Machiel J. Hardonk

Human immune response to pneumococcal polysaccharides : Complement-mediated localization preferentially on CD21-positive splenic marginal zone B cells and follicular dendritic cells

A functionally intact spleen with a marginal zone, containing B cells with high density of surface C3d-receptors (CD21), is essential for the ability to induce a primary immune response to thymus-independent type 2 (TI-2) antigens. Main representatives of natural TI-2 antigens are capsular pneumococcal polysaccharides (PPSs). In this study the localization of different types of PPS antigen is determined in human spleen tissue. Our findings indicate that a main type of TI-2 antigen, PPS, localizes preferentially in the marginal zone. PPSs show co-localization with C3, presumably C3d, at the surface of strongly CD21+ B cells equipped for rapid activation. This enables a rapid primary humoral response. The other main PPS localization at follicular dendritic cells in germinal centers, relevant for isotype switching of anti-PPS antibodies, does not seem to be dependent on the presence of specific immunoglobulin. This may explain the finding of specific IgG in an early stage after antigenic challenge. It seems likely that complement C3 fragments (likely C3d), bound to PPSs, enable PPS localization at B-cell and follicular dendritic cell surfaces by binding to CD21, the C3d receptor.

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.

ISOLATION AND CHARACTERIZATION OF CANALICULAR AND BASOLATERAL PLASMA-MEMBRANE FRACTIONS FROM HUMAN LIVER

A method is described for the isolation of subfractions from human liver plasma membranes, enriched in canalicular domains (cLPM) and basolateral domains (blLPM), respectively, and the results are compared to those obtained with rat liver. The studies were performed in 18 human livers. The cLPM (isolated at densities 1.103-1.127 for human and 1.036-1.127 for rat cLPM) from human as well as rat liver showed a lower density than the blLPM (1.141-1.161 for human and 1.151-1.172 for rat blLPM). Human and rat blLPM were characterized by increased levels of (Na+/K+)-ATPase (relative enrichment 33 and 21, respectively). Both human and rat cLPM showed high specific activities of leucine aminopeptidase; relative enrichment factors were 42 and 31, respectively. Mg(2+)-ATPase and alkaline phosphatase, specific canalicular enzymes in rat liver, were only slightly enriched in the cLPM of human liver, which indicates that these enzymes are not suitable as marker enzymes for human liver cLPM. Both cLPM and blLPM of human and rat origin were only slightly contaminated with mitochondria, lysosomes, Golgi membranes and endoplasmic reticulum. Total recoveries of cLPM and blLPM were 0.02 mg protein/g liver each for the human membrane preparations, compared to 0.07 and 0.16 mg protein/g liver for the membranes prepared from rat liver. Analysis of membrane fluidity revealed that the human liver cLPM were more rigid than blLPM (mean difference in fluorescence polarization PDPH 0.024). They contained more cholesterol (0.43 vs. 0.30 mumol/mg protein) and phospholipids (0.54 vs. 0.39 mumol/mg protein, respectively), which was compatible to rat liver plasma membrane fractions. This study shows that besides similarities, there are several differences between human and rat liver plasma membrane fractions.

The Characteristic Distribution of Alkaline Phosphatase in the Full-Term Human Placenta

The ultrastructural localization of alkaline phosphatase activity was studied in the full-term human placenta. Alkaline phosphatase activity is not only found along the plasma membrane lining the micr

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.

Glycosyl receptors in macrophage subpopulations of rat spleen and lymph node

SummaryWe have developed an immunohistochemical method for the in vivo and in vitro detection of glycosyl receptors in rat spleen and lymph nodes by using neoglycoproteins. The receptor in both organs recognized mannose coupled to bovine serum albumin (mannose-BSA), fuscose-BSA, N-acetylglucosamine-BSA and to a lesser extent glucose-BSA, but not galactose-BSA or N-acetylgalactosamine-BSA. In vitro neoglycoprotein-receptor binding was Ca2+ dependent and could be inhibited by mannan but not by mannose. Simultaneous staining with the monoclonal antibodies ED1, ED2 or ED3 revealed that only ED1-and ED3-positive macrophages were involved in the binding of neoglycoproteins. In the spleen, the marginal-zone macrophages and a subpopulation of the marginal metallophils possess glycosylbinding receptors. In the lymph nodes, the medullary sinus macrophages and a subpopulation of the outercortex macrophages are able to bind neoglycoproteins.

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

SummaryThe 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.

Hepatobiliary transport of drugs: do periportal and perivenous hepatocytes perform the same job?

Abstract Drug clearance functions of the liver are mainly performed by the hepatocytes. Geny Groothuis and colleagues propose that a heterogeneity of the hepatocytes exists with respect to the uptake and excretion of drugs. This proposal is based on observed concentration gradients in the acinus, the structural microcirculatory unit of the liver, and intrinsic differences between hepatocytes from the periportal and perivenous zones.

Morphological studies on selective acinar liver damage by N-hydroxy-2-acetylaminofluorene and carbon tetrachloride

SummaryHeterogeneity of rat hepatocytes with respect to transport function can in principle by studied by selective acinar damage of periportal (acinar zone 1) and perivenous (acinar zone 3) cells. Meaningful conclusions from such studies can be drawn only if the acinar selectivity of the toxins employed is clearly demonstrated. Therefore, transmission and scanning electron microscopy, enzyme histochemistry and determination of the increase of the activities in serum of glutamate-pyruvate transaminase, glutamate dehydrogenase and alkaline phosphatase were performed 24 h after the administration of 90 μmol/kg N-hydroxy-2-acetylamino-fluorene (N-OH-AAF) to damage zone 1 and 2.1 mmol/kg carbon tetrachloride (CCl4) to damage zone 3.N-OH-AAF administration resulted in strongly elevated serum enzyme activities. Histochemically, a decrease of enzyme activities in a very limited number of cells in zone 1 (5–20% of the acinus) was found. The remaining cells of zone 1 showed either increased or normal activities, and zone 3 cells appeared normal. Ultrastructurally, zone 3 cells were intact, but zone 1 cells exhibited several signs of damage: among others, induction of RER into fingerprints, numerous small vesicles, and widened bile canaliculi; some necrotic cells were present.CCl4 administration resulted in a relatively smaller rise of serum enzyme activities than N-OH-AAF, and in a decrease of histochemically detectable enzyme activities in zone 3 (20–50% of the acinus), while zone 1 cells appeared normal. Ultrastructurally, no changes were observed in zone 1, but zone 3 cells were necrotic or revealed swollen ER occupying most of the cytoplasmic space. Scanning EM showed no damage to the sinusoidal lining after both N-OH-AAF and CCl4-pretreatment. Bile canaliculi were normal after CCl4-pretreatment. It is concluded that administration of these doses of N-OH-AAF and CCl4 results in damage that is restricted to zone 1 and zone 3 respectively and therefore, enables further studies concerning the heterogeneity of hepatocytes with respect to transport functions. This study introduces N-OH-AAF as a new tool for the selective destruction of zone 1 of the liver acinus, with a better reproducibility of the hepatic lesion and a lower general toxicity compared with previously used toxins such as allylalcohol.

Application of enzymehistochemical methods to isolated subcellular fractions and to sucrose-ficoll density gradients

To compare histochemical and biochemical determinations of enzyme activities, enzymehistochemical procedures are applied to sections of pellets of subcellular fractions. These investigations are of value to determine the subcellular localization of histochemically demonstrable enzyme activities and to test the homogeneity of an isolated fraction. In homogenating duckling liver a great part of the endothelial cells is not destructed and consequently is found in the nuclear fraction. Kupffer cell lysosomes land in the heavy mitochondrial fraction, whereas hepatocyte lysosomes are chiefly found in the light mitochondrial fraction. β-Glucuronidase activity shows a preferentially microsomal localization. Application of enzymehistochemical staining reactions to discontinuous gradients and comparison with biochemical data provides additional information about the validity of an enzymehistochemical reaction. In rat liver the tetrazolium reductases show a distinctly dual localization: activity in the mitochondrial band and in microsomal bands. As to their localization in different bands of the gradients non-specific esterases demonstrate a clear pH-dependency.SummaryTo compare histochemical and biochemical determinations of enzyme activities, enzymehistochemical procedures are applied to sections of pellets of subcellular fractions. These investigations are of value to determine the subcellular localization of histochemically demonstrable enzyme activities and to test the homogeneity of an isolated fraction. In homogenating duckling liver a great part of the endothelial cells is not destructed and consequently is found in the nuclear fraction. Kupffer cell lysosomes land in the heavy mitochondrial fraction, whereas hepatocyte lysosomes are chiefly found in the light mitochondrial fraction. β-Glucuronidase activity shows a preferentially microsomal localization. Application of enzymehistochemical staining reactions to discontinuous gradients and comparison with biochemical data provides additional information about the validity of an enzymehistochemical reaction. In rat liver the tetrazolium reductases show a distinctly dual localization: activity in the mitochondrial band and in microsomal bands. As to their localization in different bands of the gradients non-specific esterases demonstrate a clear pH-dependency.