Decreased susceptibility of mice to infection with Listeria monocytogenes in the absence of interleukin-18

The induction of proinflammatory cytokines such as gamma interferon (IFN-gamma) and tumor necrosis factor alpha is crucial for the early control of bacterial infections. Since interleukin-18 (IL-18) acts as a potent inducer of IFN-gamma, it might play an important role in the induction of a protective immune response in listeriosis. We used a murine model of systemic Listeria monocytogenes infection to study the immune response to these intracellular bacteria in the absence of IL-18. For this purpose, IL-18-deficient mice and mice treated with anti-IL-18 neutralizing antibody were infected with L. monocytogenes, and their innate and adaptive immune responses were compared to those of control mice. Unexpectedly, we found that mice deficient in IL-18 were partially resistant to primary infection with L. monocytogenes. At day 3 after infection, the numbers of listeriae in the livers and spleens of control mice were up to 500 times higher than those in IL-18-deficient or anti-IL-18 antibody-treated mice. In addition, the level of proinflammatory cytokines was markedly reduced in IL-18-deficient mice. Enhanced resistance to L. monocytogenes infection in IL-18-deficient mice was accompanied by increased numbers of leukocytes and reduced apoptosis in the spleen 48 to 72 h after infection. In contrast, control and IL-18-deficient mice showed no significant differences in their abilities to mount a protective L. monocytogenes-specific T-cell response.     

Impact of intratumoral heterogeneity of breast cancer tissue on quantitative metabolomics using high‐resolution magic angle spinning 1H NMR spectroscopy

High‐resolution magic angle spinning (HR MAS) nuclear magnetic resonance (NMR) spectroscopy is increasingly being used to study metabolite levels in human breast cancer tissue, assessing, for instance, correlations with prognostic factors, survival outcome or therapeutic response. However, the impact of intratumoral heterogeneity on metabolite levels in breast tumor tissue has not been studied comprehensively. More specifically, when biopsy material is analyzed, it remains questionable whether one biopsy is representative of the entire tumor. Therefore, multi‐core sampling (n = 6) of tumor tissue from three patients with breast cancer, followed by lipid (0.9‐ and 1.3‐ppm signals) and metabolite quantification using HR MAS ¹H NMR, was performed, resulting in the quantification of 32 metabolites. The mean relative standard deviation across all metabolites for the six tumor cores sampled from each of the three tumors ranged from 0.48 to 0.74. This was considerably higher when compared with a morphologically more homogeneous tissue type, here represented by murine liver (0.16–0.20). Despite the seemingly high variability observed within the tumor tissue, a random forest classifier trained on the original sample set (training set) was, with one exception, able to correctly predict the tumor identity of an independent series of cores (test set) that were additionally sampled from the same three tumors and analyzed blindly. Moreover, significant differences between the tumors were identified using one‐way analysis of variance (ANOVA), indicating that the intertumoral differences for many metabolites were larger than the intratumoral differences for these three tumors. That intertumoral differences, on average, were larger than intratumoral differences was further supported by the analysis of duplicate tissue cores from 15 additional breast tumors. In summary, despite the observed intratumoral variability, the results of the present study suggest that the analysis of one, or a few, replicates per tumor may be acceptable, and supports the feasibility of performing reliable analyses of patient tissue.     

Wilms' Tumor 1 Gene Expression Using a Standardized European LeukemiaNet-Certified Assay Compared to Other Methods for Detection of Minimal Residual Disease in Myelodysplastic Syndrome and Acute Myelogenous Leukemia after Allogeneic Blood Stem Cell Transplantation

Overexpression of the Wilms' tumor 1 (WT1) gene is informative in many patients with acute myelogenous leukemia (AML) and myelodysplastic syndromes (MDS) and is measurable in peripheral blood (PB). Despite these advantages, WT1 has not broadly been established as a marker for minimal residual disease (MRD) monitoring after allogeneic hematopoietic stem cell transplantation (allo-HSCT) due to limited patient numbers, differing sample sources, and nonstandardized in-house methods. To estimate the value of WT1 as an MRD marker, we serially quantified PB WT1 expression using a standardized European LeukemiaNet-certified assay in 59 patients with AML and MDS after allo-HSCT. We compared its performance with routine methods such as chimerism, XY-fluorescence in situ hybridization (FISH), disease-specific cytogenetic, and molecular analyses, which were accessible in 100%, 34%, 68%, and 37%, respectively. Twenty-four patients (41%) relapsed within a median of 126 days after allo-HSCT, and 20 of them showed at least 1 elevated WT1 value above the validated cutoff. The other 35 patients (59%) remained in complete remission, and only 1 patient had a transient increase in WT1 expression. This reflects a sensitivity of 83% and a specificity of 97% for WT1 and appears to be favorable compared with the sensitivities and specificities observed for chimerism (33% and 91%), XY-FISH (67% and 73%), cytogenetic (33% and 77%), and molecular (78% and 85%) analyses. Further supporting its predictive impact, elevated WT1 expression prompted an earlier BM biopsy and consecutively the diagnosis of relapse in 62% of patients. The results of this real-life experience imply that PB WT1 expression is measurable by a standardized assay and predicts imminent relapse after allo-HSCT with high sensitivity and specificity in most patients with AML and MDS.     

Modifying the Implicit Illness-Related Self-Concept in Patients with Somatoform Disorders May Reduce Somatic Symptoms

Background

According to dual process theories, not only do explicit but also implicit cognitive processes play a major role in the development and maintenance of somatoform disorders (SFDs). Recent evidence [1] suggests that patients with SFDs have a stronger implicit illness-related self-concept, which is related to the experience of medically unexplained symptoms.

Purpose

The current study was designed to investigate a possible causal link between biased implicit associations and symptoms in SFD patients by experimentally modifying the implicit illness-related self-concept.

Methods

Twenty-nine patients with SFDs (according to the DSM-IV) initially completed an Implicit Association Test (IAT) for assessing the implicit illness-related self-concept. Two weeks later, they underwent an evaluative conditioning task to modify the implicit self-concept.

Results

After this procedure, a change toward a healthier implicit self-concept was apparent in the follow-up IAT. A reduction in symptom severity and changes in health- and body-related cognitions were observed 13 days after the training in the follow-up questionnaires.

Conclusions

The findings suggest that a biased implicit self-concept may be causally relevant for symptom experiences in patients with SFDs. Existing cognitive behavioral treatments for SFDs might benefit from targeting implicit cognitive processes more directly.     

O-Antigen-Negative Salmonella enterica Serovar Typhimurium Is Attenuated in Intestinal Colonization but Elicits Colitis in Streptomycin-Treated Mice

Lipopolysaccharide (LPS) is a major constituent of the outer membrane and an important virulence factor of Salmonella enterica subspecies 1 serovar Typhimurium (serovar Typhimurium). To evaluate the role of LPS in eliciting intestinal inflammation in streptomycin-treated mice, we constructed an O-antigen-deficient serovar Typhimurium strain through deletion of the wbaP gene. The resulting strain was highly susceptible to human complement activity and the antimicrobial peptide mimic polymyxin B. Furthermore, it showed a severe defect in motility and an attenuated phenotype in a competitive mouse infection experiment, where the ΔwbaP strain (SKI12) was directly compared to wild-type Salmonella. Nevertheless, the ΔwbaP strain (SKI12) efficiently invaded HeLa cells in vitro and elicited acute intestinal inflammation in streptomycin-pretreated mice. Our experiments prove that the presence of complete LPS is not essential for in vitro invasion or for triggering acute colitis.Salmonella spp. are a common cause of bacterial food-borne infections. Diseases caused by Salmonella spp. range from gastrointestinal symptoms such as fever, diarrhea, abdominal pain, and nausea to severe systemic infections. Salmonella enterica subspecies 1 serovar Typhimurium (serovar Typhimurium) is one of the most frequent enteropathogens, causing large numbers of diarrheal infections worldwide by colonizing the gut and triggering mucosal inflammation (33). The type III secretion system 1 (TTSS-1) and TTSS-2 encoded on Salmonella pathogenicity island 1 (SPI1) and SPI2 on the Salmonella genome are employed by the pathogen for mediating bacterial entry into the gut mucosa (SPI1) as well as the intracellular survival followed by systemic spread of the bacteria (SPI2) (9). Acute enteric serovar Typhimurium infection and the mechanisms leading to intestinal inflammation can be analyzed using a well-defined mouse model for Salmonella colitis: streptomycin-pretreated, naïve mice develop a vigorous local inflammation of the large intestine upon intragastric infection with serovar Typhimurium (3).Besides the SPI1- and SPI2-encoded TTSSs, serovar Typhimurium requires numerous additional virulence factors for colonizing the host, resisting host immune defense, and finally, triggering disease. One key virulence factor for serovar Typhimurium is lipopolysaccharide (LPS), a major surface component (42). It contributes to the stability of the outer membrane, serves as a permeability barrier, and protects the bacterium against environmental challenges (34). LPS is composed of three domains. The lipid A part, also known as endotoxin, anchors LPS molecules in the outer membrane with its fatty acid chains. It is connected through the inner core consisting of heptoses and Kdo (3-deoxy-d-manno-octulosonic acid), with the outer core containing hexoses and N-acetylhexoses. Linked to the last glucose of the outer core is the polymeric O-antigen region. This region is composed of 16 to >100 repeats of an oligosaccharide structure containing four to six monosaccharides (27).The endotoxic properties of LPS are mediated by the lipid A moiety, which can be recognized by Toll-like receptor 4 and thus triggers an innate immune response (16, 32). The O antigen, in combination with the inner and outer cores, serves as protection against complement antimicrobial peptides, detergents, and certain antibiotics. Furthermore, the O-antigen region is a key determinant for recognition by the adaptive immune response (40).A number of studies have established an important role for O-antigen side chains in Salmonella virulence. A signature-tagged mutagenesis screening by Morgan and coworkers proved that mutations in genes for enzymes involved in the biosynthesis of O-antigen side chains attenuated bacteria in their ability to colonize chick and calf intestines (25). Interestingly, a mutant in wbaP, the phosphogalactosyltransferase starting O-antigen biosynthesis, was able to colonize calves but showed an attenuated phenotype in chicks (25). Moreover, screening for Salmonella genes required for long-term systemic infection after intraperitoneal injection showed negative selection for mutants in O-antigen biosynthesis (21). Coinfection experiments by Nevola et al. show that mutants lacking O antigen are still able to colonize the murine intestine but are attenuated in competitive infection experiments (30). Furthermore, a recent in vitro study with Salmonella enterica serovar Typhi showed that O-antigen side chains are not necessary for adhesion to and invasion of epithelial cells. However, mutants lacking the complete outer core are severely attenuated (14). In general, the loss of core structures seems more detrimental than the loss of O-antigen side chains. However, it had remained unclear whether the O-antigen side chains are required for triggering intestinal inflammation.We wanted to analyze the role of O-antigen side chains in a well-established mouse model for enteric infections (3) and in an in vitro cellular invasion assay (36). Thus, we deleted the gene encoding the phosphogalactosyltransferase WbaP. This enzyme adds phosphogalactose to undecaprenylphosphate, the first step in O-antigen side chain biosynthesis in the cytoplasm of serovar Typhimurium (35, 43, 44). Streptomycin-pretreated mice were orally infected with the wbaP mutant strain (SKI12), and in line with published work, we found that the ΔwbaP mutant strain (SKI12) was significantly attenuated in a competitive infection assay. In spite of this, the wbaP mutant alone was able to trigger acute colitis. This demonstrates that serovar Typhimurium permits substantial manipulation of the O-antigen structure without losing its ability to trigger mucosal inflammation.     

Anti‐retroviral effects of type I IFN subtypes in vivo

Type I IFN play a very important role in immunity against viral infections. Murine type I IFN belongs to a multigene family including 14 IFN‐α subtypes but the biological functions of IFN‐α subtypes in retroviral infections are unknown. We have used the Friend retrovirus model to determine the anti‐viral effects of IFN‐α subtypes in vitro and in vivo. IFN‐α subtypes α1, α4, α6 or α9 suppressed Friend virus (FV) replication in vitro, but differed greatly in their anti‐viral efficacy in vivo. Treatment of FV‐infected mice with the IFN‐α subtypes α1, α4 or α9, but not α6 led to a significant reduction in viral loads. Decreased splenic viral load after IFN‐α1 treatment correlated with an expansion of activated FV‐specific CD8⁺ T cells and NK cells into the spleen, whereas in IFN‐α4‐ and ‐α9‐treated mice it exclusively correlated with the activation of NK cells. The results demonstrate the distinct anti‐retroviral effects of different IFN‐α subtypes, which may be relevant for new therapeutic approaches.     

TLR8‐mediated activation of human monocytes inhibits TL1A expression

TLR play important roles in inflammation and innate immune response to pathogens. TLR8 recognizes ssRNA and induces NF‐κB via MyD88 signaling. TL1A is a member of the TNF superfamily that markedly enhances IFN‐γ production by IL‐12/IL‐18‐stimulated peripheral and mucosal CD4⁺ T cells. TL1A expression is increased in the mucosa of patients with inflammatory bowel disease and is considered a key mediator of Crohn's disease (CD). We have previously shown that TL1A is strongly induced by immune complexes (IC) but not TLR ligands in antigen‐presenting cells. However, a potential interaction between these pro‐inflammatory signaling pathways has not been investigated. IC‐induced TL1A expression of monocytes was potently inhibited by a TLR8 or TLR7/8 ligand (R848) in a dose‐dependent manner. Furthermore, when co‐cultured with CD4⁺ T cells, TLR8 ligands inhibited TL1A production, resulting in almost complete inhibition of IFN‐γ production by the CD4⁺ T cells. Furthermore, we demonstrate that IFN‐α is not required for this suppressive effect by TLR8 signaling. Our data demonstrate for the first time a direct interaction between TLR and TL1A signaling pathways. TLR8 activation may be an important, novel pathway for targeted treatment of Th1‐mediated diseases, such as CD.