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M J Hardonk

Publications and source records attributed to M J Hardonk.

At least 55 records · Page 3Linked to original sources
PubMed1989

Specific demonstration of ribonucleic acid by chemical bromination and immunohistochemistry.

In this report we describe a specific staining procedure for detection of ribonucleic acid (RNA), based on bromination of uracil and subsequent immunohistochemical visualization of 5-bromouracil in RNA. This method is applicable for both cryostat and glycol methacrylate (GMA)-embedded sections. Cryostat sections must be fixed in formaldehyde, whereas tissue pieces to be embedded in GMA are fixed in cold acetone. Before bromination, sections must be treated with trypsin. Bromination was performed in a solution of bromine in potassium bromide. After bromination, excess bromine was removed with sodium bisulfite. The monoclonal antibody MoBu-1 specifically bound to brominated RNA. Ribonuclease digestion, in contrast to deoxyribonuclease digestion, abolished staining. This method makes possible precise localization of RNA, especially well demonstrated in plastic-embedded sections.

Animals↗
PubMed1989

Cerium-based demonstration of phosphatase activity in plastic-embedded sections: a comparison with conventional methods.

Cerium-based methods have been used for the demonstration of several phosphatases at alkaline, acid and neutral pH in low temperature acetone-fixed, plastic-embedded sections. At alkaline pH calcium is used as capturing agent and the precipitated calcium phosphate converted to cerium phosphate. At neutral and acid pH cerium is used directly as capturing agent. Cerium phosphate is subsequently visualized using the H2O2-DAB method. A comparison has been made with conventional calcium-cobalt and lead methods. It appeared that calcium-cobalt methods are more susceptible to improvement than lead methods.

5'-Nucleotidase↗
PubMed1989

Experimental endotoxemia in pregnancy: in situ glomerular microthrombus formation associated with impaired glomerular adenosine diphosphatase activity.

The mechanism of increased sensitivity for endotoxin in pregnancy as reflected by the formation of microthrombi in renal glomeruli is unknown. It has been shown that reduced glomerular diphosphatase (ADPase) activity in the rat kidney greatly increases the intraglomerular thrombotic tendency. We now studied experimental intraglomerular thrombosis ex vivo in association with glomerular ADPase activity in pregnant and nonpregnant control rats after infusion of either endotoxin or saline solution. Each animal (Wistar rat) was equipped with a permanent vena jugularis catheter and received either endotoxin (1.0 micrograms/kg body weight) (n = 6) or saline solution (n = 5) 7 days before being killed; nonpregnant rats were also treated with endotoxin (n = 5) or saline solution (n = 4). On day 21, before the animals were to be killed, they were anesthetized and their left kidneys were perfused with adenosine diphosphate solution (10 micrograms/ml) and platelet-rich plasma (1 x 10(9) cells/ml). Perfused kidneys were processed for light microscopy, electron microscopy, enzyme cytochemistry at the ultrastructural level, and immunohistology. The results showed decreased ADPase activity exclusively in the glomerular basement membrane of kidneys of pregnant rats treated with endotoxin in contrast to the findings in control rats. In addition, exclusively in the group of endotoxin-treated pregnant rats, significantly increased intraglomerular platelet aggregation could be detected after alternate perfusion ex vivo. We suggest that, in the present model, enhanced susceptibility of glomerular ADPase for endotoxin is due to pregnancy-associated factors that have yet to be identified. This increased susceptibility may promote in situ formation of intraglomerular microthrombi.

Animals↗
PubMed1988

Separate transport systems for biliary secretion of sulfated and unsulfated bile acids in the rat.

Biliary secretion of 3 alpha-sulfated bile acids has been studied in Wistar rats with an autosomal recessive defect in the hepatic transport of bilirubin. Liver function, established by measurement of various enzymes in plasma, by enzyme histochemical methods, and by electron microscopy, appeared to be normal in these rats. Serum levels of unconjugated, monoglucuronidated, and diglucuronidated bilirubin were 0.62, 1.62, and 6.16 mumol/liter, respectively, compared with 0.17, 0.08, and 0.02 mumol/liter in control rats. Biliary bilirubin secretion was strongly reduced in the mutant animals: 0.21 +/- 0.03 vs. 0.39 +/- 0.03 nmol/min per 100 g body wt in control rats. Despite normal biliary bile acid output, bile flow was markedly impaired in the mutant animals, due to a 53% reduction of the bile acid-independent fraction of bile flow. The transport maximum for biliary secretion of dibromosulphthalein (DBSP) was also drastically reduced (-53%). Biliary secretion of intravenously administered trace amounts of the 3 alpha-sulfate esters of 14C-labeled taurocholic acid (-14%), taurochenodeoxycholic acid (-39%), taurolithocholic acid (-73%), and glycolithocholic acid (-91%) was impaired in the jaundiced rats compared with controls, in contrast to the biliary secretion of the unsulfated parent compounds. Hepatic uptake of sulfated glycolithocholic acid was not affected in the jaundiced animals. Preadministration of DBSP (15 mumol/100 g body wt) to normal Wistar rats significantly impaired the biliary secretion of sulfated glycolithocholic acid, but did not affect taurocholic acid secretion. We conclude that separate transport systems in the rat liver exist for biliary secretion of sulfated and unsulfated bile acids; the sulfates probably share secretory pathways with the organic anions bilirubin and DBSP. The described genetic defect in hepatic transport function is associated with a reduced capacity to secrete sulfated bile acids into bile; this becomes more pronounced with a decreasing number of hydroxyl groups on the sulfated bile acid's molecule.

Animals↗
PubMed1988

Double immunohistochemical demonstration of antigen expression and DNA-incorporated 5-bromodeoxyuridine in frozen and plastic embedded sections.

In this study a double immunohistochemical staining procedure is described for the simultaneous visualization of antigen expressing cells and replicating cells. Cell surface antigen expression was marked with a monoclonal antibody against I a (His 19) or a monoclonal antibody against a membrane component of the cells of the monocyte-macrophage lineage (ED2). Replicating cells were detected by the incorporation of 5-bromodeoxyuridine. The method was applied sequentially. On frozen sections two peroxidase labeled reagents were used with two different substrates yielding a red and a dark-blue black reaction product. On plastic-embedded sections a peroxidase and an alkaline phosphatase labeled reagent were applied resulting in a brown and a blue reaction product.

Animals↗
PubMed1987

Simultaneous immunohistochemical demonstration of antigen expression and 5-bromodeoxyuridine incorporation in plastic embedded sections.

In this study a double immunohistochemical staining procedure is described for the simultaneous demonstration of antigen expressing cells and replicating cells in rat thymus. As markers for cell surface antigen expression a monoclonal antibody against Ia-expressing cells (His 19) and a monoclonal antibody against cells of the monocyte-macrophage lineage (ED2) were used. Replicating cells were demonstrated by the incorporation of 5-bromodeoxyuridine (BrdUrd). Tissue pieces were fixed in a periodate-lysine-paraformaldehyde fixative and embedded in glycol methacrylate. To demonstrate Ia-expressing cells or ED2-positive macrophages in plastic embedded sections a digestion with trypsin is necessary. The staining procedure was applied sequentially and was performed with a peroxidase and an alkaline phosphatase labeled reagent yielding respectively a brown and a blue reaction product. Results with this staining procedure on plastic embedded sections of rat thymus, an organ with a high DNA synthesizing capacity, showed incorporation of BrdUrd predominantly in the cortex. ED2-positive macrophages were only found in the cortex. The Ia-positive epithelial reticular cells demonstrated extremely well their stellate form.

Animals↗
PubMed1987

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.

Adenosine Triphosphatases↗
PubMed1987

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.

Adenosine Diphosphate↗
PubMed1986

Immunohistochemical demonstration of DNA-incorporated 5-bromodeoxyuridine in frozen and plastic embedded sections.

The application of an immunohistochemical method in the detection of replicating cells, that have incorporated 5-bromodeoxyuridine (BrdUrd), was studied on frozen and plastic embedded sections of different rat tissues. Hydrolysis conditions employed in the Feulgen procedure are essential in making the incorporated BrdUrd accessible to the monoclonal anti-BrdUrd antibodies. To demonstrate the incorporated BrdUrd in plastic embedded sections a subsequent etching with xylene and digestion with protease is necessary. Data obtained with this method are completely comparable with those found by the tritiated thymidine method. In comparison with the thymidine method, the BrdUrd method is much less time consuming and does not require precautions in working with radioactivity. The BrdUrd-method enables a more precise localization as is especially shown in the plastic embedded sections.

Animals↗
PubMed1986

Immunohistochemical demonstration of FC receptors in rat tissues using immune complexes as ligand.

To demonstrate the presence and localization of Fc receptors, rat liver cryostat sections were incubated with heterologous and autologous immune complexes (ICx) and immunoglobulin (Ig) aggregates. Binding was demonstrated using the immunoperoxidase technique. Autologous and heterologous ICx as well as aggregates from human and rat Ig appeared to bind to the sinusoidal wall. ICx bind in preference to aggregates. Monomeric Ig and aggregated Ig from swine and rabbit did not bind. The results demonstrated that ICx and rat and human Ig aggregates were bound via an Fc receptor. This Fc receptor was still intact in livers from carbontetra chloride and galactosamine treated rats. The receptor could also be demonstrated on spleen macrophages and on kidney interstitial cells. This method turned out to be an useful functional histochemical method to localize Fc receptors and to demonstrate their affinity and species specificity in tissues.

Animals↗
PubMed1986

The effect of doxycycline on polyvinylpyrrolidone-induced granuloma formation in the rat liver.

The 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 resistent to the deleterious effects of PVP as they survive 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.

Animals↗
PubMed1986

Evidence for a migratory capability of rat Kupffer cells to portal tracts and hepatic lymph nodes.

The present study concerns the migratory ability of Kupffer cells in the rat. Phagocytic cells were labeled with colloidal carbon or gold, these markers being administered intravenously either into a tail vein, which resulted in generalized reticuloendothelial uptake, or in low dose into the portal vein, which produced uptake by Kupffer cells alone. Cells containing marker were observed in the portal tracts and in hepatic lymph nodes from 1 to 3 days after injection into the portal vein. The direct movement of single marker particles to the portal tracts could be excluded. Since injection of marker into the portal vein labeled Kupffer cells exclusively, whereas blood cells, splenic and bone marrow macrophages remained unlabeled, the labeled cells in the portal tracts and hepatic lymph nodes appeared to be former Kupffer cells migrating which had migrated to these sites.

Animals↗
PubMed1986

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.

Acute Disease↗
PubMed1986

Studies on the mechanism of binding and uptake of immune complexes by various cell types of rat liver in vivo.

Soluble heterologous immune complexes (IC) were used to study the mechanism of IC binding to rat liver in vivo. Binding of IC to the various cell types of the liver, endothelial cells, hepatocytes, and Kupffer cells, was only inhibited by aggregated swine immunoglobulins. Binding was not inhibited by the absence of complement components. Intravenous injection of asialoglycoproteins, to block the galactose receptor, could not prevent IC binding. We conclude that Fc receptors play an important role in the binding of soluble heterologous IC to hepatocytes, endothelial cells, and Kupffer cells.

Animals↗
PubMed1986

Hepatic uptake of autologous immune complexes in the rat.

The hepatic uptake of autologous immune complexes (ICx) was studied in Wistar rats immunized with bovine serum albumin (BSA). After attaining a suitable serum titre of rat anti-BSA Ig (RatABSA) they were injected intravenously with 2 mg BSA. The antibody levels were measured with an ELISA and the rate of disappearance of BSA from the circulation was studied by labelling the antigen with FITC. At various times after injection of BSA the uptake of ICx in the different types of liver cells was studied with the immunoperoxidase technique at the light microscope and ultrastructural levels. Within 5 min of injection with BSA ICx appeared to be bound and internalized by Kupffer cells, endothelial cells, and hepatocytes. After 2 h hepatocytes showed many large vacuoles containing ICx, whereas most microvilli had disappeared from the sinusoidal plasma membrane of these cells. In rats with a high antibody titre BSA had disappeared almost completely (92%) from the blood within 2 h. Rats with low antibody titre showed a lower disappearance rate of BSA, whereas a considerable amount of antibody was still present in the blood after 2 h. We conclude that autologous ICx are bound and endocytosed by Kupffer cells, endothelial cells and hepatocytes of the liver. The affinity for the antigen from the circulation. Uptake of autologous ICx by hepatocytes occurs only in the presence of large amounts of circulating ICx.

Animals↗
PubMed1985

Biliary excretion of FITC metabolites after administration of FITC labeled asialo orosomucoid as a measure of lysosomal proteolysis.

An isolated perfused rat liver system was used to study the hepatic uptake and degradation of asialo orosomucoid (asialo alpha 1-acid glycoprotein). To this aim we coupled the fluorochrome FITC to the asialoglycoprotein. The covalent attachment of FITC to the glycoprotein did not affect its perfusate disappearance. The disappearance rate was characterized by a t1/2 approximately equal to 6.1 min, the clearance being 11.2 ml/min. The internalized ligand was probably extensively degraded in the lysosomes as demonstrated by the appearance of low molecular weight fluorescent compounds in the bile, having a higher fluorescence yield than the native conjugate. Lysosomal degradation of ASOR-FITC was shown to be the rate limiting step in FITC excretion into the bile. Treatment of a perfused liver with varying doses of the protease inhibitor leupeptin did not influence the perfusate disappearance rate of the protein. However, leupeptin inhibited the biliary output of FITC metabolites in a dose dependent fashion, half maximal inhibition occurring at 210 nM (in the perfusion medium), corresponding with a dose of 0.05 mg leupeptin per 10 g liver. It is concluded that the rate of lysosomal degradation of proteins in vivo can be determined by measuring the biliary excretion of fluorescent material originating from fluorescent probes covalently coupled to the particular protein.

Animals↗
PubMed1985

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.

Animals↗