Alteration of cholesterol content and oxygen level in intestinal organoids after infection with Staphylococcus aureus.

The pathogenicity elicited by Staphylococcus (S.) aureus, one of the best-studied bacteria, in the intestine is not well understood. Recently, we demonstrated that S. aureus infection induces alterations in membrane composition that are associated with concomitant impairment of intestinal function. Here, we used two organoid models, induced pluripotent stem cell (iPSC)-derived intestinal organoids and colonic intestinal stem cell-derived intestinal organoids (colonoids), to examine how sterol metabolism and oxygen levels change in response to S. aureus infection. HPLC quantification showed differences in lipid homeostasis between infected and uninfected cells, characterized by a remarkable decrease in total cellular cholesterol. As the altered sterol metabolism is often due to oxidative stress response, we next examined intracellular and extracellular oxygen levels. Three different approaches to oxygen measurement were applied: (1) cell-penetrating nanoparticles to quantify intracellular oxygen content, (2) sensor plates to quantify extracellular oxygen content in the medium, and (3) a sensor foil system for oxygen distribution in organoid cultures. The data revealed significant intracellular and extracellular oxygen drop after infection in both intestinal organoid models as well as in Caco-2 cells, which even 48 h after elimination of extracellular bacteria, did not return to preinfection oxygen levels. In summary, we show alterations in sterol metabolism and intra- and extracellular hypoxia as a result of S. aureus infection. These results will help understand the cellular stress responses during sustained bacterial infections in the intestinal epithelium.

Possibilities and efficiency of MSC co-transfection for gene therapy.

Mesenchymal stem/stromal cells (MSCs) are not only capable of self-renewal, trans-differentiation, homing to damaged tissue sites and immunomodulation by secretion of trophic factors but are also easy to isolate and expand. Because of these characteristics, they are used in numerous clinical trials for cell therapy including immune and neurological disorders, diabetes, bone and cartilage diseases and myocardial infarction. However, not all trials have successful outcomes, due to unfavourable microenvironmental factors and the heterogenous nature of MSCs. Therefore, genetic manipulation of MSCs can increase their prospect. Currently, most studies focus on single transfection with one gene. Even though the introduction of more than one gene increases the complexity, it also increases the effectivity as different mechanism are triggered, leading to a synergistic effect. In this review we focus on the methodology and efficiency of co-transfection, as well as the opportunities and pitfalls of these genetically engineered cells for therapy.

The Dynamics of Circulating Heparin-Binding Protein: Implications for Its Use as a Biomarker.

Heparin-binding protein (HBP) is a promising biomarker for the development and severity of sepsis. To guide its use, it is important to understand the factors that could lead to false-positive or negative results, such as inappropriate release and inadequate clearance of HBP. HBP is presumably released only by neutrophils, and the organs responsible for its elimination are unknown. In this study, we aimed to determine whether non-neutrophil cells can be a source of circulating HBP and which organs are responsible for its removal. We found that in two cohorts of neutropenic patients, 12% and 19% of patients in each cohort, respectively, had detectable plasma HBP levels. In vitro, three leukemia-derived monocytic cell lines and healthy CD14+ monocytes constitutively released detectable levels of HBP. When HBP was injected intravenously in rats, we found that plasma levels of HBP decreased rapidly, with a distribution half-life below 10 min and an elimination half-life of 1-2 h. We measured HBP levels in the liver, spleen, kidneys, lungs, and urine using both ELISA and immunofluorescence quantitation, and found that the majority of HBP was present in the liver, and a small amount was present in the spleen. Immunofluorescence imaging indicated that HBP is associated mainly with hepatocytes in the liver and monocytes/macrophages in the spleen. The impact of hematologic malignancies and liver diseases on plasma HBP levels should be explored further in clinical studies.