Toll-like receptor 9 signaling mediates the anti-inflammatory effects of probiotics in murine experimental colitis

BACKGROUND & AIMS: We tested whether the attenuation of experimental colitis by live probiotic bacteria is due to their immunostimulatory DNA, whether toll-like receptor (TLR) signaling is required, and whether nonviable probiotics are effective. METHODS: Methylated and unmethylated genomic DNA isolated from probiotics (VSL-3), DNAse-treated probiotics and Escherichia coli (DH5 alpha) genomic DNA were administered intragastrically (i.g.) or subcutaneously (s.c.) to mice prior to the induction of colitis. Viable or gamma-irradiated probiotics were administered i.g. to wild-type mice and mice deficient in different TLR or in the adaptor protein MyD88, 10 days prior to administration of dextran sodium sulfate (DSS) to their drinking water and for 7 days thereafter. RESULTS: Intragastric and s.c. administration of probiotic and E. coli DNA ameliorated the severity of DSS-induced colitis, whereas methylated probiotic DNA, calf thymus DNA, and DNase-treated probiotics had no effect. The colitis severity was attenuated to the same extent by i.g. delivery of nonviable gamma-irradiated or viable probiotics. Mice deficient in MyD88 did not respond to gamma-irradiated probiotics. The severity of DSS-induced colitis in TLR2 and TLR4 deficient mice was significantly decreased by i.g. administration of gamma-irradiated probiotics, whereas, in TLR9-deficient mice, gamma-irradiated probiotics had no effect. CONCLUSIONS: The protective effects of probiotics are mediated by their own DNA rather than by their metabolites or ability to colonize the colon. TLR9 signaling is essential in mediating the anti-inflammatory effect of probiotics, and live microorganisms are not required to attenuate experimental colitis because nonviable probiotics are equally effective.     

Improving the Quality of EDTA-treated Blood Specimens from Mice

Nonterminal blood sampling in laboratory mice is a very common procedure. With the goal of improving animal welfare, different sampling sites and methods have been compared but have not achieved a consensus. Moreover, most of these studies overlooked the quality of blood specimens collected. The main preanalytical concern with EDTA-treated blood specimens for hematology analyses is platelet aggregation, which is known to cause analytical errors. Our objective was to find a nonterminal blood sampling method with minimal adverse effects on mice and few or no platelet aggregates. We tested and compared 2 collection sites, 4 sampling methods, and 3 antithrombotic drugs in 80 C57BL6/j male and female mice by evaluating platelet aggregates on blood smears and platelet, WBC, and RBC counts. In addition, the blood collection process was carefully evaluated, and adverse effects were recorded. Platelet aggregation was lower in specimens collected from the jugular vein than from the facial vein, with no effect of the sampling device or the presence of an antithrombotic additive. Highly aggregated specimens were significantly associated with lower platelet counts, whereas aggregation had no effect on WBC or RBC counts. Adverse events during sampling were significantly associated with more numerous platelet aggregates. The jugular vein is thus a satisfactory sampling site in mice in terms of both animal welfare and low platelet aggregation. Using antithrombotic agents appears to be unnecessary, whereas improving sampling conditions remains a key requirement to ensure the quality of EDTA-treated blood specimens from mice.

Industrial and academic research often require hematology analyses of mouse blood. Consequently, many terminal and nonterminal techniques have become available for blood sampling in mice.^{12,21,27,40,42,53} Preanalytical variation in clinical pathology is known to be a major issue.^5,45,49 Although the effects of the blood sampling method on animal welfare have been the subject of many preanalytical hematology and biochemical analyses,^{1,6,8,9,15,16,18,24-26,36,47,50-52,54} no agreement has been reached regarding the optimal method for nonterminal blood collection in mice and, to our knowledge, only a few investigations^1,8,15,16,18 have addressed the quality of the resulting blood specimens.Our own experience of hematology measurements from nonterminal mouse EDTA-blood specimens is that some specimens show both visible clots and platelet aggregation, the latter being detected only from microscopic examination of blood smears.³³ Whereas specimens with visible clots can be eliminated, microscopic platelet aggregates can also interfere with hematology analyses or cause analytical errors, as has been reported in other species including cats.^13,22,31,39 These abnormalities require repeat sampling when possible; otherwise, the number of validated results is decreased. EDTA-treated mouse blood is especially prone to platelet aggregation and clotting.^14,28,43 This characteristic leads to errors in platelet counts (pseudothrombocytopenia) and possible misidentification of platelet aggregates as eosinophils, resulting in false leukocytosis and eosinophilia.¹⁴ In vitro platelet aggregation in mice is due to high platelet counts^34,43 and is influenced by numerous preanalytical factors including the sampling method, collection site, specimen processing, anticoagulant used, the blood:anticoagulant ratio, the mouse strain and genetic alterations.^19,28,30,43 The literature on the influence of preanalytical factors on the quality of CBC analyses in mice is scant,⁴³ and no agreement has yet been reached regarding the optimal method for nonterminal blood collection in mice. In humans and various animal species, platelet aggregation can be reduced by adding platelet aggregation inhibitors that act at different steps of aggregation. To our knowledge, the addition of such inhibitors to mouse whole blood has not been tested as a means to improve the quality of mice EDTA-treated blood specimens.The aim of this study was therefore to identify the best preanalytical conditions for nonterminal blood collection in mice, based on animal welfare, scores of platelet aggregation, and platelet, RBC, and WBC counts. The hypotheses we tested were that 1) adding an antithrombotic drug (or multiple such drugs) to the EDTA-treated blood specimen would prevent or at least significantly lower platelet aggregation, 2) the site and the method of collection influence in vitro platelet aggregation, and 3) high-quality blood sampling is a key to reducing platelet aggregation in blood specimens.     

Zygomycosis and diabetes mellitus

Zygomycoses are severe angio-invasive fungal infections that develop in immunocompromised and diabetic patients. Any episode of sinusitis not responding to short-term antibacterial therapy should evoke the diagnosis of zygomycosis in the latter population, especially in cases of a surrounding necrotic area. Appropriate diagnosis is obtained after careful direct examination of the sample and culture. Current therapy underscores the need to control glycaemia and acidosis in addition to the need for urgent administration of high-dose liposomal amphotericin B in combination with extensive surgery.     

Strong time dependence of the 76-gene prognostic signature for node-negative breast cancer patients in the TRANSBIG multicenter independent validation series.

PURPOSE: Recently, a 76-gene prognostic signature able to predict distant metastases in lymph node-negative (N(-)) breast cancer patients was reported. The aims of this study conducted by TRANSBIG were to independently validate these results and to compare the outcome with clinical risk assessment. EXPERIMENTAL DESIGN: Gene expression profiling of frozen samples from 198 N(-) systemically untreated patients was done at the Bordet Institute, blinded to clinical data and independent of Veridex. Genomic risk was defined by Veridex, blinded to clinical data. Survival analyses, done by an independent statistician, were done with the genomic risk and adjusted for the clinical risk, defined by Adjuvant! Online. RESULTS: The actual 5- and 10-year time to distant metastasis were 98% (88-100%) and 94% (83-98%), respectively, for the good profile group and 76% (68-82%) and 73% (65-79%), respectively, for the poor profile group. The actual 5- and 10-year overall survival were 98% (88-100%) and 87% (73-94%), respectively, for the good profile group and 84% (77-89%) and 72% (63-78%), respectively, for the poor profile group. We observed a strong time dependence of this signature, leading to an adjusted hazard ratio of 13.58 (1.85-99.63) and 8.20 (1.10-60.90) at 5 years and 5.11 (1.57-16.67) and 2.55 (1.07-6.10) at 10 years for time to distant metastasis and overall survival, respectively. CONCLUSION: This independent validation confirmed the performance of the 76-gene signature and adds to the growing evidence that gene expression signatures are of clinical relevance, especially for identifying patients at high risk of early distant metastases.     

The Growth Factor Midkine Antagonizes VEGF Signaling In Vitro and In Vivo

Midkine (MDK) is a heparin-binding growth factor involved in growth, survival, migration, and differentiation of various target cells and dysregulation of MDK signaling is implicated in a variety of inflammatory diseases and cancers. Although MDK has been reported to act on endothelial cells and to have proangiogenic effects, the exact role of MDK in angiogenesis is poorly defined. Here, we report that MDK is actually a modulator of angiogenesis and that it can abrogate the vascular endothelial growth factor A (VEGF-A)-induced proliferation of human microvascular endothelial cells in vitro through the downregulation of proangiogenic cytokines and through the upregulation of the antiangiogenic factor, tissue inhibitor of metalloproteinase 2. Phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2) and of downstream signaling molecules, such as phosphatidylinositol-3-kinase and mitogen-activated protein kinases, is also impaired. Moreover, MDK downregulates VEGF-A-induced neovascularization and vascular permeability in vivo. We propose a model in which MDK is a new modulator of the VEGF-A-VEGFR-2 axis.