Differential metabolism and trafficking of sphingolipids in differentiated versus undifferentiated HT29 cells.

Trafficking and metabolism of sphingolipids were examined in undifferentiated (G+) and differentiated (G+ reversed) HT29 human colon adenocarcinoma cell lines. Metabolic experiments employing a fluorescently labeled sphingolipid precursor, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylceramide++ + (C6-NBD-ceramide) revealed that both qualitative and quantitative differences exist in sphingolipid synthesis between the 2 cell lines. One of the C6-NBD-sphingolipids synthesized in G+ cells is not found in the G+ reversed cells. Furthermore, the ratio of the 2 main products, C6-NBD-glucosylceramide and C6-NBD-sphingomyelin, differs: in G+ cells glucosylceramide is by far the main product, whereas G+ reversed cells synthesize C6-NBD-sphingomyelin in slight excess. Once established, these ratios of sphingolipids are quickly restored metabolically when distortion of the ratio is caused by experimental manipulation. This indicates that they represent a true metabolic equilibrium situation of the 2 sphingolipids in these cells, while the distinct ratios are mainly determined by the NBD-lipid pool in the plasma membrane. Preferential synthesis and transfer of glucosylceramide from its site of synthesis to the cell surface do not occur when the plasma membrane pool of glucosylceramide is selectively removed. This suggests that instantaneous replenishment via specific signalling is probably not involved as a mechanism in re-establishing perturbed lipid pools. In conjunction with observations on distinct lipid trafficking pathways of glucosylceramide in G+ and G+ reversed cells, the present metabolic studies emphasize a relation between the expression of this glycolipid and the state of differentiation of HT29 cells.

Our approach towards developing a specific tumour-targeted MRI contrast agent for the brain.

This review presents various aspects of the technological development, and their assessment in the design of a contrast agent for MRI, tailored to visualise tumours in the brain. First, it was demonstrated that magnetite as a contrast agent exhibited a much stronger relaxivity than gadolinium. The prepared magnetite particles bound to dextran, were also shown to be of appropriate size by electron microscopy. After their intravenous injection into rats with blood-brain barrier disruption, the lesion was strongly enhanced by T2-shortening. Furthermore, monoclonal antibodies directed against small cell lung carcinoma, proved to be able to penetrate into tumours, which had been raised by implantation of the small cell lung carcinoma cells into the brains of nude rats. As to the essential step, it was demonstrated in vitro that magnetite particles coupled to monoclonal antibodies by the biotin-streptavidin binding, could be bound to the target cells of the antibody, changing the relaxation rates of the latter. Finally it could be shown in vitro that an alternative approach, using lymphocytes to be targeted to tumour cells, also proved feasible, in that these lymphocytes could be labelled with magnetite that had been incorporated into liposomes. Further developments will be the in vivo assessment of the acquired progress in experimental animals, before clinical application is warranted.

Ultrastructural changes of the basement membrane zone in benign lesions of the vocal folds.

The basement membrane zone (BMZ) of the epithelium of the vocal folds was investigated electron microscopically in 10 patients suffering from various benign lesions and in 3 controls. Various defects were observed: a thickening by deposition of electron dense material, a loss of normal architecture, and a near absence of normal hemidesmosomes and anchoring fibers. Beside these previously reported phenomena, many vesicles carrying electron dense material were found near the plasma membrane. The vesicles were observed at various stages of fusion with the plasma membrane, on the other side of which their content was discharged. In the cytoplasm an increase of mitochondria was seen. The amount of condensed chromatin decreased while the nucleoli increased in comparison with the controls. These observations are suggestive of a hyperactivity of the basal cells of the epithelium in response to vibratory stress.

Selective MR imaging of labeled human peripheral blood mononuclear cells by liposome mediated incorporation of dextran-magnetite particles.

Human peripheral blood mononuclear cells (PBMCs) were incubated with large unilamellar vesicles (LUV) containing encapsulated dextran-magnetite particles (DMP). This resulted in an efficient incorporation of DMP. Electron microscopy revealed the presence of DMP in cells mainly in phagosomes and secondary lysosomes. DMP-labeled PBMCs showed a strong increase of the transverse relaxation rate (up to 16.6 s-1 for 5 x 10(7) cells/ml) and, accordingly, a great loss of signal intensity in MR imaging. The fraction of DMP containing PBMCs could be enriched by magnetic cell separation. The major population of the DMP containing cells proved to be monocytes. When PBMCs depleted of monocytes were used for labeling, DMP uptake was observed also in the peripheral blood lymphocytes. The labeling of PBMCs presented here may be used in future studies of selective MR imaging of in vivo cell migration in a variety of immunologically compromised tissue states, e.g., tumors, transplantations, and abscesses.

Heterogeneity of rat liver and spleen macrophages in gadolinium chloride-induced elimination and repopulation.

Blockade of phagocytosis and selective elimination of macrophages (m phi s) are generally accepted procedures for gaining knowledge about the function of m phi s in vivo. This study demonstrates that intravenous injection of gadolinium chloride (GdCl3) not only blocks phagocytosis by rat liver m phi s (Kupffer cells) but also selectively eliminates the large m phi s situated in the periportal zone of the liver acinus. Repopulation of m phi s starts at 4 days after injection. During repopulation, m phi s are less vulnerable to GdCl3. When repopulation is complete, the new m phi s show the same vulnerability as the original ones. Splenic m phi s are less vulnerable to GdCl3 because only some of the red pulp m phi s transiently disappear. The white pulp m phi s are not affected. Repopulation occurs sooner than in liver. These results indicate that administration of GdCl3 is a suitable approach to studying the in vivo function of large Kupffer cells.

Electron microscopical demonstration of alkaline phosphatase activity with the cerium-based method in citrate-containing medium at pH 9.3 and the influence of glutaraldehyde fixation.

A cerium-based incubation medium, developed for the light microscopical demonstration of alkaline phosphatase activity, was tried out for the electron microscopical demonstration of this enzyme in kidney and heart muscle of the rat. The medium is very stable and the pH is in the optimum range of the enzyme. The medium consists of 14 mM CeCl3, 11 mM Na-citrate, 4 mM MgCl2, 10 mM p-nitrophenyl phosphate, 0.18 M glycine/NaOH buffer, pH 9.3. Other concentrations of cerium and citrate were tried out as well but 14 mM CeCl3, and 11 mM Na-citrate gave the best results with a small amount of non-specific reaction product in the nucleus that can be largely avoided by postincubation rinsing in cerium-containing buffer. In the kidney reaction product was only present along the microvilli of the proximal tubular epithelial cells. In the glomerulus no reaction product could be found whereas light microscopical cryotome sections contained activity in the glomerulus. Replacement of glutaraldehyde by formaldehyde fixatives resulted in reaction product in glomerular and tubular basement membranes, on podocyte plasma membranes and in tubular basal infoldings. In glutaraldehyde-fixed heart muscle, reaction product was present in the basement membranes and on lateral plasma membranes of endothelial cells of blood capillaries.

Specific MR imaging of human lymphocytes by monoclonal antibody-guided dextran-magnetite particles.

Human lymphocytes were labeled with biotinylated anti-lymphocyte-directed monoclonal antibodies, to which streptavidin and subsequently biotinylated dextran-magnetite particles were coupled. This labeling resulted in a strong and selective negative contrast enhancement of lymphocyte suspensions at 2.0 T, caused predominantly by the specific increase of R2 with a small but significant specific increase of R1. The R1 was found to decrease with increasing field strength. The immunolabeling procedure described here may be used for the selective signal depletion of target cells in MR imaging.

Significance of the peri-insular extracellular matrix for islet isolation from the pancreas of rat, dog, pig, and man.

The presence and distribution in the peri-insular region of extracellular matrix, and in particular basement membrane, was investigated in a comparative study comprising pancreata of rat, dog, pig, and man. Basement membrane markers, collagen type-IV and laminin, were determined immunohistochemically. Additional information pertaining to the structural relationships between endocrine and exocrine pancreas, in particular cell-to-cell and cell-to-matrix contacts, was obtained by electron microscopy. In pig, very little peri-insular capsule is present, and the structural integration of the porcine islet in the exocrine pancreas almost exclusively depends on cell-to-cell adhesion. In the canine pancreas, the islets are almost completely encapsulated with very little direct exocrine-to-endocrine cell-to-cell contact. In rat and man, the situation is intermediate with a tendency towards predominance of cell-to-matrix adhesion. The intra-insular adhesion mechanisms depend largely on cell-to-cell adhesion in all four species. The ultrastructural results suggest that collagenase preparations employed in islet isolation procedures should be of high purity as to preserve the protease-sensitive intra-islet cell-to-cell adhesion. Under these conditions, however, the endocrine-to-exocrine cell-to-cell contacts will be conserved also, resulting in an exocrine-tissue contamination of the islets of Langerhans. Consequently, additional steps for the effective removal of exocrine tissue and the purification of islets are required.

Cerium as amplifying agent--an improved cerium-perhydroxide-DAB-nickel (Ce/Ce-H2O2-DAB-Ni) method for the visualization of cerium phosphate in resin sections.

A new visualization (Ce/Ce-H2O2-DAB-Ni) procedure for cerium (Ce III) phosphate in semithin and ultrathin plastic sections (Epon 812, Lowicryl K4M, glycol methacrylate) of rat kidney tissues that had been incubated before embedding for the demonstration of phosphatases (alkaline and acid phosphatase, 5(1)-nucleotidase, Mg-dependent ATPase) is described. For this purpose the hydrophobic Epon resin was removed in NaOH-ethanol solution, whereas the hydrophilic Lowicryl and methacrylate sections did not required any etching. The primary reaction product Ce III-phosphate was amplified in a Ce III-citrate solution, subsequently oxidized with H2O2 and then visualized in a H2O2 containing DAB-nickel medium (Ce IV-perhydroxy induced DAB polymerization principle). The method yielded a very clear localization of enzyme activity. The final reaction product (DAB-nickel polymers) in 0.5 - 2.0 microns semithin sections is blue-black; the background staining is completely prevented. An increase of the staining contrast was obtained by posttreatment with OsO4 (osmium black formation). Furthermore, the enzyme reaction product could be demonstrated in 40 nm thick ultrathin sections by silver intensification, which utilized the high argyrophilia of the polymerized DAB-nickel complexes. This procedure replaces the earlier published technique.

Survey of the occurrence of adenosine polyphosphatase in extracellular matrix of rat tissues. A cytochemical investigation.

The extracellular presence of adenosine polyphosphatase was investigated in a number of rat tissues. The enzyme was demonstrated in basement membranes of epithelial cells of duodenum, urinary bladder, tongue, choroid plexus, submandibular salivary gland, lung and kidney, as well as in basement membranes of capillaries in these tissues. Furthermore adenosine polyphosphatase was demonstrated on collagen fibrils and in the cytoplasm of fibroblasts of all investigated tissues. It appears that the presence of adenosine polyphosphatase in basement membranes is a widespread phenomenon. Since extracellular ADP and ATP are known to promote respectively platelet aggregation and inflammation, the presence of extracellular ADP and ATP-hydrolyzing activity might contribute to inhibit these processes.

Light microscopical visualization of phosphatase activities demonstrated with the cerium method in resin sections of the kidney with the microwave oven.

The light microscopically invisible reaction product cerium phosphate in resin sections of rat kidney, that had been incubated for the demonstration of phosphatase activities before embedding, was converted into a visible reaction product by incubation for 10 min at 80 degrees C in alkaline lead citrate in a microwave oven. This method offers the possibility to study phosphatase activities with the cerium method in semithin Epon sections. Furthermore it is a suitable method to select areas with phosphatase activity to be studied with the electron microscope.

The cerium perhydroxide-diaminobenzidine (Ce-H2O2-DAB) procedure. New methods for light microscopic phosphatase histochemistry and immunohistochemistry.

New light microscopic visualization methods were developed for the histochemical detection of non-specific alkaline and acid phosphatase, Mg-, Ca- and Na, K-dependent adenosine triphosphatase, myosin adenosine triphosphatase, glucose-6-phosphatase, 5'-nucleotidase and thiamine pyrophosphatase with cerium ions as trapping agents in cryostat and plastic sections. The techniques are based on the conversion of cerium phosphate into cerium perhydroxide by H2O2 which decomposes at 55 degrees-60 degrees C into cerium hydroxide and oxygen radicals. These radicals are able to oxidize diaminobenzidine (DAB) to DAB brown. Addition of nickel ions to the DAB-H2O2 mixture generates bluish-black stained nickel-DAB complexes. Compared with the classical metal precipitation, azo, azoindoxyl and tetrazolium procedures the H2O2-DAB and especially the H2O2-DAB-nickel methods provided identical or superior results in catalytic phosphatase histochemistry and immunohistochemistry when using non-specific alkaline phosphatase as the enzyme label.

Decreased ATPase activity in adriamycin nephrosis is independent of proteinuria.

In previous studies from this laboratory it has been shown that ATP-ase activity in situ in the glomerular basement membrane (GBM) is clearly reduced in rats rendered nephrotic after treatment with adriamycin (ADR). The question was raised whether this reduction of ATP-ase activity in the GBM is due to toxic activity of ADR or rather a result of the nephrotic condition per se. Therefore, we studied ATP-ase activity using the cerium-based method in kidneys from ADR-treated rats without proteinuria (48 hr after ADR injection), or with proteinuria (approximately 150 mg/24 hr) several weeks after ADR injection. Also kidneys from rats rendered nephrotic by surgical ablation and from non-nephrotic rats treated with local X-irradiation (2000 rads) as well as from normal control rats were studied. The results show that in the GBM of ADR-treated or irradiated rats, clear reduction of ATP-ase activity is observed irrespective of their proteinuria, whereas in the GBM of rats rendered nephrotic by renal ablation (approximately 156 mg/24 hr mean protein excretion) no reduction of enzyme activity is found. It is concluded that decreased ATP-ase activity of the glomerular filtration barrier in ADR-treated rats is due to an early toxic activity of this drug and not a result of the nephrotic state per se. In view of the identical results in X-irradiated rats, it is likely that ADR may act through production of toxic radicals leading to damage of this membrane-associated enzyme system.

Antithrombotic activity of glomerular adenosine diphosphatase in the glomerular basement membrane of the rat kidney.

Activity of nucleoside polyphosphatases (including adenosine diphosphatase [ADPase]) in the glomerular basement membrane (GBM) of the rat kidney can be demonstrated in situ by using cytochemical methods at the ultrastructural level. To study the possible influence of glomerular ADPase activity on experimentally induced intraglomerular platelet aggregation, we carried out alternate perfusion experiments with human platelets and adenosine diphosphate (ADP) solution in rat kidneys ex vivo. This was done in rats with reduced glomerular phosphatase activity induced by either an intravenous injection of doxorubicin (8.5 mg/kg body weight) or local x-irradiation (2000 rads) as well as in rats with normal glomerular enzyme activity, that is, untreated rats or rats injected intravenously with aminonucleoside of puromycin (PAN) (15 mg/kg body weight). It is shown that in kidneys of both doxorubicin-treated and x-irradiated rats intraglomerular platelet aggregation occurs in approximately 50% of the glomeruli, whereas in PAN-treated or control rats no platelet aggregation could be detected by light microscopy. Activated platelets (by electron microscopy) and beta-thromboglobulin or platelet factor 4 (immunofluorescence microscopy) could be detected with appropriate fluorescinated antibodies along the GBM exclusively in kidneys with reduced ADPase activity caused by doxorubicin or x-irradiation treatment. Because glomerular ADPase activity in contrast to other putative antithrombotic molecules in the GBM, that is, heparan sulfate proteoglycans, is clearly affected by doxorubicin or x-irradiation treatment, it is suggested that the activity of glomerular ADPase may reflect an important antithrombotic principle in the GBM of the rat kidney.

Interaction of immunoglobulin-coupled liposomes with rat liver macrophages in vitro.

The interaction between liposomes coated with covalently linked rabbit immunoglobulin (RbIg-liposomes), and rat liver macrophages (Kupffer cells) in monolayer culture was studied biochemically with radioactive tracers and morphologically by electron microscopy. The attachment of immunoglobulin (Ig) to liposomes caused a five-fold increase in liposome uptake by the Kupffer cells at 37 degrees C, in comparison with uncoated liposomes. The uptake was linear with time for at least 4 h and linear with liposome concentration up to a lipid concentration of 0.2 mM. At 4 degrees C uptake, probably representing cell surface-bound liposomes, was reduced to a level of approx. 20% of the 37 degrees C values. Involvement of the Fc receptor in the uptake process was indicated by the reduction of RbIg-liposome uptake by more than 75% as a result of preincubating the cells with heat-aggregated human or rabbit Ig at concentrations (less than 2 mg/ml) at which bovine serum albumin (BSA) had virtually no effect on uptake. At high concentrations (10-35 mg/ml), however, albumin also reduced liposome uptake significantly (20-30%), which suggests an interaction of the RbIg-liposomes with the Kupffer cells that is partially non-specific. RbIg-liposome uptake was dependent on the amount of RbIg coupled to the liposomes. Maximal uptake values were reached at about 200 micrograms RbIg/mumol liposomal lipid. Electron microscopic observations on cells incubated with horseradish peroxidase-containing RbIg-liposomes demonstrated massive accumulation of peroxidase reaction product in intracellular vacuoles, showing that the uptake observed by label association represents true internalization.

Phosphatase cytochemistry with cerium as trapping agent. Verification of acid phosphatase and glucose-6-phosphatase reactive sites.

Lead is prevalently replaced by cerium as trapping agent in phosphatase cytochemistry to prevent non-specific precipitation. Recently, substrate specific but artefactual lead precipitates have been described in the nuclear envelope (NE) and rough endoplasmic reticulum (RER) due to a local matrix effect. In the present study a verification was carried out of the localization of acid phosphatase and glucose-6-phosphatase in the NE and RER of rat peritoneal macrophages and hepatocytes respectively with cerium. It appeared that precipitates of cerium phosphate in NE and RER of peritoneal macrophages do not represent sites of acid phosphatase activity but are due to the matrix effect. However, in rat hepatocytes these organelles demonstrate true reactive sites for glucose-6-phosphatase.

Cell surface characteristics of coagulase-negative staphylococci and their adherence to fluorinated poly(ethylenepropylene).

The ability of 21 nonencapsulated and 15 encapsulated coagulase-negative staphylococci (CNS) to adhere to xylene in xylene-water emulsions and to fluorinated poly(ethylenepropylene) (FEP) films revealed remarkable differences. Nonencapsulated CNS strains adhered well to FEP, whereas their adherence to xylene ranged widely. Encapsulated strains with low adherence to xylene showed slight adherence to FEP. Encapsulated strains which adhered well to xylene ranged widely in their adherence to FEP. It was concluded that results obtained from the xylene adherence test were not predictive of the adherence of CNS to the hydrophobic FEP surface. The number of nonwashed, slime-producing CNS strains adhering to FEP was similar to that of washed bacteria of the same strains. Bacterial adherence to FEP was decreased when FEP films were exposed to a solution containing extracellular products (EP) obtained from a slime-producing CNS strain. Bacterial adherence to xylene also decreased when the bacterial suspensions contained EP. Apparently, initial adherence of CNS to FEP and xylene is hampered by EP. Nonencapsulated and encapsulated CNS pretreated with proteolytic enzymes failed to adhere to xylene and FEP, indicating that intact surface proteins or constituents associated with surface proteins mediated their adherence to xylene and FEP. Freeze-etch replicas of a CNS strain adhering to FEP showed a smooth, flattened area on the bacterial surface at the contact site of the bacteria with the FEP, indicating that an external layer was present at the bacterial surface.

Graft-versus-host disease in the rat: cellular changes and major histocompatibility complex antigen expression in the liver.

Cellular changes in the liver were studied during an acute lethal graft-versus-host (GVH) disease in relation to the expression of major histocompatibility complex (MHC) antigens on different liver cells. Screening for MHC antigen expression revealed that control livers contained very few Ia+ cells: mainly cells in the portal tract interstitium and a small percentage of the Kupffer cells. The changes during an ongoing GVH reaction could be separated into those related to the sinusoid-associated cells, including the liver parenchyma, and those related to the portal-tract-associated cells, including periportal hepatocytes. In the sinusoids an increase in the number of Kupffer cells was seen, now all expressing Ia antigens. No damage to hepatocytes or other sinusoid-associated cells was observed. It is postulated that the increase in both number and Ia expression of the Kupffer cells is most probably due to an increased phagocytic uptake of blood-borne cellular debris and is not a result of extensive damage to hepatocytes. In the portal tracts expanding infiltrates were found composed of Ia+ T cells and macrophages (ratio 2:1). These infiltrates are probably due to a local accumulation of lymphocytes and macrophages as a result of an interaction of migrating donor-type alloreactive T cells with recipient type Ia+ cells present in the portal tract interstitium, which also interfered with normal recipient lymphocyte and macrophage traffic. Damage to portal-tract-associated cells was slight and confined to bile duct epithelial cells, which now expressed Ia antigens, and to periportal hepatocytes. In conclusion, these data do not indicate that damage to liver parenchyma plays a major role in the pathogenesis of an acute GVH reaction.