHA-fed groups. However, intake of ethanol-supplemented drinking-water induced increases in oesophageal labelling indices in rats fed a BHA-free diet. Thus 14 days of simultaneous ethanol administration has no effect on BHA-induced alterations in cell kinetics in the oesophagus, glandular stomach or forestomach of rats. In the forestomach and colorectal tissue, a high-cellulose diet resulted in a significant decrease in the BHA-induced elevation of labelling indices. Thus dietary cellulose provides a partial protection against the proliferation-enhancing effects of BHA in the rat gastro-intestinal tract.

In this study we investigated cellular proliferation of T cells and macrophages at the site of rat cardiac allografts and determined the influence of immunosuppressive therapy on the proliferative characteristics of these cell types. A bromodeoxyuridine-labeling technique was used that allowed both the accurate detection of proliferative activity and the phenotypic characterization of cellular infiltrates within grafted tissues. In untreated recipients (BN----Lewis), T cytotoxic/suppressor cells as well as T helper cells showed proliferative activity at the site of the graft. The percentage of OX8-positive cells within the graft that showed proliferation ranged from 15% to 37%. The percentage of W3/25-positive cells within the graft that showed proliferation ranged from 25% to 30%. In contrast, macrophages hardly showed proliferative activity within the graft; only 1-4% of the macrophages stained positive for bromodeoxyuridine. From these observations it is concluded that the graft serves as a nonlymphoid tissue site, wherein lymphocytes can freely proliferate and expand. To study the influence of immunosuppressive therapy on cellular proliferation, the steroid budesonide, 120 micrograms/kg/day, was administered for 13 days (MST, 20 days). During effective immunosuppressive therapy, still a remarkable amount of infiltrating cells was present within the grafts. Moreover, immunosuppressive treatment did not primarily appear to affect the proliferative capacity of the individual cell types. OX8-positive cells as well as W3/25-positive cells clearly showed proliferative activity within the treated grafts. However, despite the presence of these proliferative cells, signs of graft destruction were absent during immunosuppressive therapy. This finding may shed new light on the effect of steroids at the site of the graft and their role in the prevention of tissue destruction.

Groups of ten 5-week old male Wistar rats were fed a diet containing 0, 0.25, 0.50, 0.75, 1.0 or 2.0% butylated hydroxyanisole (BHA) ad libitum for 2 weeks; another group of rats served as a pair-fed control (PFC) group for the 2% BHA-fed animals. Subsequently, rats were injected i.p. with the thymidine-analogue bromodeoxyuridine (BrdU) which was incorporated into the DNA of cells during DNA synthesis. Cell kinetic parameters in gastro-intestinal tract tissues were determined by means of bivariate BrdU/DNA analysis applying flow-cytometry to randomized tissue samples or by applying immunohistology to randomized tissue sections. In the forestomach, glandular stomach, small intestine and colon/rectum, mean tissue labelling index (LI) was significantly increased in rats on a diet supplemented with 2% BHA, in comparison with rats fed the basal diet (0% BHA) ad libitum or restricted to the mean daily intake of 2% BHA-fed rats (PFC). In the oesophagus of rats fed 2% BHA, the LI was significantly higher in comparison with their PFC group, but not with the group of rats fed 0% BHA ad libitum. In rat forestomach, an apparent no observed effect level for ad libitum fed rats was found at 0.5% BHA (LI) and at 0.75% BHA (potential doubling time). Thus, the oesophagus, glandular stomach, small intestine and large bowel, in addition to the forestomach, are possible target tissues in rats for the proliferation enhancing effects of BHA. At the time of termination of the experiment, plasma BHA concentrations were dose dependently increased and were in the range that is easily attained in man after ingestion of a dose equal to the acceptable daily intake for BHA (0.5 mg/kg).

We used the nude mouse tumour xenograft model to study the pathogenesis of mucosa alterations in the large bowel surrounding a carcinoma. In mouse colonic mucosa overlying HT-29 colonic carcinoma xenografts in the caecum, the crypts were elongated in comparison with those in distant mucosa and also frequently showed a shift towards sialomucin production. These features, which are comparable with socalled transitional mucosa (TM) in man, were absent in control animals inoculated with Indian Ink instead of HT-29 cells. Although the localization of the proliferative cell compartment in mouse colonic mucosa overlying HT-29 xenografts appeared to be confined to the lower half of the crypt as in normal mucosa, the relative length of the DNA synthesizing cell compartment along the crypts was slightly elongated. These data strongly suggest that TM should be regarded as a secondary phenomenon rather than a premalignant change in large intestinal epithelium and that higher proliferative activity of epithelial cells contributes little to the elongation of crypts in TM.

When recA protein binds cooperatively to single-stranded DNA to form filamentous nucleoprotein complexes, it becomes competent to hydrolyze ATP. No correlation exists between the ends of such complexes and the rate of ATP hydrolysis. ATP hydrolysis is not, therefore, restricted to the terminal subunits on cooperatively bound recA oligomers, but occurs throughout the complex. Similarly, during recA protein-promoted branch migration (during DNA strand exchange), ATP hydrolysis is not restricted to recA protein monomers at the branch point. DNA cofactors of lengths varying from 16 bases to over 12,000 bases support ATP hydrolysis. The maximum value of kcat at infinite DNA concentration is about 29/min independent of the length of the DNA cofactor. The apparent dissociation constant, however, is a strong function of DNA length, providing evidence for a minimum site size of 30-50 bases for efficient binding of recA protein.

In the present study we have evaluated the prognostic significance of ploidy levels and proliferative activity in 279 cases of large bowel carcinomas which were included in a surgical prospective randomized trial. Ploidy levels and proliferative activity were determined on nuclei isolated from paraffin-embedded tissues of 279 colorectal carcinoma patients, with a mean follow-up of 51.9 months. Product limit survival analysis demonstrated a borderline significant association (P = 0.0689 by generalized Breslow; P = 0.0336 by generalized Savage) between ploidy and survival, with a 75th quantile survival of 49.8 months for patients with diploid tumors and 35.9 months for patients with aneuploid tumors. After stratification for staging, Dukes' C cases showed a statistically significant association between tumor ploidy and survival (P = 0.0224 by generalized Breslow, P = 0.0110 by generalized Savage). Product limit survival analysis for proliferative activity and survival showed a similar outcome with the strongest association in Dukes's C stage of disease (75th quantile survival of 38.9 months for low proliferative and 18.0 months for high proliferative tumors).

Four groups of six male Wistar rats (85 +/- 7 g) were fed a diet containing 0% (control) or 2% of the carcinogenic food antioxidant butylated hydroxyanisole (BHA) for 2 weeks. In this experiment, feeding 2% BHA is equivalent to a dose of 2.1 +/- 0.3 g BHA/kg/day. One 0% BHA and one 2% BHA-fed group of rats were daily injected i.p. with the cytochrome P-450 inducer phenobarbital (PB; 60 mg/kg) in saline. These two groups were encoded 0PB and 2PB respectively. Simultaneously, two control groups of rats were injected i.p. with saline only (0 and 2 respectively). PB administration increased relative weight, cytochrome P-450 content and ethoxycoumarin-0-deethylase activity of livers as compared to control rats. In addition, cytochrome P-450-mediated oxidative demethylation of BHA into tert-butyl-hydroquinone (TBHQ), monitored as urinary TBHQ excretion, was significantly increased in PB-induced rats as compared to non-induced rats (0.59 +/- 0.19 versus 0.37 +/- 0.09%; P less than 0.05). The mean labelling index (LI) and potential doubling time (Tpot) in rat forestomach were significantly (P less than 0.01) altered in groups of rats fed 2% BHA as compared to their appropriate control groups. No differences in cell kinetic parameters between either the two control groups (0, 0PB) or between the 2% BHA-fed groups (2, 2PB) was observed. Thus, although an increase in oxidative demethylation of BHA as a response to PB administration is evident, biotransformation of BHA into TBHQ is not correlated to changes in cell kinetic parameters in rat forestomach. Moreover, in rats oxidative cytochrome P-450-mediated demethylation of BHA into TBHQ appears not to be related to the oral dose of BHA. This indicates that oxidative cytochrome P-450-mediated biotransformation of BHA does not contribute to the tumorigenicity of BHA in rat forestomach.

In order to study the interaction between tumors and host environmental factors, we xenografted cells from the human colonic carcinoma cell lines HT29 and 5583-S in the subcutis, cecum, spleen and liver of nude mice and compared growth characteristics, metastatic potential and some phenotypic features of the xenografts in these sites. No remarkable differences were observed between the tumors at different inoculation sites in regard of their expression of carcinoembryonic antigen and secretory component or type of mucin produced. Also the proportion of DNA synthesizing cells as determined by bromodeoxyuridine incorporation appeared to be comparable in the studied implantation sites. Local invasive growth characteristics and metastatic potential, however, showed marked differences. Subcutaneous and cecal xenografts frequently showed tumor cell invasion into the surrounding tissue, vasoinvasive growth and discontinuous basement membrane deposition, whereas splenic and hepatic implants demonstrated more encapsulation, no invasion of blood vessels and more continuous basement membrane deposition. Subcutaneous xenografts produced no metastasis. With HT29 cells liver and lymph node metastases occurred frequently from the splenic as well as the cecal xenografts. 5583 cells regularly produced liver metastases from the splenic xenografts, whereas no metastasis from cecal xenografts were observed. We conclude that although the patterns of invasive growth and the metastatic potential differ for various implantation sites, antigen expression and cell kinetic features of tumor implants are hardly influenced by the site of inoculation.

As a first step in DNA strand exchange, recA protein forms a filamentous complex on single-stranded DNA (ssDNA), which contains stoichiometric (one recA monomer per four nucleotides) amounts of recA protein. recA protein monomers within this complex hydrolyze ATP with a turnover number of 25 min-1. Upon introduction of linear homologous duplex DNA to initiate strand exchange, this rate of ATP hydrolysis drops by 33%. The decrease in rate is complete in less than 2 min, and the rate of ATP hydrolysis then remains constant during and subsequent to the strand exchange reaction. This drop is completely dependent upon homology in the duplex DNA. In addition, the magnitude of the drop is linearly dependent upon the length of the homologous region in the linear duplex DNA. Linear DNA substrates in which pairing is topologically restricted to a paranemic joint also follow this relationship. Taken together, these properties imply that all of the available homology in the incoming duplex DNA is detected very early in the DNA strand exchange reaction, with the linear duplex DNA paired paranemically with the homologous ssDNA in the complex throughout its length. The results indicate that paranemic joints can extend over thousands of base pairs. We note elsewhere [Pugh, B. F., & Cox, M. M. (1987b) J. Biol. Chem. 262, 1337-1343] that this duplex acquires resistance to digestion by DNase with a much slower time course (30 min), which parallels the progress of strand exchange. Together these results imply that the duplex DNA is paired with the ssDNA but remains outside the nucleoprotein filament. Finally, the results also support the notion that ATP hydrolysis occurs throughout the recA nucleoprotein filament.

Groups of five male Wistar rats weighing 306 +/- 17 g were fed a diet containing 2% 2-(3)-tert-butyl-4-hydroxyanisole (BHA) or basal diet (control group) for 2 weeks. Subsequently, rats received an i.p. injection of 25 mg/kg bromodeoxyuridine (BrdU), a thymidine analogue, and were killed after 4 h. The gastro-intestinal tract was removed and fixed in 70% ethanol. After pepsin digestion of the fixed tissues, labelled cell nuclei were visualized by means of a monoclonal anti-BrdU antibody technique. Cell kinetic parameters were determined by means of bivariate BrdU/DNA analysis using flow cytometry. Mean labelling index and potential doubling time of forestomach cells were significantly increased (P less than 0.002) in the 2% BHA group as compared to control rats. Cell kinetic parameters in other organs--glandular stomach, ileum, caecum and colon--were not affected by short-term consumption of BHA.

A method is described for the accurate determination of the superhelical density (omega) of highly underwound circular DNA molecules. Using this method, duplex DNA bound by RecA protein in the presence of ATP at pH 7.5 is found to be underwound by 39.6% (omega = -0.396), corresponding to a helical periodicity of 17.4 base-pairs per turn. The underwinding is increased to 41% (17.9 base-pairs per turn) in the presence of low levels of ATP gamma S, in good agreement with the 18.6 base-pairs per turn reported previously. In spite of the extensive underwinding, the distribution of DNA topoisomers produced by RecA protein binding is small. This indicates a high degree of structural uniformity among RecA-double-stranded DNA complexes in the presence of ATP.

A multivariate analysis of the pathologic data of 350 patients with primary colorectal cancer was performed. In addition to conventional parameters such as shape and size of the primary tumor, central node involvement, angioinvasive growth, grade, and stage, new variables such as the immunoreactivity patterns of carcinoembryonic antigen (CEA), CA 19-9, mucin, serotonin, secretory component (SC), and the DNA index were tested for their potential prognostic value. Every variable except CA 19-9, serotonin, and DNA showed significant prognostic information in univariate analysis. However, in the multivariate analysis stage was the predictive factor with the highest hazard ratio, but absence of central node involvement, tumors with diameters between 3.5 cm and 6 cm, exophytic tumor growth, well-differentiated tumors, tumors with CEA immunoreactivity, absence for staining with serotonin, and diploid tumors also were included in the relative risk model. Thus, the aforementioned variables appear to play a role in the establishment of a prognostic index.

The cytokinetic behavior of isolated hepatocytes transplanted into the spleen of syngenic normal Wistar rats was studied. Hepatocyte transplantation (HTX) was performed by the intrasplenic injection of 10(7) isolated hepatocytes. The proliferation index (PI) of intrasplenic donor hepatocytes was assessed by immunocytochemical visualization of DNA-synthesizing cells after pulse-labeling with bromodeoxyuridine (BrdU), a thymidine analogue. A method for determination of intrasplenic liver mass based on tissue glutamate dehydrogenase content was developed. The spontaneous PI of donor hepatocytes at 12 and at 20 weeks post-HTX amounted to around 3%. A significant increase of intrasplenic liver mass was demonstrated between the 12th and 20th week post-HTX (from 8.1 +/- 0.8% to 10.8 +/- 0.8% of spleen weight, P less than 0.05). After partial hepatectomy (PH) at 12 weeks post-HTX, the PI of liver cells in the spleen showed a transient increase up to about 10%, which rapidly declined to the "spontaneous" level of 3%. However, PH did not cause an additional increase in intrasplenic liver mass. This study shows that continuous mitotic activity of intrasplenic hepatocytes results in an actual increase of liver mass in spleen. Although a short-lived increase of proliferative activity of ectopically grafted hepatocytes was shown to occur after PH in the HTX-treated rat, this procedure did not result in an additional increase of intrasplenic liver tissue.

Assay systems for the evaluation of carcinogen interaction with human tissues are essential for assessing cancer risk. Human hair roots (HHR) are a readily obtainable epithelial tissue source that have been employed for investigating inherited enzyme activities. In this study HHR were found to possess cytochrome P-450-dependent 7-ethoxyresorufin-O-deethylase (ERD) activity which measures cytochrome P-450 isoenzymes that are highly specific (in the order of greater than 95%) markers for the metabolic activation of many environmental carcinogenic substances such as the polycyclic aromatic hydrocarbons (PAHs). Topical application of PAHs (in liquor carbonis detergens) to the scalp of human volunteers was found to enhance the activity of this enzyme in freshly plucked hair roots. Oral and topical administration of ketoconazole to the same subjects resulted in an appreciable (up to 73%) inhibition of detectable enzyme activity. Our data suggest that measurement of ERD in HHR may be a useful marker for the study of toxifying species of cytochrome P-450 isozymes in human populations.

Nuclei, isolated from paraffin-embedded tissue, were stained with propidium iodide (PI) and found suitable for DNA analysis by flow cytometry (FCM). DNA-derived fluorescence intensity, however, was always decreased and had a much higher intersample variability as compared to results obtained with fresh material. Using chicken red blood cells (CRBC) as a model system, we found the lower fluorescence intensity to be due to the formalin fixation step in tissue processing. The intersample variability was found to be at least partly caused by variations in the duration of fixation. Overnight trypsinization improved the fluorescence intensity but did not reduce the intersample variability. Under all conditions tested PI binding to CRBC appeared to be saturable. Since fresh diploid or red blood cells could not be used to standardize DNA histograms, an alternative approach was developed in which nuclei from paraffin-embedded normal and tumor tissue of the same specimen were mixed. With this method DNA indices (DI) of 24 colorectal cancers were found to be closely correlated (r = 0.9877, P less than 0.001) with DI obtained with fresh tumor tissue from the same patients. The correlation of the percentages of S-phase nuclei between paraffin-extracted and fresh samples (r = 0.5875, P less than 0.05) was as high as could be expected, taking sampling differences into account. This method is an important tool for the retrospective analysis of FCM-derived DNA parameters in relation to diagnosis and prognosis of neoplasms.

Cultured human endothelial cells were infected with human cytomegalovirus AD 169 and Kerr. The infection resulted in the appearance of viral antigens in the nuclei of about 10% of the endothelial cells and in the concomitant disappearance of vWF from the infected cells. No differences were observed between endothelial cells from different sources (umbilical cord veins or arteries, adult veins).

In order to test the contention that metastasis is a selective process and that therefore metastases might show a more restricted pattern of phenotypic and genotypic characteristics than primary tumors, we compared the expression of carcinoembryonic antigen, Ca 19-9, secretory component, serotonin, and mucin production as well as flow cytometric data on DNA content and percentage of S-phase cells in 87 primary large bowel carcinomas and their lymph node metastases. In a majority of the cases primary tumors and their metastases were largely identical with regard to the examined phenotypic features. In discrepant cases, however, metastases did not invariably show a more restricted pattern than primary tumors, indicating high differentiational plasticity of primary and metastatic colorectal cancer cells. In contrast, in a number of cases genotypic discrepancies were observed. We conclude that phenotypic characteristics of colorectal cancer cells cannot be used to study the pathogenesis of lymph node metastasis. Genotypic studies, however, suggest that lymphogenic metastasis may be a selective event.

Detection of liver cell membrane autoantibodies is routinely performed by immunofluorescence testing of patient sera on rabbit hepatocyte suspensions. We have investigated the possible use of cells from the PLC/PRF/5 human hepatoma cell-line. These cells were employed as substrate in an immunofluorescence test which was compared with the conventional rabbit hepatocyte assay. We found a close correlation between the results obtained with these different substrates on visual reading. We furthermore compared visual reading of immunofluorescence preparations with flow-cytometrical analysis of immunostained cell suspensions. The results with these different methods were largely confirmatory. The PLC/PRF/5 cells are easily available and should therefore be regarded as a highly valuable new substrate for detection of liver cell membrane autoantibodies. Flow cytometry appears to be a technically simple and reliable method for quantitative analysis.

Colonic mucosa was implanted subcutaneously into nude mice in order to investigate the potential use of xenografts as an in vivo model system for the study of colonic epithelial proliferation and differentiation. The xenografts were followed for 5 weeks. Proliferation was studied by bromodeoxyuridine (BrdU) incorporation followed by visualization of the DNA-synthesizing cells with an anti BrdU monoclonal antibody. Differentiated cells were visualized by histochemical staining of goblet- and enterochromaffin cells and of columnar cells by immunoperoxidase staining for carcinoembryonic antigen (CEA), secretory component, and serotonin. In different strains of mice different observations were made. Less immunocompetent strains (Balb c nu/nu, NMRI nu/nu) developed abscesses at the site of the xenograft as well as wasting disease. These phenomena almost never occurred in CD-1 nu/nu mice. The success rate was highest in CD-1 nu/nu mice. After about 1 week, only crypt base cells remained vital and started to repopulate crypts with epithelial cells showing normal colonic differentiation features such as CEA, secretory component, and serotonin immunoreactivity and mainly sulphomucin production. After longer periods of time, crypts started to form cyst-like structures due to accumulation of secretion products and dead cells. DNA-synthesizing cells were seen in the basal areas of recognizable crypts and in the cyst-like structures in a random distribution. These results indicate that normal colon mucosa xenografted into nude mice maintains its proliferative and differentiating capacity for at least 5 weeks.

RecA protein promoted formation of paranemic joints, in which a recA-ssDNA complex and a nicked circular dsDNA molecule are homologously aligned without net cross-strand interwinding, is accompanied by extensive underwinding of the dsDNA molecule. When the nick is sealed by DNA ligase, a highly negatively superhelical DNA molecule is formed. This underwinding has the following properties: (a) it occurs within 2 min; (b) it is completely homology dependent; (c) it does not require a homologous free DNA end. The resulting underwound DNA species is comprised of a heterogeneous population of topoisomers. The degree of unwinding exhibits a strong dependence on the fractional length of homology in the dsDNA molecule and indicates that paranemic joints can extend for at least 2900 base pairs. The rapid underwinding associated with paranemic joint formation is followed by a longer phase in which the dsDNA molecule is more extensively underwound.

A mouse monoclonal IgM antibody reactive with dendritic cells (DC) from the Brown Norway (BN) rat was prepared. This antibody (1F119) binds to a membrane-bound antigen present on DC from thoracic duct lymph, spleen, thymus, and lymph node. The antigen is present only in low density on 5% of splenic macrophages (M phi) and absent from peritoneal M luminal diameter. In situ, the antibody exhibits a strong reactivity towards DC in the thymic medulla, whereas no reaction is observed with cortical cells. Furthermore, cells positive for 1F119 can be identified in T-cell areas of spleen, lymph node, and Peyers' patches. 1F119 was genetically restricted in that a strong reactivity was found with DC from rats of the RT1n and RT1u haplotypes, an intermediate reactivity with the RT1c haplotype, only a weak reactivity with the RT1l and RT1b haplotypes, and no reactivity with the RT1a and RT1k haplotypes. The relatively weak reactivity of 1F119 with respect to the RT1l haplotype also appeared from a weak binding of 1F119 to DC from Lewis rats, as was assessed by FACS analysis. This result was comparable to the binding of OX3 (RT1.Bl and RT1.Bu) to DC from BN rats. Studies performed on thymus sections of recombinant rat strains indicate that 1F119, despite its apparent specificity for DC, reacts with a polymorphic RT1.B product.

In a series of 61 primary colorectal carcinomas, we attempted to determine which primary tumor characteristics correlated with the possibility to continuously maintain tumor cells in vitro or in vivo, and to what extent the characteristics of a primary tumor were maintained in vitro or as xenograft. Four continuous cell lines and 10 serially transplantable tumors were obtained. Only one cell line could be maintained both in vitro and in vivo. Tumors that had metastized and tumors in the proximal colon showed a higher success for in vivo and in vitro growth. DNA analysis showed that in most xenografts the DNA index was identical to that the primary tumor. However, in some cases tumor cell subpopulations were lost or genetically variant new subpopulations were generated. In general, the degree of differentiation in the xenografts corresponded with the least differentiated areas in the primary tumor. Xenografts appeared to display comparable of antigen expression.

A panel of crossreactive anti-human, -mouse and -rat MHC class II monoclonal antibodies (Mabs) was used to examine MHC class II antigen expression on canine T lymphocytes by cytofluorometry. The presence of MHC class II antigens was demonstrated on activated T lymphoblasts as well as on non-stimulated peripheral blood T lymphocytes. A number of anti-MHC class II Mabs reacted only with activated T lymphoblasts. Immunoprecipitation studies confirmed the Ia-like or MHC class II molecular character of the antigens on canine T cells. The expression of MHC class II antigens on peripheral blood T lymphocytes appeared to be not induced by stimulation of the T cells, as purified T lymphocytes of specific pathogen free dogs reacted with anti-MHC class II Mabs. Moreover, the study indicates that MHC class II antigen expression is present in the neonatal thymus. Lectin stimulated and allogeneically stimulated T lymphoblasts showed a stronger expression of MHC class II antigens in comparison with non-stimulated T cells.