Enteropathogenic Escherichia coli and enterohemorrhagic E. coli cause an inflammatory colitis in human patients characterized by neutrophil infiltration, proinflammatory cytokine expression, and crypt hyperplasia. Citrobacter rodentium causes a similar colitis in mice and serves as a model for enteropathogenic E. coli infection in humans. C. rodentium induces systemic T-cell-dependent antibody production that facilitates clearance of the bacteria and protects the host from reinfection. The role of innate immune cells in infectious colitis, however, is less well understood. In this study, we have determined the role of mast cells in the inflammatory response and disease induced by C. rodentium. Mice deficient in mast cells exhibit more severe colonic histopathology and have a higher mortality rate following infection with C. rodentium than do wild-type animals. Despite unimpaired neutrophil recruitment and lymphocyte activation, mast cell-deficient mice have a disseminated infection evident in crucial organ systems that contributes to sepsis. Importantly, mast cells also have the capacity to directly kill C. rodentium. Together, these results suggest that mast cells protect the host from systemic infection by reducing the bacterial load and preventing dissemination of the bacterium from the colon.Enteric pathogens such as enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) attach to and colonize the host gastrointestinal tract and cause weight loss, diarrhea, crypt hyperplasia, and transient colitis. EPEC is a major contaminant in food and water sources in developing countries and causes significant increases in morbidity and mortality, primarily among children. EHEC contaminates food and water sources in industrialized nations and causes both diarrhea and hemolytic-uremic syndrome. A related pathogen, Citrobacter rodentium, has only about 50% of its genes in common with EPEC and EHEC but nevertheless induces disease in rodents that is virtually identical to that caused by EPEC and EHEC in humans. C. rodentium infection in mice is widely used as an animal model of pathogenic E. coli infection in humans (7, 9, 10, 18-21, 34, 42, 43, 45-47).EPEC, EHEC, and C. rodentium all induce a characteristic lesion in the intestine of the host that is characterized by intimate attachment to the host intestinal epithelial cells and effacement of microvilli. Such attaching and effacing (A/E) lesions result from translocation of virulence proteins from the bacterium into the host cell (13,22,23,26). These factors induce formation of membranous protrusions, called pedestals, beneath the bacterium, and anchor the bacterium to the host cell (reviewed in reference 36). Pedestal formation is required for subsequent disease (31, 37). EPEC, EHEC, and C. rodentium all contain a 35-kb pathogenicity island called LEE (locus of enterocyte effacement) that encodes ϳ41 virulence factors that are essential for the formation of A/E lesions and for disease. The virulence factors from the LEE loci of EPEC, EHEC, and C. r...

Infection of CD4- cells by HIV-1 is well documented, but the mechanism responsible remains a matter of discussion. Previously we modified an HIV-1 virus strain, NL4-3, by deleting the Env proteins (gp41 and gp120) and inserting the enhanced green fluorescent protein (EGFP), and found that the Env(-) virus infects several types of CD4- cells. Here, we have prepared Env(-) virus from both the CD4- cell line, 293T, and the CD4+ cell lines, CEM and SUPT1, and found that HIV-1 Env(-) virus from either cell type is infectious for both CD4+ and several CD4- cell lines. Replication of HIV-1 Env(-) virus-infected cells was demonstrated by p24 gag protein assays and real-time reverse transcriptase polymerase chain reaction (RT-PCR) of the culture medium from infected cells. Virus collected from the HIV-1-Env(-) infected cultures proved infectious to several CD4- cell lines. Our results suggest that HIV-1 infects both CD4- and CD4+ cells using a gp120-independent mechanism. This infection mechanism may provide new explanations for HIV-1 latency and persistent infection in patients.

The HIV-1 envelope glycoprotein (Env) undergoes conformational changes while driving entry. We hypothesized that some of the intermediate Env conformations could be represented in tethered constructs where gp120 and the ectodomain of gp41 are joined by flexible linkers. Tethered Envs with long linkers (gp140-14 with 15 aa and gp140-24 with 26 aa) were stable and recognized by conformationally dependent anti-gp120 and anti-gp41 monoclonal antibodies (mAbs). Surprisingly, these proteins potently inhibited membrane fusion mediated by R5, X4, and R5X4 Envs with 5-100-fold lower IC50 than a tethered Env with short linker (gp140-4 with 4 aa), gp120, gp140, soluble CD4, or DP178 (T20). Compared to gp140, gp140-14,24 exhibited increased binding to anti-gp41 cluster II mAbs but not to cluster I mAbs. Cluster II mAbs but not cluster I, IV, or V mAbs reversed the inhibitory effect of gp140-14,24 suggesting a role of exposed conserved gp41 structures for the mechanism of inhibition. These findings suggest the existence of conserved gp41 structures that are important for HIV-1 entry and can be stably exposed in the native environment of the Env even in the absence of receptor-mediated activation. Thus, tethered Envs with long linkers may not only be important as HIV-1 inhibitors but also for elucidation of viral entry mechanisms and development of novel vaccine immunogens.