Extracellular secretion of products is the major mechanism by which Gram-negative pathogens communicate with and intoxicate host cells. Vesicles released from the envelope of growing bacteria serve as secretory vehicles for proteins and lipids of Gram-negative bacteria. Vesicle production occurs in infected tissues and is influenced by environmental factors. Vesicles play roles in establishing a colonization niche, carrying and transmitting virulence factors into host cells, and modulating host defense and response. Vesicle-mediated toxin delivery is a potent virulence mechanism exhibited by diverse Gram-negative pathogens. The biochemical and functional properties of pathogen-derived vesicles reveal their potential to critically impact disease.In nearly every case, virulence factors of Gram-negative pathogens are secreted products that enhance the survival of the bacteria and/or damage the host. Secretion of virulence factors by Gram-negative pathogens is complicated by the fact that the bacterial envelope consists of two lipid bilayers, the inner and outer membrane, and the periplasm in between. Gram-negative pathogens have developed many strategies, some specific to pathogens, to enable active virulence factors to gain access to the extracellular environment, typically the tissues or bloodstream of the host organism (Henderson et al. 2004). The Type II and Type V secretion systems are two-step processes in which proteins are transported first through the inner membrane (IM) and then through the outer membrane (OM). For secretion via the Type I, Type III, and Type IV secretion systems, the material is transferred directly into the extracellular milieu or into another cell. The Type III system is specific for the transport of factors by pathogenic bacteria. All of these secretion systems secrete individual proteins or small complexes. This review examines secretion via OM vesicles, a distinct "Type VI" mechanism that enables bacteria to secrete a large, complex group of proteins and lipids into the extracellular milieu.Both pathogenic and nonpathogenic species of Gram- Vesicles are a means by which bacteria interact with prokaryotic and eukaryotic cells in their environment. Some of the best-characterized vesicles are those produced by pathogens. Biochemical analysis and functional characterization of pathogen-derived outer membrane vesicles demonstrate that this secretory pathway has been usurped by pathogens for the transport of active virulence factors to host cells (Table 1). Naturally produced OM vesicles from pathogenic bacteria contain adhesins, toxins, and immunomodulatory compounds, and they directly mediate bacterial binding and invasion, cause cytotoxicity, and modulate the host immune response. By participating in such diverse aspects of the host-pathogen interaction, OM vesicles are potent bacterial virulence factors. Formation of bacterial OM vesiclesNaturally produced bacterial vesicles are discrete, closed OM blebs produced by growing cells, not products of cell lysis or cell death (Mug-Opstelte...

Enterotoxigenic Escherichia coli (ETEC) is a prevalent cause of traveler's diarrhea and infant mortality in thirdworld countries. Heat-labile enterotoxin (LT) is secreted from ETEC via vesicles composed of outer membrane and periplasm. We investigated the role of ETEC vesicles in pathogenesis by analyzing vesicle association and entry into eukaryotic cells. Fluorescently labeled vesicles from LT-producing and LT-nonproducing strains were compared in their ability to bind adrenal and intestinal epithelial cells. ETEC-derived vesicles, but not control nonpathogenderived vesicles, associated with cells in a time-, temperature-, and receptor-dependent manner. Vesicles were visualized on the cell surface at 41C and detected intracellularly at 371C. ETEC vesicle endocytosis depended on cholesterol-rich lipid rafts. Entering vesicles partially colocalized with caveolin, and the internalized vesicles accumulated in a nonacidified compartment. We conclude that ETEC vesicles serve as specifically targeted transport vehicles that mediate entry of active enterotoxin and other bacterial envelope components into host cells. These data demonstrate a role in virulence for ETEC vesicles.

Gram-negative bacteria shed outer membrane vesicles composed of outer membrane and periplasmic components. Since vesicles from pathogenic bacteria contain virulence factors and have been shown to interact with eukaryotic cells, it has been proposed that vesicles behave as delivery vehicles. We wanted to determine whether heterologously expressed proteins would be incorporated into the membrane and lumen of vesicles and whether these altered vesicles would associate with host cells. Ail, an outer membrane adhesin/invasin from Yersinia enterocolitica, was detected in purified outer membrane and in vesicles from Escherichia coli strains DH5␣, HB101, and MC4100 transformed with plasmidencoded Ail. In vesicle-host cell co-incubation assays we found that vesicles containing Ail were internalized by eukaryotic cells, unlike vesicles without Ail. To determine whether lumenal vesicle contents could be modified and delivered to host cells, we used periplasmically expressed green fluorescent protein (GFP). GFP fused with the Tat signal sequence was secreted into the periplasm via the twin arginine transporter (Tat) in both the laboratory E. coli strain DH5␣ and the pathogenic enterotoxigenic E. coli ATCC strain 43886. Pronase-resistant fluorescence was detectable in vesicles from Tat-GFP-transformed strains, demonstrating that GFP was inside intact vesicles. Inclusion of GFP cargo increased vesicle density but did not result in morphological changes in vesicles. These studies are the first to demonstrate the incorporation of heterologously expressed outer membrane and periplasmic proteins into bacterial vesicles.Gram-negative bacteria secrete proteins solubly and in association with outer membrane vesicles. All Gram-negative bacteria studied to date, including Escherichia coli, Neisseria meningitidis, Pseudomonas aeruginosa, Helicobacter pylori, Borrelia burgdorferi, Shigella flexneri and Actinobacillus actinomycetemcomitans, produce outer membrane vesicles (1-8). Vesicles were first observed by electron microscopy and range in size from 20 -250 nm in diameter. Gram-negative bacteria are bounded by an inner and outer membrane that encloses the periplasmic space. During vesiculation, the outer membrane pinches off (1), resulting in a closed proteoliposome composed of outer membrane lipids and proteins and periplasmic components, but not inner membrane or cytosolic components. Virulence factors such as VacA, shiga toxin, heat-labile enterotoxin (LT), 1 leukotoxin, and ClyA are associated with vesicles from pathogenic bacteria (2, 4 -6, 8 -10).Bacterial outer membrane vesicles interact with both eukaryotic cells and other bacteria via surface-expressed factors to deliver vesicle components and virulence factors (5,6,8,(11)(12)(13)(14)(15)(16).2 For example, LT associated with lipopolysaccharide on the surface of enterotoxigenic E. coli (ETEC) vesicles triggers internalization via caveolae and delivers not only catalytically active LT, which intoxicates the eukaryotic cell, but also other bacterial vesicle components.2 Other s...

Background:A dichotomous index combining two gene expression assays, HOXB13 : IL17BR (H : I) and molecular grade index (MGI), was developed to assess risk of recurrence in breast cancer patients. The study objective was to demonstrate the prognostic utility of the combined index in early-stage breast cancer.Methods:In a blinded retrospective analysis of 588 ER-positive tamoxifen-treated and untreated breast cancer patients from the randomised prospective Stockholm trial, H : I and MGI were measured using real-time RT–PCR. Association with patient outcome was evaluated by Kaplan–Meier analysis and Cox proportional hazard regression. A continuous risk index was developed using Cox modelling.Results:The dichotomous H : I+MGI was significantly associated with distant recurrence and breast cancer death. The >50% of tamoxifen-treated patients categorised as low-risk had <3% 10-year distant recurrence risk. A continuous risk model (Breast Cancer Index (BCI)) was developed with the tamoxifen-treated group and the prognostic performance tested in the untreated group was 53% of patients categorised as low risk with an 8.3% 10-year distant recurrence risk.Conclusion:Retrospective analysis of this randomised, prospective trial cohort validated the prognostic utility of H : I+MGI and was used to develop and test a continuous risk model that enables prediction of distant recurrence risk at the patient level.

These results support continued evaluation of pramlintide as a potential treatment for obesity.

OBJECTIVE -To assess long-term weight loss efficacy and safety of pramlintide used at different dosing regimens and in conjunction with lifestyle intervention (LSI).RESEARCH DESIGN AND METHODS -In a 4-month, double-blind, placebocontrolled, dose-ranging study, 411 obese subjects were randomized to receive pramlintide (six arms: 120, 240, and 360 g b.i.d. and t.i.d.) or placebo in conjunction with a structured LSI program geared toward weight loss. Of the 4-month evaluable subjects (n ϭ 270), 77% opted to continue preexisting treatment during an 8-month single-blind extension (LSI geared toward weight maintenance).RESULTS -At month 4, mean weight loss from baseline in the pramlintide arms ranged from 3.8 Ϯ 0.7 to 6.1 Ϯ 0.8 kg (2.8 Ϯ 0.8 kg with placebo). By month 12, initial 4-month weight loss was regained in the placebo group but was maintained in all but the 120-g b.i.d. group. Placebo-corrected weight loss with 120 g t.i.d. and 360 g b.i.d. averaged 3.2 Ϯ 1.2 kg (3.1 Ϯ 1.1% body wt) and 3.3 Ϯ 1.1 kg (3.1 Ϯ 1.0% body wt), respectively, at month 4 (both P Ͻ 0.01; 4-month evaluable n ϭ 270) and 6.1 Ϯ 2.1 kg (5.6 Ϯ 2.1% body wt) and 7.2 Ϯ 2.3 kg (6.8 Ϯ 2.3% body wt), respectively, at month 12 (both P Ͻ 0.01; 12-month evaluable n ϭ 146). At month 12, 40 and 43% of subjects treated with 120 g t.i.d. and 360 g b.i.d., respectively, achieved Ն10% weight loss (vs. 12% for placebo). Nausea, the most common adverse event with pramlintide in the 4-month study (9 -29% pramlintide vs. 2% placebo), was generally mild to moderate and occurred in Ͻ10% of subjects during the extension. CONCLUSIONS -When used over 12 months as an adjunct to LSI, pramlintide treatment, with low-dose three-times-daily or higher-dose two-times-daily regimens, helped obese subjects achieve greater initial weight loss and enhanced long-term maintenance of weight loss.

Evidence from rodent studies indicates that the β-cell-derived neurohormone amylin exerts multiple effects on eating behavior, including reductions in meal size, intake of highly palatable foods, and stress-induced sucrose consumption. To assess the effect of amylin agonism on human eating behavior we conducted a randomized, blinded, placebo-controlled, multicenter study investigating the effects of the amylin analog pramlintide on body weight, 24-h caloric intake, portion sizes, “fast food” intake, and perceived control of eating in 88 obese subjects. After a 2-day placebo lead-in, subjects self-administered pramlintide (180 μg) or placebo by subcutaneous injection 15 min before meals for 6 wk without concomitant lifestyle modifications. Compared with placebo, pramlintide treatment elicited significant mean reductions from baseline in body weight on day 44 (−2.1 ± 0.3 vs. +0.1 ± 0.4%, P < 0.001), 24-h caloric intake (−990 ± 94 vs. −243 ± 126 kcal on day 3, P < 0.0001; −680 ± 86 vs. −191 ± 161 kcal on day 43, P < 0.01), portion sizes, and caloric intake at a “fast food challenge” (−385 ± 61 vs. −109 ± 88 kcal on day 44, P < 0.05). Pramlintide treatment also improved perceived control of eating, as demonstrated by a 45% placebo-corrected reduction in binge eating scores ( P < 0.01). The results of this translational research study confirm in humans various preclinical effects of amylin agonism, demonstrating that pramlintide-mediated weight loss in obese subjects is accompanied by sustained reductions in 24-h food intake, portion sizes, fast food intake, and binge eating tendencies.

Accurate determination of cancer origin is necessary to guide optimal treatment but remains a diagnostic challenge. Gene expression profiling technologies have aided the classification of tumors and, therefore, could be applied in conjunction with clinicopathologic correlates to improve accuracy. We report an expanded version of the previously