Close Membrane Contact Research Articles

Gap junctions: historical discoveries and new findings in the Caenorhabditiselegans nervous system.

ABSTRACTGap junctions are evolutionarily conserved structures at close membrane contacts between two cells. In the nervous system, they mediate rapid, often bi-directional, transmission of signals through channels called innexins in invertebrates and connexins in vertebrates. Connectomic studies from Caenorhabditis elegans have uncovered a vast number of gap junctions present in the nervous system and non-neuronal tissues. The genome also has 25 innexin genes that are expressed in spatial and temporal dynamic pattern. Recent findings have begun to reveal novel roles of innexins in the regulation of multiple processes during formation and function of neural circuits both in normal conditions and under stress. Here, we highlight the diverse roles of gap junctions and innexins in the C. elegans nervous system. These findings contribute to fundamental understanding of gap junctions in all animals.

High‐throughput deterministic single‐cell encapsulation and droplet pairing, fusion, and shrinkage in a single microfluidic device

In this article, we present a microfluidic device capable of successive high-yield single-cell encapsulation in droplets, with additional droplet pairing, fusion, and shrinkage. Deterministic single-cell encapsulation is realized using Dean-coupled inertial ordering of cells in a Yin-Yang-shaped curved microchannel using a double T-junction, with a frequency over 2000 Hz, followed by controlled droplet pairing with a 100% success rate. Subsequently, droplet fusion is realized using electrical actuation resulting in electro-coalescence of two droplets, each containing a single HL60 cell, with 95% efficiency. Finally, volume reduction of the fused droplet up to 75% is achieved by a triple pitchfork structure. This droplet volume reduction is necessary to obtain close cell-cell membrane contact necessary for final cell electrofusion, leading to hybridoma formation, which is the ultimate aim of this research.

The 5-HT immunoreactive innervation of theHelixprocerebrum

In the procerebrum of terrestrial snails, 5-HT is a key modulatory substance of the generation of synchronous oscillatory activity and odor learning capability. In this study, we have analyzed the characteristics of the 5-HT-immunoreactive (5-HT-IR) innervation of the distinct anatomical regions of the procerebrum of Helix pomatia, applying correlative light- and electron microscopic immunocytochemistry. A dense network of 5-HT-IR innervation was demonstrated in the cell body layer, meanwhile a varicose fiber system of different density occurred in the different neuropil regions. At the ultrastructural level, labeled varicosities were found to contact both procerebral cell bodies, and different unlabeled axon profiles in the neuropils. The labeled structures established mostly close non-specialized membrane contacts with the postsynaptic profiles. The overall dense distribution of 5-HT-IR innervation supports a general modulatory role of 5-HT in processing different olfactory events.

Snare-Mediated Fusion Between Highly Curved and Un-Curved Membranes

During neurotransmitter release, curved synaptic vesicles fuse with the un-curved pre-synaptic plasma membrane, leading to the merger of two lipid bilayers and the release of neurotransmitters, a process that requires SNARE proteins in both membranes. Our previous modeling of this system employed two populations of highly curved vesicles (SUV), while others have used two populations of vesicles having ill-defined but likely not high curvature. In our studies, the v-SNARE synaptobrevin (SB), the t-SNARE syntaxin (SX), and SNAP-25 linked vesicles via a SNARE complex, but were unable to promote fusion without poly ethylene glycol (PEG) to force close membrane contact. We hypothesized that the geometry of the membranes may contribute to native synaptic vesicle fusion. Here we reconstitute SB into SUVs and SX into relatively uncurved (LUV) vesicles, whose composition, DOPC/DOPE/sphingomeylin/DOPS/cholesterol (32/25/15/8/20), models that of the native membranes. Lipid mixing (LM), contents mixing (CM) and leakage (L) time courses were fitted globally to 3- or 4-state sequential models, from which we obtained estimates of rate constants for conversion between states as well as probabilities of LM, CM and L for each state (Biophys. J., 2007, 92; 4012). In the absence of SNAREs, the mismatched curvature of the LUV-SUV system promotes more efficient and productive fusion events than fusion between SUVs (Biophys. J., 2010, 98, S1; 674a). LUVs containing SX (1:2250 P/L) and SUVs containing SB (1:950 P/L) still did not fuse in the absence of PEG. However with 6% PEG, the probability of CM was greatly shifted to the first step in the fusion process. The results suggest that with mismatched curvature, SNAREs may enhance rapid transient pore formation that precedes fully LM and final pore formation. Supported by NIGMS grants GM000678 to UNC and GM32707 to BRL.

Atrial granular cells of the snailAchatina fulicarelease proteins into hemolymph after stimulation of the heart nerve

The atrium of the gastropod mollusc Achatina fulica receives rich innervation and contains numerous granular cells (GCs). We studied the atrial innervation and discovered that axon profiles typical in appearance of peptidergic neurons form close unspecialized membrane contacts with GCs. Then, we investigated, at both morphological and biochemical levels, the effect of electrical stimulation of the heart nerve on GCs of Achatina heart perfused in situ. The ultrastructural study demonstrated changes in granule morphology consistent with secretion. These events included alteration of granule content, intracellular granule fusion and formation of complex degranulation channels, within which the granule matrix solubilized. It was shown that electrical stimulation resulted in a significant increase of the total protein concentration in the perfusate. Furthermore, SDS-PAGE analysis of the perfusate revealed three new proteins with molecular masses of 16, 22, and 57 kDa. Affinity-purified polyclonal antibodies against the 16 kDa protein were obtained; the whole-mount immunofluorescence technique revealed the presence of this protein in the granules of atrial GCs. In GCs of the stimulated atrium, a progressive loss of their granular content was observed. The results suggest that the central nervous system can modulate the secretory activity of the atrial GCs through non-synaptic pathways.

The ultrastructure of bladder lamina propria nerves in healthy subjects and patients with detrusor hyperreflexia.

Detrusor hyperreflexia is a common finding in patients with neurological disease involving the spinal cord. In animal models it has been attributed to an emergent reflex mediated mostly by unmyelinated C-fibers. We describe and measure ultrastructural features of these nerves in the lamina propria in healthy subjects and patients with detrusor hyperreflexia. Flexible cystoscopic bladder biopsies were obtained from 51 patients (8 controls, 8 with tropical spastic paraparesis, 23 with multiple sclerosis and 12 with spinal cord disease). Electron micrographs were obtained of every nerve profile seen in the midpoint of the biopsy specimen, and in each nerve profile a number of variables were measured and recorded. The mean nerve profile diameter was greater in patients with tropical spastic paraparesis (mean 2.19 microm.) compared to controls (1.59 microm.) and patients with multiple sclerosis (1.55 microm.) (p <0.001). We observed a sparse urothelial innervation by naked axonal varicosities but similar bare varicosities were more frequent in the superficial layer of the lamina propria. In deeper layers close membrane contacts between axonal varicosities and cells with cytological characteristics of myofibroblasts were seen. We described and measured ultrastructural characteristics of human bladder lamina propria nerves. The mean profile diameter is larger in patients with tropical spastic paraparesis compared to controls and patients with multiple sclerosis. This study provides a baseline to which other bladder disorders can be compared and may allow the effect of intravesical treatments on these nerves to be assessed. Some possible functional aspects of observed structural interrelationships are presented.

The Ultrastructure of Bladder Lamina Propria Nerves in Healthy Subjects and Patients With Detrusor Hyperreflexia

Detrusor hyperreflexia is a common finding in patients with neurological disease involving the spinal cord. In animal models it has been attributed to an emergent reflex mediated mostly by unmyelinated C-fibers. We describe and measure ultrastructural features of these nerves in the lamina propria in healthy subjects and patients with detrusor hyperreflexia. Flexible cystoscopic bladder biopsies were obtained from 51 patients (8 controls, 8 with tropical spastic paraparesis, 23 with multiple sclerosis and 12 with spinal cord disease). Electron micrographs were obtained of every nerve profile seen in the midpoint of the biopsy specimen, and in each nerve profile a number of variables were measured and recorded. The mean nerve profile diameter was greater in patients with tropical spastic paraparesis (mean 2.19 microm.) compared to controls (1.59 microm.) and patients with multiple sclerosis (1.55 microm.) (p <0.001). We observed a sparse urothelial innervation by naked axonal varicosities but similar bare varicosities were more frequent in the superficial layer of the lamina propria. In deeper layers close membrane contacts between axonal varicosities and cells with cytological characteristics of myofibroblasts were seen. We described and measured ultrastructural characteristics of human bladder lamina propria nerves. The mean profile diameter is larger in patients with tropical spastic paraparesis compared to controls and patients with multiple sclerosis. This study provides a baseline to which other bladder disorders can be compared and may allow the effect of intravesical treatments on these nerves to be assessed. Some possible functional aspects of observed structural interrelationships are presented.

Multiple local contact sites are induced by GPI-linked influenza hemagglutinin during hemifusion and flickering pore formation.

Membrane fusion intermediates induced by the glycosylphosphatidylinositol-linked ectodomain of influenza hemagglutinin (GPI-HA) were investigated by rapid freeze, freeze-substitution, thin section electron microscopy, and with simultaneous recordings of whole-cell admittance and fluorescence. Upon triggering, the previously separated membranes developed numerous hourglass shaped points of membrane contact (approximately 10-130 nm waist) when viewed by electron microscopy. Stereo pairs showed close membrane contact at peaks of complementary protrusions, arising from each membrane. With HA, there were fewer contacts, but wide fusion pores. Physiological measurements showed fast lipid dye mixing between cells after acidification, and either fusion pore formation or the lack thereof (true hemifusion). For the earliest pores, a similar conductance distribution and frequency of flickering pores were detected for both HA and GPI-HA. For GPI-HA, lipid mixing was detected prior to, during, or after pore opening, whereas for HA, lipid mixing is seen only after pore opening. Our findings are consistent with a pathway wherein conformational changes in the ectodomain of HA pull membranes towards each other to form a contact site, then hemifusion and pore formation initiate in a small percentage of these contact sites. Finally, the transmembrane domain of HA is needed to complete membrane fusion for macromolecular content mixing.

Surface Tongue-and-groove Contours on Lens MIP Facilitate Cell-to-cell Adherence

The lens major intrinsic protein (MIP, AQP0) is known to function as a water and solute channel. However, MIP has also been reported to occur in close membrane contacts between lens fiber cells, indicating that it has adhesive properties in addition to its channel function. Using atomic force and cryo-electron microscopy we document that crystalline sheets reconstituted from purified ovine lens MIP mostly consisted of two layers. MIP lattices in the apposing membranes were in precise register, and determination of the membrane sidedness demonstrated that MIP molecules bound to each other via their extracellular surfaces. The surface structure of the latter was resolved to 0.61 nm and revealed two protruding domains providing a tight “tongue-and-groove” fit between apposing MIP molecules. Cryo-electron crystallography produced a projection map at 0.69 nm resolution with a mirror symmetry axis at 45 ° to the lattice which was consistent with the double-layered nature of the reconstituted sheets. These data strongly suggest an adhesive function of MIP, and strengthen the view that MIP serves dual roles in the lens.

Ultrastructural aspects of peptidergic modulation in the peripheral nervous system of Helix pomatia.

The ultrastructural characteristics of peptidergic peripheral contacts in the snail, Helix pomatia, were investigated, with special attention to the innervation of the heart, buccal mass, and salivary gland by Mytilus inhibitory peptide-immunoreactive neurons. Following the application of correlative light- and electron-microscopic pre-embedding immunocytochemistry, the peripheral tissues reveal a rich innervation by Mytilus inhibitory peptide-immunoreactive elements. These neurons establish three types of neuromuscular contacts in the heart and buccal mass: (1) close (16-20 nm) unspecialized membrane contacts; (2) contacts with a relative wide (40-100 nm) intersynaptic cleft; and (3) labeled varicosties located freely in the extracellular space, far (0. 5-several microm) from the muscle cells. In the salivary gland, the immunoractive profiles contact both the muscular and glandular elements with close (type 1) and wider (type 2) membrane attachments. The great majority of Mytilus inhibitory peptide-immunoreactive profiles contain an ultrastructurally uniform population of large (120-150 nm) electron dense granules. The ultrastructural features of the innervation by Mytilus inhibitory peptide-immunoreactive elements are compared with those established by immunogold labelled FMRFamide-containing profiles in the heart and salivary gland. These latter display similarities in forming the different kinds of intercellular contacts, and differences in the morphological variability of the content of granules in the immunolabeled profiles. The results suggest diverse, non-synaptic modulatory roles of neuropeptides in the peripheral nervous system of Helix pomatia, including localized membrane effects and neurohormonal-like remote global controls, that may also be of significance in orchestrating the effects of neuropeptides released at the same time on different targets.

Ultrastructural observations of adherent cell pairs in hydra vulgaris

Previous morphological studies of cell sorting in Hydra vulgaris have considered only clusters of cells. Here, we present ultrastructural observations on the adherent region of cell pairs brought into contact (following dissociation) using a three-dimensional laser manipulator. There was a much larger area of close membrane contact between endodermal cell pairs in comparison with ectodermal cell pairs. Separation distances between membranes were categorized into three classes: closest distance (<4 nm); medium distance (5-25 nm); and cleavage (>25 nm). The sum of distances in the closest and medium categories as a proportion of total contact length was significantly greater (P<0.01) for endodermal cells (49.0+/-6.5 %) than for ectodermal cells (26.7+/-4. 4 %). In intact Hydra, this sum of distances was also significantly greater for endodermal cells, indicating that newly adherent cells, even after adhesion for only 10 min, display similar morphological characteristics to cells in intact Hydra. This suggests that close membrane contacts contribute to differential cell adhesion, which may form the basis of the cell sorting process.

Resynthesis of phosphatidylinositol in permeabilized neutrophils following phospholipase Cβ activation: transport of the intermediate, phosphatidic acid, from the plasma membrane to the endoplasmic reticulum for phosphatidylinositol resynthesis is not dependent on soluble lipid carriers or vesicular transport

Receptor-mediated phospholipase C (PLC) hydrolysis of phosphoinositides is accompanied by the resynthesis of phosphatidylinositol (PI). Hydrolysis of phosphoinositides occurs at the plasma membrane, and the resulting diacylglycerol (DG) is converted into phosphatidate (PA). Two enzymes located at the endoplasmic reticulum (ER) function sequentially to convert PA back into PI. We have established an assay whereby the resynthesis of PI could be followed in permeabilized cells. In the presence of [γ-32P]ATP, DG generated by PLC activation accumulates label when converted into PA. The 32P-labelled PA is subsequently converted into labelled PI. The formation of labelled PI reports the arrival of labelled PA from the plasma membrane to the ER. Cytosol-depleted, permeabilized human neutrophils are capable of PI resynthesis following stimulation of PLCβ (in the presence of phosphatidylinositol-transfer protein), provided that CTP and inositol are also present. We also found that wortmannin, an inhibitor of endocytosis, or cooling the cells to 15 °C did not stop PI resynthesis. We conclude that PI resynthesis is dependent neither on vesicular transport mechanisms nor on freely diffusible, soluble transport proteins. Phosphatidylcholine-derived PA generated by the ADP-ribosylation-factor-stimulated phospholipase D pathway was found to accumulate label, reflecting the rapid cycling of PA to DG, and back. This labelled PA was not converted into PI. We conclude that PA derived from the PLC pathway is selected for PI resynthesis, and its transfer to the ER could be membrane-protein-mediated at sites of close membrane contact.

Resynthesis of phosphatidylinositol in permeabilized neutrophils following phospholipase Cβ activation: transport of the intermediate, phosphatidic acid, from the plasma membrane to the endoplasmic reticulum for phosphatidylinositol resynthesis is not dependent on soluble lipid carriers or vesicular transport

Receptor-mediated phospholipase C (PLC) hydrolysis of phosphoinositides is accompanied by the resynthesis of phosphatidylinositol (PI). Hydrolysis of phosphoinositides occurs at the plasma membrane, and the resulting diacylglycerol (DG) is converted into phosphatidate (PA). Two enzymes located at the endoplasmic reticulum (ER) function sequentially to convert PA back into PI. We have established an assay whereby the resynthesis of PI could be followed in permeabilized cells. In the presence of [gamma-32P]ATP, DG generated by PLC activation accumulates label when converted into PA. The 32P-labelled PA is subsequently converted into labelled PI. The formation of labelled PI reports the arrival of labelled PA from the plasma membrane to the ER. Cytosol-depleted, permeabilized human neutrophils are capable of PI resynthesis following stimulation of PLCbeta (in the presence of phosphatidylinositol-transfer protein), provided that CTP and inositol are also present. We also found that wortmannin, an inhibitor of endocytosis, or cooling the cells to 15 degrees C did not stop PI resynthesis. We conclude that PI resynthesis is dependent neither on vesicular transport mechanisms nor on freely diffusible, soluble transport proteins. Phosphatidylcholine-derived PA generated by the ADP-ribosylation-factor-stimulated phospholipase D pathway was found to accumulate label, reflecting the rapid cycling of PA to DG, and back. This labelled PA was not converted into PI. We conclude that PA derived from the PLC pathway is selected for PI resynthesis, and its transfer to the ER could be membrane-protein-mediated at sites of close membrane contact.

Close anatomical relationships between nerve fibers and MHC class II-expressing dendritic cells in the rat liver and extrahepatic bile duct.

The anatomical relationships between immunocytochemically identified nerve fibers and MHC class II-expressing antigen presenting dendritic cells were investigated in the rat hepatobiliary system using immunocytochemistry, confocal laser scanning, and electron microscopy. Close proximity of nerve fiber varicosities immunostained for PGP 9.5 and MHC class II-expressing dendritic cells was frequently observed in the wall of extrahepatic bile ducts, in Glisson's area, around central and hepatic veins, and in the liver capsule. Contacts between nerve fibers staining for substance P, calcitonin gene-related peptide, calretinin, and vasoactive intestinal polypeptide and dendritic cells were more often observed around extrahepatic bile ducts than in Glisson's area. Nerve fibers immunostaining for tyrosine hydroxylase and neuropeptide Y were numerous both in the wall of extrahepatic bile ducts and in Glisson's area and frequently contacted dendritic cells there. At the ultrastructural level, close membrane contacts between bare axolemmal areas of unmyelinated nerve fibers and processes of MHC class II-expressing cells were observed. These results demonstrate close anatomical relationships of nerve fibers from various sources with antigen presenting dendritic cells in the visceral domain and suggest modulation of antigen presentation by the autonomic nervous system.

Fusion and metabolism of plant cells as affected by microgravity.

Plant cell protoplasts derived from leaf tissue of two different tobacco species (Nicotiana tabacum., N. rustica L.) were exposed to short-term (sounding rocket experiments) and long-term (spacelab) microgravity environments in order to study both (electro) cell fusion and cell metabolism during early and later stages of tissue regeneration. The period of exposure to microgravity varied from 10 min (sounding rocket) to 10 d (space shuttle). The process of electro fusion of protoplasts was improved under conditions of microgravity: the time needed to establish close membrane contact between protoplasts (alignment time) was reduced (5 as compared to 15 s under 1 g) and numbers of fusion products between protoplasts of different specific density were increased by a factor of about 10. In addition, viability of fusion products, as shown by the ability to form callus, increased from about 60% to more than 90%. Regenerated fusion products obtained from both sounding-rocket and spacelab experiments showed a wide range of intermediate properties between the two parental plants. This was verified by isozyme analysis and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). In order to address potential metabolic responses, more general markers such as the overall energy state (ATP/ADP ratio), the redox charge of the diphosphopyridine nucleotide system (NADH/NAD ratio), and the pool size of fructose-2,6-bisphosphate (Fru 2,6 bisp), a regulator of the balance between glycolysis and gluconeogenesis, were determined. Responses of these parameters were different with regard to short-term and long-term exposure. Shortly after transition to reduced gravitation (sounding rocket) ratios of ATP/ADP exhibited strong fluctuation while the pool size of NAD decreased (indicating an increased NADH/NAD ratio) and that of Fru 2,6 bisp increased. As similar changes can be observed under stress conditions, this response is probably indicative of a metabolic stress compensation. Samples taken for up to 7 d of exposure to microgravity showed the opposite effect. Here, the ratios of ATP/ADP and of NADH/NAD, and the pool size of Fru 2,6 bisp were decreased. We take this an an indication of metabolic relaxation, i.e. decreased metabolic turnover. As rates of protoplast regeneration and cell division were obviously similar to 1-g controls, we conclude that under conditions of microgravity regenerating tobacco mesophyll protoplasts need less metabolic energy for the same effort.

Muscle development in the tambaqui, an important Amazonian food fish

Eggs of the tambaqui Colossoma macropomum were incubated at 28 and 31) C. Somitogenesis started shortly after the formation of the neural plate and notochord. New somites were added at the rate of one every 13 min at 28) C and one every 11 min at 31) C. Myogenesis started in the most rostral myotomes at the 9-somite stage and proceeded in a caudal direction. Mononuclear myotubes with the morphological characteristic of muscle pioneer cells were observed lateral to the notochord. The majority of myotubes were formed from the fusion of 3–6 spindle-shaped myoblasts. Myofibril synthesis started soon after cell fusion at the periphery of myotubes. Close membrane contacts and ‘gap’-type junctions were observed between myotubes, immature muscle fibres and at the inter-somite boundary, suggesting that the cells were electrically coupled. Embryos exhibited rhythmic movements at the 20-somite stage, and hatched at the 29–30-somite stage 15–18 h post-fertilisation (PFT) at 28° C and 11 h PFT at 31° C. Larvae hatched at a comparatively early stage of development prior to the completion of somitogenesis and the formation of eye pigment, pectoral fins and jaws. The myotomes comprised a single superficial layer of well-differentiated muscle fibres which contained abundant mitochondria, overlying an inner core of myotubes (presumptive white muscle layer). Differentiation and growth during the larval stages was extremely rapid, and the juvenile stage was reached after little more than 6 days at 28° C.

Muscle development in the tambaqui, an important Amazonian food fish

Eggs of the tambaqui Colossoma macropomum were incubated at 28 and 31) C. Somitogenesis started shortly after the formation of the neural plate and notochord. New somites were added at the rate of one every 13 min at 28) C and one every 11 min at 31) C. Myogenesis started in the most rostral myotomes at the 9‐somite stage and proceeded in a caudal direction. Mononuclear myotubes with the morphological characteristic of muscle pioneer cells were observed lateral to the notochord. The majority of myotubes were formed from the fusion of 3–6 spindle‐shaped myoblasts. Myofibril synthesis started soon after cell fusion at the periphery of myotubes. Close membrane contacts and ‘gap’‐type junctions were observed between myotubes, immature muscle fibres and at the inter‐somite boundary, suggesting that the cells were electrically coupled. Embryos exhibited rhythmic movements at the 20‐somite stage, and hatched at the 29–30‐somite stage 15–18 h post‐fertilisation (PFT) at 28° C and 11 h PFT at 31° C. Larvae hatched at a comparatively early stage of development prior to the completion of somitogenesis and the formation of eye pigment, pectoral fins and jaws. The myotomes comprised a single superficial layer of well‐differentiated muscle fibres which contained abundant mitochondria, overlying an inner core of myotubes (presumptive white muscle layer). Differentiation and growth during the larval stages was extremely rapid, and the juvenile stage was reached after little more than 6 days at 28° C.

EFFECTS OF LYMPHOKINE-ACTIVATED KILLER-CELLS ON MELANOMA NODULES MAINTAINED IN 3-DIMENSIONAL CULTURE

Lymphokine activated killer (LAK) cells were cocultivated from 2 to 6 days with WM266 metastatic melanoma cells maintained as nodules in organotypic culture. The LAK cells in suspension were allowed to deposit freely on the nodule surface from where they could infiltrate spontaneously into the nodules. Immunohistochemical studies were done to localize the LAK cells as well as electron microscopical observations for effector/target membrane contacts. Proliferation of the nodules was tested and also that of the LAK cells after coculturing using tritiated thymidine incorporation into DNA. Cell death was determined by arrest of thymidine incorporation and total nodule disintegration. Infiltration rate of LAK cells into the nodules was low: after coculturing 5% of the nodule cells were LAK cells. Although close membrane contacts and cytoplasmic fusions between effector and target cells leading to tumor cell apoptosis were observed, this direct cytolytic process seemed to be too infrequent for the induction of total nodule disintegration at day 6. Therefore, the indirect pathway to cytolysis might be predominant implying, among other cytokines, soluble TNF. On the other hand, LAK cell proliferation diminished strongly after coculturing (down to 11%) but the cytotoxicity was significantly enhanced (18% higher) suggesting an enhancement of differentiation. This might account for the peculiar efficacy of LAK cells on melanomas in vivo and it would be of interest to study this phenomenon further.

Peripheral monocyte and naive T-cell recruitment and activation in Crohn's disease

Background & Aims : Transmural perivascular mononuclear cell infiltrates are a feature of Crohn's disease. The aim of this study was a molecular characterization of the mechanisms leading to the formation of these infiltrates. Methods : Endothelial cell and leukocyte expression of the adhesion molecules directing leukocyte transendothelial migration were studied in situ by immunohistochemical analysis of 10 samples from patients with Crohn's disease and 10 samples from normal controls. Double-staining methods were used to characterize the cells forming the infiltrates. Results : CD11a + and l-selectin-positive mononuclear cells seemed to be the major component of perivascular infiltrates. The vast majority of these cells were CD68 +, CD31 + monocytes/macrophages surrounded by CD3 +, l-selectin-positive, CD31 +, CD45RA +, and/or CD45RO + T lymphocytes. T lymphocytes within the vessels expressed both CD45RA and CD45RO markers. Endothelial cells were intercellular adhesion molecule 1 positive and mostly CD34 +. Strong adhesion between l-selectin-positive and CD11a + intravascular mononuclear cells and CD34 + and intercellular adhesion molecule 1-positive endothelial cells were observed. Conclusions : Data indicate that peripheral mononuclear cells are actively recruited in the submucosa of Crohn's disease tissue; endothelial cells express adhesion molecules highly permissive for transendothelial migration of monocytes and both naive and memory T cells contributing to infiltrates generation; and close membrane contact between migrated macrophages and naive T cells leads to the T-cell transition from naive to memory phenotype within Crohn's disease.

Experimental pulmonary schistosomiasis: lack of morphological evidence of modulation in schistosomal pulmonary granulomas.

Numerous pulmonary schistosome egg granulomas were present in mice submitted to partial portal vein ligation (Warren's model). The granulomas were characterized by cellular aggregations formed within alveolar tissue. Main cellular types were macrophages (epithelioid cells), eosinophils, plasma cells and lymphocytes. These cells were supported by scanty fibrous stroma and exhibited close membrane contact points amongst themselves, but without forming specialized adhesion apparatus. When granulomas involved arterial structures, proliferation of endothelial and smooth muscle cells occurred and fibrosis associated with angiogenesis became more evident. Granulomas formed around mature eggs in the pulmonary alveolar tissue presented approximately the same size and morphology regardless of the time of infection, the latter being 10, 18 and 25 weeks after cercarial exposure. This persistence of morphological appearance suggests that pulmonary granulomas do not undergo immunological modulation, as is the case with the granulomas in the liver and, to a lesser extent, in the intestines. Probably, besides general immunological factors, local (stromal) factors play an important role in schistosomal granuloma modulation.