DataSheet1_Primary sarcopenia is associated with elevated spontaneous NET formation.pdf

Introduction: Sarcopenia is a frequent complication of liver cirrhosis, but it can also occur independently as a result of any underlying cause. The immune system plays an important role in the pathogenesis of both liver cirrhosis and sarcopenia. Neutrophil function, including neutrophil extracellular trap (NET) formation, is linked to chronic inflammation; however, it has not been extensively studied in patients with sarcopenia. Here, we aim to study if main neutrophil functions, such as phagocytosis, reactive oxygen species (ROS) production, and NET formation, are altered in patients with sarcopenia with or without liver cirrhosis.Methods: Neutrophils from 92 patients (52 patients with liver cirrhosis and sarcopenia, 25 patients with liver cirrhosis without sarcopenia, and 15 patients with sarcopenia without liver cirrhosis) and 10 healthy controls were isolated and stimulated with heat-inactivated E. coli (250 bacteria/cell), phorbol 12-myristate 13-acetate (PMA) (100 nM), or incubation medium in duplicates for 2 h at 37°C. Cells were fixed with paraformaldehyde and stained with 4′,6-diamidino-2-phenylindole (DAPI). Pictures of 10 random fields of vision per slide were taken with an Olympus BX51 fluorescence microscope (Olympus, Shinjuku, Tokyo, Japan) at 600x total magnification. The DNA Area and NETosis Analysis (DANA) algorithm was used to quantify the percentage of NET formation per patient. Phagocytosis and ROS production were assessed with the PhagotestTM kit and PhagoburstTM kit (Glycotope, Heidelberg, Germany) in 92 patients and 21 healthy controls, respectively.Results: Spontaneous NET formation was significantly elevated in patients with only sarcopenia compared to patients with cirrhosis and sarcopenia (p = 0.008) and healthy controls (p = 0.039). NET formation in response to PMA was significantly decreased in patients with cirrhosis (p = 0.007), cirrhosis and sarcopenia (p Discussion: Spontaneous NET formation might contribute to chronic inflammation and sarcopenia pathogenesis. This, however, does not result in the impairment of the NET formation function of neutrophils in response to a bacterial stimulus and, therefore, cannot be not linked with the increased risk of bacterial infections neither in sarcopenia nor in cirrhosis. The semi-automated NET formation analysis can be successfully implemented to analyze the vast amount of data generated within clinical studies. This approach opens up the possibilities to develop an NET formation-based biomarker in different diseases including sarcopenia and implement NET formation analysis into clinical settings. Phagocytosis and ROS production were not affected in patients with sarcopenia. Further research is needed to explore the mechanism of NET formation in patients with sarcopenia and its potential as a biomarker in sarcopenia.</p

Alterations in Gut Microbiome Composition and Barrier Function Are Associated with Reproductive and Metabolic Defects in Women with Polycystic Ovary Syndrome (PCOS): A Pilot Study

<div><p>Background</p><p>Polycystic ovary syndrome (PCOS) is a common female endocrinopathy of unclear origin characterized by hyperandrogenism, oligo-/anovulation, and ovarian cysts. Women with PCOS frequently display overweight, insulin resistance, and systemic low-grade inflammation. We hypothesized that endotoxemia resulting from a leaky gut is associated with inflammation, insulin resistance, fat accumulation, and hyperandrogenemia in PCOS. In this pilot study, we compared the stool microbiome, gut permeability, and inflammatory status of women with PCOS and healthy controls.</p><p>Methods</p><p>16S rRNA gene amplicon sequencing was performed on stool samples from 24 PCOS patients and 19 healthy controls. Data processing and microbiome analysis were conducted in mothur and QIIME using different relative abundance cut-offs. Gut barrier integrity, endotoxemia, and inflammatory status were evaluated using serum and stool markers and associations with reproductive, metabolic, and anthropometric parameters were investigated.</p><p>Results</p><p>The stool microbiome of PCOS patients showed a lower diversity and an altered phylogenetic composition compared to controls. We did not observe significant differences in any taxa with a relative abundance>1%. When looking at rare taxa, the relative abundance of bacteria from the phylum Tenericutes, the order ML615J-28 (phylum Tenericutes) and the family S24-7 (phylum Bacteroidetes) was significantly lower and associated with reproductive parameters in PCOS patients. Patients showed alterations in some, but not all markers of gut barrier function and endotoxemia.</p><p>Conclusion</p><p>Patients with PCOS have a lower diversity and an altered phylogenetic profile in their stool microbiome, which is associated with clinical parameters. Gut barrier dysfunction and endotoxemia were not driving factors in this patient cohort, but may contribute to the clinical phenotype in certain PCOS patients.</p></div

PCOS diagnostic criteria and phenotypes.

<p>Serum total testosterone (A) and DHEAS (B) levels in PCOS patients and controls. C. Prevalence of hirsutism, OA, and PCOM in PCOS patients and controls. D. Prevalence of phenotypes within PCOS patients according to the Rotterdam Criteria. PCOM: polycystic ovarian morphology, OA: oligo-/amenorrhoea, HA: hyperandrogenism (clinical and/or biochemical).</p

Alpha rarefaction curves of stool samples from PCOS patients and controls.

<p>Faith's phylogenetic diversity (A) and the number of observed OTUs (B). All samples were rarefied to the smallest observed number of reads (24,991). Median and IQR are plotted.</p

Principal coordinate analysis (PCoA) plots of stool samples from PCOS patients and controls.

<p>PCoA plots of weighted (A) and unweighted (B) UniFrac distance matrices. Each dot represents the bacterial community composition of one individual stool sample. Axis titles indicate the percentage variation explained.</p

Association of differentially abundant taxa with bacterial diversity and reproductive parameters.

<p>The number of samples with detectable levels of the relevant taxa (read count ≥10) was 12/43 for Tenericutes, 5/43 for ML615J-28, and 10/43 for S24-7. Median and IQR are plotted for continuous variables.</p

Study subject characteristics.

<p>Study subject characteristics.</p

Association of microbiome parameters with parameters of gut barrier integrity and inflammation.

<p>LPS: lipopolysaccharide (= endotoxin), hsCRP: high-sensitivity C-reactive protein. Dots represent individual study subjects. Median and IQR are plotted. The number of samples with detectable levels of the relevant taxa (read count ≥10) was 12/43 for Tenericutes, 5/43 for ML615J-28, and 10/43 for S24-7.</p

Relative abundances of bacterial genera and phyla that differed significantly between PCOS patients and controls.

<p>Relative abundances of bacterial genera and phyla that differed significantly between PCOS patients and controls.</p

Markers of gut barrier integrity and inflammation in PCOS patients and controls.

<p>DAO: diamine oxidase, sCD14: soluble CD14 (endotoxin co-receptor), LBP: lipopolysaccharide binding protein, LPS: lipopolysaccharide (= endotoxin), hsCRP: high-sensitivity C-reactive protein. Dots represent individual study subjects. Bars represent median and IQR.</p