Induction of endothelial cell apoptosis by the antivascular agent 5,6-dimethylxanthenone-4-acetic acid

5,6-Dimethylxanthenone-4-acetic acid, synthesised in this laboratory, reduces tumour blood flow, both in mice and in patients on Phase I trial. We used TUNEL (TdT-mediated dUTP nick end labelling) assays to investigate whether apoptosis induction was involved in its antivascular effect. 5,6-Dimethylxanthenone-4-acetic acid induced dose-dependent apoptosis in vitro in HECPP murine endothelial cells in the absence of up-regulation of mRNA for tumour necrosis factor. Selective apoptosis of endothelial cells was detected in vivo in sections of Colon 38 tumours in mice within 30 min of administration of 5,6-Dimethylxanthenone-4-acetic acid (25 mg kg−1). TUNEL staining intensified with time and after 3 h, necrosis of adjacent tumour tissue was observed. Apoptosis of central vessels in splenic white pulp was also detected in tumour-bearing mice but not in mice without tumours. Apoptosis was not observed in liver tissue. No apoptosis was observed with the inactive analogue 8-methylxanthenone-4-acetic acid. Positive TUNEL staining of tumour vascular endothelium was evident in one patient in a Phase I clinical trial, from a breast tumour biopsy taken 3 and 24 h after infusion of 5,6-Dimethylxanthenone-4-acetic acid (3.1 mg m−2). Tumour necrosis and the production of tumour tumour necrosis factor were not observed. No apoptotic staining was seen in tumour biopsies taken from two other patients (doses of 3.7 and 4.9 mg m−2). We conclude that 5,6-Dimethylxanthenone-4-acetic acid can induce vascular endothelial cell apoptosis in some murine and human tumours. The action is rapid and appears to be independent of tumour necrosis factor induction

Young women's use of a microbicide surrogate: The complex influence of relationship characteristics and perceived male partners' evaluations

This is the post-print version of the article. The official published version can be found at the link below.Currently in clinical trials, vaginal microbicides are proposed as a female-initiated method of sexually transmitted infection prevention. Much of microbicide acceptability research has been conducted outside of the United States and frequently without consideration of the social interaction between sex partners, ignoring the complex gender and power structures often inherent in young women’s (heterosexual) relationships. Accordingly, the purpose of this study was to build on existing microbicide research by exploring the role of male partners and relationship characteristics on young women’s use of a microbicide surrogate, an inert vaginal moisturizer (VM), in a large city in the United States. Individual semi-structured interviews were conducted with 40 young women (18–23 years old; 85% African American; 47.5% mothers) following use of the VM during coital events for a 4 week period. Overall, the results indicated that relationship dynamics and perceptions of male partners influenced VM evaluation. These two factors suggest that relationship context will need to be considered in the promotion of vaginal microbicides. The findings offer insights into how future acceptability and use of microbicides will be influenced by gendered power dynamics. The results also underscore the importance of incorporating men into microbicide promotion efforts while encouraging a dialogue that focuses attention on power inequities that can exist in heterosexual relationships. Detailed understanding of these issues is essential for successful microbicide acceptability, social marketing, education, and use.This study was funded by a grant from National Institutes of Health (NIHU19AI 31494) as well as research awards to the first author: Friends of the Kinsey Institute Research Grant Award, Indiana University’s School of HPER Graduate Student Grant-in-Aid of Research Award, William L. Yarber Sexual Health Fellowship, and the Indiana University Graduate and Professional Student Organization Research Grant

Epithelial p38α Controls Immune Cell Recruitment in the Colonic Mucosa

Intestinal epithelial cells (IECs) compose the first barrier against microorganisms in the gastrointestinal tract. Although the NF-κB pathway in IECs was recently shown to be essential for epithelial integrity and intestinal immune homeostasis, the roles of other inflammatory signaling pathways in immune responses in IECs are still largely unknown. Here we show that p38α in IECs is critical for chemokine expression, subsequent immune cell recruitment into the intestinal mucosa, and clearance of the infected pathogen. Mice with p38α deletion in IECs suffer from a sustained bacterial burden after inoculation with Citrobacter rodentium. These animals are normal in epithelial integrity and immune cell function, but fail to recruit CD4+ T cells into colonic mucosal lesions. The expression of chemokines in IECs is impaired, which appears to be responsible for the impaired T cell recruitment. Thus, p38α in IECs contributes to the host immune responses against enteric bacteria by the recruitment of immune cells

Conditional Stat1 Ablation Reveals the Importance of Interferon Signaling for Immunity to Listeria monocytogenes Infection

Signal transducer and activator of transcription 1 (Stat1) is a key player in responses to interferons (IFN). Mutations of Stat1 cause severe immune deficiencies in humans and mice. Here we investigate the importance of Stat1 signaling for the innate and secondary immune response to the intracellular bacterial pathogen Listeria monocytogenes (Lm). Cell type-restricted ablation of the Stat1 gene in naïve animals revealed unique roles in three cell types: macrophage Stat1 signaling protected against lethal Lm infection, whereas Stat1 ablation in dendritic cells (DC) did not affect survival. T lymphocyte Stat1 reduced survival. Type I IFN (IFN-I) signaling in T lymphocytes reportedly weakens innate resistance to Lm. Surprisingly, the effect of Stat1 signaling was much more pronounced, indicating a contribution of Stat1 to pathways other than the IFN-I pathway. In stark contrast, Stat1 activity in both DC and T cells contributed positively to secondary immune responses against Lm in immunized animals, while macrophage Stat1 was dispensable. Our findings provide the first genetic evidence that Stat1 signaling in different cell types produces antagonistic effects on innate protection against Lm that are obscured in mice with complete Stat1 deficiency. They further demonstrate a drastic change in the cell type-dependent Stat1 requirement for memory responses to Lm infection

O-Antigen Delays Lipopolysaccharide Recognition and Impairs Antibacterial Host Defense in Murine Intestinal Epithelial Cells

Although Toll-like receptor (TLR) 4 signals from the cell surface of myeloid cells, it is restricted to an intracellular compartment and requires ligand internalization in intestinal epithelial cells (IECs). Yet, the functional consequence of cell-type specific receptor localization and uptake-dependent lipopolysaccharide (LPS) recognition is unknown. Here, we demonstrate a strikingly delayed activation of IECs but not macrophages by wildtype Salmonella enterica subsp. enterica sv. (S.) Typhimurium as compared to isogenic O-antigen deficient mutants. Delayed epithelial activation is associated with impaired LPS internalization and retarded TLR4-mediated immune recognition. The O-antigen-mediated evasion from early epithelial innate immune activation significantly enhances intraepithelial bacterial survival in vitro and in vivo following oral challenge. These data identify O-antigen expression as an innate immune evasion mechanism during apical intestinal epithelial invasion and illustrate the importance of early innate immune recognition for efficient host defense against invading Salmonella

TRIM27 Negatively Regulates NOD2 by Ubiquitination and Proteasomal Degradation

NOD2, the nucleotide-binding domain and leucine-rich repeat containing gene family (NLR) member 2 is involved in mediating antimicrobial responses. Dysfunctional NOD2 activity can lead to severe inflammatory disorders, but the regulation of NOD2 is still poorly understood. Recently, proteins of the tripartite motif (TRIM) protein family have emerged as regulators of innate immune responses by acting as E3 ubiquitin ligases. We identified TRIM27 as a new specific binding partner for NOD2. We show that NOD2 physically interacts with TRIM27 via the nucleotide-binding domain, and that NOD2 activation enhances this interaction. Dependent on functional TRIM27, ectopically expressed NOD2 is ubiquitinated with K48-linked ubiquitin chains followed by proteasomal degradation. Accordingly, TRIM27 affects NOD2-mediated pro-inflammatory responses. NOD2 mutations are linked to susceptibility to Crohns disease. We found that TRIM27 expression is increased in Crohns disease patients, underscoring a physiological role of TRIM27 in regulating NOD2 signaling. In HeLa cells, TRIM27 is partially localized in the nucleus. We revealed that ectopically expressed NOD2 can shuttle to the nucleus in a Walker A dependent manner, suggesting that NOD2 and TRIM27 might functionally cooperate in the nucleus. We conclude that TRIM27 negatively regulates NOD2-mediated signaling by degradation of NOD2 and suggest that TRIM27 could be a new target for therapeutic intervention in NOD2-associated diseases.Funding Agencies|German Research Foundation (DFG)|SFB670-NG01|Swedish Society of Medicine||Regional Research Council of South-East Sweden (FORSS)||Swedish Research Council division of Medicine||Gustav V 90th anniversary foundation||Italian Telethon Foundation||DFG|SE 1122/2-1|</p

The Hemorrhagic Coli Pilus (HCP) of Escherichia coli O157:H7 Is an Inducer of Proinflammatory Cytokine Secretion in Intestinal Epithelial Cells

Enterohemorrhagic Escherichia coli (EHEC) O157:H7, the causative agent of hemorrhagic colitis and the hemolytic uremic syndrome (HUS), produces long bundles of type IV pili (TFP) called hemorrhagic coli pili (HCP). HCP are capable of mediating several phenomena associated with pathogenicity: i) adherence to human and bovine epithelial cells; ii) invasion of epithelial cells; iii) hemagglutination of rabbit erythrocytes; iv) biofilm formation; v) twitching motility; and vi) specific binding to laminin and fibronectin. HCP are composed of a 19 kDa pilin subunit (HcpA) encoded by the hcpA chromosomal gene (called prepilin peptidase-dependent gene [ppdD] in E. coli K-12).In this study we investigated the potential role of HCP of E. coli O157:H7 strain EDL933 in activating the release of pro- and anti-inflammatory cytokines from a variety of host epithelial cells. We found that purified HCP and a recombinant HcpA protein induced significant release of IL-8 and TNF-alpha, from cultured polarized intestinal cells (T84 and HT-29 cells) and non-intestinal HeLa cells. Levels of proinflammatory IL-8 and TNF-alpha, but not IL-2, IL6, or IL-10 cytokines, were increased in the presence of HCP and recombinant HcpA after 6 h of incubation with >or=50 ng/ml of protein, suggesting that stimulation of IL-8 and TNF-alpha are dose and time-dependent. In addition, we also demonstrated that flagella are potent inducers of cytokine production. Furthermore, MAPK activation kinetics studies showed that EHEC induces p38 phosphorylation under HCP-producing conditions, and ERK1/2 and JNK activation was detectable after 3 h of EHEC infection. HT-29 cells were stimulated with epidermal growth factor stimulation of HT-29 cells for 30 min leading to activation of three MAPKs.The HcpA pilin monomer of the HCP produced by EHEC O157:H7 is a potent inducer of IL-8 and TNF-alpha release, an event which could play a significant role in the pathogenesis of hemorrhagic colitis caused by this pathogen