New histochemical and ultrastructural observations on normal bovine tonsils.

Samples of normal bovine palatine tonsils were examined by light and electron microscopy. Like human tonsils, they were composed of crypts, subepithelial areas, follicles, and T-dependent zones, but their well-developed capsule subdivided the lymphoid tissue by connective septa. B cells formed the major lymphoid component. The follicles and T-dependent zones had morphological and histochemical features typical of peripheral lymph organs. Follicular dendritic cells were isolated and shown to be similar to human follicular dendritic cells.

Uptake of long-circulating immunoliposomes, directed against colon adenocarcinoma cells, by liver metastases of colon cancer.

Radiolabeled ([3H]cholesteryloleyl ether) immunoliposomes directed against rat colon adenocarcinoma CC531 cells were prepared by random coupling of a tumor cell-specific antibody, CC52, via a thio ether bond. In vitro binding experiments demonstrated a saturable and specific interaction of CC52-immunoliposomes, which could be inhibited by free non-coupled CC52 but not by irrelevant antibodies. The in vivo targeting potential of CC52-immunoliposomes, which were pegylated to achieve prolonged circulation times, was tested in an established rat liver CC531 metastasis model. Twenty-four hours after injection of the liposomes, 25% of the CC52-immunoliposomes were still present in the blood, which was comparable with the control liposomes (either with or without antibody). Liposomes were mainly taken up from the blood by the liver and the spleen, although hepatic uptake of the immunoliposomes was higher and splenic uptake was lower as compared to liposomes without antibody. Within the metastatic tumor nodules in the liver, uptake of both the CC52-immunoliposomes and non-specific immunoliposomes was significantly higher than that of control liposomes without antibody. Visualization of fluorescently or gold labeled CC52-immunoliposomes revealed that, although targeting to liver metastases was achieved, the immunoliposomes were mostly not associated with tumor cells but rather localized in tumor associated cells, probably macrophages.

Selective transfer of a lipophilic prodrug of 5-fluorodeoxyuridine from immunoliposomes to colon cancer cells.

A monoclonal antibody against the rat colon carcinoma CC531 was covalently coupled to liposomes containing a dipalmitoylated derivative of the anticancer drug FUdR as a prodrug in their bilayers. We investigated the in vitro interaction of these liposomes with CC531 target cells and the mechanism by which they deliver the active drug FUdR intracellularly to the cells by monitoring the fate of the liposomal bilayer markers cholesterol-[(14)C]oleate and [(3)H]cholesteryloleylether as well as the (3)H-labeled prodrug and colloidal gold as an encapsulated liposome marker. After binding of the immunoliposomes to the cell surface, only limited amounts were internalized as demonstrated by a low level of hydrolysis of liposomal cholesterol ester and by morphological studies employing colloidal gold-labeled immunoliposomes. By contrast, already within 24 h immunoliposome-incorporated FUdR-dP was hydrolyzed virtually completely to the parent drug FUdR intracellularly. This process was inhibited by a variety of endocytosis inhibitors, indicating that the prodrug enters and is processed by the cells by a mechanism involving an endocytic process, resulting in intracellular FUdR concentrations up to 3000-fold higher than those in the medium. Immunoliposomes containing poly(ethyleneglycol) (PEG) chains on their surface, with the antibody coupled either directly to the bilayer or at the distal end of the PEG chains were able to deliver the prodrug into the tumor cells at the same rate as immunoliposomes without PEG. Based on these observations, we tentatively conclude that during the interaction of the immunoliposomes with the tumor cells the lipophilic prodrug FUdR-dP is selectively transferred to the cell surface and subsequently internalized by constitutive endocytic or pinocytic invaginations of the plasma membrane, thus ultimately delivering the prodrug to a lysosomal compartment where hydrolysis and release of parent drug takes place. This concept allows for an efficient delivery of a liposome-associated drug without the need for the liposome as such to be internalized by the cells.

Different intrahepatic distribution of phosphatidylglycerol and phosphatidylserine liposomes in the rat.

Liposomes with diameters of 200 to 400 nm containing phosphatidylserine (PS) or phosphatidylglycerol (PG) were injected intravenously into rats. Two hours after injection, 75% of the injected dose of PS liposomes was found in the liver and only 10% found in the spleen, while 35% of the PG liposomes was found in the liver and as much as 40% was found in the spleen. Cell-isolation experiments revealed the following remarkable difference in the intrahepatic distribution between the two liposome formulations: the PS liposomes distributed in about equal amounts to Kupffer cells and hepatocytes, despite their size (200-400 nm) exceeding that of the endothelial fenestrae (average 150 nm), whereas the PG liposomes were only taken up by the Kupffer cells and not at all by the hepatocytes. Double-label studies, using liposomes in which the lipid-moiety was radio labeled with [3H]cholesteryloleylether ([3H]CE) and the water phase with [14C]sucrose, showed that the liposomes were taken up as intact particles. These observations were confirmed through electron microscopy by determining the in situ localization of liposome-encapsulated colloidal gold particles in thin sections of liver and spleen. The differences in organ distribution are ascribed to differences in opsonization patterns of the two liposomal surfaces. For the difference in intrahepatic distribution, we offer the following two explanations: the exploitation of the blood cell-mediated forced sieving concept and the indication of a PS-specific pharmacological effect on the dimensions of the fenestrations.

Responses of Haemophilus pleuropneumoniae to iron restriction: changes in the outer membrane protein profile and the removal of iron from porcine transferrin.

Outer membranes from Haemophilus pleuropneumoniae grown under iron-replete and iron-restricted conditions in vitro were analysed by means of SDS-PAGE and immunoblotting. Iron restriction resulted in the appearance of two or more novel polypeptides in the molecular size range of 96-102 kD and an increased amount of a 79 kD polypeptide. These polypeptides were recognized by porcine immune sera indicating their production by H. pleuropneumoniae during growth in vivo. Although soluble siderophore production could not be detected, growth of the organisms on an iron-restricted medium was enhanced by the presence of porcine transferrin but not by bovine or human transferrin. The results suggest that H. pleuropneumoniae possesses a specific transferrin receptor, perhaps in the form of an iron-regulated outer membrane protein.

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.

The specificity of nephritogenic antibodies. IV. Binding of monoclonal antithymocyte antibodies to rat kidney.

Polyclonal rabbit antirat thymocyte serum (ATS) has been shown to form in situ glomerular immune aggregates following perfusion into normal rat kidney ex vivo. This may be due to the presence of T-cell-like epitopes in the rat kidney, or it may be a result of contaminating anti-connective-tissue antibodies in ATS. To exclude the latter possibility we investigated binding to the rat kidney of three different (mouse) monoclonal antirat thymocyte antibodies (anti-T-cell MoAbs), directed to Thy 1.1 antigen, as well as (control) anti-B-cell MoAbs. The MoAbs were incubated in vitro with kidney sections or perfused into the blood-free rat kidney ex vivo. It was shown using immunofluorescence (IF) and immunoelectron microscopy (IEM) (peroxidase technique) that the anti-T-cell MoAbs used, in contrast to anti-B-cell MoAbs, are able to bind with glomerular capillary walls, and with mesangial structures after incubation in vitro or perfusion ex vivo. Although staining patterns are not completely identical, the reaction product is clearly demonstrated throughout the glomerular basement membrane (GBM) and along the plasma membranes of endothelial and epithelial cells, after contact with either of the three anti-T-cell MoAbs used. It is concluded that the presence of T-cell-like epitopes in the rat kidney may lead to immune complex formation following contact with anti-T-cell MoAbs. The nephritogenicity of rabbit ATS, as well as of some batches of clinically used ATS, may also be explained by this mechanism rather than by the usually assumed presence of contaminating antibodies in these polyclonal antisera.

The specificity of nephritogenic antibodies. II. Immune complex glomerulopathy in rats induced by heterologous antithymocyte serum.

Injection of rabbit anti-rat thymocyte serum (ATS) i.v into rats induces a transient glomerulopathy characterized by immune aggregates localized in the mesangium and along the glomerular capillary wall, as detected by immunofluorescence (IF) techniques. Neither light microscopical alterations in the kidney, nor proteinuria could be detected in these animals and no autologous IgG could be observed in the glomeruli during the observation period (45 days). Results from ex vivo perfusion studies showed identical localization of immune aggregates--which, in electron microscopy, appear to be localized subepithelially. The ATS used, was monospecific in that no other specificities could be detected after thorough absorption with rat tissue extracts including tubular brushborder antigens, so it is concluded from these data that ATS is able to participate in the formation of immune complexes in situ by recognizing epitopes both in the mesangium and at the epithelial side of the glomerular basement membrane.