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Biomedical subjects

J H Resau

Publications and source records attributed to J H Resau.

At least 91 records · Page 5Linked to original sources
PubMed1988

Localization of keratin mRNA in human tracheobronchial epithelium and bronchogenic carcinomas by in situ hybridization.

An in situ hybridization technique was applied to detect expression of keratin mRNAs in xenotransplanted human tracheobronchial epithelium and lung carcinomas. Tissues from eight tracheas repopulated with cells from five different noncancerous donors and 15 squamous cell carcinomas were used. Using a K6 (56 kd) human keratin cDNA (KA-1) and a K14 (50 kd) cDNA (KB-2) as probes, radiolabeled by nick-translation with 3H-dATP/TTP, the specificity and significant differences in the levels of silver grains on various epithelial lesions in formalin-fixed, paraffin-embedded tissue sections were demonstrated. In situ hybridization with either KA-1 or KB-2 probe showed similar localization of silver grains in all histologic types in consecutive tissue sections. In xenotransplanted tracheobronchial epithelia, very few grains were seen over cells of simple, pseudostratified, or stratified epithelia two to three cell layers thick. Nonkeratinizing stratified hyperplastic epithelia of more than three cell layers showed uniform localization of numerous grains throughout the lesions. In contrast, epidermoid metaplasias exhibited a dense and localized pattern of grains on the basal and parabasal cell layers with a decrease in grain density toward the surface layers. Carcinoma cells from bronchogenic squamous cell carcinomas showed a higher density and more uniform localization of grains. Well-differentiated carcinoma cells contained more keratin mRNAs than moderately to poorly differentiated carcinoma cells. This evidence obtained with the KA-1 and KB-2 probes demonstrates the different localization patterns of keratin mRNAs in different epithelial lesions. In addition, the levels of mRNA expressed show a positive correlation with the degree of squamous differentiation. It was of particular interest that an ordered program of keratin mRNA expression proportional to the level of cellular differentiation was observed in epidermoid metaplasias. Both of these probes serve as keratinization markers of human tracheobronchial epithelial lesions.

Animals↗
PubMed1988

Unesterified cholesterol-rich lipid particles in atherosclerotic lesions of human and rabbit aortas.

Unesterified cholesterol-rich lipid particles were isolated from human and cholesterol-fed rabbit aortas. These particles have been previously reported to constitute the initial lipid deposition in atherosclerotic lesion development. Purification of the particles was accomplished with microfiltration, gel filtration chromatography, and density gradient centrifugation. Particles from both human and rabbit aortas had a density of between 1.02 g/ml and 1.08 g/ml with a peak at d = 1.036 g/ml. These particles had a high molar ratio of unesterified cholesterol to phospholipid (2.4:1 in rabbit, 2.6:1 in human) and a high percentage of their cholesterol in an unesterified form (82% in rabbit, 76% in human). The particles had diameters between 700 and 3000 A and showed unilamellar and multilamellar structures. Freeze-fractured particles had smooth fracture faces and sometimes contained a smooth-surfaced core. Upon incubation with filipin, particles showed typical filipin-sterol complexes, demonstrating the presence of unesterified cholesterol. The particles we have isolated may constitute an early pathologic form of accumulated cholesterol in developing lesions and may represent a degradation product of infiltrated plasma low-density lipoprotein.

Animals↗
PubMed1988

Histogenesis and clonality of pancreatic tumors induced by v-myc and v-raf oncogenes in NFS/N mice.

Newborn NFS/N mice were inoculated with pseudotypes of murine retroviruses containing murine v-raf, avian v-myc, or both v-raf and v-myc within a single construct. Foci of dysplastic acinar cells, similar to those observed in rats given chemical carcinogens, were induced in 77% of mice inoculated with the raf/myc construct with a latency as short as 15 days. However, all animals given this construct also developed fibrosarcomas, erythroblastosis, and lymphomas and died within 70 days of infection, before pancreatic acinar carcinomas developed. Dysplastic foci were also observed in mice infected with viruses containing v-raf or v-myc alone with latencies of 3-4 weeks, and carcinomas were seen after an average latency of 150 days in 31% of mice infected with either of two viruses expressing v-myc alone. Two primary carcinomas were transplanted in mice, and in vitro cell lines were developed from one of the transplants. DNA prepared from seven primary carcinomas, the two transplanted tumors, and the in vitro cell lines was hybridized with a v-myc probe. Each tumor had a unique pattern of proviral integrations that was retained, with the gain or loss of single sites, in the transplants and derivative cell lines. The clonal nature of the advanced pancreatic acinar carcinomas is discussed in relation to their histogenesis and the transforming potentials of the raf and myc oncogenes.

Animals↗
PubMed1987

Cell injury and regeneration of human epithelium in organ culture.

Human esophageal, tracheal, and pancreatic ductal fragments were collected at autopsy after a postmortem interval of 12 hours or less and maintained in explant organ culture for 30 days. The viability and growth of the explants was assessed by morphology, LDH enzyme release, and cellular outgrowth. The viability and growth of the bronchial explant epithelium was directly related to the postmortem interval. Esophageal epithelial regeneration followed the desquamation of the superficial cell layers. Pancreatic epithelia appeared to grow more slowly and with less outgrowth than the other tissues. Epithelial cell growth along the explant surface and onto the culture dish appeared to proceed through the well-characterized process that follows cell injury, i.e., flattening, migration, replication, and differentiation. Thus, sufficient numbers of viable epithelial cells capable of regeneration were present in routine autopsy epithelium, but there was considerable variation from tissue to tissue and case to case. The most effective and accurate approach to follow when evaluating and predicting the growth and viability of these explants is by using a combination of morphologic, enzymatic and biologic assays. Errors in the interpretation of viability are possible when only one assay method is utilized. These tissues grown in explant organ culture are suitable for studies on the mechanism and response of epithelia to cell injury, recovery and wound healing.

Bronchi↗
PubMed1986

Effects of B-retinyl acetate on human breast epithelium in explant culture.

Explants of human breast (obtained aseptically from ten women ages 18-37) were examined and the vitamin A compound B-retinyl acetate was tested (at 3 X 10(-5) and 3 X 10(-6) M) for its effects on the fine structure and growth of the epithelium. In the absence of B-retinyl acetate, cells growing out from the explant (outgrowth cells) underwent squamous metaplasia, began to accumulate many intermediate filaments (tonofilaments), and revealed large desmosomes after 2 weeks in culture. In the presence of either concentration of B-retinyl acetate, the epithelial cells were largely prevented from undergoing squamous metaplasia. The glandular epithelium inside the explant maintained a typical secretory appearance for 2 weeks in control cultures (without the retinoid) and then began to show increased numbers of lysosomes and a loss of secretory granules. These glandular epithelial cells did not undergo squamous metaplasia, but they contained increased numbers of intermediate filaments. In contrast, glandular epithelium cultured in either concentrations of B-retinyl acetate appeared secretory for as long 6 weeks in culture, and intermediate filaments were not obvious. Autoradiographs demonstrated that both concentrations of B-retinyl acetate inhibited cell division in the outgrowth epithelium and in the (internal) glandular epithelium. The ability of B-retinyl acetate to reverse squamous metaplasia in the outgrowth cells was also tested. Outgrowth cells reversed to a more normal ductlike appearance after 6 weeks culture in standard medium followed by only 1 week in 20 micrograms/ml B-retinyl acetate. After 7 weeks in standard medium and 1 week in 10 micrograms/ml B-retinyl acetate, the cells showed a partial reversal of the squamous metaplasia.

Adolescent↗
PubMed1986

Explant organ culture of hamster alimentary tract epithelium.

Adult Syrian Golden hamster alimentary tract maintained as explants in organ culture was studied using the model system for hamster pancreas described by Resau et al. (1983a). Explants of esophagus, stomach, duodenum and colon were maintained in organ culture on Gelfoam sponge rafts in a high-oxygen atmosphere with serum-supplemented CMRL-1066 medium. All of the tissues were observed to show evidence of sublethal acute cell injury during the first several days of culture. Subsequently, the epithelial tissues recovered from this injury, repopulated the denuded areas of the explants and replicated within the sponge matrix. Explants were maintained in a differentiated state for 30+ days and sampled for morphology to examine to process of cell injury, repair, differentiation and replication which occurs in mucosal epithelia. The percentage of basement membrane covered by epithelia in the explants from various tissues was compared to the level of LDH in the media to reveal the relationship between viability determined by biochemical and by morphological methods. Restitution of the mucosal surface occurred in all of the explants. We conclude that adequate populations of replicating cells are maintained within the epithelium of the hamster alimentary tract tissues in vitro so that restitution can occur through migration and subsequent differentiation of the epithelial cells within the mucosa of the explants.

Animals↗
PubMed1986

Contact smear cytologic preparations of organ culture explant tissue.

The effects of cytologic sampling by contact-smear on the viability and morphology of epithelial tissues maintained as explants in organ culture were studied. Tracheal and pancreatic tissues from Syrian golden hamsters were maintained for 30 days in culture, and sampled cytologically at weekly intervals. Analysis of sequential Papanicolaou-stained smears demonstrates that explant tissues can be evaluated cytologically over time in vitro without apparent effects on either the tissue viability, histochemistry or morphology. Microscopic examination further reveals that cellular samples retain diagnostically significant cytomorphologic features of the in vivo tissues. These observations indicate that this technique is useful for understanding the long-term response to carcinogens of epithelial tissue maintained in explant organ culture.

Animals↗
PubMed1986

Rapid detection of xenotransplanted human tissues using in situ hybridization.

A rapid and sensitive method for the identification of human tissues xenotransplanted in nude mice was developed. An in situ hybridization technique made it possible to distinguish between cells of human origin and cells of murine origin in formalin-fixed paraffin sections. High-molecular-weight DNAs extracted from human or mouse tissues were sonicated, nick-translated with 32P-dCTP, and used as hybridization probes. Dot blot hybridization of 32P-labeled probes revealed clear species-specific signals. Formalin-fixed paraffin-embedded tissue samples from repopulated tracheal transplants, containing either human tracheal epithelial cells or human renal tubular cells, were used. Cells of human and murine origin were distinguishable by in situ hybridization with sonicated DNA probes. This method has several advantages; simple preparation of probes, high sensitivity, and applicability to formalin-fixed paraffin-embedded tissue sections. In situ hybridization with sonicated DNA probes should provide a powerful tool for verifying the human origin of xenotransplanted tissues in nude mice.

Animals↗
PubMed1985

What's new in in vitro studies of exocrine pancreatic cell injury?

Exocrine pancreas in vitro models are useful for the study of pancreatic differentiation, secretion mechanisms, cell injury, and lysosomal processing of secretory product. Syrian hamster pancreas in explant organ culture undergoes a series of morphologic changes which parallel in vitro acinar cell injury, differentiation, and phenotypic alteration. Within 48 hours, the cultured acinar cells show morphologic evidence of sublethal cell injury. Autophagy and crinophagy are particularly striking. The autophagic processes can be inhibited by the addition of the protein synthesis inhibitor cycloheximide or by culture at lowered temperatures (20 degrees C). Acinar cells lethally damaged show pyknotic nuclei, high amplitude swelling, and necrosis. Approximately 25% of each explant is viable after 72 hr in culture and the viability remains constant at 25-35% for up to 60 days of culture. The morphological changes of the explants are consistent with many of the features of pancreatitis and carcinoma of the exocrine pancreas. There is an increase in the ductal elements and a decrease in acini over time in culture. This may be due to: (a) an increased replication of ductal epithelial cells concomitant with necrosis of acinar epithelial cells and/or (b) phenotypic alteration of acinar cells to ductal cells. Acinar cell necrosis and phenotypic alterations may in part be due to the activation of lysosomal degradation pathways. Processes which inhibit lysosomal activation proved protective against these alterations, while processes which promote zymogen activation were deleterious.

Animals↗
PubMed1985

Studies on the mechanisms of altered exocrine acinar cell differentiation and ductal metaplasia following nitrosamine exposure using hamster pancreatic explant organ culture.

Syrian golden hamster pancreatic organ explants were treated with either methylnitrosourea (MNU) or N-methyl-N-nitroso-N'-nitroguanidine (MNNG). In control explants treated with only dimethylsulfoxide, there was evidence of autophagy and crinophagy in acinar cells. Carcinogen-exposed explants showed increased numbers of autophagic and crinophagic vacuoles. In long-term cultured explants there was an increase in the number of ducts over zero time control tissues. Eosinophilic cells similar to hepatocyte-like cells were seen in 90% of the carcinogen-treated explant experiments and in 45% of the controls. Nitrosamine exposure did not induce an increase in the overall amount of necrosis measured morphometrically. Nitrosamine exposure in vitro appears to lead to a sequence of events that follow carcinogen metabolism by the acinar cells. The changes that follow include altered cell morphology and toxic cell injury evidenced by autophagic and crinophagic processes, regeneration of ductal appearing cells, the appearance of hepatocyte-like cells and an overall increase in the amount of ductal metaplasia. Within some of these ductal foci, several ductules show atypical features.

Animals↗
PubMed1984

Cytological studies of carcinogen-treated bovine pancreatic ductal organ explants.

In order to develop a model to study cytological alterations in human pancreatic cancer, we have exposed bovine pancreatic ductal organ explants to a single dose of 2.5 micrograms per ml of N-methyl-N-nitroso-N'- nitroguanidine (MNNG). At serial time intervals, contact cytology smears of the explants were prepared and stained with the Papanicolaou stain. Control samples contained classic tall columnar and cuboidal ductal cells. Cells were uniform in size, shape, staining characteristics, and nuclear morphology. Ductal explants exposed to MNNG progressed through a series of dysplastic and atypical stages during the first 5 to 12 days in culture. Chromatin became increasingly more granular and nucleoli increased in both number and size. Exposed cells were larger than controls and had many dysplastic features. From day 15 to 30, the cells underwent changes morphologically consistent with those of human pancreatic adenocarcinomas .

Adenocarcinoma↗
PubMed1984

Degradation of zymogen granules by lysosomes in cultured pancreatic explants.

Pancreatic explants from Syrian hamsters were maintained in culture for determination of the fate of the zymogen granules in the acinar cells. By 6 hours of culture the zymogen granule matrix in many cells becomes nonhomogeneous, and electron-lucent spaces appear. Autoradiography performed 48 hours after pulse labeling shows that several of the altered zymogen granules contain radioactively labeled proteins. Hence these altered granules are mature zymogen granules undergoing regression. Ultrastructural analyses indicate that the zymogen granules are degraded intracellularly in the lysosomes by autophagy, by direct fusion between the lysosomes and the zymogen granules, as well as by engulfment of intact zymogen granules by the lysosomes. Budding of cytoplasm into zymogen granules is also frequently observed. Acid phosphatase histochemistry and anti-amylase immunohistochemistry were used for demonstration of presence of hydrolases and of secretory material in the degenerating zymogen granules. Cycloheximide and low temperature inhibit the degradation of zymogen granules and enhance the short-term viability of the explant. Secretagogues stimulate secretion but have little or no effect on the degradation of zymogen granules or on explant viability. The lysosomes participate in the intracellular degradation of zymogen granules in acinar cells of pancreatic explants. The induction of these lysosomal catabolic processes correlates with the structural alteration of pancreatic acinar cells. The intracellular degradation of secretory membranes and products by the lysosomes may play a role in the adaptation of pancreatic acinar cells to injury.

Acid Phosphatase↗
PubMed1983

Organ explant culture of adult Syrian golden hamster pancreas.

An organ explant culture system has been developed for long term maintenance of adult pancreatic tissue from the Syrian golden hamster. Gastric and duodenal lobe explants of up to 0.5 cm2 size were placed in tissue culture dishes (60 mm2) on Gelfoam sponge rafts to which was added 5 ml of CMRL medium 1066 supplemented with heat inactivated newborn bovine serum, L-glutamine, hydrocortisone, insulin, and antibiotics. Dishes were placed in a controlled atmosphere chamber, which was gassed with 45% O2, 50% N2, and 5% CO2 and incubated at 36.5 degrees C. Viability of the tissues was determined by light and electron microscopy as well as by [3]thymidine incorporation. Explants were viable for up to 70 d. Zymogen granule-containing cells characteristic of acinar cells and mucus-containing cells characteristic of ductal cells were present throughout this period. However, endocrine cells were only present for the 1st wk in culture.

Animals↗
PubMed1983

The cell kinetics of the adaptation of the human esophagus to organ culture.

The adaptation of normal human esophageal explants to organ culture for the first 33 d of in vitro growth was evaluated using histomorphology and [3H]TdR autoradiography combined with mitotic blockade. On the 3rd d in culture, extensive desquamation of superficial cells reduced the epithelium to about four cell layers. Thereafter, the epithelium remained atrophic, with a relative increase in basal and suprabasal cells. The percentage of cells synthesizing DNA was greatest from Day 4 through 8, just after desquamation, and reached a maximum on Day 4 (24 h [3H]TdR labeling index of 62%). The labeling index (LI) fluctuated, thereafter, but remained high (26% on Day 33). During the last 6 h of each [3H]TdR labeling interval, mitosis was blocked by colcemid. The 6 h mitotic rate (MR) was a reasonably constant fraction of the LI (maximum at 4 d: MR = 1.44%), but was much lower than predicted by [3H]TdR labeling indicating the loss of large numbers of cells after DNA synthesis but before or during mitosis. Unlabeled mitotic figures appeared between Days 1 to 3 and 6 to 33, suggesting that the epithelium initially contained a considerable population of cells arrested or delayed in G2 and continued to generate cells that remained in premitosis longer than 24 h. These results indicate that the atrophy observed in vitro is characterized by a relative increase in the basal and suprabasal cell category, a high replication rate, initial recruitment of cells arrested in premitosis, and rapid cell turnover with significant loss of cells at the premitotic or mitotic step, or both. Thus it seems that human esophageal epithelium grown in organ culture is a satisfactory substrate for experimentation (for example, in vitro carcinogenesis) that requires cell replication. However, there are major differences between the kinetics of esophageal epithelium in vivo and in vitro.

Cell Division↗
PubMed1981

Aryl hydrocarbon hydroxylase in human prostate.

Aryl hydrocarbon hydroxylase (AHH) was present in explant cultures of human prostate obtained from surgery of benign prostatic hyperplasia and was inducible by benz[a]anthracene (BA). The induction of AHH ranged from 14- to 150-fold when compared with control values and 10-fold variation of AHH inducibility among individuals was observed. Epithelial cells grown from human prostate tissue also contained measurable AHH activity and AHH was inducible by BA and 7,12-dimethylbenz[a]anthracene (DMBA). Inducibility of AHH by BA ranged from 2- to 63-fold. The inducibility of AHH by DMBA was always less than that by BA. In cells treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), there were no changes in AHH activity. These findings support the view that the human prostate is susceptible to environmental polycyclic hydrocarbon carcinogens and that environmental and occupational factors might contribute to the etiology of human prostatic carcinoma.

9,10-Dimethyl-1,2-benzanthracene↗
PubMedDate not supplied

Mass spectrometric analysis of tobacco-specific nitrosamine-DNA adducts in smokers and nonsmokers.

A gas chromatography, negative ion chemical ionization mass spectrometry (GC-NICI-MS) based assay for tobacco-specific nitrosamine adducts of DNA is described. The assay is based on the observation that acid hydrolysis of DNA from animals treated with tobacco-specific nitrosamines releases 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB). HPB and the internal standard [4,4-D2]HPB are derivatized with pentafluorobenzoyl chloride and the resulting HPB-pentafluorobenzoate is purified by high-performance liquid chromatography prior to GC-NICI-MS analysis. DNA from human peripheral lung and tracheobronchial tissue, collected at autopsy, was analyzed for acid-released HPB. The mean HPB level (fmol/mg of DNA) for peripheral lung DNA was 11 +/- 16 (SD, n = 9) for smokers and 0.9 +/- 2.3 (n = 8) for nonsmokers. Mean adduct levels in tracheobronchus were 16 +/- 18 (n = 4) for smokers and 0.9 +/- 1.7 (n = 4) for nonsmokers. These are the first measurements of tobacco-specific nitrosamine-DNA adducts in humans. Further studies comparing the levels of DNA and globin adducts will provide a better understanding of the metabolic activation of tobacco-specific nitrosamines in humans and may provide a more accurate indication of an individual's risk of developing tobacco-related cancer.

Adolescent↗
PubMedDate not supplied

Reproducible isolation of type II pneumocytes from fetal and adult rat lung using nycodenz density gradients.

Isolating fresh, relatively pure type II pneumocytes from the lung, particularly of fetal origin, is a difficult process. Separation by buoyant density gradient centrifugation has been used successfully to isolate adult type II cells. There is concern, however, that Percoll, a gradient medium that is commonly used for type II cell isolation, may be toxic to cells. We evaluated a new gradient medium, Nycodenz, that is (1) a true solution, (2) transparent, (3) not metabolized by cells, and (4) nontoxic to cells. Type II pneumocytes were isolated from 19- and 21-day gestation fetal and adult rat lung by elastase digestion and separated on preformed isotonic Nycodenz gradients (2 mL each of 27.6, 20.7, 13.8, and 4.6 (w/v) solutions). Type II pneumocytes were recovered from the density range 1.057-1.061 and identified by binding of FITC-conjugated and gold-complexed Maclura pomifera lectin. Cells derived from 19-day fetal lung contained abundant glycogen and reacted with a monoclonal antibody to the cytokeratins 8 and 18, which are markers of the fetal type II cell. Adult type II cells reacted with antibodies to cytokeratins 8, 18, and 19. Type II cell purity was 79.7 +/- 2.4%, 83.8 +/- 2.8%, and 82.6 +/- 1.8% (means +/- SEM) for 19- and 21-day gestation fetal and adult lung preparations, respectively. Cell viability was greater than 95%. The final cell yield for adult preparations was 17.8 +/- 2.7 x 10(6)/rat (means +/- SEM). To determine if the freshly isolated type II pneumocytes were functionally active, the incorporation of [3H]choline into phosphatidylcholine was measured. The percent saturation of phosphatidylcholine was high for both populations of freshly isolated cells. However, adult type II pneumocytes incorporated [3H]choline into phosphatidylcholine more rapidly than 21-day gestation fetal cells (5.97 x 10(-3) dpm/10(6) cells/h vs. 0.32 x 10(-3) dpm/10(6) cells/h, P less than .005). We have demonstrated that, using the Nycodenz isolation method, it is possible to obtain a high yield of relatively pure viable type II cells from fetal and adult lung that can be used for immediate study or cultured with an excellent plating efficiency (39 +/- 6.3%).

Animals↗