Conversion of rutin, a prevalent dietary flavonol, by the human gut microbiota

The gut microbiota plays a pivotal role in the conversion of dietary flavonoids, which can affect their bioavailability and bioactivity and thereby their health-promoting properties. The ability of flavonoids to stimulate the activity of the microbiota has, however, not been systematically evaluated. In the present study, we used a fluorescence-based single-cell activity measure [biorthogonal non-canonical ammino acid-tagging (BONCAT)] combined with fluorescence-activated cell-sorting (FACS) to determine which microorganisms are stimulated by the flavonoid rutin. We performed anaerobic incubations of human fecal microbiota amended with rutin and in the presence of the cellular activity marker L-Azidohomoalanine (AHA) to detect rutin-stimulated cells. We found that 7% of cells in the gut microbiota were active after a 6 h incubation and 23% after 24 h. We then sorted BONCAT-positive cells and observed an enrichment of Lachnospiraceae (Lachnoclostridium, and Eisenbergiella), Enterobacteriaceae, Tannerellaceae and Erysipelotrichaceae species in the rutin-responsive fraction of the microbiota. There was marked inter-individual variability in the appearance of rutin conversion products after incubation with rutin. Consistent with this, there was substantial variability in the abundance of rutin-responsive microbiota among different individuals. Specifically, we observed that Enterobacteriaceae were associated with conversion of rutin into quercetin-3-glucoside and Lachnospiraceae were associated with quercetin production. This suggests that individual microbiotas differ in their ability to metabolize rutin and utilize different conversion pathways

Accelerated Tryptophan Degradation Predicts Poor Survival in Trauma and Sepsis Patients

Immune system activation and inflammation accompanies immune dysfunction in trauma and sepsis patients. Immunodeficiency may develop in such patients as one consequence of an activated chronic pro-inflammatory response. According to recent data, degradation of L-tryptophan (TRP) via the kynurenine (KYN) pathway by the cytokine-inducible enzyme indoleamine 2,3-dioxygenase (IDO) could represent an important contributor to the deficient responsiveness of immunocompetent cells. Compared to healthy controls, patients post trauma or with sepsis had increasing KYN concentrations and KYN to TRP ratios (KYN/TRP) whereas TRP concentrations decreased. Likewise, concentrations of cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and of immune activation marker neopterin increased in patients (all p < 0.001). Furthermore in patients KYN/TRP, KYN and neopterin concentrations were further increasing (all p < 0.001), whereas the changes of TRP, TNF-α and IL-6 concentrations were not significant. Compared to the survivors, the non-survivors had a higher concentration of KYN, neopterin, TNF-α and IL-6 as well as a higher KYN/TRP ratio. KYN/TRP correlated with neopterin (p < 0.001) and also with TNF-α (p < 0.01) and IL-6 concentrations (p < 0.05) and inversely with the in vitro response of stimulated monocytes. We conclude that increased TRP degradation in patients post trauma is closely associated with immune activation. Cytokines released during the pro-inflammatory response may induce the activity of IDO and thus accelerate TRP degradation. Thus, increased IDO activity most likely represents a result of host response to pro-inflammation in patients. Data support a possible role of inflammation-induced IDO in the diminished immunoresponsiveness in patients

Cord Blood Derived CD4+CD25high T Cells Become Functional Regulatory T Cells upon Antigen Encounter

Background: Upon antigen exposure, cord blood derived T cells respond to ubiquitous environmental antigens by high proliferation. To date it remains unclear whether these “excessive” responses relate to different regulatory properties of the putative T regulatory cell (Treg) compartment or even expansion of the Treg compartment itself

Differential Interaction of Platelet-Derived Extracellular Vesicles With Circulating Immune Cells: Roles of TAM Receptors, CD11b, and Phosphatidylserine

Secretion and exchange of biomolecules by extracellular vesicles (EVs) are crucial in intercellular communication and enable cells to adapt to alterations in their microenvironment. EVs are involved in a variety of cellular processes under physiological conditions as well as in pathological settings. In particular, they exert profound effects on the innate immune system, and thereby are also capable of modulating adaptive immunity. The mechanisms underlying their interaction with their recipient cells, particularly their preferential association with monocytes and granulocytes in the circulation, however, remain to be further clarified. Surface molecules exposed on EVs are likely to mediate immune recognition and EV uptake by their recipient cells. Here, we investigated the involvement of Tyro3, Axl, and Mer (TAM) tyrosine kinase receptors and of integrin CD11b in the binding of platelet-derived EVs, constituting the large majority of circulating EVs, to immune cells in the circulation. Flow cytometry and Western Blotting demonstrated a differential expression of TAM receptors and CD11b on monocytes, granulocytes, and lymphocytes, as well as on monocyte subsets. Of the TAM receptors, only Axl and Mer were detected at low levels on monocytes and granulocytes, but not on lymphocytes. Likewise, CD11b was present on circulating monocytes and granulocytes, but remained undetectable on lymphocytes. Differentiation of monocytes into classical, intermediate, and non-classical monocyte subsets revealed distinct expression patterns of Mer and activated CD11b. Co-incubation of isolated monocytes and granulocytes with platelet-derived EVs showed that the binding of EVs to immune cells was dependent on Ca++. Our data do not support a particular role for TAM receptors or for activated CD11b in the association of platelet-derived EVs with monocytes and granulocytes in the circulation, as anti-TAM antibodies did not interfere with EV binding to isolated immune cells, as binding was not dependent on the presence of TIM4 acting synergistically with TAM receptors, and as neither low levels of Gas6, required as a linker between phosphatidylserine (PS) on the EV surface and TAM receptors on immune cells, nor masking of PS on the EV surface did interfere with EV binding

Tissue-regenerative potential of the secretome of γ-irradiated peripheral blood mononuclear cells is mediated via TNFRSF1B-induced necroptosis.

Peripheral blood mononuclear cells (PBMCs) have been shown to produce and release a plethora of pro-angiogenetic factors in response to γ-irradiation, partially accounting for their tissue-regenerative capacity. Here, we investigated whether a certain cell subtype of PBMCs is responsible for this effect, and whether the type of cell death affects the pro-angiogenic potential of bioactive molecules released by γ-irradiated PBMCs. PBMCs and PBMC subpopulations, including CD4+ and CD8+ T cells, B cells, monocytes, and natural killer cells, were isolated and subjected to high-dose γ-irradiation. Transcriptome analysis revealed subpopulation-specific responses to γ-irradiation with distinct activation of pro-angiogenic pathways, cytokine production, and death receptor signalling. Analysis of the proteins released showed that interactions of the subsets are important for the generation of a pro-angiogenic secretome. This result was confirmed at the functional level by the finding that the secretome of γ-irradiated PBMCs displayed higher pro-angiogenic activity in an aortic ring assay. Scanning electron microscopy and image stream analysis of γ-irradiated PBMCs revealed distinct morphological changes, indicative for apoptotic and necroptotic cell death. While inhibition of apoptosis had no effect on the pro-angiogenic activity of the secretome, inhibiting necroptosis in stressed PBMCs abolished blood vessel sprouting. Mechanistically, we identified tumor necrosis factor (TNF) receptor superfamily member 1B as the main driver of necroptosis in response to γ-irradiation in PBMCs, which was most likely mediated via membrane-bound TNF-α. In conclusion, our study demonstrates that the pro-angiogenic activity of the secretome of γ-irradiated PBMCs requires interplay of different PBMC subpopulations. Furthermore, we show that TNF-dependent necroptosis is an indispensable molecular process for conferring tissue-regenerative activity and for the pro-angiogenic potential of the PBMC secretome. These findings contribute to a better understanding of secretome-based therapies in regenerative medicine

Immunomodulation to Prevent or Treat Neonatal Sepsis: Past, Present, and Future

Despite continued advances in neonatal medicine, sepsis remains a leading cause of death worldwide in neonatal intensive care units. The clinical presentation of sepsis in neonates varies markedly from that in older children and adults, and distinct acute inflammatory responses results in age-specific inflammatory and protective immune response to infection. This review first provides an overview of the neonatal immune system, then covers current mainstream, and experimental preventive and adjuvant therapies in neonatal sepsis. We also discuss how the distinct physiology of the perinatal period shapes early life immune responses and review strategies to reduce neonatal sepsis-related morbidity and mortality. A summary of studies that characterize immune ontogeny and neonatal sepsis is presented, followed by discussion of clinical trials assessing interventions such as breast milk, lactoferrin, probiotics, and pentoxifylline. Finally, we critically appraise future treatment options such as stem cell therapy, other antimicrobial protein and peptides, and targeting of pattern recognition receptors in an effort to prevent and/or treat sepsis in this highly vulnerable neonatal population

Influenza A Virus Induces an Immediate Cytotoxic Activity in All Major Subsets of Peripheral Blood Mononuclear Cells

A replication defective influenza A vaccine virus (delNS1 virus) was developed. Its attenuation is due to potent stimulation of the innate immune system by the virus. Since the innate immune system can also target cancer cells, we reasoned that delNS1 virus induced immune-stimulation should also lead to the induction of innate cytotoxic effects towards cancer cells.Peripheral blood mononuclear cells (PBMCs), isolated CD56+, CD3+, CD14+ and CD19+ subsets and different combinations of the above subsets were stimulated by delNS1, wild type (wt) virus or heat inactivated virus and co-cultured with tumor cell lines in the presence or absence of antibodies against the interferon system. Stimulation of PBMCs by the delNS1 virus effectively induced cytotoxicity against different cancer cell lines. Surprisingly, virus induced cytotoxicity was exerted by all major subtypes of PBMCs including CD56+, CD3+, CD14+ and CD19+ cells. Virus induced cytotoxicity in CD3+, CD14+ and CD19+ cells was dependent on virus replication, whereas virus induced cytotoxicity in CD56+ cells was only dependent on the binding of the virus. Virus induced cytotoxicity of isolated cell cultures of CD14+, CD19+ or CD56+ cells could be partially blocked by antibodies against type I and type II (IFN) interferon. In contrast, virus induced cytotoxicity in the complete PBMC preparation could not be inhibited by blocking type I or type II IFN, indicating a redundant system of activation in whole blood.Our data suggest that apart from their well known specialized functions all main subsets of peripheral blood cells also initially exert a cytotoxic effect upon virus stimulation. This closely links the innate immune system to the adaptive immune response and renders delNS1 virus a potential therapeutic tool for viro-immunotherapy of cancer

A worldwide survey on incidence, management and prognosis of oesophageal fistula formation following atrial fibrillation catheter ablation: The POTTER-AF study.

AIMS Oesophageal fistula represents a rare but dreadful complication of atrial fibrillation catheter ablation. Data on its incidence, management and outcome are sparse. METHODS AND RESULTS This international multicenter registry investigates the characteristics of oesophageal fistulae after treatment of atrial fibrillation by catheter ablation. A total of 553,729 catheter ablation procedures (radiofrequency: 62.9%, cryoballoon: 36.2%, other modalities: 0.9%) were performed at 214 centers in 35 countries. In 78 centers 138 patients (0.025%, radiofrequency: 0.038%, cryoballoon: 0.0015% (p<0.0001)) were diagnosed with an oesophageal fistula. Periprocedural data were available for 118 patients (85.5%). Following catheter ablation, the median time to symptoms and the median time to diagnosis were 18 (7.75, 25; range: 0-60) days and 21 (15, 29.5; range: 2-63) days, respectively. The median time from symptom onset to oesophageal fistula diagnosis was 3 (1, 9; range: 0-42) days. The most common initial symptom was fever (59.3%). The diagnosis was established by chest computed tomography in 80.2% of patients. Oesophageal surgery was performed in 47.4% and direct endoscopic treatment in 19.8%, and conservative treatment in 32.8% of patients. The overall mortality was 65.8%. Mortality following surgical (51.9%) or endoscopic treatment (56.5%) was significantly lower as compared to conservative management (89.5%) (odds ratio 7.463 (2.414, 23.072) p<0.001). CONCLUSIONS Oesophageal fistula after catheter ablation of atrial fibrillation is rare and occurs mostly with the use of radiofrequency energy rather than cryoenergy. Mortality without surgical or endoscopic intervention is exceedingly high

Extracellular Vesicles in Autologous Cell Salvaged Blood in Orthopedic Surgery

(1) Background: Cell salvage is highly recommended in orthopedic surgery to avoid allogeneic transfusions. Preparational steps during cell salvage may induce extracellular vesicle (EV) formation with potential thrombogenic activity. The purpose of our study was to assess the appearance of EVs at retransfusion. (2) Methods: After ethics committee approval and informed consent, blood was withdrawn from the autotransfusion system (Xtra, Sorin, Germany) of 23 patients undergoing joint arthroplasty. EVs were assessed by flow cytometry in two times centrifugated samples. EVs were stained with specific antibodies against cellular origins from platelets (CD41), myeloid cells (CD15), monocytes (CD14), and erythrocytes (CD235a). The measured events/µL in the flow cytometer were corrected to the number of EVs in the retransfusate. (3) Results: We measured low event rates of EVs from platelets and myeloid origin (<1 event/µL) and from monocytic origin (<2 events/µL). Mean event rates of 17,042 events/µL (range 12–81,164 events/µL) were found for EVs from red blood cells. (4) Conclusion: Retransfusate contains negligible amounts of potentially thrombogenic EVs from platelet and monocytic origin. Frequent EVs from erythrocytes may indicate red blood cell destruction and/or activation during autologous cell salvage. Further research is needed to investigate the clinical relevance of EVs from salvaged red blood cells