The Ras-related GTPase Rap1 promotes cell adhesion and migration. Although the significance of Rap1 contribution to cell migration is increasingly being recognized, little is known about the biochemical mechanisms driving this process. In the present study, we discovered a previously unidentified regulatory role of insulin-like growth factor type I (IGF-I) receptor (IGF-IR) in CRK Src homology 3 (SH3)-binding guanine-nucleotide-releasing protein (C3G)-Rap1-fascin-actin axis promoting cell movement. We demonstrate that a burst of Rap1 activity, rather than presumed hyperactivation, is imperative for the onset of cell movement. We show that while autophosphorylated IGF-IR signals to C3G to activate Rap1, subsequent IGF-IR internalization promotes gradual inactivation of Rap1 by putative Rap1 GTPase-activating protein (GAP). Additionally, IGF-IR signalling recruits active Rap1 at sites of cell motile protrusions. C3G depletion prevents IGF-I-induced fascin accumulation at actin microspikes and blocks protrusions. In the absence of IGF-IR activity, the wild-type (WT) Rap1 and the constitutively active V12Rap1 mutant remain in cell-cell contacts. Forced inactivation of Rap1 signalling by overexpressing dominant negative N17Rap1, Rap1GAP or by silencing C3G has a detrimental effect on filamentous (F)-actin and cell adhesion irrespective of IGF-IR signalling. We conclude that the basal levels of Rap1 activity holds up cell adhesion, whereas sequential regulation of C3G and GAP by IGF-IR reverses the labile Rap1 function from supporting adhesion to promoting migration.

Both the role of inducible nitric oxide synthase (iNOS) in the development of inflammatory bowel disease (IBD) as well as the molecular details governing its mucosal induction remain unclear. In the present study we evaluated the role of the residing intestinal microflora in the induction of epithelial iNOS upon transfer of CD45RB CD4 T cells to SCID mice. To this end, CB-17 SCID mice were reared with conventional flora (CNV) or germfree CB-17 SCID mice were monoassociated with Helicobacter.muridarum, act A (-) mutant L. monocytogenes, segmented filamentous bacteria (SFB), or O. anthropi. Within 2 weeks CNV SCID mice injected with CD45RB CD4 T cells showed a focal, epithelial iNOS expression on the apical site of villi that preceded the infiltration of CD4 cells and cytokine production followed by extension of this expression to the entire surface along the whole crypt axis as the colitis progressed. SCID mice monoassociated with H.muridarum developed a severe colitis and showed high epithelial iNOS expression. CNV-SCID mice without T cells and SCID mice mono-associated with SFB did not show any iNOS expression, whereas SCID mice mono-associated with act A(-) mutant Listeria. monocytogenes and Ochrobactrum. anthropi showed some scattered epithelial iNOS staining on the apical site of a few villi, but none of these mice developed colitis. These findings demonstrate that the expression of epithelial iNOS is highly bacterium-specific and correlates with the severity of disease, suggesting an important role for this enzyme in the development of IBD. Epithelial iNOS expression in the CD45RB transfer model of colitis 103 5.

Background: Both the role of inducible nitric oxide synthase (iNOS) in the development of inflammatory bowel disease (IBD) as well as the molecular details governing its mucosal induction remain unclear.Methods: In the present study we evaluated the role of the residing intestinal microflora in the induction of epithelial iNOS upon transfer of CD45RB(high) CD4(+) T cells to SCID mice. CB-17 SCID mice were reared with conventional flora (CNV) or germfree CB-17 SCID mice were monoassociated with Helicobacter muridarum, act A(-) mutant Listeria monocytogenes, segmented filamentous bacteria (SFB), or Ochrobactrum anthropi.Results: Within 2 weeks CNV SCID mice injected with CD45RB high CD4+ T cells showed a focal, epithelial iNOS expression on the apical site of villi that preceded the infiltration of CD4(+) T cells and cytokine production followed by extension of this expression to the entire surface along the whole crypt axis as the colitis progressed. SCID mice monoassociated with H. muridarum developed a severe colitis and showed high epithelial iNOS expression. CNV-SCID mice without T cells and SCID mice monoassociated with SFB did not show any iNOS expression, whereas SCID mice monoassociated with act A(-) mutant L. monocytogenes and O. anthropi showed some scattered epithelial iNOS staining on the apical site of a few villi, but none of these mice developed colitis.Conclusions: These findings demonstrate that the expression of epithelial iNOS is highly bacterium-specific and correlates with the severity of disease, suggesting an important role for this enzyme in the development of IBD.

SummaryThe goal of vaccination is the generation of immune memory, an immune state that permits rapid and intense recall responses to a pathogen. Considerable effort is being made to understand the nature of memory T cells. We report here that by extending the length of in vitro culture following a single restimulation with specific peptide, preparations of highly enriched, highly active antigen‐specific CD8+ memory T cells could be obtained. These cultures were begun with splenocytes from mice primed by infection either with an attenuated strain of Listeria monocytogenes or vaccinia virus, both expressing the human immunodeficiency virus‐1‐gag gene. In the cultures, antigen‐specific cytotoxic T lymphocyte (CTL) activity reached a maximum at about 9 days and thereafter fell to negligible values. Concomitant with the fall of CTL activity, however, we observed enrichment for a subset of CD11ahigh antigen‐specific gag‐tetramerpos CD8+ T cells. The cells showed little or no 4‐hr CTL activity, but had high delayed (18‐hr) CTL activity, and very high cytolytic activity after restimulation. They rapidly expressed interferon‐γ production. Their growth and survival after sorting was completely dependent on interleukin‐2 or ‐15. As few as 5000 of the fluorescence‐activated cell sorting‐purified cells protected recipients against challenge 3 months after transfer. In response to the challenge, the cells repopulated lymphoid and non‐lymphoid organs and showed a sizeable increase in number. The cells therefore demonstrate high protective activity for long periods of time. These cultured cells are thus a potential source of enriched natural memory T cells for reperfusion studies and in which the mechanisms that underlie the generation, differentiation and persistence of memory can be examined.

ABSTRACT CD8+ T cells are a major component of the adaptive response of a host to infections by viruses and other intracellular pathogenic agents. However, because of the intrinsic immaturity of the immune system of neonatal animals, neonates are highly sensitive to a variety of pathogens and may be unable to respond in a protective manner. Here we explore whether a hyperattenuated strain of Listeria monocytogenes that can be used as a live vaccine vector in adults is safe and able to induce an effective response in neonates. We answer both questions affirmatively.

BACKGROUND & AIMSRecently, a number of animal models for different aspects of inflammatory bowel disease (IBD) have been developed. The aim of this study was to use one of these to determine whether particular, ostensibly innocuous, intestinal bacteria could provoke or exacerbate IBD.METHODSConventionally reared C.B17 SCID mice were compared with germ-free and gnotobiotic mice, monoassociated with 1 of 5 intestinal bacteria, after transfer of CD45RB(high) CD4(+) T cells from conventionally reared congenic BALB/c mice. Recipient mice were monitored over 7-12 weeks for clinical signs of IBD, and tissues were analyzed by histology/flow cytometry for abnormal inflammation and CD4(+) T cell outgrowth.RESULTSNeither germ-free mice nor mice monoassociated with segmented filamentous bacteria, Ochrobactrum anthropi, a nonpathogenic mutant of Listeria monocytogenes, or Morganella morganii developed any signs of IBD. In contrast, mice monoassociated with Helicobacter muridarum displayed an accelerated development of IBD in 5-6 weeks compared with 8-12 weeks observed in conventionally reared mice. The outgrowth of CD4(+) T cells in spleen and large intestine of H. muridarum monoassociated mice, as well as in conventionally reared mice was significantly higher than that in the other monoassociated mice.CONCLUSIONSAmong the intestinal bacteria tested, H. muridarum can serve as a provocateur of IBD in this model.

ABSTRACTStudies utilizing various immunodeficient mouse models of rotavirus (RV) infection demonstrated significant roles of RV-specific secretory immunoglobulin A (IgA), CD4+T cells, and CD8+T cells in the clearance of RV and protection from secondary infection. Secretion of small but detectable amounts of IgA in RV-infected αβ T-cell receptor knockout mice (11) and distinctive anatomical localization and physiology of B1 cells suggested that B1 cells might be capable of producing RV-specific intestinal IgA in a T-cell-independent fashion and, therefore, be responsible for ablation of RV shedding. We investigated the role of B1 cells in the resolution of primary RV infection using a SCID mouse model. We found that the adoptive transfer of unseparated peritoneal exudate cells ablates RV shedding and leads to the production of high levels of RV-specific intestinal IgA. In contrast, purified B1 cells do not ablate RV shedding and do not induce a T-cell-independent or T-cell-dependent, RV-specific IgA response but do secrete large amounts of polyclonal (total) intestinal IgA. Cotransfer of mixtures of purified B1 cells and B1-cell-depleted peritoneal exudate cells differing in IgA allotypic markers also demonstrated that B2 cells (B1-cell-depleted peritoneal exudate cells) and not B1 cells produced RV-specific IgA. To our knowledge, this is the first observation that B1 cells are unable to cooperate with CD4+T cells and produce virus-specific intestinal IgA antibody. We also observed that transferred CD4+T cells alone are capable of resolving RV shedding, although no IgA is secreted. These data suggest that RV-specific IgA may not be obligatory for RV clearance but may protect from reinfection and that effector CD4+T cells alone can mediate the resolution of primary RV infection. Reconstitution of RV-infected SCID mice with B1 cells results in the outgrowth of contaminating, donor CD4+T cells that are unable to clear RV, possibly because their oligoclonal specificities may be ineffective against RV antigens.