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PubMedDate not supplied

[Rat Kupffer cells cultures and their applications to experimental hepatology (author's transl)].

Non-parenchymal cells were obtained by perfusion of rat liver with 0.05% collagenase followed by incubation at 37 degrees C of liver fragments in collagenase in a rotating water-bath. Kupffer cells were separated from endothelial cells by centrifugal elutriation and could be kept alive for several days. They had the same morphology as in vivo and retained the same properties of phagocytosis and pinocytosis. Rat Kupffer cells cultures could be used to investigate a number of unsolved problems of hepatic pathophysiology.

Animals↗
PubMed1979

Regeneration of cerebral microvessels: a morphologic and histochemical study after local freeze-injury.

The sequential changes that occur during regeneration of cerebral microvessels was studied in young mice after a local freeze-injury. The immediate breakdown in the blood-brain barrier to horseradish peroxidase was followed by dissolution of cells in the vessel wall although the basal lamina was preserved. This residual basal lamina formed the pathway for the orderly regeneration of microvessels. Three days after injury, reactive endothelial cells with prominent pinocytosis and patent interendothelial channels were present at the edge of the lesion. By 5 days, endothelialization of necrotic vessels was in progress with patent vascular channels and tight junction formation. Establishment of astrocytic contacts or perivascular fibrosis by 7 days was generally followed by reconstitution of normal vascular morphology and integrity at 15 to 35 days after injury.

Animals↗
PubMed1993

Endo-lysosomal acidification in Dictyostelium discoideum amoebae. Effects of two endocytosis inhibitors: caffeine and cycloheximide.

Fluid-phase endocytosis (pinocytosis) is highly active in amoebae of the cellular slime mould Dictyostelium discoideum as it provides an efficient entry of nutrients in axenic strains. Detailed kinetic analyses were conducted using fluorescein-labeled dextran (FITC-dextran) as fluid-phase marker and pH probe. Cells were first pulsed with FITC-dextran during a short period then chased by suspension in probe-free medium. Chase kinetics were characterized by a lag phase of about 40 min before pseudo-first order FITC-dextran efflux and thus reflected the progression of the probe cohort through the various endosomal compartments along the endosomal pathway. Temporal evolution of endo-lysosomal pH showed a rapid acidification (T1/2 approximately 10 min) to pH 5.0 followed by an increase up to pH 6.2 to 6.3. The effects of cycloheximide and caffeine, two inhibitors of endocytosis in Dictyostelium amoebae, on the evolution of endosomal pH during fluid-phase endocytosis, have been investigated. Cycloheximide fully blocked the cellular transit of FITC-dextran but acidification of endo-lysosomal compartments still took place. Caffeine increased endo-lysosomal pH, probably as a consequence of an elevation of cytosolic [Ca2+]. Furthermore, it allowed the functional identification of a caffeine-insensitive terminal segment of the endocytic pathway. It corresponded to a recycling, postlysosomal compartment at pH 6.2 to 6.3 with an apparent volume of 160 microns 3/amoebae.

Animals↗
PubMed1995

In vitro effects of mannan and cytochalasin B on the uptake of horseradish peroxidase and [14C]sucrose by Trypanosoma cruzi epimastigotes.

Ultrastructural and quantitative studies were conducted to determine the in vitro effects of mannan and cytochalasin B (CB) on the transport of horseradish peroxidase (HRP) and [14C]sucrose by epimastigotes of Trypanosoma cruzi (strain Y). Time-dependent changes in HRP uptake were observed in cells incubated with the actin inhibitor CB. After 60 min incubation in CB, HRP and sucrose uptakes were inhibited by 48 +/- 15.4% and 16.5 +/- 3.96%, respectively. Morphological changed included HRP reaction product on the cell surface and a reduction in the number of HRP-positive reservosomes when compared to controls. After 120 min incubation, no inhibition was measured for either molecule. However, electron microscopy revealed HRP reaction product on the surface of the cells and in the cytosol. Also, perturbation of the plasma membrane was evident, suggesting that CB compromised the integrity of the plasma membrane, allowing HRP and sucrose to diffuse into the cytosol, giving misleading quantitative results. Mannan displayed a concentration-dependent inhibitory effect on HRP uptake but had little effect on sucrose uptake. Electron microscopic analysis revealed no change in the number of reservosomes per cell but reduction in the amount of HRP in reservosomes concomitant with mannan concentration. These results suggest that T. cruzi epimastigotes transport HRP by receptor-mediated and fluid-phase pinocytosis.

Animals↗
PubMed1976

[ Perfecting a simple method of organotypic thyroid gland culture in Xenopus laevis (Amphibia, Anoura)].

The larval thyroid of Xenopus cultured in an adequate medium, keeps its morphological differentiations at an ultrastructural scale and reacts against TSH added to the culture medium, through the development of apical microvilli, which is a sign of an active pinocytosis of the colloid, and also of ergastoplasmic lamellar and vesicular structures suggesting an active protein synthesis.

Animals↗
PubMed1993

Sodium chloride stress induced morphological and ultrastructural changes in Aspergillus repens.

Halotolerant fungus, A. repens, showed a considerable difference in its growth rate, morphology, ultrastructural and molecular composition under NaCl stress as compared to control i.e. non-stressed condition. Light microscopic observations revealed significant differences in their mycelial thickness, their branching and septa. Transmission electron microscopic observations of both the conditions depicted significant differences in the qualitative and quantitative changes in mitochondria. Frequent pinocytotic vesiculation (vacuoles) of plasma membrane was observed in fungus under stress but no such vesiculation in control. The multivesiculate structures observed under stress with their origin from the cell membranes and subsequent release into vacuoles have not been reported in fungi under normal physiological conditions. The observations on pinocytosis are discussed in relation to ion compartmentation and salt tolerance in A. repens.

Aspergillus↗
PubMed1977

Attachment and ingestion of mycoplasmas by mouse macrophages. II. Scanning electron microscopic observations.

Mycoplasmas adhere closely to the central region of the surface of mouse peritoneal macrophages in vitro. They do not appear connected to each other or the macrophage membrane, and they induce no change in the surface of the cell. After addition of antimycoplasma antibody, mycoplasmas show interconnections and the cell shows an increase occurrence of ruffled membrane and folding over the mycoplasmas. Large and small lacunae appear in the membrane at sites other than those taking in organisms, and the cell develops a diffusely granular appearance. These changes are associated with an increase in pinocytosis of horseradish peroxidase that is 85% above controls. Five minutes after addition of antibody, the macrophage appears contracted and engorged and has persistent membrane changes consisting of pits, openings, and membrane folds. Trypsin causes slow ingestion of surface mycoplasmas without the obvious membrane folding over organisms but with evidence of a predominantly invaginating process of phagocytosis. The macrophage surface has numerous microprojections, but is does not have the granular appearance seen after addition of antibody. Trypsin and Mycoplasma pulmonis antigen do not enhance macrophage pinocytic rates. (Am J Pathol 87:347-358, 1977).

Animals↗
PubMed1996

Modulation of macrophage scavenger receptor transport by protein phosphorylation.

The identification of three highly conserved phosphorylation sites in the cytoplasmic domain of each of the monomeric subunits of the macrophage scavenger receptor suggests that protein phosphorylation may regulate this receptor pathway. To investigate this, mouse peritoneal macrophages were pretreated with either the protein phosphatase inhibitor okadaic acid or the protein kinase inhibitor staurosporine to modulate cellular protein phosphorylation and their effects on the metabolism of acetyl-LDL were measured. Both okadaic acid and staurosporine inhibited the degradation of acetyl-low density lipoprotein (LDL) without affecting cellular lactic dehydrogenase (LDH) levels. The inhibition by okadaic acid was due to a 70% decrease in acetyl-LDL binding whereas post-receptor processing was minimally affected. Calyculin A, another serine/threonine phosphatase inhibitor, also reduced acetyl-LDL binding, whereas lithium chloride, an inositol phosphatase inhibitor, did not. Okadaic acid did not decrease steady state receptor mRNA levels nor decrease the number of total cellular receptors, consistent with a posttranslational mechanism of action. Interestingly, protease sensitivity studies showed that the receptors were still located on the cell surface. These studies suggest that okadaic acid inhibits acetyl-LDL binding by causing the redistribution of surface receptors into a sequestered compartment or inactivating the receptors. In contrast, staurosporine produced a paradoxical increase in receptor expression (30%) but slowed post-receptor processing (2.3-fold decrease). The latter was due to an inhibition of ligand internalization (2.9-fold decrease) via a protein kinase C-independent mechanism. Macrophage pinocytosis was also slowed by staurosporine (38% decrease); however, this does not appear to account for the inhibition of scavenger receptor internalization. Direct receptor phosphorylation was also slowed by staurosporine (38% decrease); however, this does not appear to account for the inhibition of scavenger receptor internalization. Direct receptor phosphorylation was also investigated and it was established that the receptor can be phosphorylated; however, changes in receptor function did not correlate with changes in the degree of receptor phosphorylation. Together these studies demonstrate that changes in cellular protein phosphorylation affect the expression, surface transport, and internalization of the macrophage scavenger receptor and suggest that the regulated phosphorylation/dephosphorylation of cellular proteins may be an important biochemical mechanism that controls normal processing of ligands by this receptor pathway.

Animals↗
PubMed1997

Chitin particle-induced cell-mediated immunity is inhibited by soluble mannan: mannose receptor-mediated phagocytosis initiates IL-12 production.

Previous studies showed that mouse spleen cells produced IL-12, TNF-alpha, and IFN-gamma when stimulated with phagocytosable-size chitin particles (N-acetyl-D-glucosamine polymers). To dissect the mechanisms of the cytokine production in this study, spleen cells from BALB/c mice were cultured with 1 to 10 microm chitin particles, heat-killed Corynebacterium parvum vaccine, zymosan, and mannan (a mannose polymer)-coated latex beads (1 microm) at 1, 10, or 100 microg/ml. We found that these particles induced IL-12, TNF-alpha, and IFN-gamma. However, these cytokines were not produced when spleen cells were cultured with soluble chitin, mannan, or laminarin (a polymer of beta-glucan), 1 to 10 microm beta-glucan particles, laminarin-coated latex beads, 1 microm latex beads, 50 to 100 microm chitin particles, or 50 to 100 microm mannan-coated beads. Soluble mannan, but not soluble laminarin, inhibited cytokine production following stimulation with 1 to 10 microm chitin particles, zymosan, or heat-killed C. parvum. In addition, cytochalasin D also inhibited cytokine production. The treatments with soluble mannan or with cytochalasin D, in sharp contrast, did not inhibit LPS-induced IL-12/IFN-gamma production or exogenous IL-12-induced IFN-gamma production. Finally, spleen cells from C3H/HeJ mice also showed comparable levels of IL-12/TNF-alpha/IFN-gamma production when induced by 1 to 10 microm chitin particles. Taken together, our results indicate that mannose receptor-mediated phagocytosis, but not the receptor-mediated pinocytosis, is highly associated with the production of IFN-gamma-inducing extracellular signaling factors such as IL-12 and TNF-alpha. The novel mechanism of phagocytosis-dependent IL-12 production appears to be distinct from that of LPS-induced cytokine production.

Actins↗
PubMed1998

Effect of endocytosis inhibitors on Coxiella burnetii interaction with host cells.

The obligate intracellular rickettsia Coxiella burnetii has previously been reported to reach the intravacuolar compartment of host cells by phagocytosis. With the aim to further examine the mechanisms of C. burnetii internalisation, macrophage monolayers were treated with well characterised inhibitors of endocytosis. The treatment with two general inhibitors, colchicine and methylamine, resulted in a pronounced dose-dependent decrease of radiolabelled phase II rickettsiae retained from the intracellular fraction. A third inhibitor used, amiloride, has been reported to reduce effectively clathrin-independent pinocytic pathways. The internalisation of C. burnetii was shown to be substantially reduced also by amiloride and the effect was dependent on its concentration. The passive role of C. burnetii in the internalisation was verified by using heat-killed C. burnetii. Host cells treated with either of the three inhibitors (amiloride, colchicine and methylamine) showed a similar reduction of intracellular C. burnetii after exposure to killed as well as live organisms. The data presented indicate that different endocytic mechanisms, pinocytosis as well as phagocytosis, may mediate the uptake of C. burnetii by a host cell.

Amiloride↗
PubMed1999

Multiple signal transduction pathways regulate TNF-induced actin reorganization in macrophages: inhibition of Cdc42-mediated filopodium formation by TNF.

TNF is known to regulate macrophage (Mphi) migration, but the signaling pathways mediating this response have not been established. Here we report that stimulation of the 55-kDa TNF receptor (TNFR-1) induced an overall decrease in filamentous actin (F-actin), inhibited CSF-1- and Cdc42-dependent filopodium formation, and stimulated macropinocytosis. Using a panel of TNFR-1 mutants, the regions of the receptor required for each of these responses were mapped. The decrease in F-actin required both the death domain and the membrane proximal part of the receptor, whereas inhibition of filopodium formation and increased pinocytosis were only dependent upon a functional death domain. When the TNF-induced decrease in F-actin was inhibited using either receptor mutants or the compound D609, TNF-stimulated actin reorganization at the cell cortex became apparent. This activity was dependent upon the FAN-binding region of TNFR-1. We conclude that different domains of TNFR-1 mediate distinct changes in the Mphi cytoskeleton, and that the ability of TNF to inhibit Mphi chemotaxis may be due to decreased filopodium formation downstream of Cdc42.

Actins↗
PubMed1998

Cytochemical evidence for potassium-dependent p-nitrophenylphosphatase activity in pavement cells of Rana esculenta mesentery.

BACKGROUND: We previously reported that during hibernation in Rana esculenta, various organs (i.e., skin, urinary bladder, kidney) change their osmoregulatory activity. Here, we considered the possible role of the frog mesentery in the ion transport, evaluating morphological and cytochemical (K+-p-nitrophenylphosphatase activity) aspects. METHODS: Pieces of mesentery from Rana esculenta collected in their natural environment during April, June, October, and January were processed to reveal ultrastructural morphology and K+-p-NPPase activity, using cerium as capture agent. RESULTS: The mesenteric mesothelium contained three types of cells: pavement, mitochondria-rich, and ciliated. Only the pavement cells expressed intense reactivity on the basolateral membranes and in the adjacent pinocytotic vesicles; some reaction product also was found on the apical membranes. Moreover, morphological and cytochemical characteristics of the pavement cells appeared to be very seasonal. CONCLUSIONS: The presence of mitochondria-rich cells and ciliated cells, generally found in structures involved in the transport of liquids, as well as K+-p-NPPase activity and pinocytosis in pavement cells, is consistent with the hypothesis that frog mesentery may be involved in seasonally variable osmoregulation.

4-Nitrophenylphosphatase↗
PubMed1998

Antitumor activity of methotrexate-albumin conjugates in rats bearing a Walker-256 carcinoma.

We have recently reported that albumin accumulates in solid tumors and serves there as a source of nitrogen and energy. Methotrexate-albumin conjugates [MTX(I)-RSA] derivatized at a molar ratio of 1:1 differ favorably from original MTX in terms of plasma presence and tumor uptake. The purpose of this study was to evaluate the therapeutic efficacy of these novel conjugates in a comparative study with low m.w. MTX is Sprague-Dawley rats bearing a Walker-256 carcinoma. The maximum tolerated dose (MTD) for MTX and MTX(I)-RSA was determined (2 mg/kg based on MTX injected on days 1, 3 and 8). The tumor-bearing rats received injections of either the MTD or MTD/2 of MTX, MTX-albumin or mixtures containing the MTD/2 or MTD/4 of both MTX and MTX-albumin. No toxic side effects were observed. Cure rate and tumor growth retardation were slightly better for the conjugate compared with MTX alone in the MTD group (16 complete remissions vs. 14 of 20 rats). The best results were achieved for the combination treatment with MTX and MTX-albumin, with complete remission in all 20 rats. In conclusion, MTX-albumin conjugates show therapeutic activity in vivo without toxic side effects. Additive effects were observed for a combination of MTX-albumin and MTX. These effects might be caused by the much longer tumor exposition time of the conjugate in conjunction with a different route of uptake (pinocytosis for MTX-albumin vs. folate receptors for MTX).

Albumins↗
PubMed1996

Src, ras, and rac mediate the migratory response elicited by NGF and PMA in PC12 cells.

The combination of nerve growth factor (NGF) and phorbol 12-myristate 13-acetate (PMA) rapidly induced the locomotion of PC12 cells by sequentially stimulating lamellar spreading, ruffling with pinocytosis, and polarization by retraction from the substratum. During migration, cells acquired long processes as a result of several undisrupted cell-substratum attachment points. The effect of NGF on PC12 migration was blocked by K-252a, a selective inhibitor of the trk family of receptor tyrosine kinases. When PMA was added to cells expressing pp60v-src, the cells displayed the same morphological behavior as they did with NGF and PMA addition. Activated ras only partially substituted for the effects of NGF; but, when ras was inhibited, the number of migrating cells decreased significantly due to a defect in spreading and retraction. Expression of an activated form of rac stimulated spontaneous growth of lamellipodia and enhanced cell migration in response to PMA. Expression of a dominant negative form of rac inhibited cell spreading and motility. Also, as a later effect, rac-inhibited cells extended much shorter neurites than wild type cells in response to NGF alone. These results indicate that the cytoarchitectural changes induced by NGF and PMA in PC12 cells are mediated by src, ras, and rac. Whereas ras and rac activation affect lamellipodia extension and retraction but not pinocytotic ruffling, src activation is involved in all three events.

Animals↗
PubMed1998

Regulation of smooth muscle cell proliferation using paclitaxel-loaded poly(ethylene oxide)-poly(lactide/glycolide) nanospheres.

Available data suggest that drugs should be delivered to a vascular lesion at a high concentration over an extended period of time to control vascular smooth muscle cell (VSMC) proliferation. This study was undertaken to formulate a paclitaxel, an antimicrotubule agent, into a biodegradable poly (ethylene oxide)-poly(lactide/glycolide) (PEO-PLGA) nanosphere as a sustained drug delivery system and to study its effects on VSMC in culture. The paclitaxel-loaded nanospheres (PT/NS), prepared by an emulsion-solvent evaporation method, had an average diameter of approximately 150 nm and showed a sustained release profile over 4 weeks. The PT/NS exhibited antiproliferative effects comparable to those observed with free paclitaxel. The cellular internalization of nanospheres was visualized using confocal fluorescence microscopy, and from a flow cytometry study the progressive cellular uptake profile, uptake inhibition at low temperature, and saturation uptake kinetics (concentration dependency) were observed. These suggest that (adsorptive) pinocytosis is a major uptake mechanism of the nanospheres. The sustained drug release profile and cellular internalization results suggest that nanospheres loaded with paclitaxel may potentially be used as an endocytizable, local sustained drug delivery system for the prevention of restenosis.

Animals↗
PubMed1980

Neoplastic human colon cells in studies on the translocation of dimeric IgA.

The translocation of dimeric IgA across epithelial cells was studied by immunoelectron microscopy in an in vitro system with cultured neoplastic human colon cells (HT-29). Ultrastructurally, the cells were found to be well-polarized epithelial cells connected by intercellular junctions. Secretory component (SC) was localized to the basolateral plasma membranes. Dimeric human IgA, when reacted with the cells at 0 C, bound selectively to SC. When incubated at 37 C, the bound dimeric IgA was internalized by pinocytosis and transported apically through the cytoplasm in vesicles. The vesicles were opened to the lumen at the apical surfaces or discharged into the lumen. We conclude that the translocation of dimeric IgA across intestinal epithelial cells has been defined at the ultrastructural level in cultured neoplastic colon cells in vitro.

Biological Transport, Active↗
PubMed2000

Actin polymerization in response to different chemoattractants is reduced in granulocytes from chronic myeloid leukemia patients.

Chronic myeloid leukemia (CML), a hematopoietic stem cell disorder, is characterized by the presence of Philadelphia chromosome (Ph1). Earlier studies have shown that various functions, such as chemotaxis, fluid phase pinocytosis, phagocytosis, and degranulation in response to chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP), were defective in polymorphonuclear leukocytes (PMNL) from CML patients. These functions depend on actin microfilaments (MF). Further studies showed that fMLP-induced actin polymerization was lower in CML PMNL. To see if this defect is specific to stimulation by fMLP alone or is a global phenomenon involving other chemoattractant receptors, chemotaxis and actin polymerization were studied in response to fMLP, an analog of fMLP, formyl-methionine-1 aminocyclooctane 1 carboxylic acid-phenyalanine-O-methionine (FACC8), platelet-activating factor (PAF), and leukotriene B4 (LTB4). These compounds bind to different chemoattractant receptors. Chemotaxis and actin polymerization in response to all four chemoattractants were significantly lower in CML PMNL compared with PMNL from normal subjects and were differentially affected for the different chemoattractants. These results suggest a global phenomenon involving all four chemoattractant-stimulated pathways. This lower amount of F-actin may be responsible for the defective chemotaxis seen in these cells.

Actin Cytoskeleton↗
PubMed2002

CpG-DNA aided cross-presentation of soluble antigens by dendritic cells.

For cross-presentation immature dendritic cells (DC) require enhanced antigen (Ag) uptake and a maturation signal to prime for MHC class I-restricted CTL responses in vivo. While immunostimulatory CpG-DNA provides, via TLR9, the maturation signal, CpG-DNA linked to Ag augments cellular Ag uptake. In this study we show that CpG-DNA ovalbumin (OVA) conjugates trigger in vivo peptide-specific CTL responses at tenfold lower Ag doses compared to a mixture of CpG-DNA plus OVA. We provide evidence that CpG-DNA-OVA conjugates shift OVA uptake by immature DC from the presumably inefficient fluid phase pinocytosis to efficient DNA receptor-mediated endocytosis. Since the DNA-binding receptor mediating endocytosis lacks any sequence specificity, cellular uptake of OVA conjugated with either stimulatory or non-stimulatory oligonucleotides (ODN) is equally enhanced. As a consequence cross-linking of OVA with either stimulatory or non-stimulatory DNA yields, via enhanced OVA uptake, efficient generation and presentation of the dominant OVA-CTL epitope SIINFEKL. However, only stimulatory CpG-ODN cross-linked to OVA provide the DC maturation signal required to trigger robust primary CTL responses towards the cross-presented MHC class I complexed T cell epitope SIINFEKL. Our studies show that stimulatory CpG-ODN linked to Ag fulfill a dual role: enhancement of Ag uptake yielding efficient Ag cross-presentation by DC and in addition, their activation into professional DC.

Adjuvants, Immunologic↗