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Stem cells generate rapidly dividing transit-amplifying cells that have lost the capacity for self-renewal but cycle for a number of times until they exit the cell cycle and undergo terminal differentiation. We know very little of the type of signals that trigger the earliest steps of stem cell differentiation and mediate a stem cell to transit-amplifying cell transition. We show that in normal intestinal epithelium, endoplasmic reticulum (ER) stress and activity of the unfolded protein response (UPR) are induced at the transition from stem cell to transit-amplifying cell. Induction of ER stress causes loss of stemness in a Perk-eIF2α-dependent manner. Inhibition of Perk-eIF2α signaling results in stem cell accumulation in organoid culture of primary intestinal epithelium. Our findings show that the UPR plays an important role in the regulation of intestinal epithelial stem cell differentiation., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Ex vivo, human mesenchymal stem cells (hMSCs) undergo spontaneous cellular senescence after a limited number of cell divisions. Intranuclear structures of the nuclear lamina were formed in senescent hMSCs, which are identified by the presence of Hayflick-senescence-associated factors. Notably, spatial changes in lamina shape were observed before the Hayflick senescence-associated factors, suggesting that the lamina morphology can be used as an early marker to identify senescent cells. Here, we applied quantitative image-processing tools to study the changes in nuclear architecture during cell senescence. We found that centromeres and telomeres colocalised with lamina intranuclear structures, which resulted in a preferred peripheral distribution in senescent cells. In addition, telomere aggregates were progressively formed during cell senescence. Once formed, telomere aggregates showed colocalization with gamma-H2AX but not with TERT, suggesting that telomere aggregates are sites of DNA damage. We also show that telomere aggregation is associated with lamina intranuclear structures, and increased telomere binding to lamina proteins is found in cells expressing lamina mutants that lead to increases in lamina intranuclear structures. Moreover, three-dimensional image processing revealed spatial overlap between telomere aggregates and lamina intranuclear structures. Altogether, our data suggest a mechanical link between changes in lamina spatial organization and the formation of telomere aggregates during senescence of hMSCs, which can possibly contribute to changes in nuclear activity during cell senescence.

Treatment strategies for superficial mycosis caused by the dermatophyte Trichophyton rubrum consist of the use of topical or oral antifungal preparations. We have recently discovered that T. rubrum is susceptible to photodynamic treatment (PDT), with 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) as a photosensitizer. The susceptibility appeared to depend on the fungal growth stage, with PDT efficacy higher with microconidia when compared to mycelia. The aim of this study was to investigate, with the use of scanning electron microscopy, the morphological changes caused by a lethal PDT dose to T. rubrum when grown on isolated human stratum corneum. Corresponding dark treatment and light treatment without photosensitizer were used as controls. A sub-lethal PDT dose was also included in this investigation The morphologic changes were followed at various time points after the treatment of different fungal growth stages. Normal fungal growth was characterized by a fiber-like appearance of the surface of the hyphae and microconidia with the exception of the hyphal tips in full mycelia and the microconidia shortly after attachment to the stratum corneum. Here, densely packed globular structures were observed. The light dose (108 J/cm2) in the absence of Sylsens B, or the application of the photosensitizer in the absence of light, caused reversible fungal wall deformations and bulge formation. However, after a lethal PDT, a sequence of severe disruptions and deformations of both microconidia and the mycelium were observed leading to extrusion of cell material and emptied fungal elements. In case of a non-lethal PDT, fungal re-growth started on the remnants of the treated mycelium.

The RNA replication complexes of mammalian positive-stranded RNA viruses are generally associated with (modified) intracellular membranes, a feature thought to be important for creating an environment suitable for viral RNA synthesis, recruitment of host components, and possibly evasion of host defense mechanisms. Here, using a panel of replicase-specific antisera, we have analyzed the earlier stages of severe acute respiratory syndrome coronavirus (SARS-CoV) infection in Vero E6 cells, in particular focusing on the subcellular localization of the replicase and the ultrastructure of the associated membranes. Confocal immunofluorescence microscopy demonstrated the colocalization, throughout infection, of replicase cleavage products containing different key enzymes for SARS-CoV replication. Electron microscopy revealed the early formation and accumulation of typical double-membrane vesicles, which probably carry the viral replication complex. The vesicles appear to be fragile, and their preservation was significantly improved by using cryofixation protocols and freeze substitution methods. In immunoelectron microscopy, the virus-induced vesicles could be labeled with replicase-specific antibodies. Opposite to what was described for mouse hepatitis virus, we did not observe the late relocalization of specific replicase subunits to the presumed site of virus assembly, which was labeled using an antiserum against the viral membrane protein. This conclusion was further supported using organelle-specific marker proteins and electron microscopy. Similar morphological studies and labeling experiments argued against the previously proposed involvement of the autophagic pathway as the source for the vesicles with which the replicase is associated and instead suggested the endoplasmic reticulum to be the most likely donor of the membranes that carry the SARS-CoV replication complex.

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. EAV particles contain seven structural proteins: the nucleocapsid protein N, the unglycosylated envelope proteins M and E, and the N-glycosylated membrane proteins GP(2b) (previously named G(S)), GP(3), GP(4), and GP(5) (previously named G(L)). Proteins N, M, and GP(5) are major virion components, E occurs in virus particles in intermediate amounts, and GP(4), GP(3), and GP(2b) are minor structural proteins. The M and GP(5) proteins occur in virus particles as disulfide-linked heterodimers while the GP(4), GP(3), and GP(2b) proteins are incorporated into virions as a heterotrimeric complex. Here, we studied the effect on virus assembly of inactivating the structural protein genes one by one in the context of a (full-length) EAV cDNA clone. It appeared that the three major structural proteins are essential for particle formation, while the other four virion proteins are dispensable. When one of the GP(2b), GP(3), or GP(4) proteins was missing, the incorporation of the remaining two minor envelope glycoproteins was completely blocked while that of the E protein was greatly reduced. The absence of E entirely prevented the incorporation of the GP(2b), GP(3), and GP(4) proteins into viral particles. EAV particles lacking GP(2b), GP(3), GP(4), and E did not markedly differ from wild-type virions in buoyant density, major structural protein composition, electron microscopic appearance, and genomic RNA content. On the basis of these results, we propose a model for the EAV particle in which the GP(2b)/GP(3)/GP(4) heterotrimers are positioned, in association with a defined number of E molecules, above the vertices of the putatively icosahedral nucleocapsid.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.