Ce Hulstaert

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

SummaryThe 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 periinsular 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.

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

SummaryAcid 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 contranst in comparison with solely osmium tetroxide postfixation. This facilitates the precize localization of the reaction product.

Interaction of liposomes with Kupffer cells in vitro.

We investigated the interaction of liposomes with rat Kupffer cells in monolayer maintenance culture. The liposomes (large unilamellar vesicles, LUV) were composed of 14C-labelled phosphatidylcholine, cholesterol and phosphatidylserine (molar ratio 4:5:1) and contained either 3H-labelled inulin or 125I-labelled bovine serum albumin as a non-degradable or a degradable aqueous space marker, respectively. After 2-3 days in culture the cells exhibited optimal uptake capacity. The uptake process showed saturation kinetics, maximal uptake values amounting to 2 nmol of total liposomal lipid/h/10(6) cells. This is equivalent to 1500 vesicles per cell. The presence of fetal calf serum (FCS) during incubation increased uptake nearly two-fold, whereas freshly isolated rat serum had no effect. The binding of the liposomes to the cells caused partial release of liposomal contents (about 15-20%) both at 4 degrees C and at 37 degrees C. In the presence of metabolic inhibitors the uptake at 37 degrees C was reduced to about 20% of the control values. Inulin and lipid label became cell-associated at similar rates and extents, whereas the association of albumin label gradually decreased after attaining a maximum at relatively low values. When, after 1 h incubation, the liposomes were removed continued incubation for another 2 h in absence of liposomes led to an approx. 30% release of cell-associated lipid label into the medium in water-soluble form. Under identical conditions as much as 90% of the cell-associated albumin label was released in acid-soluble form. Contrarily, the inulin label remained firmly cell-associated under these conditions. From these results we conclude that Kupffer cells in monolayer culture take up liposomes primarily by way of an adsorptive endocytic mechanism. This conclusion was confirmed by morphological observations on cells incubated with liposomes containing fluorescein isothiocyanate (FITC) dextran or horseradish peroxidase as markers for fluorescence microscopy and electron microscopy, respectively.

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.

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.

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

SummaryThe 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°C in the complete incubation medium could not prevent the penetration hindrance. However, when employing 30 μm vibratome sections from tissue blocks that were frozen in melting nitrogen, the penertration hindrance was prevented.

The cerium perhydroxide-diaminobenzidine (Ce-H2O2-DAB) procedure

SummaryNew 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°–60° 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

SummaryLead is prevalently replaced by cerium as trapping agent in phosphatase cytochemistry to prevent nonspecific precipitation. Recently, substrate specific but artefactual lcad 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.

Survey of the occurrence of adenosine polyphosphatase in extracellular matrix of rat tissues

SummaryThe 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.

THE EFFECT OF DOXYCYCLINE ON POLYVINYLPYRROLIDONE-INDUCED GRANULOMA-FORMATION IN THE RAT-LIVER

SummaryThe tetracyclines specifically inhibit mitochondrial protein synthesis when present at the same low concentrations as used for their antibacterial action. Inhibition of mitochondrial protein synthesis leads to decrease in the oxidative energy-generating capacity of cells. Therefore, the presence of tetracyclines may result in proliferation arrest.In the present study we show that continuous intravenous administration of polyvinylpyrrolidone (PVP) induces the formation of granulomas in the normal rat liver; the rats usually die within 2 weeks of continuous PVP treatment. Athymic (nude) rats appear to be more résistent to the deleterious effects of PVP as they survivce the treatment for at least 5 weeks. Although the livers of the PVP-treated nude rats are heavily infiltrated with phagocytic cells, they seldom show granulomas. Reconstitution of nude rats with syngenic thymocytes leads, on the other hand, to extensive granuloma formation. Normal rats treated continuously with PVP plus doxycycline, however, all survive, their livers showing only a few very small granulomas and the normal low number of phagocytic cells.We conclude that the formation of granulomas induced by PVP is a process which is mediated by T-lymphocytes. Because doxycycline prevents this kind of granuloma formation it seems likely that doxycycline not only impairs the proliferation and differentiation of T-lymphocytes but also of monocytes and macrophages.