Escherichia coli strains producing Shiga toxins (Stx) 1 and 2 colonize the lower gastrointestinal tract in humans and are associated with gastrointestinal and systemic diseases. Stx are detectable in the feces of infected patients, and it is likely that toxin passes from the intestinal tract lumen to underlying tissues. The objective of this study was to develop an in vitro model to study the passage of Stx across intact, polarized cell monolayers. Translocation of biologically active Stx was examined in four cell lines grown on polycarbonate filters. Stx1 translocated across intestinal cell monolayers (CaCo2A and T84 cells) in an energy-requiring and saturable manner, while the monolayers maintained a high level of electrical resistance. Stx1 had no effect on electrical resistance or inulin movement across these cell lines for at least 24 h. Induction of specific Stx receptors with sodium butyrate reduced the proportion of toxin translocated across CaCo2A monolayers but had no major effect on the movement of horseradish peroxidase or [ 3 H]inulin. We have shown that biologically active Stx1 is capable of moving across intact polarized intestinal epithelial cells without apparent cellular disruption, probably via a transcellular pathway. The data also suggest that the presence of Stx receptors on the apical surface of intestinal epithelial cells may offer some protection against the absorption of luminal Stx1.

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The antigenic diversity within a panel of 63 Haemophilus ducreyi isolates was examined by Western blot (immunoblot) analysis with a pool of 238 well-characterized human antisera. When a serum pool adsorbed on a mixture of Haemophilus influenzae, H. parainfluenzae, and H. parahaemolyticus was used, the immunoprotiles suggested that prominent antigenic proteins involved in the human immune response have apparent molecular masses of 63, 42, 34 to 30, and 28.5 to 28 kDa. Preliminary subcellular localization revealed that these antigens are associated with the cellular membrane. Two subsets of antigens were discriminated by detergent extraction. There was no evidence that the antigen composition is altered by changing the growth conditions. With a serum pool adsorbed on the Haemophilus spp. mixture supplemented with Actinobacillus actinomycetemcomitans, Pasteurella ureae, Neisseria gonorrhoeae, and Escherichia coli, antigenic determinants more specific for H. ducreyi were identified. An immunodominant 28.5to 28-kDa protein was expressed by all H. ducreyi isolates. In the range from 34 to 30 kDa, 56 isolates revealed a dominant protein with variable molecular mass. By using both proteins (28.5 to 28 kDa and 34 to 30 kDa) as immunotypic markers, seven different immunopatterns were identified. Antigenic diversity among isolates from different geographical origins as well as from a single area was observed.Haemophilus ducreyi is a poorly known and fastidious microorganism causing chancroid, a sexually transmitted disease (STD) that is characterized by genital ulcers and by abcedation of the inguinal lymph nodes (6). Recently, chancroid has received more interest because it is emerging as the major cause of genital ulcer disease in several parts of the developing world, and it is reappearing in poor minority populations in Europe and the United States (10,14,16). In addition, several reports suggest that genital ulcer disease, in particular chancroid, is a risk factor for the sexual transmission of the human immunodeficiency virus (9). Little is known about the antigenic composition of H. ducreyi, and the nature of the immune response upon infection has been poorly studied. Serum immunoglobulin G (IgG) and IgM antibodies have been detected in patients with a clinical diagnosis of chancroid both by dot immunobinding and by an enzyme immunoassay (8,13). Western blot analysis of sera from normal and immunized rabbits has shown that rabbits immunized with H. ducreyi respond with a humoral immune response in which multiple antigenic polypeptides are detected. The most prominent antigens are reported to have apparent molecular masses of 79, 62, 55, 49, and 26 kDa (7) and of 67, 42, 22.5, and 20 kDa (12). However, the specificity of the antigens towards H. ducreyi has not been demonstrated.In the present study, the antigenic diversity occurring within a panel of H. ducreyi isolates was examined by Western blot (immunoblot) analysis. Prominent proteins involved in the human immune response and H. ducreyispecific antigens were iden...

Shiga-like toxin (SLT)-producing Escherichia coli (SLTEC) is the leading cause of acute renal failure among children. SLTEC are most commonly ingested from contaminated food, and because cattle are a major reservoir, ground beef and milk have been a significant source of contamination associated with multiperson outbreaks. While serotype O157:H7 has been principally identified in the United States there are many other SLTEC serotypes associated with human disease. We have therefore examined the utility of an enzyme immunoassay (EIA) for Shiga-like toxins as a means of detecting the presence of low levels of multiple SLTEC serotypes in ground beef and milk. In the present study we demonstrated that it is possible to detect low levels (approximately 1 SLTEC per g of ground beef) in both small-scale (2 g of beef per 5 ml) and standard large-scale (25 g of beef per 225 ml) food microbial cultures. The EIA was also capable of allowing detection of SLTEC in nonspiked retail ground beef samples: we were able to recover SLTEC isolates (O113:Hu; O22:H-; O82:H8) from 3 of 12 ground beef samples. The EIA detected SLTs produced in spiked milk samples when as few as 1 SLTEC per ml was added. Overall the EIA proved to be a highly sensitive way to detect the presence of SLTEC in either ground beef or milk samples after overnight enrichment culturing in an appropriate broth and should provide a rapid and convenient method for the detection of multiple pathogenic SLTEC serotypes.

Fatty Acid Synthase (FASN) is a multi-domain protein that carries out de novo fatty acid (palmitate) synthesis from acetate and malonate in mammalian cells. FASN is up-regulated in cancer cells, providing fatty acid building blocks for rapid cell growth and cell division. Increased FASN expression is correlated with disease progression and poor prognosis in many cancers including prostate, breast, ovary, colon, and lung. FASN has been demonstrated to play an important role in carcinogenesis by protecting cells from apoptosis. Herein we report a new series of potent, selective and orally bioavailable FASN inhibitors. Recent publications disclose several FASN inhibitor chemotypes that share a common pharmacophore, wherein an aromatic group and an acylated cyclic amine are attached to a central scaffold. We postulated that a spirocyclic imidazolinone core would be an acceptable and drug-like scaffold, inspired by the precedent of irbesartan, an approved antihypertensive drug in which a spirocyclopentyl-imidazolinone core replaces the substituted imidazole ring of losartan, an older approved agent from the same drug class. This hypothesis led to a new spirocyclic imidazolinone based FASN inhibitors. Extensive SAR efforts resulted in FASN inhibitors with potent enzyme and cell activity, selectivity, and oral bioavailability exemplified by JNJ-54302833. JNJ-54302833 is a potent inhibitor of human FASN (IC50 = 28 nM) and also potently inhibits proliferation of A2780 ovarian cells (IC50 = 13 nM) in lipid-reduced medium. This cellular activity can be rescued by addition of palmitate, demonstrating on-target effects. JNJ-54302833 is also potent in many other cells, including PC3M (IC50 = 25 nM) and LnCaP-Vancouver prostate cells (IC50 = 66 nM), and is highly bioavailable (F 61%) with good exposures. In a pharmacodynamics study in H460 lung xenograft-bearing mice, oral treatment with JNJ-54302833 resulted in elevated tumor levels of malonyl-CoA and decreased tumor levels of palmitate. This novel series potently inhibits the FASN KR domain (IC50 = 54 nM for JNJ-54302833); specific binding to KR was confirmed by crystal structures.In summary, we have designed and discovered a new series of FASN inhibitors that are potent both in enzyme and in cell proliferation assays, are highly bioavailable, and bind to KR domain. Additionally, palmitate rescue of lipid-reduced cellular activity suggests selectivity and pharmacodynamics studies confirm target engagement. Citation Format: Tianbao Lu, Richard Alexander, Gilles Bignan, James Bischoff, Peter Connolly, Max Cummings, Sabine De Breucker, Norbert Esser, Erwin Fraiponts, Ron Gilissen, Bruce Grasberger, Boudewijn Janssens, Donald Ludovici, Lieven Meerpoel, Christophe Meyer, Michael Parker, Danielle Peeters, Carsten Schubert, Karine Smans, Luc Van Nuffel, Peter Vermeulen. Design and synthesis of a series highly potent and bioavailable FASN KR domain inhibitors for cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4747. doi:10.1158/1538-7445.AM2014-4747