Geertruida Harms

IMMUNOHISTOCHEMICAL DEMONSTRATION OF DNA-INCORPORATED 5-BROMODEOXYURIDINE IN FROZEN AND PLASTIC EMBEDDED SECTIONS

SummaryThe 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.

Formaldehyde treated albumin contains monomeric and polymeric forms that are differently cleared by endothelial and kupffer cells of the liver : evidence for scavenger receptor heterogeneity

Formaldehyde treated albumin (F-HSA) was found to consist of a monomeric and a polymeric fraction. Both fractions were primarily endocytosed by rat liver sinusoidal cells. However, immunohistochemical staining of endocytosed material showed that the relative contribution of the endothelial and Kupffer cells in uptake of the monomer and the polymer differed significantly, with the monomer mainly having an endothelial cell- and the polymer predominantly having a Kupffer cell pattern of distribution. To directly confirm these heterogeneous patterns, we injected in vivo the 125I-labeled F-HSA fractions and isolated the endothelial and Kupffer cells by centrifugal elutriation. 73.7% of the monomeric F-HSA was found in endothelial cells and only 14.9% was found in Kupffer cells. In contrast, the polymeric F-HSA (1500 kD) was mainly endocytosed by Kupffer cells (71%), whereas the endothelial cells contributed only for 24% in hepatic uptake. In vivo studies and isolated perfused rat liver experiments showed that endocytosis of both monomer and polymer was inhibited by co-administration of polyinosinic acid, a well known inhibitor for scavenger receptors, indicating that these receptors on endothelial and Kupffer cells are mainly involved in this uptake process.

Spleen autotransplantation provides restoration of functional splenic lymphoid compartments and improves the humoral immune response to pneumococcal polysaccharide vaccine

After splenectomy, patients have an increased risk of overwhelming post‐splenectomy infection (OPSI) or sepsis involving encapsulated bacteria such as pneumococs. The value of spleen autotransplantation after splenectomy because of trauma has long been questioned. Much attention has been given to the restoration of mononuclear phagocyte system (MPS) function, which appeared to be similar to that of splenectomized individuals. The presence of specific anti‐pneumococcal antibodies may enhance phagocytosis of opsonized bacteria by other parts of the MPS, as present in the liver. Therefore, in the present study we have evaluated the restoration of the humoral immune response after spleen autotransplantation, especially to pneumococcal capsular polysaccharides (PPS). Wistar rats were divided into three groups which were operated as follows: splenectomy, splenectomy followed by autotransplantation, and sham operation. After 12 weeks the rats were vaccinated with 23‐valent pneumococcal vaccine. Blood samples were taken after 3 days, 3 and 6 weeks for anti‐PPS IgM and IgG ELISA against types 3, 4, 6, 9, 14 and 23. In addition, immunohistological studies were performed on the autotransplants. Significant antibody titre rises were found in a main proportion of the autotransplanted rats, comparable to sham‐operated rats. Splenectomized rats showed as well a significantly lower increase in immunoglobulin levels, as significant differences in the proportion of rats showing a minimum two‐fold increase of antibody level, considered to represent an adequate response. The titres were highest 3 days after vaccination. Immunohistochemical studies demonstrated structurally functional autotransplants, including an intact marginal zone. Considering this significant anti‐ pneumococcal antibody response, spleen autotransplants can be expected to enable an improved humoral response to PPS, and to contribute to protection against OPSI after splenectomy.

Zonal heterogeneity of rat hepatocytes in the in vivo uptake of 17 nm colloidal gold granules

SummaryThe in vivo uptake in hepatocytes of intravenously injected colloidal gold granules with a diameter of 17 nm or 79 nm and coated with bovine serum albumin or with polyvinyl-pyrrolidone was studied. Irrespective of coating only the 17 nm granules were taken up in hepatocytes. Perivenous hepatocytes did take up much more gold granules than periportal hepatocytes. The gold granules were found in lysosomes around bile canaliculi. Two hours after injection hepatocytes contained the maximal amount of granules. At least a portion of the granules was discharged into the bile. The observed zonal gradient in the uptake of 17 nm gold granules might be caused by the greater supply of granules to the perivenous hepatocytes as a combined result of the higher porosity of the endothelial lining and the smaller number of Kupffer cells with a low endocytic activity in this zone.

Proliferation rate of colonic mucosa in normal subjects and patients with colonic neoplasms: a refined immunohistochemical method.

An increased colonic epithelial proliferation rate and an increase of the cryptal proliferative zone are probable markers of increased susceptibility to colonic cancer. In this study an immunohistochemical method using 5-bromo-deoxyuridine (BrdUrd) to measure the proliferation rate of colonic mucosa in vitro was used. Fresh endoscopic colonic biopsy specimens were incubated with BrdUrd and then processed for immunohistochemistry using a monoclonal antibody. Essential procedures with respect to the equal distribution of nuclei stained with BrdUrd in the biopsy specimens proved to be the cutting of the specimens before incubation and the use of a microwave oven at the beginning of incubation. The use of the procedure of the running average showed that 12 length cut crypts are sufficient to determine reliably the proliferation rate, expressed as the labelling index (LI). This was determined in the biopsy specimens of 10 subjects without organic colonic disease, eight patients with adenomatous colonic polyps, and in six patients with (recent) colonic carcinoma. Mean LI in the controls was significantly lower than in patients with colonic polyps and in those with colon cancer. It is concluded that this method is promising for screening persons at risk for colon cancer and will be of great potential in performing dietary intervention studies in these subjects.

IMMUNOHISTOCHEMICAL ANTIGEN DEMONSTRATION IN PLASTIC-EMBEDDED LYMPHOID-TISSUE

We describe a method for post-embedding immunohistochemical demonstration of a wide range of antigens in glycol methacrylate-embedded tissue. Rat spleen and thymus tissues were fixed by immersion in fixatives containing different concentrations of paraformaldehyde, washed in sucrose phosphate buffer, dehydrated in acetone, infiltrated in a glycol methacrylate mixture in which the commonly used softener 2-butoxyethanol was replaced by butaandiol monoacrylate, and embedded. Trypsin was used to re-expose the masked antigenicity. Excellent results were obtained with a panel of monoclonal antibodies (MoAbs) directed against T-cells, B-cells, Ia-positive cells, macrophages, follicular dendritic cells, and leucocyte common antigen-bearing cells. The method described combines exact localization of antigens with optimal tissue morphology.

Complement dependency of splenic localization of pneumococcal polysaccharide and conjugate vaccines

The immune response to polysaccharides is initiated when polysaccharides bind complement factor C3d, and these polysaccharide–C3d complexes subsequently localize on splenic marginal zone B cells strongly expressing CD21 (complement receptor 2). Infants and children under the age of 2 years have low or absent expression of CD21 on their marginal zone B cells, and consequently do not adequately respond to polysaccharides. In contrast, polysaccharide–protein conjugate vaccines are able to induce antibodies at this young age. Conjugate vaccines apparently overcome the necessity for CD21–C3d interaction for an antipolysaccharide immune response.

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

SummaryIn 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 la-positive epithelial reticular cells demonstrated extremely well their stellate form.

A HISTOCHEMICAL-STUDY ABOUT THE INVOLVEMENT OF RAT-LIVER CELLS IN THE UPTAKE OF HETEROLOGOUS IMMUNE-COMPLEXES FROM THE CIRCULATION

SummaryIntravenously injected immune complexes (ICx) composed of bovine serum albumin (BSA) and rabbit anti-BSA were taken up by the liver. Insoluble complexes, made in antibody excess, were rapidly taken up by Kupffer cells and were metabolized within 24 h.Soluble complexes, made in antigen excess, were only partly taken up by Kupffer cells. In addition these complexes were found, taken up and metabolized by endothelial cells. Until 2 h after injection soluble complexes could also be observed along the microvilli of hepatocytes. No signs of endocytosis in hepatocytes could be observed. It is concluded, that ICx can be taken up by Kupffer cells as well as by endothelial cells. The physical state of the complexes, soluble or insoluble, determines the cell type in which uptake occurs.

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.