Effects of liposome dose and the presence of lymphosarcoma cells on blood clearance and tissue distribution of large unilamellar liposomes in mice.

Large unilamellar liposomes (50 to 500 mumol of lipid per kg) were injected i.v. or i.p. into normal and lymphosarcoma-bearing mice. The percentage of the dose remaining in the blood and that accumulated in liver, spleen, and various other organs was measured 4 hr after injection. The results indicate that liposomes cause a dose-dependent saturation of the hepatic and splenic clearance capacities. One day after injection of 10(6) lymphosarcoma cells, the capacity of the tumor-bearing mice to eliminate liposomes from the blood (in a 4-hr period) was inhibited 30 to 50%. This could be ascribed to a decreased activity of the reticuloendothelial system caused by the tumor cells, as was indicated by the simultaneous inhibition of carbon clearance. Six days after injection of the lymphosarcoma cells, the elimination of liposomes from the blood in tumor-bearing mice was restored to the value in normal mice. The possible involvement of tumor cells in the uptake of liposomes by the liver was investigated morphologically after i.v. injection of peroxidase-containing liposomes into lymphosarcoma-bearing mice. Liposome-entrapped peroxidase activity was never observed in the tumor cells. The results presented here indicate that the lymphosarcoma cells do not directly participate in the hepatic accumulation of liposomes, although their mere presence may have significant indirect effects on the elimination of liposomes from the blood and on their tissue distribution.

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.

Plasma membrane specialization and intracellular polarity of freshly isolated rat hepatocytes.

It was investigated whether rat hepatocytes maintain their plasma membrane specialization (sinusoidal, lateral and bile canalicular sites) and their intracellular polarity (peribiliary region, rich in lysosomes and poor in mitochondria) after isolation. The morphology of the hepatocytes and the cytochemical localization of marker enzymes for the bile canalicular membrane (alkaline phosphatase, adenosine triphosphatase and 5' nucleotidase), for the lysosomes (acid phosphatase) and for the mitochondria (beta-hydroxybutyrate dehydrogenase and succinate dehydrogenase) were studied in situ and directly after isolation using both light and electron microscopy. The morphology of the cells and the cytochemical activity of acid phosphatase, succinate dehydrogenase and beta-hydroxybutyrate dehydrogenase showed that in isolated cells, as in situ, the lysosomes were concentrated in bands, devoid of mitochondria. Unlike in situ the reaction product of alkaline phosphatase, adenosine triphosphatase and 5'nucleotidase was evenly distributed along the entire plasma membrane of the isolated cells. Morphologically, no tight or gap junctions or desmosomes could be detected in the isolated cells, while the plasma membrane appeared to be homogeneously covered with uniform microvilli. In conclusion it can be stated that during isolation the hepatocytes loose their distinct plasma membrane specialization, but maintain their peribiliary region rich in lysosomes and poor in mitochondria.

Selenium in in vitamin-E-deficient diets and the occurrence of myopathy as a symptom of vitamin E deficiency.

The occurrence of myopathy in vitamin-E-deficient ducklings, which is used for the determination of the degree of vitamin E deficiency, is depending on the selenium content of the diet. The selenium content of a semi-synthetic diet and its constituents was determined by neutron activation analysis, which showed to be an adequately sensitive and precise method of analysis for selenium. The casein compound was the main source of the selenium in the diet. Myopathy occurred with diets containing about 50 ppb Se; diets containing about 100 ppb Se failed to induce any signs of myopathy.

Changes of relaxation times T1 and T2 in rat tissues after biopsy and fixation.

NMR spectroscopical measurements of relaxation times were conducted on muscle, intestine, fatty tissue and cerebral cortex and white matter of the rat at various time intervals following removal of the tissue. It appeared that most tissues can be stored at 4 degrees C up to 24 hours without noticeable effects on NMR relaxation parameters. Exceptions are the T2 of muscle and the T1 and T2 of intestine, which tended to change in the first hour after biopsy. Relaxation parameters change considerably after fixation of the tissues. Therefore the effects of fixation have to be taken into account when carrying out NMR measurements on fixed tissues.

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.

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.

A rapid method for obtaining monomeric glutaraldehyde.

A new method for the distillation of glutaraldehyde to obtain the monomeric form is presented. The monomer is obtained after only one distillation and it has a purification index (Pi) smaller than 0.20.

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.

Prevention of penetration hindrance in cerium-based glucose-6-phosphatase cytochemistry by freezing tissue in melting nitrogen.

The demonstration of the ultrastructural localization of glucose-6-phosphatase in rat liver is hampered by penetration problems of the medium as appears from ultrathin cross-sections of vibratome sections. Besides the plasma membrane, also the cytoplasm forms a serious barrier for the penetration of the medium constituents. Prolonged preincubation for as long as 48 h at 4 degrees C in the complete incubation medium could not prevent the penetration hindrance. However, when employing 30 micron vibratome sections from tissue blocks that were frozen in melting nitrogen, the penetration hindrance was prevented.

Cytochemical demonstration of phosphatases in the rat liver by a cerium-based method in combination with osmium tetroxide and potassium ferrocyanide postfixation.

Acid phosphatase, alkaline phosphatase, glucose-6-phosphatase, Mg-activated adenosine triphosphatase and 5'nucleotidase were demonstrated in the rat liver using a cerium-based method. This method can be applied routinely and yields better results than the lead-based method. The tissue was postfixed in osmium tetroxide and potassium ferrocyanide which considerably enhances the membrane contrast in comparison with solely osmium tetroxide postfixation. This facilitates the precise localization of the reaction product.

Nutritional myopathy in ducklings: a growth rate-dependent symptom of "tissue peroxidosis' due to a net nutritional shortage of vitamin E plus selenium in skeletal muscle.

Although low dietary vitamin E is an absolute prerequisite for the occurrence of nutritional myopathy in ducklings, it was found that the myopathy can be prevented if the vitamin E depletion does not start before 2 weeks of age and if the dietary selenium content is not too low (greater than 74 ppb). Under these circumstances growth remained normal and the animals stayed alive during the experimental period of 14 weeks, despite low levels of vitamin E in serum, liver, and muscle. The weekly growth rate of the nondeficient duckling, as derived from the growth curve, is extremely high during the first 2 weeks of age with a distinct peak during the 2nd week followed by a steep fall during the 3rd week. In vitamin E-deficient ducklings tissue vitamin E dilution curves were calculated by mathematic transformation of the growth curve. Th results suggest that a critical net nutritional shortage of vitamin E plus selenium in skeletal muscle causes myopathy in ducklings. It is proposed that nutritional myopathy should be considered as a symptom of "tissue peroxidosis' and not as a symptom of vitamin E deficiency per se.

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.

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.

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.

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.

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.