Transport of chitosan microparticles for mucosal vaccine delivery in a human intestinal M-cell model.

Uptake of particulate antigen carrier systems by specialized M-cells of the gut-associated lymphoid tissue is still a limiting step in inducing efficient immune responses after oral vaccination. Although transport of soluble drugs over the epithelial barrier of the gut is extensively studied in vitro by using the Caco-2 cell model, this was for long time not possible for particles due to the absence of M-cells. By co-culturing Caco-2 cells with cultured human B-lymphocytes (Raji-cells), cells which are morphologically and functionally similar to M-cells can be induced. This human M-cells model makes it possible to study the uptake of microparticles for oral vaccine delivery. In this way, chitosan microparticles, which have demonstrated to target the Peyer's patches efficiently in vivo, could be tested in vitro. The development of this M-cells model facilitates the optimization of the microparticles in order to target them even more efficiently to the M-cells in the gut. In this study, the integrity of the human M-cell model was investigated by determining the transepithelial electrical resistance (TEER), 14C-mannitol transport and morphology using scanning electron microscopy. The uptake of particles was investigated by measuring transport of both fluorescently labeled microspheres (Fluospheres) and chitosan microparticles using flowcytometry. No discontinuities or abnormalities could be found in the co-culture. Scanning electron microscopy showed that morphologically different cells were present in the human M-cell model. Both commercially available Fluospheres (size 0.2 microm) and chitosan microparticles (size 1.7 microm) for oral vaccine delivery were transported at a significantly higher amount by the human M-cell model compared to the transport by the Caco-2 cell monoculture. Since chitosan microparticles were proven to be taken up by Peyer's patches in mice as well, this human M-cell model is able to predict the M-cell uptake of microparticles for oral vaccine delivery. This M-cell model is a new tool, which can be used to scan, develop and optimize microparticles for oral vaccine delivery. Since the M-cell uptake can now be studied in vitro, the targeting of these cells can be studied more efficiently and can now be done in cells from human origin.

Observations at the articular surface of hip prostheses: an analytical electron microscopy study on wear and corrosion.

We used scanning electron microscopy in combination with X-ray microanalysis to evaluate Co-, Cr-, and Mo-based human femoral hip prostheses. In total, 23 retrieved implants and four new implants were included in this study. Scanning electron microscopy of the polished surface of all arthroplasties showed, in addition to the polishing marks, small round and angular holes or pits. Other types of surface irregularities were interpreted as wear or corrosion of the metal compound. In all cases studied, corrosion propagated from holes at the surface of the polished prosthesis heads, in some cases also along phase boundaries. X-ray microanalysis of the intact prosthetic surface showed a relative composition of the elements Co, Cr, and Mo, which was in agreement with the manufacturer's information (63:33:4%). However, X-ray microanalysis spot analysis of the surface holes showed deviation in the relative composition of the elements Co, Cr, and Mo and also the presence of Si, Ti, and Al. Furthermore, Ti and Al could be traced back at an artificially made fracture plane of a new prosthesis. Therefore, Ti and Al have to be present during the manufacturing process. These impurities in the metal prosthesis alloy may create a galvanic element with the Co, Cr, Mo alloy of the implant. If this is the case, such a galvanic element in combination with the electrolyte environment formed by body fluids, can induce galvanic corrosion with release of metal particles.

Myosin V colocalizes with melanosomes and subcortical actin bundles not associated with stress fibers in human epidermal melanocytes.

Mutations of the gene encoding myosin V can produce a dilute or silvery hair color and various neurologic defects in mice and patients with Griscelli syndrome, leading to speculations that the myosin V motor protein plays a critical role in transporting melanosomes within melanocytes and neurosecretory vesicles within neurons. Therefore, we investigated the in vitro expression of myosin V in cultured normal human melanocytes, keratinocytes, and dermal fibroblasts using reverse transcriptase-polymerase chain reaction and northern blot analysis. Subcellular distribution of myosin V and proximity to actin bundles and melanosomes were determined by double indirect immunofluorescence labeling and immunogold electron microscopy. In all studied cells myosin V is expressed and treatment of melanocytes with the cyclic AMP-inducer 3-isobutyl-1-methylxanthine causes an induction of the myosin V message. In all cells myosin V colocalizes with actin bundles, concentrating in the subcortical cell zone. In the melanocyte it is closely associated with melanosomes. Quantitative analysis of myosin V labeling in melanocytes reveals a significantly higher (p < 0.005) presence of myosin V in the periphery of dendrites. These results suggest that myosin V is important in melanosome transport in human melanocytes. Possible roles in the other skin cells remain to be elucidated.

Mannose receptor-mediated uptake of antigens strongly enhances HLA class II-restricted antigen presentation by cultured dendritic cells.

Dendritic cells (DC) efficiently take up antigens by macropinocytosis and mannose receptor-mediated endocytosis. Here we show that endocytosis of mannose receptor-antigen complexes takes place via small coated vesicles, while non-mannosylated antigens were mainly present in larger vesicles. Shortly after internalization the mannose receptor and its ligand appeared in the larger vesicles. Within 10 min, the mannosylated and non-mannosylated antigens co-localized with typical markers for major histocompatibility complex class II-enriched compartments and lysosomes. In contrast, the mannose receptor appeared not to reach these compartments, suggesting that it releases its ligand in an earlier endosomal structure. Moreover, we demonstrate that mannosylation of protein antigen and peptides resulted in a 200-10,000-fold enhanced potency to stimulate HLA class II-restricted peptide-specific T cell clones compared to non-mannosylated peptides. Our results indicate that mannosylation of antigen leads to selective targeting and subsequent superior presentation by DC which may be applicable in vaccine design.

Mannose receptor mediated uptake of antigens strongly enhances HLA-class II restricted antigen presentation by cultured dendritic cells.

Dendritic cells (DCs) use macropinocytosis and mannose receptor mediated endocytosis for the uptake of exogenous antigens. Here we show that the endocytosis of the mannose receptor and mannosylated antigen is distinct from that of a non-mannosylated antigen. Shortly after internalization, however, both mannosylated and non-mannosylated antigen are found in an MIIC like compartment. The mannose receptor itself does not reach this compartment, and probably releases its ligand in an earlier endosomal structure. Finally, we found that mannosylation of peptides strongly enhanced their potency to stimulate HLA class II-restricted peptide-specific T cell clones. Our results indicate that mannosylation of antigen leads to selective targeting and subsequent superior presentation by DCs which may be useful for vaccine design.

Immunoelectron microscopy reveals significant granule fusion upon stimulation of electropermeabilized human neutrophils.

Although electropermeabilization has become an important tool for studying the signal requirements of exocytosis, relatively little is known about the morphological changes accompanying this response in electropermeabilized cells. In this study, we determined that electropermeabilization of human neutrophils by itself caused only minor changes in the morphology as determined by transmission electron microscopy. The structure of the plasma membrane did not show detectable changes, whereas the cytoplasm was more electron lucent as compared to intact cells. Activation of intact neutrophils with formyl-methionyl-leucyl-phenylalanine (FMLP), in the presence of cytochalasin-B, caused the development of invaginations of the plasma membrane. In contrast, activation of electropermeabilized cells with 1 microM Ca2+ and/or 50 microM GTP-gamma-S caused the development of vacuoles that did not seem to be in contact (or had previously been in contact) with the extracellular environment. However, fusion of azurophilic and specific granules with these vacuoles clearly had taken place. The response characteristics of this fusion induced by Ca2+ and GTP-gamma-S were quite similar to those of the direct fusion of granules with the plasma membrane. We conclude that in electropermeabilized human neutrophils, two processes involving granule fusion can be distinguished. First, a direct fusion of granules with the plasma membrane. Secondly, the fusion of granules leading to the formation of vacuoles, not in contact with the extracellular space.

Evidence for endocytosis of E-selectin in human endothelial cells.

E-selectin is an inducible adhesion molecule on endothelial cells. The internalization of this glycoprotein was investigated on tumor necrosis factor (TNF)-activated cultured human umbilical vein endothelial cells (HUVEC). Kinetics of intercellular adhesion molecule-1 (ICAM-1) were studied in parallel experiments. Internalization studies were performed with radioiodinated antibodies in an acid elution endocytosis assay, and by immunohistology; both approaches gave equivalent results. [125I]ENA1, a monoclonal antibody (mAb) specific for E-selectin, was internalized at a rate of approximately 1.7% of the membrane-bound [125I]mAb per minute. In contrast, less than 0.1% of membrane-bound [125I]RR1/1, an mAb specific for ICAM-1, was internalized per minute. TNF-activated HUVEC were immunostained and examined by light microscopy (LM) and electron microscopy (EM). LM revealed the presence of ENA1, but not RR1/1, after 30 minutes of incubation with these mAb in cytoplasmic vesicles, which were characterized as multivesicular bodies by EM. Without previous mAb exposure of the endothelial cells, both high amounts of E-selectin and bovine serum albumin complexed to colloidal gold, used as a marker for fluid-phase internalization, were detected in the same organelles, thus arguing against mAb interaction-induced E-selectin internalization. Furthermore, the amount of E-selectin surface expression was not influenced by ongoing mAb presence, also arguing against mAb interference with normal E-selectin kinetics. Taken together, these results indicate that TNF-activated HUVEC constitutively internalize E-selectin. Physiological significance of E-selectin internalization in the regulation of E-selectin membrane expression, and in clearing E-selectin ligands from the circulation, needs further investigation.

The human neutrophil FcRIII (CD16) on the plasma membrane can be replenished from an intracellular source.

The internalization of peroxidase-antiperoxidase (PAP) immune complexes by human neutrophil granulocytes was studied at an ultrastructural level. PAP initially bound to the plasma membrane at 4 degrees C accumulated in endosomes within 5 min of internalization. By that time, all of the ligand bound to the plasma membrane had been removed from the cell surface and the cells were able to bind newly added PAP. Pre-embedding labelling of FcRIII on these cells showed that this receptor was present on the cell surface, indicating involvement of FcRIII in the rebinding of PAP in neutrophils. The origin of FcRIII present on the plasma membrane after Fc receptor-mediated internalization of PAP was investigated in another series of experiments. Incubation of the cells with pronase eliminated the epitope on the Fc receptor recognized by anti-FcRIII. After the pronase treatment hardly any Fc receptors were detected on the plasma membrane. However, incubation of the cells for only 5 min in a protease-free medium after the pronase treatment led to an abundance of FcRIII on the plasma membrane of the neutrophils. These findings support the hypothesis that FcRIII on the plasma membrane of human neutrophil granulocytes is replenished from an internal source of free Fc receptors and suggest that at least some of the receptors present on the cell surface after the binding and internalization of PAP originate from this source in the cytoplasm.

Membrane surface antigen expression on neutrophils: a reappraisal of the use of surface markers for neutrophil activation.

Neutrophil research relies largely on studies with highly purified cells. Yet the isolation procedures induce changes in surface expression of several proteins. We used a large panel of monoclonal antibodies (MoAbs) to characterize in detail the phenotypic changes during isolation and stimulation of human neutrophils. Centrifugation on density gradients appears to be the crucial step that causes an increase in expression of antigens not detectable on neutrophils in whole blood samples (cytochrome b558 recognized by MoAb 7D5; and CD10) or expressed at significantly lower levels (CD11a, CD11b, CD11c, CD13, CD16, CD45, and CD67). Other antigens were unaffected by the density gradient centrifugation step (CD32, CD54, CD58, Leu-8, HLA class I). Upregulation of antigens was also determined by stimulation of purified neutrophils. Upregulation of CD63 was an excellent marker for release from azurophil granules. We subsequently related the surface antigen expression to functional activities of purified neutrophils. From these experiments, we concluded that 7D5-as "early activation" marker--does not necessarily discriminate between primed or resting neutrophils with respect to NADPH oxidase activity.

Localization of the low-Mr subunit of cytochrome b558 in human blood phagocytes by immunoelectron microscopy.

Cytochrome b558 is a membrane-bound component of the NADPH-oxidase system in phagocytes and consists of a low-Mr subunit of 22 to 23 Kd and a high-Mr subunit of 75 to 90 Kd. The present study on the subcellular localization of the low Mr subunit of cytochrome b558 (p22-phox) in resting human peripheral blood phagocytes was based on immunogold labeling with monoclonal antibody (MoAb) 449, recently characterized. In post-embedding labeled neutrophils, this subunit was found mainly in the membrane of the specific granules. This conclusion was supported by a quantitative analysis of the results obtained in immunogold double-labeled sections with a polyclonal antiserum against lactoferrin (LF) as a marker for specific granules and a polyclonal antiserum against myeloperoxidase (MPO) used to identify azurophil granules. No labeling of the plasma membrane was observed, because of limited penetration of the antibody into the cryosections, preventing the detection of low antigen concentrations. Pre-embedding labeling of digitonin-permeabilized neutrophils, which has the advantage of a better penetration of the antibody into the cells, showed intense immunoreactivity on the cytoplasmic side of intact granules and low labeling on the inner surface of the plasma membrane. These complementary findings indicate that in resting neutrophils the epitope of p22-phox, recognized by MoAb 449, is present on the cytoplasmic side of the membrane of specific granules and the plasma membrane. Similar observations were made in eosinophils, where MoAb 449 reacted strongly with the cytoplasmic side of numerous small granules, and a low level of labeling was observed on the inner surface of the plasma membrane. In monocytes, MoAb 449 labeling also occurred on the inner surface of plasma membrane, of endocytotic compartments, and the outer surface of relatively small granules differing from peroxidase-containing lysosomes, as shown by immunogold double-labeling with MPO.

Differences in low density lipoprotein receptor expression in the suprabasal layer of normal and psoriatic epidermis.

Previous morphological experiments on the distribution of binding sites for low density lipoprotein (LDL) on normal and psoriatic epidermis in situ, done with the LDL-gold technique [Mommaas-Kienhuis AM, et al. J Invest Dermatol 89: 513-517, 1987.] showed an unequivocal correlation between the ability to bind LDL-gold complexes and the state of keratinocyte differentiation. To determine the involvement of the LDL receptor in this phenomenon, we applied immunoelectronmicroscopical methods in conjunction with a monoclonal anti-LDL receptor antibody. Biopsy specimens of normal and psoriasis skin were fixed before being embedded in Lowicryl K4M. Ultrathin sections were incubated first with the anti-LDL receptor antibody, and then with a second antibody conjugated to colloidal gold. On basal cells of both normal and psoriatic epidermis the LDL receptor was distributed evenly between the cell surface and the cytoplasm. No obvious differences in the density of LDL receptors were observed. However, cells from the suprabasal layer showed two striking differences in the localization of the LDL receptor: 1) normal epidermis showed fewer LDL receptor molecules, whereas in psoriasis epidermis the number increased relative to those on basal cells; and 2) in normal suprabasal cells most of the LDL receptors were located inside the cell, but in psoriasis the majority was found on the cell surface. Both phenomena are discussed and we postulate that the higher expression of LDL receptors in psoriasis suprabasal cells and the high expression of the receptor on the cell surface is connected with the hyperproliferative state of the disorder.

On the origin of peritoneal resident macrophages. I. DNA synthesis in mouse peritoneal resident macrophages.

Mouse resident macrophages were isolated from the unstimulated peritoneal cavity at various intervals after one intravenous injection of tritiated thymidine. EM autoradiography showed that 15 min after the injection, peritoneal resident macrophages, with the characteristic localization of the peroxidatic activity in the rough endoplasmic reticulum and the nuclear envelope, had already incorporated tritiated thymidine. In the peripheral blood, monocytes with silver grains over the nuclei were seen from 12 h onwards with a maximum at 48 h. In the peritoneal cavity labelled monocytes appeared at 24 h. The peak for labelled blood monocytes was not followed by a peak for labelled peritoneal resident macrophages. The conclusion is drawn that resident macrophages from the unstimulated peritoneal cavity are able to proliferate locally.

Expression of 5'nucleotidase activity and wheat-germ agglutinin binding sites in mononuclear phagocytes from bone marrow cultures.

The question as to whether the various types of mononuclear phagocyte found in bone marrow cultures and recognized by specific peroxidatic (PO) activity patterns differ in the expression of binding sites for the lectin wheat-germ agglutinin (WGA) and the activity of the ectoenzyme 5'nucleotidase (5'N) was investigated. Monoblasts, promonocytes, monocytes, and/or exudate macrophages, and exudate-resident macrophages generally showed a high level of WGA binding, and a considerably lower level was found in the PO-negative cells and in resident macrophages. 5'N activity was absent in monoblasts, promonocytes, and in the great majority of the monocytes and/or exudate macrophages, but was demonstrable in exudate-resident macrophages and resident macrophages, as well as in PO-negative macrophages after 4 days of culture. On the basis of the successive occurrence of the above-mentioned phenotypes in cultures, the possibility that this diversity in WGA binding and 5'N activity is related to modulation during cell differentiation is discussed. The present findings led to the conclusion that the PO-negative macrophages, whose origin was previously not entirely certain, are precursors of resident macrophages.

Heterogeneity in 5'-nucleotidase activity of mouse peritoneal macrophages. An EM-cytochemical and biochemical study.

After stimulation of the mouse peritoneal cavity with newborn calf serum (NBCS), four types of monocyte and macrophage were distinguished on the basis of peroxidase (PO) patterns. Cytochemically, these cells showed strong heterogeneity in 5'-nucleotidase (5'N) activity. Monocytes and monocyte-derived macrophages with PO activity in granules lacked 5'N activity. Resident macrophages (with PO activity in RER and nuclear envelope) generally had significant 5'N activity on the plasma membrane, the pattern showing close correlation with the biochemical findings. The group of PO-negative macrophages comprised both 5'N-negative and 5'N-positive cells. These findings suggest two possibilities, i.e., that monocytes (5'N-)transform via PO-negative cells (5'N -/+) into resident macrophages (5'N +), or that the monocytes and monocyte-derived macrophages and the resident macrophages represent separate lineages. The fourth type of macrophage, the exudate-resident cell (with PO activity both in granules and in the RER and nuclear envelope), occurred only in low numbers and very late after NBCS stimulation, and is therefore considered not to be a transitional cell between monocytes and resident macrophages.

A cytochemical method for the demonstration of 5'-nucleotidase in mouse peritoneal macrophages, with cerium ions used as trapping agent.

A method was developed for the demonstration of 5'-nucleotidase in murine peritoneal resident macrophages. The cells are incubated cytochemically without agitation and cerium chloride is used as a trapping agent. Under these conditions, the great majority of the macrophages in the unstimulated peritoneal cavity show enzyme activity in the plasma membrane. In the presence of AMP-S (an AMP analogue inhibiting 5'-nucleotidase, as shown biochemically) there was a decrease in both the number of positive macrophages and the amount of reaction product on the plasma membranes. This indicates that the enzyme activity detected by our cytochemical procedure is attributable to 5'-nucleotidase.

The effect of colchicine on the intracellular transport of 3H-fucose-labelled glycoproteins in the absorptive cells of cultured human small-intestinal tissue. An autoradiographical and biochemical study.

The effect of colchicine on the intracellular transport of 3H-fucose-labelled glycoproteins in the absorptive cells of cultured biopsy specimens of the human intestine was investigated by light- and electron-microscopical autoradiography and by biochemical methods. The results showed a decrease in the radioactivity of the cell coat on the microvilli and an increase in the Golgi apparatus and in the apical vesicles and tubules. This divergence is attributed to a colchicine-induced impairment of the normal transport of cell-coat glycoproteins from the Golgi apparatus, via the apical vesicles and tubules, to the apex of the cell. The radioactivity of the lysosome-like bodies in the absorptive cells cultured with colchicine also increased. This finding supports a crinophagic function of these organelles in the degradation of excess cell-coat material.

Resolution of a gold latensification-elon ascorbic acid developer for Ilford L4 emulsion.

The electron-microscopical autoradiographical resolution of a gold latensification-elon ascorbic acid (GEA) developer for Ilford L4 emulsion was determined experimentally, using radioactive line sources of tritiated albumin (Heijnen and Geuze, 1977). For sections with a thickness of 62 nm (SD +/- 11), which were covered with a carbon layer about 5 nm thick and a slightly overlapping monolayer of L4 silver bromide crystals, the measured half-distance (HD) of resolution was 115 nm. This improvement in resolution, the high efficiency of the GEA developer for L4 emulsion (Wisse and Tates, 1968), and the excellent visibility of the cellular structures under the small silver grains, mean that the L4-GEA combination deserves preverence as a method for quantitative electron-microscopical autoradiography.

Transport of radiolabelled glycoprotein to cell surface and lysosome-like bodies of absorptive cells in clutured small-intestinal tissue from normal subjects and patients with a lysosomal storage disease.

The transport of 3H-fucose- and 3H-glucosamine-labelled glycoproteins in the absorptive cells of cultured human small-intestinal tissue was investigated with light- and electron-microscopical autoradiography. The findings showed that these glycoproteins were completed in the Golgi apparatus and transported in small vesicular structures to the apical cytoplasm of these cells. Since this material arrived in the cell coat on the microvilli and in the lysosome-like bodies simultaneously, a crinophagic function of these organelles in the regulation of the transport or secretion of cell-coat material was supported. In the absorptive cells of patients with fucosidosis or Hunter's type of lysosomal storage disease, a smiliar transport of cell-coat material to the lysosome-like bodies and a congenital defect of a lysosomal hydrolase normally involved in the degradation of cell-coat material, can explain the accumulation of this material in the dense bodies.