Susumu Ito

Communicable ulcerative colitis induced by T-bet deficiency in the innate immune system.

Inflammatory bowel disease (IBD) has been attributed to overexuberant host immunity or the emergence of harmful intestinal flora. The transcription factor T-bet orchestrates inflammatory genetic programs in both adaptive and innate immunity. We describe a profound and unexpected function for T-bet in influencing the behavior of host inflammatory activity and commensal bacteria. T-bet deficiency in the innate immune system results in spontaneous and communicable ulcerative colitis in the absence of adaptive immunity and increased susceptibility to colitis in immunologically intact hosts. T-bet controls the response of the mucosal immune system to commensal bacteria by regulating TNF-alpha production in colonic dendritic cells, critical for colonic epithelial barrier maintenance. Loss of T-bet influences bacterial populations to become colitogenic, and this colitis is communicable to genetically intact hosts. These findings reveal a novel function for T-bet as a peacekeeper of host-commensal relationships and provide new perspectives on the pathophysiology of IBD.

A Critical Period for Social Experience–Dependent Oligodendrocyte Maturation and Myelination

A Critical Period for Glia The brain develops in fits and starts—while one system is completed, another system may still be under construction. Such transient states are known as critical periods, and during these specific aspects of brain development may become more sensitive to outside agents than they would be later. Makinodan et al. (p. 1357) observed the effect of environmental conditions on the brains of mice bioengineered to develop fluorescent oligodendrocytes. The mice were exposed to a variety of social conditions during rearing, ranging from isolation to a normal laboratory cage setting, or to settings enriched with extra buddies and a steady rotation of new play toys. The results show that social isolation leaves a developmental trace that persists into adulthood. Specifically, they found that oligodendrocytes, which produce the myelin that insulates neurons, were underdeveloped, suggesting that there may be a critical period that governs development of these glial oligodendrocyte cells. In mice, early social experience regulates prefrontal cortex myelination that is essential for normal cognitive development. Early social isolation results in adult behavioral and cognitive dysfunction that correlates with white matter alterations. However, how social deprivation influences myelination and the significance of these myelin defects in the adult remained undefined. We show that mice isolated for 2 weeks immediately after weaning have alterations in prefrontal cortex function and myelination that do not recover with reintroduction into a social environment. These alterations, which occur only during this critical period, are phenocopied by loss of oligodendrocyte ErbB3 receptors, and social isolation leads to reduced expression of the ErbB3 ligand neuregulin-1. These findings indicate that social experience regulates prefrontal cortex myelination through neuregulin-1/ErbB3 signaling and that this is essential for normal cognitive function, thus providing a cellular and molecular context to understand the consequences of social isolation.

Gastrin deficiency results in altered gastric differentiation and decreased colonic proliferation in mice

BACKGROUND & AIMSnGastrin is a peptide hormone important in the regulation of both acid secretion and differentiation of oxyntic mucosal cells of the stomach. To further elucidate the role of gastrin in the growth and development of the gastrointestinal tract, we have generated mice that are deficient in gastrin.nnnMETHODSnGastrin-deficient mice were generated through targeted gene disruption. Gastric and colonic architecture were determined by routine histology and immunohistochemical techniques. Proliferation was assessed by 5-bromo-2-deoxyuridine incorporation.nnnRESULTSnTargeted disruption of the gastrin gene resulted in mice incapable of expressing gastrin messenger RNA (mRNA) or producing gastrin peptide. This deficiency led to a marked change in gastric architecture, with a decrease in number of parietal and enterochromaffin-like cells and an increase in number of mucous neck cells. There was no difference in the proliferation labeling index of the stomach in gastrin-deficient mice (3.04% +/- 0.33%) compared with wild-type littermates (3.15% +/- 0.18%). The colon of gastrin-deficient mice seemed normal histologically, although there was a decreased proliferation labeling index (2.97% +/- 0.52%) compared with wild-type littermates (4.71% +/- 0.44%; P < 0.01).nnnCONCLUSIONSnGastrin is important in regulating the differentiation of the gastric mucosa and is a trophic factor for the colonic mucosa.

Mice lacking secretory phospholipase A2 show altered apoptosis and differentiation with Helicobacter felis infection

BACKGROUND & AIMSnInfection with Helicobacter pylori uniformly leads to a chronic superficial gastritis that may progress to atrophic gastritis, a premalignant process. A mouse model of Helicobacter felis infection was used to study possible genetic determinants of the response to infection.nnnMETHODSnThree inbred mouse strains with known secretory phospholipase A2 (sPLA2) genotypes [BALB/c (+/+), C3H/HeJ (+/+), and C57BL/6 (-/-)] were orally infected with H. felis and examined longitudinally using routine histology, immunocytochemistry, electron microscopy, proliferating cell nuclear antigen, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, and Northern and Western blot studies.nnnRESULTSnOnly the C57BL/6 strain showed increased gastric fundic proliferation and apoptosis in response to infection. In addition, the C57BL/6 mouse showed a marked loss of parietal and chief cells, along with a marked expansion of an aberrant gastric mucous cell lineage that stained positive for spasmolytic polypeptide. In contrast, no significant change in these cell types was observed in BALB/c and C3H/HeJ strains. Increased expression of sPLA2 was observed in BALB/c and C3H/HeJ after H. felis infection, whereas sPLA2 expression was absent in C57BL/6 mice.nnnCONCLUSIONSnH. felis infection leads to increased apoptosis and altered cellular differentiation in the C57BL/6 mouse, a strain that lacks gastric sPLA2 expression. Because sPLA2 has been identified recently as the MOM1 (modifier of MIN) locus that influences polyp formation in the colon, these studies suggest that sPLA2 may also influence the gastric epithelial response to Helicobacter infection.

Morphology of Rat Gastric Mucosal Damage, Defense, and Restitution in the Presence of Luminal Ethanol

The morphology of ligated rat stomachs filled with absolute ethanol for 1, 7, 15, 30, and 60 min was studied. With interrupted blood circulation, the entire mucosa was destroyed within minutes. With intact circulation, the mucosae remained macroscopically normal or developed visible lesions. Areas with lesions had extensive surface and gland cell disruption and were hemorrhagic and hyperemic. Histologically, nonlesion sites had about 92% of the mucous cells on the surface and upper gastric pits destroyed within 1 min. Detached necrotic cells and an accumulation of an exudate with fibrin fibers formed a thick layer over the damaged mucosal surface. Intact cells from the gastric pits started migrating over the denuded basal lamina and after 7 min about 4% of the surface was reepithelialized. Within the next 8 min there was a sharp increase in mucosal restitution to about 54%. After 30 min 84% of the mucosa was restituted and there was only a slight increase to about 87% after 1 h of continuous exposure to ethanol. After 30 min the ethanol was diluted to about 40% vol/vol. Focal geyserlike eruptions of plasma containing polymerized fibrin were visible at sites of epithelial discontinuity. Restitution of the mucosal surface in the presence of luminal ethanol may be facilitated by the favorable microenvironment created by the thick unstirred layer formed by the mucoid coat of necrotic cells, fibrin, and continued plasma shedding.

Gastrointestinal Cell Plasma Membrane Wounding and Resealing In Vivo

Previous studies in vitro have shown that various mechanical methods used to wound plasma membranes allow normally impermeant, water-soluble markers, such as fluorescein dextran or horseradish peroxidase, to enter the cytosol. Subsequent membrane resealing traps these nontoxic fluorescent or electron microscopic markers within living, surviving wounded cells. The present report is the first, to our knowledge, to use this strategy to study cell membrane wounding and resealing in the intact animal. We show that gut cells wounded in vivo by mechanical forces are capable of resealing disruptions of their plasma membranes. More importantly, we show that wounding of cell membranes, followed by resealing, occurs not only in mechanically injured gut but also in normal, experimentally undisturbed gut. A variety of cell types were wounded and resealed membrane wounds in the mechanically injured stomach: surface mucous, endothelial, fibroblastic, parietal, and chief cells. Mucous cells successful at resealing membrane wounds apparently became active participants in the motile events of stomach repair. In undisturbed gut, cell membrane wounding and resealing was most frequently observed in the colon, but was also observed in the esophagus, stomach, duodenum, and ileum. Surface epithelial cells in undisturbed gut were retained for greater than 48 h after surviving membrane wounds. Two important roles are suggested for membrane resealing in gut: (a) preservation of motile cells nearest epithelial discontinuities requiring repair after injury, and (b) maintenance of epithelial integrity in normally functioning gut. Our finding of cell wounding in undisturbed gut may explain, in part, why rapid, continual cell turnover is characteristic of gut epithelia. We propose that membrane disruption, or wounding, is a normal and common occurrence in vivo, and that a biologically significant function of the plasma membrane is to reseal such wounds. The occurrence of in vivo cell membrane wounding and resealing suggests an unrecognized route for molecular traffic into and out of cytoplasm.

Hepatitis C Virus-like Particles Synthesized in Insect Cells as a Potential Vaccine Candidate

BACKGROUND & AIMSnHepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. Successful vaccine development is crucial in controlling global HCV infection. We have previously described the generation of HCV-like particles (HCV-LPs) in insect cells using a recombinant baculovirus containing the complementary DNA of the HCV structural proteins. These HCV-LPs had similar morphological and biophysical properties as the putative virions. In this study, we analyzed the structural features, antigenic composition, seroreactivity, and immunogenicity of purified HCV-LPs.nnnMETHODSnHCV-LPs were analyzed by electron microscopy and antibody immunolabeling and precipitation. An enzyme-linked immunosorbent assay (ELISA) using HCV-LPs was developed. The humoral response to HCV-LPs in mice was studies by core and envelope ELISAs, Western immunoblotting, and immunofluorescence.nnnRESULTSnStructural and antigenic compositions of HCV-LPs were shown to be similar to those of putative HCV virions. Using the HCV-LP ELISA, high-titer anti-HCV antibodies were detected in individuals infected with various HCV genotypes. In vivo, HCV-LPs elicited a humoral response broadly directed against HCV structural proteins.nnnCONCLUSIONSnHCV-LPs resemble HCV virions and are capable of inducing a humoral response targeted against various regions of HCV structural proteins, suggesting that HCV-LPs may be promising as a potential vaccine candidate.

Binding of Hepatitis C Virus-Like Particles Derived from Infectious Clone H77C to Defined Human Cell Lines

ABSTRACT Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. The study of viral entry and infection has been hampered by the inability to efficiently propagate the virus in cultured cells and the lack of a small-animal model. Recent studies have shown that in insect cells, the HCV structural proteins assemble into HCV-like particles (HCV-LPs) with morphological, biophysical, and antigenic properties similar to those of putative virions isolated from HCV-infected humans. In this study, we used HCV-LPs derived from infectious clone H77C as a tool to examine virus-cell interactions. The binding of partially purified particles to human cell lines was analyzed by fluorescence-activated cell sorting with defined monoclonal antibodies to envelope glycoprotein E2. HCV-LPs demonstrated dose-dependent and saturable binding to defined human lymphoma and hepatoma cell lines but not to mouse cell lines. Binding could be inhibited by monoclonal anti-E2 antibodies, indicating that the HCV-LP-cell interaction was mediated by envelope glycoprotein E2. Binding appeared to be CD81 independent and did not correlate with low-density lipoprotein receptor expression. Heat denaturation of HCV-LPs drastically reduced binding, indicating that the interaction of HCV-LPs with target cells was dependent on the proper conformation of the particles. In conclusion, our data demonstrate that insect cell-derived HCV-LPs bind specifically to defined human cell lines. Since the envelope proteins of HCV-LPs are presumably presented in a virion-like conformation, the binding of HCV-LPs to target cells may allow the study of virus-host cell interactions, including the isolation of HCV receptor candidates and antibody-mediated neutralization of binding.

AFRICAN TRYPANOSOME INTERACTIONS WITH AN IN VITRO MODEL OF THE HUMAN BLOOD–BRAIN BARRIER

The neurological manifestations of sleeping sickness in man are attributed to the penetration of the blood–brain barrier (BBB) and invasion of the central nervous system by Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. However, how African trypanosomes cross the BBB remains an unresolved issue. We have examined the traversal of African trypanosomes across the human BBB using an in vitro BBB model system constructed of human brain microvascular endothelial cells (BMECs) grown on Costar Transwell™ inserts. Human-infective T. b. gambiense strain IL 1852 was found to cross human BMECs far more readily than the animal-infective Trypanosoma brucei brucei strains 427 and TREU 927. Tsetse fly–infective procyclic trypomastigotes did not cross the human BMECs either alone or when coincubated with bloodstream-form T. b. gambiense. After overnight incubation, the integrity of the human BMEC monolayer measured by transendothelial electrical resistance was maintained on the inserts relative to the controls when the endothelial cells were incubated with T. b. brucei. However, decreases in electrical resistance were observed when the BMEC-coated inserts were incubated with T. b. gambiense. Light and electron microscopy studies revealed that T. b. gambiense initially bind at or near intercellular junctions before crossing the BBB paracellularly. This is the first demonstration of paracellular traversal of African trypanosomes across the BBB. Further studies are required to determine the mechanism of BBB traversal by these parasites at the cellular and molecular level.

A Dominant Interference Collagen X Mutation Disrupts Hypertrophic Chondrocyte Pericellular Matrix and Glycosaminoglycan and Proteoglycan Distribution in Transgenic Mice

Collagen X transgenic (Tg) mice displayed skeleto-hematopoietic defects in tissues derived by endochondral skeletogenesis.(1) Here we demonstrate that co-expression of the transgene product containing truncated chicken collagen X with full-length mouse collagen X in a cell-free translation system yielded chicken-mouse hybrid trimers and truncated chicken homotrimers; this indicated that the mutant could assemble with endogenous collagen X and thus had potential for dominant interference. Moreover, species-specific collagen X antibodies co-localized the transgene product with endogenous collagen X to hypertrophic cartilage in growth plates and ossification centers; proliferative chondrocytes also stained diffusely. Electron microscopy revealed a disrupted hexagonal lattice network in the hypertrophic chondrocyte pericellular matrix in Tg growth plates, as well as altered mineral deposition. Ruthenium hexamine trichloride-positive aggregates, likely glycosaminoglycans (GAGs)/proteoglycans (PGs), were also dispersed throughout the chondro-osseous junction. These defects likely resulted from transgene co-localization and dominant interference with endogenous collagen X. Moreover, altered GAG/PG distribution in growth plates of both collagen X Tg and null mice was confirmed by a paucity of staining for hyaluronan and heparan sulfate PG. A provocative hypothesis links the disruption of the collagen X pericellular network and GAG/PG decompartmentalization to the potential locus for hematopoietic failure in the collagen X mice.