Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS)

BACKGROUND: Steroid 5 alpha-reductase is essential for the intracellular accumulation of dihydrotestosterone (DHT), which mediates androgen effects on target tissue. METHODS: In the present study, we describe the differential expression and cellular localization of 5 alpha-reductase 1 and 2 isoenzymes in the human prostate, and untreated and hormone-resistant prostatic carcinomas. The secretory epithelium of normal and hyperplastic glands showed strong nuclear 5 alpha-reductase 1 reactivity. Accordingly, the DHT forming 5 alpha-reductase process in secretory luminal cell types may be mediated predominantly by the type 1 isoenzyme. The androgen-independent basal cell layer variably expressed type 1 and 2 isoenzymes in nuclear and cytoplasmatic compartments. This suggests that circulating androgens are involved to control the basal cell layer, which represents the proliferative compartment of the human prostate. RESULTS: When compared with benign prostate tissue, increased 5 alpha-reductase reactivity was detected in prostate cancer, particularly in high-grade tumors and androgen-insensitive states of the disease. In cancerous lesions, the type 1 isoenzyme tended to shift to the cytoplasm, while the nuclear staining remained unchanged or slightly increased. Referring to the type 2 isoenzyme, increased cytoplasmatic and nuclear enzyme activity was detected in malignant cells when compared with adjacent benign prostate tissue. Even endocrine differentiated tumor cells that consistently lacked the nuclear androgen receptor variably expressed 5 alpha-reductase immunoreactivity. CONCLUSIONS: Although the functional significance of the differential subcellular localization of type 1 and 2 isoenzymes is currently unknown, the present data suggest that prostate cancer retains the DHT forming 5 alpha-reductase process in high-grade lesions and recurrent disease. Accordingly, circulating androgens may be still significant in these hormone-refractory malignancies.     

A method for processing paraffin sections of multilayer cultured tissue

A simple and rapid method is described for processing histological preparations from multilayer cultures growing in plastic Petri dishes, A covering collodion film is utilized to remove the tissue from the plastic dish and transfer it onto a paper block prior to embedding in Paraplast. To avoid any disruption by the collodion of the plasticware, the cultured tissue is first immersed in a solution of collodion and absolute alcohol (1:1) and then covered with pure collodion. All steps are carried out in the cold. This procedure allows morphological, histochemical, immunofluorescent, and autoradiographic studies to be carried out on serial sections of cultured tissue.     

Magnetic resonance imaging of the normal mouse brain: comparison with histologic sections

Details in the living animal can be compared with respective histologic section taken after death. In less than 35 min, 12 to 16 coronal and transverse images through the brain can be obtained, each with an in-plane resolution of 100 x 200 microns and a slice thickness of 900 microns. The experimental parameters can be manipulated not only to achieve different tissue contrast but also to access any desired plane.     

A comparison of immunohistochemical staining of human cultured mesothelial cells and ovarian tumour cells using epithelial and mesothelial cell markers

Atlantic salmon (Salmo salar) post-smolts were fed diets containing either Fosol (FO), a North Sea fish oil, sunflower oil (SO), linseed oil (LO) or Marinol K (MO), a southern hemisphere fish oil rich in 20:5(n-3) for 12 weeks. A macrophage-enriched leucocyte preparation was obtained from head kidney and the fatty acid compositions of the individual membrane phospholipids measured. In general phospholipids from SO- and LO-fed fish had increased 18:2(n-6), 20:2(n-6) and 20:3(n-6) compared to the fish oil treatments while LO-fed fish had lower 20:4(n-6) than any other dietary treatment. Fish fed LO also had increased 18:3(n-3), 18:4(n-3), 20:3(n-3) and 20:4(n-3). The 20:5(n-3) content of kidney macrophage-enriched leucocyte phospholipids was highest in MO-fed fish followed by FO- and LO-fed fish with the lowest level in fish fed SO. The overall effect on the ratio of eicosanoid precursors, 20:4/20:5, showed the highest value in SO-fed fish and the lowest in fish fed LO. Production of LTB5 by kidney macrophage-enriched leucocytes stimulated with A23187 was highest in MO-fed fish and lowest in those fed SO. Production of LTB4 was greatest in SO-fed fish and lowest in fish fed LO. Serum Ig levels were significantly affected by dietary treatment with highest values in fish fed FO and SO and lowest in fish fed MO and LO.     

Interaction of Escherichia coli heat-stable enterotoxin B with cultured human intestinal epithelial cells

Binding of Escherichia coli heat-stable enterotoxin B (STb) to the human intestinal epithelial cell lines T84 and HT29 and to polarized T84 cells was studied to define the initial interaction of this peptide toxin with target cells. Equilibrium and competitive binding isotherms showed that 125I-STb bound specifically to T84 and HT29 cells; however, the toxin-epithelial cell interactions could be characterized by low-affinity binding (< or = 10(5) M(-1)) to a high number of binding sites (> or = 10(6) per cell). STb binding to T84 and HT29 cells as a function of 125I-STb concentration did not approach saturation at levels well above the effective biological concentration of STb for fluid secretion. Treatment of the 125I-STb-bound T84 and HT29 cells with an acidic saline solution to remove surface-bound toxin revealed that only approximately 55% +/- 10% of 125I-STb could be removed by this treatment at 4 degrees C, suggesting that approximately half of the bound STb was stably associated with the plasma membrane and/or internalized into the cytoplasm. Similar results were obtained when binding and internalization experiments were conducted at 22 and 37 degrees C. Immunofluorescence studies demonstrated that the strongest signal for STb appeared in the plasma membrane even after acid treatment. Toxin-treated cells also displayed diffuse cytoplasmic staining, indicating that once cell bound, STb did not appear to preferentially associate with membrane vesicles or cellular organelles. Binding and subsequent internalization of 125I-STb were not affected by treatment of the cells with trypsin, endoglycosidase F/peptide N-glycosidase F, Vibrio cholerae neuraminidase, tunicamycin, or 5 mM sodium chlorate, which blocks sulfation of surface proteoglycans. In addition, the internalization process was not altered by preincubation of the cells with the cytoskeleton inhibitors cytochalasin D and colchicine or cellular perturbants (i.e., 0.45 M sucrose and 5 mM sodium azide), indicating that cell surface proteins or carbohydrates did not function as STb receptors. The binding of 125I-STb to polarized T84 cells was also examined, and the total and nonspecific binding isotherms were found to overlap, indicating that the apical surface of polarized T84 cells did not contain a specific receptor for STb. In comparison to undifferentiated cells, twice the amount of bound STb (approximately 80% +/- 10%) was removable from polarized T84 cells after treatment with acidic solution. The percentage of surface-bound STb to polarized T84 cells did not vary significantly with the transepithelial electrical resistance of the cells or when STb was applied basolaterally. Together, our results indicate that STb binds with relatively low affinity to the plasma membrane of cultured intestinal epithelial cells and polarized T84 cells, probably to membrane lipids, and becomes stably associated with the lipid bilayer. The fact that a significant portion of the bound STb becomes free in the cytoplasm, even at a low temperature, suggests that the bound toxin may directly traverse the membrane bilayer.     

Retinoic acid metabolism in cultured retinal pigment epithelial cells

PURPOSE: To examine the stability of retinoic acid administered to cultured bovine retinal pigment epithelial (RPE) cells and to determine if RPE cells metabolize retinoic acid by a cytochrome P-450 mechanism. METHODS: Retinoic acid metabolism was examined in cultured RPE cells and subcellular fractions quantitatively by a thin-layer chromatography procedure and qualitatively by normal and reverse phase high-performance liquid chromatography. RESULTS: Cultured bovine RPE cells were found to have an activity that converts retinoic acid into more polar metabolites rapidly released from the cell. The highest specific activity for this process is found in the post-mitochondrial pellet (100,000g), is induced by retinoic acid, and is inhibited by ketoconazole. The major product of the RPE cell-mediated metabolism of retinoic acid is 4-oxo-retinoic acid, a known P-450 monooxygenase product of retinoic acid. The retinoic acid metabolizing activity is greatest in primary RPE cultures and decreases with aging in culture. CONCLUSIONS: These data suggest that a cytochrome P-450 monooxygenase is involved in the metabolism of retinoic acid in RPE cells, and this is similar to the findings of other investigators using other cells and tissues. The authors' findings suggest that the RPE may be important in the deactivation of this biologically potent retinoid in the retina.     

Expression of protein kinase C isoenzymes in cultured hooded rat retinal pigmented epithelial cells: comparison with dystrophic Royal College of Surgeons rat

To our knowledge, prograde access to the left ventricle to perform a left-sided AV node (AVN) ablation has not been reported. We present the case of a patient with Noonan's syndrome and supravalve aortic stenosis who required aortic valve replacement with a mechanical valve. Due to chronic atrial fibrillation, a transvenous pacing system was implanted and transcatheter radiofrequency ablation of the AVN was performed. Attempts at right-sided AVN ablation were unsuccessful. Due to the presence of the mechanical aortic valve, a left-sided AVN ablation was performed successfully using the transseptal approach. The patient died suddenly 6 months after the procedure, and the histologic study of the heart is reported.     

Surface proteins of normal and transformed epithelial liver cells

Surface protein patterns of 12 various tumorigenic and non-tumorigenic liver epithelial cell lines (IAR series) were studied by means of lactoperoxidase catalyzed iodination followed by the gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis. These patterns were found to be very similar except a protein with an apparent mol. wt. about 150 kilodaltons which appeared in cells of tumors derived from in vitro transformed epithelial liver cells. Surface fibronectin was present in all the three non-tumorigenic lines, and in 7 of 9 tumorigenic cell lines examined. The indirect immunofluorescence revealed that in non-tumorigenic lines this protein was localized predominantly in the regions of cell-to-cell contacts, while in tumorigenic cells fibronectin often formed fibrillar networks similar to those of fibroblasts.     

Physical connections between feeder cells and recipient normal mammary epithelial cells

Amphotericin B (AmB) is the most widely used polyene antibiotic to treat systemic fungal infections which affect an increasing number of immunocompromised patients. It is generally thought that AmB forms pores within the fungi membranes by interacting with ergosterol, the main sterol of fungi. However, it also interacts with the cholesterol contained in mammalian cells, hence its toxicity. In order to have a better understanding of the interactions prevailing between AmB and sterols, differential scanning calorimetry was used to study various mixtures incorporating from 6.5 to 25 mol% of AmB in pure dipalmitoylphosphatidylcholine (DPPC) vesicles and in ergosterol- or cholesterol-containing DPPC vesicles. The sterol concentration was kept constant at 12.5 mol% with respect to the phospholipid. Our results show that three phases co-exist when AmB is dispersed in the pure phospholipid. One corresponds to the phospholipid phase alone. The two others are characterised by a broad transition at temperatures higher than the main transition temperature of the pure phospholipid, corresponding to the drug in interaction with the aliphatic chains of the lipid. The fact that the transition temperatures of these additional components are higher than that of the pure phospholipid suggests that AmB interacts strongly with the aliphatic chains of the lipid, consistent with the idea prevailing in the literature that AmB by itself may form pores in a lipid matrix. When AmB interacts with cholesterol-containing bilayers the thermograms also present three components. Upon increasing the concentration of AmB, though, an important broadening of these components is observed which is explained in terms of destabilisation of the organisation of the aliphatic chains. The situation is strikingly different if ergosterol is present in the lipid matrix. The thermograms remain unmodified as the concentration of AmB is increased and a broad transition, now involving only two components when the thermograms are decomposed, is observed. An analysis of the results shows that various interacting units, e.g. AmB+DPPC and (AmB+ergosterol)+DPPC, are present within the membrane. These units involve the phospholipid and hence contribute to its structurisation. The important differences between the thermograms obtained with the ergosterol- as compared to the cholesterol-containing bilayers, in spite of the structural similarity of these two sterols, provides strong evidence for the selectivity of interaction of AmB with ergosterol as compared to cholesterol. It is thus clear that the action of AmB on cholesterol- as compared to ergosterol-containing membranes results from different mechanisms. Finally, UV-visible spectra of AmB in pure as well as sterol-containing DPPC vesicles show the presence of absorption bands that give support to the interpretation derived from the calorimetric data.     

Human mammary cancer cell lines and other epithelial cells cultured as organoid tissue in lenticular pouches of reinforced collagen membranes

Incidence and mortality rates for lung cancer in the United States are significantly greater in blacks than in whites. This disparity cannot be explained by differences in smoking behavior. We hypothesize that the observed racial differences in risk may be due to differences in the metabolic activation or detoxification of the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). To test this, different biomarkers of NNK exposure and metabolism, including the urinary metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and the presumed detoxification product [4-(methylnitrosamino)-1-(3-pyridyl)but-1-yl]-beta-O-D-glucosiduronic acid (NNAL-Gluc), were examined along with questionnaire data on lifestyle habits and diet in a metabolic epidemiological study of 34 black and 27 white healthy smokers. Results demonstrated that urinary NNAL-Gluc:NNAL ratios, a likely indicator of NNAL glucuronidation and detoxification, were significantly greater in whites than in blacks (P < 0.02). In addition, two phenotypes were apparent by probit analysis representing poor (ratio < 6) and extensive (ratio > or = 6) glucuronidation groups. The proportion of blacks falling into the former, potentially high-risk group was significantly greater than that of whites (P < 0.05). The absolute levels of urinary NNAL, NNAL-Gluc, and cotinine were also greater in blacks than in whites when adjusted for the number of cigarettes smoked. None of the observed racial differences could be explained by dissimilarities in exposure or other sociodemographic or dietary factors. Also, it is unlikely that the dissimilarities are due to racial differences in preference for mentholated cigarettes, because chronic administration of menthol to NNK-treated rats did not result in either increases in urinary total NNAL or decreases in NNAL-Gluc:NNAL ratios. Altogether, these results suggest that racial differences in NNAL glucuronidation, a putative detoxification pathway for NNK, may explain in part the observed differences in cancer risk.     

Expression of CD1a monocytes cultured with supernatants from periodontally diseased gingival epithelial cells

MATERIAL AND METHODS: We evaluated the role of color Doppler US-guided compression in the non-invasive treatment of femoral artery pseudoaneurysms after cardiac catheterization, including 22 PTCA procedures. The diagnosis of 32 pseudoaneurysms in 32 patients was accomplished by detection of the typical US-Doppler pattern consisting of the swirling color Doppler flow and the "to and fro" pulsed Doppler waveform at a mean 3.6 days (1 to 14) after the cardiac catheterization. Thirteen patients had multiple cavity pseudoaneurysms (2 to 4). All the patients immediately underwent compression therapy. RESULTS: Treatment was successful in 42/49 cavities (86%) and 25/32 patients (78%), usually after 1 to 3 compression cycles of 6 to 8 minutes duration. Only one recurrence was noted at the 24 hour US-Doppler follow-up. In all cases, pain relief during compression was an excellent clinical sign of hemostatic plug formation and conversion from pseudoaneurysm to simple hematoma. Failures occurred among patients under high dose anticoagulants in spite of 4 to 10 compression cycles. COMMENTARY: In conclusion, color Doppler US-guided compression of post-cardiac catheterization pseudoaneurysms should be the first line therapeutic modality, even in cases of multiple cavities and among patients under effective anticoagulation therapy.     

Calcium-activated potassium conductances on cultured nontransformed dog pancreatic duct epithelial cells

Pancreatic duct epithelial cells (PDECs) mediate the pancreatic secretion of fluid and electrolytes. Membrane K+ channels on these cells regulate intracellular K+ concentration; in combination with the Na+/H+ antiport and Na+,K+ adenosine triphosphatase (ATPase), they may also mediate serosal H+ secretion, balancing luminal HCO3- secretion. We describe the K+ conductances on well-differentiated and functional nontransformed cultured dog PDECs. Through 86Rb+ efflux studies, we demonstrated Ca(2+)-activated K+ channels that were stimulated by A23187, thapsigargin, and 1-ethyl-2-benzimidazolinone, but not forskolin. These conductances also were localized on the basolateral membrane because 86Rb+ efflux was directed toward the serosal compartment. Of the K+ channel blockers, BaCl2, charybdotoxin, clotrimazole, and quinidine, but not 4-aminopyridine, apamin, tetraethylammonium, or iberiotoxin, inhibited 86Rb+ efflux. This efflux was not inhibited by amiloride, ouabain, and bumetanide, inhibitors of the Na+/H+ antiport, the Na+,K(+)-ATPase pump, and the Na+,K+,2Cl- cotransporter, respectively. When apically permeabilized PDEC monolayers were mounted in Ussing chambers with a luminal-to-serosal K+ gradient, A23187 and 1-ethyl-2-benzimidazolinone stimulated a charybdotoxin-sensitive short-circuit current (Isc) increase. Characterization of K+ channels on these cultured PDECs, along with previous identification of Cl- channels (1), further supports the importance of these cells as models for pancreatic duct secretion.     

Eosinophil adhesion to cultured airway epithelial cells from guinea pig lungs

We examined the effects of histamine, tumor necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma), indomethacin and dexamethasone on the adherence of eosinophils to airway epithelial cells. Epithelial cells were isolated from guinea pig trachea through enzymatic digestion, and cultured until they showed confluence. Eosinophils were taken by peritoneal lavage from guinea pigs sensitized with horse serum and purified. The adhesion of eosinophils to cultured epithelial cells was examined using 51Cr. The percentage adhesion was obtained by dividing counts per minute of lysed eosinophils adhered to epithelial cells by suspended eosinophils. Histamine significantly increased the adhesion of eosinophils to epithelial cells, while TNF alpha or IFN gamma did not significantly alter the adhesion. Indomethacin significantly inhibited the adhesion of eosinophils to epithelial cells in both nontreated and histamine-induced conditions. Dexamethasone also reduced adhesion, although it did not reach statistical significance. These findings indicate that histamine induced eosinophil adherence to airway epithelial cells, suggesting a role of endogenous prostaglandins in adherence.     

Bacterial phospholipase C upregulates matrix metalloproteinase expression by cultured epithelial cells

Phospholipase C (PLC) is a putative virulence factor of several pathogenic bacteria. We studied if exogenous PLC would perturb epithelial behavior in infected tissues. Gelatin and casein zymography of cell culture medium indicated that the broad-spectrum PLC of Bacillus cereus induced matrix metalloproteinase (MMP) production in epithelial cells of human skin (NHEK), human gingiva (HGE), and porcine periodontal ligament (PLE). In all three cell types, the strongest increase (ninefold) at 0.1 U/ml was seen in the MMP-9 (92-kDa gelatinase) activity, and the effect was dose dependent in the range of 0.1 to 1.0 U/ml. A relatively weaker increase (twofold) in MMP-2 (72-kDa gelatinase) was also observed in each cell type. PLC induction of MMP-3 (48-kDa stromelysin) was also seen in NHEK and HGE on gelatin and more sensitively for PLE by casein zymography (fivefold). Total gelatinolytic activity as measured by degradation of 14C-labeled denatured type I collagen increased by about 18-fold (NHEK), 12-fold (HGE), and 14-fold (PLE). Northern analysis showed a clear increase in the MMP-9, and a minor increase in MMP-3 mRNA levels but no significant increase in MMP-2 mRNA levels. Further studies with PLE revealed that MMP-9 induction by PLC progressively increased with the length of cell culture time in the absence of serum. PLC induction of MMPs was polar, with MMP-9 and MMP-3 secreted primarily in the apical direction and MMP-2 secreted mainly in the basal direction. The PLC effect was blocked by neomycin, an inhibitor of the phosphoinositol signal pathway. No significant effects were observed in MMP expression with the calcium ionophore A23187 or phospholipase A2. Morphologically, PLC treatment resulted in reduced contacts between the cultured cells and loss of the cell surface microvilli. These results suggest that PLC secreted by bacterial pathogens may disrupt epithelium of infected tissue and increase the subepithelial tissue destruction through induction of MMPs.     

Rapid progesterone actions on thymulin-secreting epithelial cells cultured from rat thymus

Many soluble factors of neural, endocrine, paracrine and autocrine origin are present in the thymus and modulate its function. Long-term effects of sex steroids have been documented for thymocytes and cells of the thymic microenvironment. In this report we examine rapid actions of progesterone upon aspects of epithelial cell physiology. Progesterone (0.1-10 microM) was applied to cultured thymulin-secreting thymic epithelial cells (TS-TEC) and changes in transmembrane potential, transmembrane current, intracellular calcium levels and thymulin secretion were assessed. Rapid changes in electrophysiology and intracellular calcium provide evidence for a membrane-bound progesterone receptor in these cells, in addition to classical cytoplasmic receptors. Application of progesterone to TS-TEC caused electrophysiological changes in 56% of cells (n = 40), activating an inward current (-24 +/- 9 pA at 1 microM, n = 7, p < 0.02) and dose-dependent depolarization (7.1 +/- 1.8 mV at 1 microM, n = 19, p < 0.01). Intracellular calcium levels, monitored by the ratiometric fluorescent calcium indicator fura-2, increased within seconds of progesterone (1 microM) application. Progesterone (1 microM) increased thymulin levels in supernatant, as measured by ELISA, above the levels in the preapplication period (142 +/- 16% of the preapplication period, n = 3, p < 0.02). This effect was reduced in the presence of cobalt chloride which blocks voltage-dependent calcium channels. In addition, TS-TEC in culture were immunoreactive to antibody AG7. This antibody was raised to a membrane-bound antigen involved in calcium influx subsequent to progesterone binding in sperm. Thus we suggest that progesterone acts upon many aspects of TS-TEC physiology through both cytoplasmic and membrane-bound receptors.     

Ultrastructural aspects of the interaction between Escherichia coli and cultured epithelial cells

Microorganisms from two Escherichia coli serotypes were allowed to interact with cultured epithelial cells and the interaction process was followed from 30 to 360 min. Destruction of the microvilli on the epithelial monolayers as well as of the cells themselves was observed only with 0111:H2 serotype.