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Dendritic cells as effector cells: gamma interferon activation of murine dendritic cells triggers oxygen-dependent inhibition of Toxoplasma gondii replication.

Toxoplasma gondii is an obligate intracellular parasite that infects a wide variety of nucleated cells in its numerous intermediate hosts, including humans. Much interest has focused on the ability of gamma interferon (IFN-gamma)-activated macrophages to prevent intracellular replication, but some other cells (e.g., fibroblasts, endothelial cells, microglial cells, astrocytes, enterocytes and retinal pigment cells) can also be activated to induce this inhibition of proliferation. Dendritic cells are generally known to be involved in the induction of immune responses, but no previous study had investigated the possibility that dendritic cells may act as effector cells of this system. Our results show that IFN-gamma-activation inhibits the replication of T. gondii in dendritic cells, with the inhibition being dose dependent. Neither nitrogen derivatives nor tryptophan starvation appears to be involved in the inhibition of parasite replication by IFN-gamma. Experiments with oxygen scavengers indicate that intracellular T. gondii replication is oxygen dependent. Our findings suggest that, in addition to their essential role in stimulating the immune system, dendritic cells probably act as effector cells in the first line of defense against pathogen invasion.

Animals↗

A novel, rapid strategy to form dendritomas from human dendritic cells and hepatocellular carcinoma cell line HCCLM3 cells using mature dendritic cells derived from human peripheral blood CD14+ monocytes within 48 hours of in vitro culture.

AIM: Dendritomas formed by fusing cancer cells to dendritic cells have already been applied to clinical treatment trial of several types of cancers. Dendritic cells for the fusion in most trials and experiments were from blood monocytes in standard 7-d protocol culture, which requires 5-7 d of culture with granulocyte-macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), followed by 2-3 d of activation with a combination of proinflammatory mediators such as tumor necrosis factoralpha (TNFalpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and prostaglandin E(2) (PGE(2)). One study showed that mature monocyte-derived dendritic cells could be obtained within 48 h of in vitro culture with the same protocol as standard 7-d culture and referred to as FastDCs. Here we aimed to fuse human hepatocellular carcinoma cell line HCCLM3 cells with mature monocyte-derived dendritic cells within 48 h of in vitro culture (FastDC). METHODS: HCCLM3 cells were cultured in RPMI 1640 with 150 mL/L fetal calf serum (FCS). CD14+monocytes from healthy human peripheral blood were purified with MACS CD14 isolation kit and cultured in six-well plates in fresh complete DC medium containing RPMI-1640, 20 mL/L heat inactivated human AB serum, 2 mmol/L L-glutamine, 100 microg/mL gentamicin, 1 000 U/mL GM-CSF and 500 U/mL IL-4 for 24 h, then proinflammatory mediators such as TNFalpha (1 000 U/mL), IL-1beta (10 ng/mL), IL-6 (10 ng/mL) and PGE(2) (1 microg/mL) were supplemented for another 24 h, and thus mature FastDCs were generated. HCCLM3 cells and FastDCs were labeled with red fluorescent dye PKH26-GL and green fluorescent dye PKH67-GL respectively. After the red fluorescent-stained HCCLM3 cells were irradiated with 50 Gy, FastDCs and irradiated HCCLM3 cells were fused in 500 mL/L polyethylene glycol(PEG)+100 mL/L dimethyl sulfoxide (DMSO) to generate novel dendritomas. The FastDCs and novel dendritomas were immunostained with anti-CD80, anti-CD86, anti-CD83, anti-HLA-DR mAbs and analyzed by fluorescence-activated cell sorting (FACS). Novel dendritomas were nucleus-stained with Hoechst 33258 and analyzed by confocal laser scanning microscopy. RESULTS: Mature FastDCs with highly expressed surface markers CD80, CD86, CD83 and HLA-DR were generated within 48 h in vitro. Novel dendritomas with dual red-green fluorescence were constructed fast and successfully, and FACS analysis showed that the fusion efficiency was 24.27% and the novel dendritomas expressed the same activation markers as FastDCs. Confocal laser scanning microscopy analysis showed representative images of dendritomas. CONCLUSION: Dendritomas can be formed fast with mature FastDCs from healthy human peripheral blood monocytes (PBMC) by incubation with GM-CSF and IL-4 for 24 h and by activation with proinflammatory mediators for an additional period of 24 h. Owing to shorter time required for in vitro DCs development, the generation of these novel dendritomas reduced labor and cost. This rapid method for formation of dendritomas may represent a new strategy for immunotherapy of hepatocellular carcinoma.

Carcinoma, Hepatocellular↗

Muscarinic regulation of dendritic and axonal outputs of rat thalamic interneurons: a new cellular mechanism for uncoupling distal dendrites.

Inhibition is crucial for sharpening the sensory information relayed through the thalamus. To understand how the interneuron-mediated inhibition in the thalamus is regulated, we studied the muscarinic effects on interneurons in the lateral posterior nucleus and lateral geniculate nucleus of the thalamus. Here, we report that activation of muscarinic receptors switched the firing pattern in thalamic interneurons from bursting to tonic. Although neuromodulators switch the firing mode in several other types of neurons by altering their membrane potential, we found that activation of muscarinic subtype 2 receptors switched the fire mode in thalamic interneurons by selectively decreasing their input resistance. This is attributable to the muscarinic enhancement of a hyperpolarizing potassium conductance and two depolarizing cation conductances. The decrease in input resistance appeared to electrotonically uncouple the distal dendrites of thalamic interneurons, which effectively changed the inhibition pattern in thalamocortical cells. These results suggest a novel cellular mechanism for the cholinergic transformation of long-range, slow dendrite- and axon-originated inhibition into short-range, fast dendrite-originated inhibition in the thalamus observed in vivo. It is concluded that the electrotonic properties of the dendritic compartments of thalamic interneurons can be dynamically regulated by muscarinic activity.

Action Potentials↗

[Presynaptic dendrites and dendro-dendritic synapses in the superior tubercles of the rat quadrigeminum].

The types of dendro-dendritic synapses and their participation in the synaptic, organization of superficial layers of the quadrigeminum superior tubercles were studied electron microscopically. In addition to simple forms of dendro-dentritic synapses the reciprocal dendro-dendritic synapses were revealed. Presynaptic dendrites formed the synaptic fields and glomerules of the superficial grey layer. The terminals of optical, cortical fibres from the visual cortex and other types of terminals terminated on presynaptic dendrites.

Animals↗

Submillisecond precision of the input-output transformation function mediated by fast sodium dendritic spikes in basal dendrites of CA1 pyramidal neurons.

The ability of cortical neurons to perform temporally accurate computations has been shown to be important for encoding of information in the cortex; however, cortical neurons are expected to be imprecise temporal encoders because of the stochastic nature of synaptic transmission and ion channel gating, dendritic filtering, and background synaptic noise. Here we show for the first time that fast local spikes in basal dendrites can serve to improve the temporal precision of neuronal output. Integration of coactivated, spatially distributed synaptic inputs produces temporally imprecise output action potentials within a time window of several milliseconds. In contrast, integration of closely spaced basal inputs initiates local dendritic spikes that amplify and sharpen the summed somatic potential. In turn, these fast basal spikes allow precise timing of output action potentials with submillisecond temporal jitter over a wide range of activation intensities and background synaptic noise. Our findings indicate that fast spikes initiated in individual basal dendrites can serve as precise "timers" of output action potentials in various network activity states and thus may contribute to temporal coding in the cortex.

Action Potentials↗

Dendritic transport. I. Colchicine stimulates the transport of lysosomal enzymes from cell bodies to dendrites.

Injection of colchicine into the lateral cerebral ventricle of the rat was found to induce a paradoxical translocation of two lysosomal enzymes, dipeptidyl peptidase II (Dpp II) and acid phosphatase, from the soma of neurons to the dendrites. Following a single injection of colchicine, neuronal somata, which normally contain the bulk of these lysosomal enzymatic activities, become depleted of these enzymes, whereas dendrites become abnormally enriched. All neurons which contained these enzymes, except those of the mesencephalic nucleus of the trigeminal nerve, displayed this phenomenon. Lysosomal enzyme translocation into dendrites was observed in the mitral cell layer within 1 hr after a colchicine injection and could be induced in most neuronal populations by injections of colchicine as low as 25 micrograms. Five days after a 100-micrograms colchicine injection, a normal pattern of enzyme distribution was observed, indicating that the effect of colchicine was reversible. Enzyme translocation was not accompanied by gross changes in cell morphology, nor did it result in the specific loss of neuronal cell bodies which contained these enzymes. The results indicate that colchicine, under conditions known to inhibit axoplasmic transport, stimulates the transport of lysosomal enzymes from the cell body to the dendrites.

Acid Phosphatase↗

Dendritic transport. II. Somatofugal movement of neuronal lysosomes induced by colchicine: evidence for a novel transport system in dendrites.

The effect of colchicine injections on the ultrastructural localization of dipeptidyl peptidase II (Dpp II) was studied in the mitral cells of the rat olfactory bulb. In control animals, electron-dense reaction product representing Dpp II activity was observed in lysosomes, lipofuscin granules, short cisternae located close to the granular endoplasmic reticulum, and dense granules. Lysosomes and lipofuscin granules were the most intensely stained organelles. Dpp II-containing organelles were localized mainly to the cell body and were randomly distributed in the perikaryal cytoplasm. Twenty-four hours after a 100-micrograms intracerebroventricular colchicine injection, the distribution of Dpp II-containing organelles was drastically altered. Short cisternae and dense granules containing Dpp II reaction product were noticeably absent in these preparations. Lysosomes and lipofuscin granules were depleted from the perikaryal cytoplasm and were concentrated in dendrites. Lysosomes were observed to extend for considerable distances in dendrites where they acquired elongated and dumbbell shapes. The shapes of some of these labeled lysosomes gave the impression that they were actively being "pulled" into the dendrites. These results indicate that microtubules sequester lysosomes to the perikaryal cytoplasm and suggest the presence of a novel transport system responsible for the movement of lysosomes from the cell body to the dendrites.

Animals↗

Three-dimensional organization of smooth endoplasmic reticulum in hippocampal CA1 dendrites and dendritic spines of the immature and mature rat.

Recent studies have shown high levels of calcium in activated dendritic spines, where the smooth endoplasmic reticulum (SER) is likely to be important for regulating calcium. Here, the dimensions and organization of the SER in hippocampal spines and dendrites were measured through serial electron microscopy and three-dimensional analysis. SER of some form was found in 58% of the immature spines and in 48% of the adult spines. Less than 50% of the small spines at either age contained SER, suggesting that other mechanisms, such as cytoplasmic buffers, regulate ion fluxes within their small volumes. In contrast, >80% of the large mushroom spines of the adult had a spine apparatus, an organelle containing stacks of SER and dense-staining plates. Reconstructed SER occupied 0.001-0.022 microm3, which was only 2-3.5% of the total spine volume; however, the convoluted SER membranes had surface areas of 0.12-2.19 microm2, which were 12 to 40% of the spine surface area. Coated vesicles and multivesicular bodies occurred in some spines, suggesting local endocytotic activity. Smooth vesicles and tubules of SER were found in continuity with the spine plasma membrane and margins of the postsynaptic density (PSD), respectively, suggesting a role for the SER in the addition and recycling of spine membranes and synapses. The amount of SER in the parent dendrites was proportional to the number of spines and synapses originating along their lengths. These measurements support the hypothesis that the SER regulates the ionic and structural milieu of some, but not all, hippocampal dendritic spines.

Aging↗

Dendrite and dendritic spine alterations in Alzheimer models.

Synaptic damage and loss are factors that affect the degree of dementia experienced in Alzheimer disease (AD) patients. Multicolor DiOlistic labeling of the hippocampus has been undertaken which allows the full dendritic arbor of targeted neurons to be imaged. Using this labeling technique the neuronal morphology of two transgenic mouse lines (J20 and APP/PS1) expressing mutant forms of the Amyloid Precursor Protein (APP), at various ages, have been visualized and compared to Wild Type (WT) littermate controls. Swollen bulbous dystrophic neurites with loss of spines were apparent in the transgenic animals. Upon quantification, statistically significant reductions in the number of spines and total dendrite area was observed in both transgenic mouse lines at 11 months of age. Similar morphological abnormalities were seen in human AD hippocampal tissue both qualitatively and quantitatively. Immunohistochemistry and DiOlistic labeling was combined so that Abeta plaques were imaged in relation to the dendritic trees. No preferential localization of these abnormal dystrophic neurites was seen in regions with plaques. DiI labeled reative astrocytes were often apparent in close proximity to A beta plaques.

Aging↗

Dendritic spinules in rat nigral neurons revealed by acetylcholinesterase immunocytochemistry and serial sections of the dendritic spine heads.

Dendritic spinules of rat nigral neurons were visualized at electron microscopic level by acetylcholinesterase immunocytochemistry and serial sections of the nigral dendrites. The spinules (at least 150 nm in length and 10-20 nm in width) which protruded from the spine heads are found in extracellular space in the neuropil and particularly between nerve terminals of the presynaptic neurons and fine glial processes. The nigral spinules are, however, not observed as invaginated processes in the nerve terminals. The dendritic spinule may be endowed with synaptic plasticity and metabolic exchange between nerve terminals and glial processes.

Acetylcholinesterase↗

Identification of avian dendritic cells in the spleen using a monoclonal antibody specific for chicken follicular dendritic cells.

BACKGROUND: In the chicken, circulating antigens enter the splenic white pulp via the Schweigger-Seidel sheaths (ellipsoids), where they are bound by cells, the ellipsoid associated cells (EAC), which are located on the periphery of the ellipsoid. There is an increasing body of evidence that these antigen-binding cells move through the PALS, to be finally located within the germinal centers, where these antigen-transporting EAC function as follicular dendritic cells (FDC). The aim of the current study was to further study the relationship between the EAC, the FDC, and the antigen-bearing EAC which migrate through the splenic white pulp. METHODS: In order to identify the splenic FDC and their presumed migrating EAC precursors in the chicken, we used a monoclonal antibody produced against chicken FDC and an antiserum anti-S-100 protein which identifies chicken dendritic cells in lymphoid organs. RESULTS: Cells reacting with the 74.3 monoclonal antibody, which identifies FDC, were found within the germinal center, around the penicilliform capillary, in the periellipsoidal white pulp, and in the periarteriolar lymphatic sheaths (PALS). S-100+ cells were found in these same locations. CONCLUSIONS: A comparison between the staining patterns obtained with both antibodies strongly suggested that the intrasplenic distribution of 74.3+ cells was identical with that of FDC, EAC, and antigen-binding EAC migrating in the PALS. Therefore, the 74.3 monoclonal antibody identified not only FDC but also the splenic precursor cells of FDC, in accordance with the hypothesis of the migration of the EAC through the white pulp. S-100+ cells were more numerous than 74.3+ cells, which is in accordance with the fact that S-100 protein antibody stains both FDC and interdigitating dendritic cells (ID). This has allowed us to suggest that 74.3- EAC may represent precursors of ID. The current findings reinforce previous investigations, which provided evidence supporting the migration of EAC through the PALS and further supported the hypothesis which considers EAC precursors of FDC.

Animals↗

Cocaine self-administration alters the morphology of dendrites and dendritic spines in the nucleus accumbens and neocortex.

We studied the influence of cocaine use on the structure of neurons in brain regions that contribute to its rewarding effects by allowing rats to self-administer cocaine (0.33 mg/infusion) for 1 h a day for 1 month. Control animals were left undisturbed or allowed to work for food for the same period of time. After an additional 1 month drug-free period the brains were processed for Golgi-Cox staining. In rats that self-administered cocaine, but not rats that worked for food, there was a significant increase in dendritic branching and in the density of dendritic spines on medium spiny neurons in the shell of the nucleus accumbens and on pyramidal cells in the prefrontal and parietal (but not occipital) cortex. There was also a 2.6-fold increase in the incidence of spines with multiple heads (branched spines) on medium spiny neurons. Finally, in the prefrontal cortex some of the apical dendrites of pyramidal cells appeared misshaped, having large bulbous structures on their terminal tips. We speculate that cocaine self-administration experience alters patterns of synaptic connectivity within limbocortical circuitry that is thought to contribute to cocaine's incentive motivational effects and may have neuropathological effects in frontal areas involved in decision making and judgment. Together, these two classes of drug-induced neuroadaptations may contribute to the development of addiction.

Animals↗

Selective recognition of rat follicular dendritic cells (dendritic reticulum cells) by a new monoclonal antibody Ki-M4R in vitro and in vivo.

Using unstimulated rat peritoneal cells as immunogen a new monoclonal antibody Ki-M4R was produced. Ki-M4R recognizes follicular dendritic cells (dendritic reticulum cells) in germinal centers of lymphoid follicles in lymphatic tissue. In addition, sinus lining cells, endothelia of postcapillary venules, as well as mesangial cells of the renal glomerula immunoreact with Ki-M4R in vitro as well as in vivo. This antibody might be useful for studying the interaction of follicular dendritic cells and B-cell immune response.

Animals↗

Three-dimensional analysis of dendritic spines. I. Quantitative observations related to dendritic spine and synaptic morphology in cerebral and cerebellar cortices.

A total of 212 dendritic spines (108 from the visual and 104 from cerebellar cortices of the mouse) were analyzed in serial sections. Dendritic spines (DS) and synaptic active zones (SAZ) were classified according to their shape, and the following quantitative data were measured: DS stalk and bulb diameters, DS length and volume, number of cisterns of the spine apparatus, DS and SAZ surface areas and their mutual proportions. Quantitative relationships between the spine apparatus and the size of DS and SAZ, between the volume and surface area of DS and between the size of DS and the size of SAZ were studied. Thin, mushroom-shaped and stubby DS with simple (circular or oval), complex (perforated, annulate or horseshoe-shaped) and multifocal SAZ were found on terminal branches of pyramidal cell apical dendrites and club-shaped DS with simple (circular or oval) SAZ on spiny branchlets of Purkinje cells. Statistically significant differences were found between all values measured on various DS types in the visual cortex. Linear dependencies of the DS surface area on DS volume and of the SAZ surface area on the DS surface area were established. Only a limited area of DS plasma membrane (7-10%) was occupied by SAZ. This finding indicates a possible functional importance of the SAZ/DS (and possibly also of the total SAZ/total postsynaptic membrane) surface ratio.

Animals↗

S-100 protein-positive dendritic cells and CD34-positive dendritic interstitial cells in palatine tonsils.

Dendritic cells (DCs) are effective antigen-presenting cells and have been shown to mature from precursor CD34-positive stromal cells (dendritic interstitial cells, DICs) or monocytes. To gain insight into the local immune response in human tonsils, we investigated immunohistochemically the presence of DCs and DICs in 17 non-hyperplastic and 13 hyperplastic tonsils. Dense infiltrates of S-100-positive DCs were noted in the majority of hyperplastic tonsils, while there were fewer in non-hyperplastic tonsils. DICs were noted specifically at the periphery in the dense hemi-capsule cap that separates the tonsil from the underlying muscle. In addition, their small number suggests that the accumulation of S-100 dendritic cells in hyperplastic palatine tonsils is achieved through migration from other sites rather than through maturation from precursors locally.

Adult↗

Serial reconstruction of microtubular arrays within dendrites of the cat retinal ganglion cell: the cytoskeleton of a vertebrate dendrite.

Serial reconstruction at the EM level of cat retinal ganglion cell dendrites reveals that: (1) the microtubular array is discontinuous, (2) microtubular endings are associated with smooth endoplasmic reticulum (SER), mitochondria, and plasma membrane, (3) individual microtubules always maintain a minimum distance from other microtubules (87 nm), SER (43 nm) and plasma membrane (69 nm), and (5) individual microtubules can 'wander' independent of adjacent microtubules throughout the dendritic volume. These observations, taken with some recent biochemical and immunohistochemical data by other workers, suggest that the microtubules are surrounded by a coat of high molecular weight, microtubular-associated proteins (HMW MAPs), which effectively creates a 90 nm tube around a central microtubular core. Our results suggest that bundles of these 'MAP-tubes' may serve as a major component of the dendritic cytoskeleton in the cat ganglion cells.

Animals↗

Age-related changes in basal dendrite and dendritic spine of hippocampal pyramidal neurons (CA1) among SAMP1TA/Ngs--quantitative analysis by the rapid Golgi method.

It has been confirmed that a substrain of the senescence-accelerated mouse SAMP1TA/Ngs develops learning disturbance-like behavior at 3 months of age, exhibits almost normal behavior at 5 months, and manifests learning disturbance at 7 months. The changes with age in basal dendrites and dendritic spines of CA1 pyramidal neurons were quantitatively evaluated by the Golgi method using male SAMP1TA/Ngs. The correlation between the change in learning ability and the morphometry was examined. The number of dendritic spines in the 3- and 7-month-old groups was significantly lower than that in the 5-month-old group. It is presumed that the disturbance in acquisition of learning ability at 3 months of age is secondary to the immaturity of neurons, while the learning disturbance at 7 months of age is due to neuronal aging. This substrain, which is characterized by the impairment of acquired learning ability due to senescence, is useful as a model for studies on human brain dysfunction associated with senescence.

Age Factors↗

A behavioral role for dendritic integration: HCN1 channels constrain spatial memory and plasticity at inputs to distal dendrites of CA1 pyramidal neurons.

The importance of long-term synaptic plasticity as a cellular substrate for learning and memory is well established. By contrast, little is known about how learning and memory are regulated by voltage-gated ion channels that integrate synaptic information. We investigated this question using mice with general or forebrain-restricted knockout of the HCN1 gene, which we find encodes a major component of the hyperpolarization-activated inward current (Ih) and is an important determinant of dendritic integration in hippocampal CA1 pyramidal cells. Deletion of HCN1 from forebrain neurons enhances hippocampal-dependent learning and memory, augments the power of theta oscillations, and enhances long-term potentiation (LTP) at the direct perforant path input to the distal dendrites of CA1 pyramidal neurons, but has little effect on LTP at the more proximal Schaffer collateral inputs. We suggest that HCN1 channels constrain learning and memory by regulating dendritic integration of distal synaptic inputs to pyramidal cells.

Animals↗