DNA synthesis in human duodenal biopsies maintained in organ culture

Incorporation of 3H-thymidine during organ culture was studied in duodenal biopsies from 14 patients. Pulse-label at various intervals disclosed active incorporations during the first 2 h in culture. Labelling index declined to low levels at 3-4 h. Thereafter incorporation increased again and persisted throughout the rest of the culture period of 11 h. The DNA synthesis rate of crypt cells between 4 and 11 h in culture was calculated in 5 patients after pulse-label and continuous labelling of explants in parallel culture. The rate of entry into DNA synthesis was about 24 cells per 1,000 crypt cells per hour in flat, coeliac biopsies, versus 9-13 in controls, Gluten did not influence DNA synthesis rate, whereas wheat germ lectin inhibited DNA synthesis. Counting of the total number of mitoses and labelled fraction of mitoses disclosed active crypt cell renewal in flat, coeliac biopsies. In normal-appearing biopsies no mitoses were labelled, indicating delayed exit from S-phase or long duration of G2-phase in these explants.     

Some ultrastructural effects of testosterone and insulin on the ventral prostate of rats in organ culture

Summary The fine structure of the secretory epithelial cells of rat's ventral prostate has been studied following organ culture. Culturing with either testosterone or insulin alone, and with the two hormones combined, were carried out to investigate how insulin modifies the action of testosterone on the maintenance of cellular integrity. After 4 days in hormone-free culture, the secretory epithelial cells showed signs of cellular atrophy and regression, involving loss of the apical microvilli, absence of the apical secretory vacuoles, atrophy of the Golgi apparatus, decrease in rough endoplasmic reticulum and the appearance of autophagic vacuoles. The presence in the medium of either testosterone or insulin alone, or combined, prevented cellular atrophy and regression. The best maintenance of cellular integrity was obtained in a culture containing both hormones. The effects of insulin was approximately equivalent to those of testosterone in the maintenance of cellular integrity.     

In vitro differentiation of myxoid liposarcomas maintained in organ culture system.

Liposarcoma falls into the differential diagnosis of myxoid malignant mesenchymal tumors. On the other hand, its relation with white or brown fat is controversial. Two cases of liposarcoma have been studied by organ culture, a method which provides cell and tissue redifferentiation in vitro. Both cases developed successively cytoplasmic glycogen granules and lipid droplets as well as a single lipidic vacuole in the late phase of cultivation as a marker of fat differentiation. Our results support the possibility of identifying myxoid liposarcomas as well as their origin from white fat tissue.     

Adherence of Bordetella bronchiseptica 276 to porcine trachea maintained in organ culture.

Two organ culture models have been adapted for porcine tracheae in order to study colonization by Bordetella bronchiseptica. Rings or segments excised from tracheae of newborn piglets were incubated overnight at 37 degrees C in a nutrient medium under 5% CO2-95% air conditions. Tracheal segments were infected with B bronchiseptica 276, and after different incubation times, bacterial counts were done. B. bronchiseptica adhered well to tracheae maintained in culture, and no statistically significant differences between the two models were observed. Noninfected tracheal mucosae maintained a normal appearance for several days, whereas infected mucosae showed typical damage caused by B. bronchiseptica, namely, loss of ciliary activity and cilia and sloughing of ciliated cells. Our data indicated that porcine tracheal organ culture could be advantageously used to study in vitro colonization by B. bronchiseptica.     

A comparison of the specificity of phosphatidylcholine synthesis by human fetal lung maintained in either organ or organotypic culture.

Human fetal lung (14-18 weeks gestation) was maintained in either organ or organotypic culture. By 4 days in organ culture or 14 days in organotypic culture, epithelial cells within both culture systems exhibited well-developed apical microvilli and possessed numerous intracellular lamellar bodies characteristic of surfactant phospholipid stores. However, analysis of the pattern of synthesis of individual molecular species of phosphatidylcholine by [14C]choline incorporation and reversed-phase h.p.l.c. showed that this apparent maturation was not paralleled by an increased synthesis of the dipalmitoyl species in either culture system. By contrast, the fractional synthesis of dipalmitoyl phosphatidylcholine, expressed as a percentage of total [14C]choline incorporation, decreased with time in both organ and organotypic culture. Moreover, these fractions were not significantly different from those measured in parallel monolayer cultures of mixed human fetal lung cells that displayed mainly fibroblast morphology. These results suggest that the synthesis pattern of phosphatidylcholine species by lung cells in culture is determined principally by their incubation conditions and not by their state of apparent maturation.     

An ultrastructural study of the effects of fluoroacetate on hepatocytes

Summary This research was undertaken in order to determine if known biochemical processes involving the conversion of fluoroacetate are reflected morphologically at the electron microscopic level. No changes were observed in the hepatocytes after 2.5 mg/kg of fluoroacetate and sacrifice at 3 hours, but with 5 mg/kg of the drug there was an increase in the amount of agranular endoplasmic reticulum, an aberrant shape of mitochondria, and a disappearance of visible glycogen. One hour after 10, 15, 20, or 30 mg/kg of fluoroacetate the results showed a tremendous increase in the amount of agranular endoplasmic reticulum, an aberration in mitochondrial structure, and a disappearance of the intramitochondrial granules and glycogen. The possible relationship of these changes to fluoroacetate poisoning is discussed.Research supported by USPHS Grants HE 12751, NS 05665, and 00690.Recipient of Career Research Development Award 1K3 GM 28064.—The authors would like to express their appreciation to Drs. E. M. Gal and R. A. Peters for supplying the samples of fluoroacetate and to Mr. J. Mascorro for technical assistance.     

RNA synthesis inhibitors on young rat adrenal in primary culture. An ultrastructural study

Adrenal cells of newborn rat adrenals in primary tissue culture were treated with actinomycin D (2, 10 and 25 micrograms/ml), alpha-amanitin (5 and 50 micrograms/ml) and cordycepin (100 micrograms/ml) and studied with the electron microscope. The most striking changes observed with the three drugs were localized on the nucleoli, and consisted, respectively, of segregation (actinomycin D), fragmentation (alpha-amanitin) and microsegregation (cordycepin). These results are consistent with the molecular sites of action of the drugs and are comparable to previous in vivo findings.     

Metabolic activation of benzo[a]pyrene in human skin maintained in short-term organ culture

The metabolic activation of benzo[a]pyrene (BP) was examined in six samples of human skin after topical application of the hydrocarbon to the skin in short-term organ culture. The results show that all of the samples were capable of metabolizing BP to water-soluble products and to ether-soluble products that included the 4,5-, 7,8- and 9,10-dihydrodiols and a product which had chromatographic properties identical with those of authentic trans-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (BP-11,12-diol). The major BP-deoxyribonucleoside adduct detected in each skin sample appeared to be formed from the reaction of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BP-7,8-diol 9,10-oxide) with deoxyguanosine residues in DNA.     

An ultrastructural study of osmiophilia in the human rectum

Synopsis Rectal biopsies from subjects with a normal rectum and from patients with various forms of inflammatory bowel disease were studied by the prolonged osmication technique. No consistent ultrastructural differences were observed between these groups, but there were striking differences between individual epithelial cells in the same biopsy and between the epithelium and the cells of the lamina propria. The Golgi apparatus was demonstrated occasionally in the epithelium, often in endothelium. Endoplasmic reticulum, perinuclear cisterna and mitochondria were variably outlined. In plasma cells, there were striking differences in osmiophilia.The underlying mechanism of the different staining patterns is not clear. The findings do not appear to help in the differential diagnosis of inflammatory bowel disease nor to shed any new light on their underlying pathogenic mechanisms.     

An ultrastructural study of embryonic chick retinal neurons in culture

Summary The differentiation of cells and synapses in explants of 9-day-old chick embryo retina has been studied by light and electron microscopy over a period of 35 days in vitro, and samples of retina from the 9-day chick foetus were directly fixed and prepared for study.At the time of explantation the retinae were poorly differentiated and no lamination was apparent. From day 14 onwards, (i) outer and inner nuclear layers (ONL, INL) separated by a layer of neuropil corresponding to the outer plexiform layer (OPL) and (ii) a layer of scattered large ganglion cells separated from the INL by a zone of neuropil resembling the inner plexiform layer (IPL) were apparent, and (iii) a well-differentiated outer limiting membrane was established close to the surface of the explants. In the oldest cultures some development of photoreceptor outer segments occurred but a distinct optic nerve fibre layer did not form.Although cell identification presented problems even in the oldest cultures, the major retinal cell types described in vivo could be identified. Photoreceptor cells developed pedicles in the OPL which became filled with synaptic vesicles and synaptic ribbons and established ribbon synapses (including triads) with and were commonly invaginated by processes from horizontal and bipolar cells. Processes of bipolar cells in the IPL formed simple and dyad synapses. At least two types of presynaptic amacrine cells were also identified in the INL, one of which contained large numbers of dense-core vesicles. The ganglion cells, though sparse, were large and well differentiated.These findings show that all the major neuronal types of the retina are capable of developing and differentiating in vitro, lagging behind the time-table of development and differentiation in vivo by approximately 7 days, but resulting in a histotypically organised retina with synaptic neuropil showing many similarities to the corresponding neuropil in vivo.     

An ultrastructural study of the human micropolycystic ovary

The basement membrane of follicles in the micropolycystic ovaries of infertile women was thickened compared to that in the ovaries of normal women or those with typical polycystic ovaries.     

An ultrastructural study on muciparous microcysts of the human adenohypophysis

The microcysts found in two adenohypophyses removed from patients with nonsecreting adenomas of this gland have been studied. In one case the cysts were completely surrounded by neoplastic tissue, whereas in the second they were located within the normal parenchyma. The cysts were surrounded by a basement membrane, and appeared to contain a finely granular weakly electron-dense substance. Their walls were lined by mostly cubic cells with microvilli, joined by junctional complexes. Intracytoplasmic microlumina could be seen in some of these cells. Cells showing cilia with a 9 + 2 fibrillar pattern and cells with mucosecretory differentiation were also present. Furthermore endocrine cells were also found in the cyst-lining epithelium. The ultrastructural aspects of the cyst cells lead to reject the hypothesis that these are derived from folliculostellate cells. Besides, intermediate aspects between cyst cells and tumor cells were never visible. Therefore it seems likely that a relationship between adenoma and cyst cells does not exist and that cyst cells originate from remnants of Rathke's pouch with a ciliate or mucosecretory differentiation. The presence of endocrine cells in the cyst-lining epithelium deserves further study, for it seems to indicate that an endocrine differentiation may occur in the adult, too. To our knowledge this is the first ultrastructural study of these structures in adult human pituitary gland.     

Theophylline effects on normal uveal melanocytes in culture: an ultrastructural study

Theophylline enhances maturation and differentiation of uveal melanocytes. By electron microscopy, we showed that theophylline changes small, dendritic melanocytes into large, platelike cells; it also enhances DOPA reaction as evidenced by increased deposition of DOPA reaction products in dilated cisternae and vesicles around the Golgi region. The effect is partially reversible in choroidal melanocytes but irreversible in iridial cells. It appears that theophylline, in addition to inducing tyrosine activity, accelerates the maturation and/or aging that normally occurs in cultured melanocytes when incubation is prolonged.     

Functional and ultrastructural effects of nontypeable haemophilus influenzae in a hamster trachea organ culture system

Summary A hamster trachea organ culture system was utilized to evaluate quantitatively the effects of a strain of nontypeableHaemophilus influenzae (NTHI) and culture supernatants of the same strain on ciliary activity. Tracheal explants were maintained in organ culture for 96 to 144 h and ciliary activity was observed daily with an inverted microscope. Explants continuously exposed to a strain of NTHI had a progressive decline in ciliary activity which was significantly lower than uninfected controls evaluated concomitantly by 48 h of exposure and thereafter. Histologic studies revealed a progressive degeneration of mucosal cells and exfoliation of ciliated cells. Scanning electron microscopy showed little adherence of NTHI to the mucosal surface. Sterile broth cultures of NTHI and supernatants of organ cultures infected with the same NTHI strain had no adverse effect on ciliary activity. Infected tracheal explants treated with ampicillin 24, 48, or 72 h after continuous bacterial challenge had no significant decline in ciliary activity compared to controls. The lack of adherence and the histologic changes observed when hamster trachea cultures were infected with NTHI suggested a toxin might mediate the damage observed. Broth and organ culture supernatants, however, produced no damage. Therefore, further studies are needed to determine the role, if any, of a toxin in the production of damage to hamster tracheal explants by NTHI. This work was supported by a Merit Review grant from the Veterans Administration and by Grant AI-19641 from the National Institute of Allergy and Infectious Diseases.