Comparison of the Skin Microbiota in the Periocular Region between Patients with Inflammatory Skin Diseases and Healthy Participants: A Preliminary Study.

(1) Background: Periocular or periorbital dermatitis is a common term for all inflammatory skin diseases affecting the area of skin around the eyes. The clear etiopathogenesis of periocular dermatitis is still not fully understood. Advances in molecular techniques for studying microorganisms living in and on our bodies have highlighted the microbiome as a possible contributor to disease, as well as a promising diagnostic marker and target for innovative treatments. The aim of this study was to compare the composition and diversity of the skin microbiota in the periocular region between healthy individuals and individuals affected by the specific entity of periocular dermatitis. (2) Methods: A total of 35 patients with periocular dermatitis and 39 healthy controls were enrolled in the study. After a skin swab from the periocular region was taken from all participants, DNA extraction and 16S rRNA gene amplicon sequencing using Illumina NovaSeq technology were performed. (3) Results: Staphylococcus and Corynebacterium were the most abundant bacterial genera in the microbiota of healthy skin. Analysis of alpha diversity revealed a statistically significant change (p < 0.05) in biodiversity based on the Faith's PD index between patients and healthy individuals. We did not observe changes in beta diversity. The linear discriminant analysis effect size (LEfSe) revealed that Rothia, Corynebacterium, Bartonella, and Paracoccus were enriched in patients, and Anaerococcus, Bacteroides, Porphyromonas, and Enhydrobacter were enriched in healthy controls. (4) Conclusions: According to the results obtained, we assume that the observed changes in the bacterial microbiota on the skin, particularly Gram-positive anaerobic cocci and skin commensals of the genus Corynebacterium, could be one of the factors in the pathogenesis of the investigated inflammatory diseases. The identified differences in the microbiota between healthy individuals and patients with periocular dermatitis should be further investigated.

Correction: Fibromyalgia-associated hyperalgesia is related to psychopathological alterations but not to gut microbiome changes.

[This corrects the article DOI: 10.1371/journal.pone.0274026.].

Modulation of the skin microbiome in cutaneous T-cell lymphoma delays tumour growth and increases survival in the murine EL4 model.

Cutaneous T-cell lymphomas (CTCL) are a group of lymphoproliferative disorders of skin-homing T cells causing chronic inflammation. These disorders cause impairment of the immune environment, which leads to severe infections and/or sepsis due to dysbiosis. In this study, we elucidated the host-microbial interaction in CTCL that occurs during the phototherapeutic treatment regime and determined whether modulation of the skin microbiota could beneficially affect the course of CTCL. EL4 T-cell lymphoma cells were intradermally grafted on the back of C57BL/6 mice. Animals were treated with conventional therapeutics such as psoralen + UVA (PUVA) or UVB in the presence or absence of topical antibiotic treatment (neomycin, bacitracin, and polymyxin B sulphate) as an adjuvant. Microbial colonisation of the skin was assessed to correlate with disease severity and tumour growth. Triple antibiotic treatment significantly delayed tumour occurrence (p = 0.026), which prolonged the survival of the mice (p = 0.033). Allocation to phototherapeutic agents PUVA, UVB, or none of these, along with antibiotic intervention, reduced the tumour growth significantly (p = 0.0327, p ≤ 0.0001, p ≤ 0.0001 respectively). The beta diversity indices calculated using the Bray-Curtis model showed that the microbial population significantly differed after antibiotic treatment (p = 0.001). Upon modulating the skin microbiome by antibiotic treatment, we saw an increase in commensal Clostridium species, e.g., Lachnospiraceae sp. (p = 0.0008), Ruminococcaceae sp. (p = 0.0001)., Blautia sp. (p = 0.007) and a significant reduction in facultative pathogens Corynebacterium sp. (p = 0.0009), Pelomonas sp. (p = 0.0306), Streptococcus sp. (p ≥ 0.0001), Pseudomonas sp. (p = 0.0358), and Cutibacterium sp. (p = 0.0237). Intriguingly, we observed a significant decrease in Staphylococcus aureus frequency (p = 0.0001) but an increase in the overall detection frequency of the Staphylococcus genus, indicating that antibiotic treatment helped regain the microbial balance and increased the number of non-pathogenic Staphylococcus populations. These study findings show that modulating microbiota by topical antibiotic treatment helps to restore microbial balance by diminishing the numbers of pathogenic microbes, which, in turn, reduces chronic inflammation, delays tumour growth, and increases survival rates in our CTCL model. These findings support the rationale to modulate the microbial milieu during the disease course of CTCL and indicate its therapeutic potential.

Human Milk Oligosaccharides in Maternal Serum Respond to Oral Glucose Load and Are Associated with Insulin Sensitivity.

(1) Background: Pregnancy presents a challenge to maternal glucose homeostasis; suboptimal adaptations can lead to gestational diabetes mellitus (GDM). Human milk oligosaccharides (HMOs) circulate in maternal blood in pregnancy and are altered with GDM, suggesting influence of glucose homeostasis on HMOs. We thus assessed the HMO response to glucose load during an oral glucose tolerance test (OGTT) and investigated HMO associations with glucose tolerance/insulin sensitivity in healthy pregnant women. (2) Methods: Serum of 99 women, collected at 0 h, 1 h and 2 h during a 75 g OGTT at 24-28 gestational weeks was analyzed for HMOs (2'FL, 3'SLN, LDFT, 3'SL) by HPLC; plasma glucose, insulin and C-peptide were analyzed by standard biochemistry methods. (3) Results: Serum 3'SL concentrations significantly increased from fasting to 1 h after glucose load, while concentrations of the other HMOs were unaltered. Higher 3'SL at all OGTT time points was associated with a generally more diabetogenic profile, with higher hepatic insulin resistance (HOMA-IR), lower insulin sensitivity (Matsuda index) and higher insulin secretion (C-peptide index 1). (4) Conclusions: Rapid increase in serum 3'SL post-oral glucose load (fasted-fed transition) indicates utilization of plasma glucose, potentially for sialylation of lactose. Associations of sialylated HMOs with a more diabetogenic profile suggest sustained adaptations to impaired glucose homeostasis in pregnancy. Underlying mechanisms or potential consequences of observed HMO changes remain to be elucidated.

Effect of Antibiotic Eye Drops on the Nasal Microbiome in Healthy Subjects-A Pilot Study.

BACKGROUND: Antibiotic eye drops are frequently used in clinical practice. Due to the anatomical connection via the nasolacrimal duct, it seems possible that they have an influence on the nasal/pharyngeal microbiome. This was investigated by using two different commonly used antibiotic eye drops. METHODS: 20 subjects were randomized to four groups of five subjects receiving eye drops containing gentamicin, ciprofloxacin, or, as controls, unpreserved povidone or benzalkonium chloride-preserved povidone. Nasal and pharyngeal swabs were performed before and after the instillation period. Swabs were analyzed by Illumina next-generation sequencing (NGS)-based 16S rRNA analysis. Bacterial culture was performed on solid media, and bacterial isolates were identified to the species level by MALDI-TOF MS. Species-dependent antimicrobial susceptibility testing was performed using single isolates and pools of isolates. RESULTS: Bacterial richness in the nose increased numerically from 163 ± 30 to 243 ± 100 OTUs (gentamicin) and from 114 ± 17 to 144 ± 45 OTUs (ciprofloxacin). Phylogenetic diversity index (pd) of different bacterial strains in the nasal microbiome increased from 12.4 ± 1.0 to 16.9 ± 5.6 pd (gentamicin) and from 10.2 ± 1.4 to 11.8 ± 3.1 pd (ciprofloxacin). Unpreserved povidone eye drops resulted in minimal changes in bacterial counts. Preservative-containing povidone eye drops resulted in no change. A minor increase (1-2-fold) in the minimal inhibitory concentration (MIC) was observed in single streptococcal isolates. CONCLUSIONS: Antibiotic eye drops could affect the nasal microbiome. After an instillation period of seven days, an increase in the diversity and richness of bacterial strains in the nasal microbiome was observed.

Human gene-engineered calreticulin mutant stem cells recapitulate MPN hallmarks and identify targetable vulnerabilities.

Calreticulin (CALR) mutations present the main oncogenic drivers in JAK2 wildtype (WT) myeloproliferative neoplasms (MPN), including essential thrombocythemia and myelofibrosis, where mutant (MUT) CALR is increasingly recognized as a suitable mutation-specific drug target. However, our current understanding of its mechanism-of-action is derived from mouse models or immortalized cell lines, where cross-species differences, ectopic over-expression and lack of disease penetrance are hampering translational research. Here, we describe the first human gene-engineered model of CALR MUT MPN using a CRISPR/Cas9 and adeno-associated viral vector-mediated knock-in strategy in primary human hematopoietic stem and progenitor cells (HSPCs) to establish a reproducible and trackable phenotype in vitro and in xenografted mice. Our humanized model recapitulates many disease hallmarks: thrombopoietin-independent megakaryopoiesis, myeloid-lineage skewing, splenomegaly, bone marrow fibrosis, and expansion of megakaryocyte-primed CD41+ progenitors. Strikingly, introduction of CALR mutations enforced early reprogramming of human HSPCs and the induction of an endoplasmic reticulum stress response. The observed compensatory upregulation of chaperones revealed novel mutation-specific vulnerabilities with preferential sensitivity of CALR mutant cells to inhibition of the BiP chaperone and the proteasome. Overall, our humanized model improves purely murine models and provides a readily usable basis for testing of novel therapeutic strategies in a human setting.

Fibromyalgia-associated hyperalgesia is related to psychopathological alterations but not to gut microbiome changes.

Fibromyalgia-syndrome (FMS) is a complex disease characterized by chronic widespread pain and additional symptoms including depression, cognitive dysfunction ("fibro-fog") and maldigestion. Our research team examined whether FMS-related pain parameters assessed by quantitative sensory testing (QST) and psychological disturbances are accompanied by alterations of the fecal microbiome. We recruited 25 patients with FMS and 26 age- and sex-matched healthy controls. Medical background, food habits, psychopathology and quality of life were assessed through questionnaires. Stool samples were analyzed by 16S rRNA gene amplification and sequencing. QST was performed according to the protocol of the German Network for Neuropathic Pain. QST showed that both lemniscal and spinothalamic afferent pathways are altered in FMS patients relative to healthy controls and that peripheral as well as central pain sensitization processes are manifest. Psychometric assessment revealed enhanced scores of depression, anxiety and stress. In contrast, neither the composition nor the alpha- and beta-diversity of the fecal microbiome was changed in FMS patients. FMS patients segregate from healthy controls in various parameters of QST and psychopathology, but not in terms of composition and diversity of the fecal microbiome. Despite consideration of several confounding factors, we conclude that the contribution of the gut microbiome to the pathophysiology of FMS is limited.

Transcriptomic underpinnings of high and low mirror aggression zebrafish behaviours.

BACKGROUND: Aggression is an adaptive behaviour that animals use to protect offspring, defend themselves and obtain resources. Zebrafish, like many other animals, are not able to recognize themselves in the mirror and typically respond to their own reflection with aggression. However, mirror aggression is not an all-or-nothing phenomenon, with some individuals displaying high levels of aggression against their mirror image, while others show none at all. In the current work, we have investigated the genetic basis of mirror aggression by using a classic forward genetics approach - selective breeding for high and low mirror aggression zebrafish (HAZ and LAZ). RESULTS: We characterized AB wild-type zebrafish for their response to the mirror image. Both aggressive and non-aggressive fish were inbred over several generations. We found that HAZ were on average more aggressive than the corresponding LAZ across generations and that the most aggressive adult HAZ were less anxious than the least aggressive adult LAZ after prolonged selective breeding. RNAseq analysis of these fish revealed that hundreds of protein-encoding genes with important diverse biological functions such as arsenic metabolism (as3mt), cell migration (arl4ab), immune system activity (ptgr1), actin cytoskeletal remodelling (wdr1), corticogenesis (dgcr2), protein dephosphorylation (ublcp1), sialic acid metabolism (st6galnac3) and ketone body metabolism (aacs) were differentially expressed between HAZ and LAZ, suggesting a strong genetic contribution to this phenotype. DAVID pathway analysis showed that a number of diverse pathways are enriched in HAZ over LAZ including pathways related to immune function, oxidation-reduction processes and cell signalling. In addition, weighted gene co-expression network analysis (WGCNA) identified 12 modules of highly correlated genes that were significantly associated with aggression duration and/or experimental group. CONCLUSIONS: The current study shows that selective breeding based of the mirror aggression phenotype induces strong, heritable changes in behaviour and gene expression within the brain of zebrafish suggesting a strong genetic basis for this behaviour. Our transcriptomic analysis of fish selectively bred for high and low levels of mirror aggression revealed specific transcriptomic signatures induced by selective breeding and mirror aggression and thus provides a large and novel resource of candidate genes for future study.

Insights into the Composition of a Co-Culture of 10 Probiotic Strains (OMNi BiOTiC® AAD10) and Effects of Its Postbiotic Culture Supernatant.

BACKGROUND: We aimed to gain insights in a co-culture of 10 bacteria and their postbiotic supernatant. METHODS: Abundances and gene expression were monitored by shotgun analysis. The supernatant was characterized by liquid chromatography mass spectroscopy (LC-MS) and gas chromatography mass spectroscopy (GC-MS). Supernatant was harvested after 48 h (S48) and 196 h (S196). Susceptibility testing included nine bacteria and C. albicans. Bagg albino (BALBc) mice were fed with supernatant or culture medium. Fecal samples were obtained for 16S analysis. RESULTS: A time-dependent decrease of the relative abundances and gene expression of L. salivarius, L. paracasei, E. faecium and B. longum/lactis and an increase of L. plantarum were observed. Substances in LC-MS were predominantly allocated to groups amino acids/peptides/metabolites and nucleotides/metabolites, relating to gene expression. Fumaric, panthotenic, 9,3-methyl-2-oxovaleric, malic and aspartic acid, cytidine monophosphate, orotidine, phosphoserine, creatine, tryptophan correlated to culture time. Supernatant had no effect against anaerobic bacteria. S48 was reactive against S. epidermidis, L. monocytogenes, P. aeruginosae, E. faecium and C. albicans. S196 against S. epidermidis and Str. agalactiae. In vivo S48/S196 had no effect on alpha/beta diversity. Linear discriminant analysis effect size (LEfSe) and analysis of composition of microbiomes (ANCOM) revealed an increase of Anaeroplasma and Faecalibacterium prausnitzii. CONCLUSIONS: The postbiotic supernatant had positive antibacterial and antifungal effects in vitro and promoted the growth of distinct bacteria in vivo.

Free threonine in human breast milk is related to infant intestinal microbiota composition.

BACKGROUND: Accumulating evidence indicates that free amino acids (FAA) might be bioactive compounds with potential immunomodulatory capabilities. However, the FAA composition in human milk is still poorly characterized with respect to its correlation to maternal serum levels and its physiological significance for the infant. Studies addressing the relation of human milk FAA to the infants' intestinal microbiota are still missing. METHODS: As part of a pilot study, maternal serum and breast milk FAA concentrations as well as infant intestinal microbiota (16S rRNA) were determined 2 months after birth. The study cohort consisted of 41 healthy mothers and their term delivered, healthy infants with normal birthweight. The relationship between maternal serum and milk FAA was determined by correlation analyses. Associations between (highly correlated) milk FAA and infant intestinal beta diversity were tested using PERMANOVA, LefSe and multivariate regression models adjusted for common confounders. RESULTS: Seven breast milk FAA correlated significantly with serum concentrations. One of these, threonine showed a negative association with abundance of members of the class Gammaproteobacteria (R2adj = 17.1%, p = 0.006; ß= - 0.441). In addition, on the level of families and genera, threonine explained 23.2% of variation of the relative abundance of Enterobacteriaceae (R2adj; p = 0.001; ß = - 0.504) and 11.1% of variability in the abundance of Escherichia/Shigella (R2adj, p = 0.025; ß = - 0.368), when adjusted for confounders. CONCLUSION: Our study is the first to suggest potential interactions between breast milk FAA and infant gut microbiota composition during early lactation. The results might be indicative of a potential protective role of threonine against members of the Enterobacteriaceae family in breast-fed infants. Still, results are based on correlation analyses and larger cohorts are needed to support the findings and elucidate possible underlying mechanisms to assess the complex interplay between breast milk FAA and infant intestinal microbiota in detail.

Colonic Medium-Chain Fatty Acids Act as a Source of Energy and for Colon Maintenance but Are Not Utilized to Acylate Ghrelin.

The capacity of microbiota to produce medium-chain fatty acids (MCFA) and related consequences for the gastrointestinal (GI) tract have never been reported before. We verified the impact of nutrition-related factors on fatty acid (FAs) production and found that caloric restriction decreased levels of most of MCFAs in the mouse cecum, whereas overnight fasting reduced the levels of acetate and butyrate but increased propionate and laurate. A diet high in soluble fibre boosted the production of short-chain fatty acids (SCFA) and caproate whereas a high-cellulose diet did not have an effect or decreased the levels of some of the FAs. Rectal infusion of caprylate resulted in its rapid metabolism for energy production. Repeated 10-day MCFA infusion impacted epididymal white adipose tissue (eWAT) weight and lipid accumulation. Repeated infusion of caprylate rectally tended to increase the concentration of active ghrelin in mice plasma; however, this increase was not statistically significant. In Caco-2 cells, caprylate increased the expression of Fabp2, Pdk4, Tlr3, and Gpr40 genes as well as counteracted TNFα-triggered downregulation of Pparγ, Occludin, and Zonulin mRNA expression. In conclusion, we show that colonic MCFAs can be rapidly utilized as a source of energy or stored as a lipid supply. Further, locally produced caprylate may impact metabolism and inflammatory parameters in the colon.

Microbial contribution to the caloric restriction-triggered regulation of the intestinal levels of glutathione transferases, taurine, and bile acid.

Recently we showed that caloric restriction (CR) triggers an increase in the levels of free taurine, taurine-conjugated bile acids (BA), and other taurine conjugates in intestinal mucosa while decreasing glutathione (GSH) levels in wild-type male mice. In the current project, we decided to investigate whether the microbiota is involved in the response to CR by depleting gut bacteria. The antibiotics treatment diminished CR-specific increase in the levels of free taurine and its conjugates as well as upregulated expression and activity of GSH transferases (GST) in the intestinal mucosa. Further, it diminished a CR-related increase in BAs levels in the liver, plasma, and intestinal mucosa. Transplant of microbiota from CR mice to ad libitum fed mice triggered CR-like changes in MGST1 expression, levels of taurine and taurine conjugates in the mucosa of the ileum. We show for the first time, that microbiota contributes to the intestinal response to CR-triggered changes in BA, taurine, and GST levels.

Land use in urban areas impacts the composition of soil bacterial communities involved in nitrogen cycling. A case study from Lefkosia (Nicosia) Cyprus.

The different types of land-use and soil lithology in urban and peri-urban areas of modern cities compose a complex mosaic of soil ecosystems. It is largely unknown how these differences result in changes in bacterial community composition and structure as well as in functional guilds involved in N cycling. To investigate the bacterial composition and the proportion of denitrifiers in agricultural, forested, schoolyard and industrial areas, 24 samples were collected from urban and peri-urban sites of Lefkosia. Bacterial diversity and the proportion of denitrifiers were assessed by NGS and qPCR, respectively. Proteobacteria, Actinobacteria, Bacteriodetes, Chloroflexi, Acidobacteria and Planctomycetes were identified as the most dominant phyla across all sites, while agricultural sites exhibited the highest bacterial diversity. Heavy metals such as Co, Pb, V and Al were identified as key factors shaping bacterial composition in industrial and schoolyard sites, while the bacterial assemblages in agricultural and forested sites were associated with Ca. Variance partitioning analysis showed that 10.2% of the bacterial community variation was explained by land use management, 5.1% by chemical elements due to soil lithology, and 1.4% by sampling location. The proportion of denitrifiers varied with land use management. In industrial and schoolyard sites, the abundance of the nosZII bacterial community increased while nirK abundance declined. Our data showed that land use and lithology have a moderate impact on the bacterial assemblages in urban and peri-urban areas of Lefkosia. As the nosZII bacterial community is important to the N2O sink capacity of soils, it would be interesting to elucidate the factors contributing to the proliferation of the nosZII clade in these soils.

Characterization of the Luminal and Mucosa-Associated Microbiome along the Gastrointestinal Tract: Results from Surgically Treated Preterm Infants and a Murine Model.

Environmental factors, including nutritional habits or birth mode, are known key determinants for intestinal microbial composition. Investigations of the intestinal microbiome in different species in a multiplicity of studies during recent decades have revealed differential microbial patterns and quantities along the gastrointestinal (GI) tract. Characterization of the microbial pattern in various aspects is a prerequisite for nutritional interventions. In this 16S rRNA amplicon-based approach, we present a characterization of the mucosa-associated microbiome in comparison with the luminal community of four infants at the time of the closure of ileostomies and perform a systematic characterization of the corresponding luminal and mucosal microbiome from jejunal, ileal and colonic regions, as well as collected feces in mice. The most dominant taxa in infant-derived samples altered due to individual differences, and in the mucosa, Enterococcus, Clostridiumsensustricto1, Veillonella, Streptococcus and Staphylococcus were the most abundant. Two less abundant taxa differed significantly between the mucosa and lumen. In murine samples, relative abundances differed significantly, mainly between the intestinal regions. Significant differences between mouse mucosa- and lumen-derived samples could be found in the observed species with a trend to lower estimated diversity in mucosa-derived samples, as well as in the relative abundance of individual taxa. In this study, we examined the difference between the mucosal and luminal bacterial colonization of the gastrointestinal tract in a small sample cohort of preterm infants. Individual differences were characterized and statistical significance was reached in two taxa (Cupriavidus, Ralstonia). The corresponding study on the different murine intestinal regions along the GI tract showed differences all over the intestinal region.

Patients with coronary heart disease, dilated cardiomyopathy and idiopathic ventricular tachycardia share overlapping patterns of pathogenic variation in cardiac risk genes.

BACKGROUND: Ventricular tachycardia (VT) is a major cause of sudden cardiac death (SCD). Clinical investigations can sometimes fail to identify the underlying cause of VT and the event is classified as idiopathic (iVT). VT contributes significantly to the morbidity and mortality in patients with coronary artery disease (CAD) and dilated cardiomyopathy (DCM). Since mutations in arrhythmia-associated genes frequently determine arrhythmia susceptibility screening for disease-predisposing variants could improve VT diagnostics and prevent SCD in patients. METHODS: Ninety-two patients diagnosed with coronary heart disease (CHD), DCM, or iVT were included in our study. We evaluated genetic profiles and variants in known cardiac risk genes by targeted next generation sequencing (NGS) using a newly designed custom panel of 96 genes. We hypothesized that shared morphological and phenotypical features among these subgroups may have an overlapping molecular base. To our knowledge, this was the first study of the deep sequencing of 96 targeted cardiac genes in Kazakhstan. The clinical significance of the sequence variants was interpreted according to the guidelines developed by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) in 2015. The ClinVar and Varsome databases were used to determine the variant classifications. RESULTS: Targeted sequencing and stepwise filtering of the annotated variants identified a total of 307 unique variants in 74 genes, totally 456 variants in the overall study group. We found 168 mutations listed in the Human Genome Mutation Database (HGMD) and another 256 rare/unique variants with elevated pathogenic potential. There was a predominance of high- to intermediate pathogenicity variants in LAMA2, MYBPC3, MYH6, KCNQ1, GAA, and DSG2 in CHD VT patients. Similar frequencies were observed in DCM VT, and iVT patients, pointing to a common molecular disease association. TTN, GAA, LAMA2, and MYBPC3 contained the most variants in the three subgroups which confirm the impact of these genes in the complex pathogenesis of cardiomyopathies and VT. The classification of 307 variants according to ACMG guidelines showed that nine (2.9%) variants could be classified as pathogenic, nine (2.9%) were likely pathogenic, 98 (31.9%) were of uncertain significance, 73 (23.8%) were likely benign, and 118 (38.4%) were benign. CHD VT patients carry rare genetic variants with increased pathogenic potential at a comparable frequency to DCM VT and iVT patients in genes related to sarcomere function, nuclear function, ion flux, and metabolism. CONCLUSIONS: In this study we showed that in patients with VT secondary to coronary artery disease, DCM, or idiopathic etiology multiple rare mutations and clinically significant sequence variants in classic cardiac risk genes associated with cardiac channelopathies and cardiomyopathies were found in a similar pattern and at a comparable frequency.

Activation of nuclear factor-kappa B subunits c-Rel, p65 and p50 by plasma lipids and fatty acids across the menstrual cycle.

This study focused on a comprehensive analysis of the canonical activation pathway of the redox-sensitive transcription factor nuclear factor-kappa B (NF-κB) in peripheral blood mononuclear cells, addressing c-Rel, p65 and p50 activation in 28 women at early (T1) and late follicular (T2) and mid (T3) and late luteal (T4) phase of the menstrual cycle, and possible relations with fasting plasma lipids and fatty acids. For the first time, strong inverse relations of c-Rel with apolipoprotein B were observed across the cycle, while those with LDL cholesterol, triglycerides as well as saturated (SFA), particularly C14-C22 SFA, monounsaturated (MUFA), and polyunsaturated fatty acids (PUFA) clustered at T2. In contrast, p65 was positively related to LDL cholesterol and total n-6 PUFA, while p50 did not show any relations. C-Rel was not directly associated with estradiol and progesterone, but data suggested an indirect C22:5n-3-mediated effect of progesterone. Strong positive relations between estradiol and individual SFA, MUFA and n-3 PUFA at T1 were confined to C18 fatty acids; C18:3n-3 was differentially associated with estradiol (positively) and progesterone (inversely). Given specific roles of c-Rel activation in immune tolerance, inhibition of c-Rel activation by higher plasma apolipoprotein B and individual fatty acid concentrations could have clinical implications for female fertility.

Effect of a Multispecies Probiotic on Intestinal and Skin Colonization by Multidrug-Resistant Gram-Negative Bacteria in Patients in a Long-Term Care Facility: A Pilot Study.

Residents in long-term care facilities (LTCFs) are frequently colonized by multidrug-resistant Gram-negative bacteria, putting them at risk for subsequent infections. We aimed to evaluate the effect of the multispecies probiotic Omnibiotic10AAD® on the intestinal and inguinal skin colonization of patients by multidrug-resistant Gram-negative bacteria in LTCFs. Patients colonized by multidrug-resistant Gram-negative bacteria received a 12 week oral course of Omnibiotic10AAD®. Inguinal swabs and stool samples were collected during and after treatment for microbiological and microbiome analysis. The median age of patients was 76 years. Twelve patients completed the pilot study. Intestinal colonization was reduced to 42% of patients 8 weeks after the end of treatment, but increased to 66% 24 weeks after the end of probiotic treatment. Colonization of inguinal skin was lowest during probiotic treatment and increased thereafter. Fecal microbiome analysis revealed statistically significant increases of the genus Enterococcus comparing start and end of probiotic treatment. In conclusion, a 12 week course of a multispecies probiotic led to a transient reduction of intestinal colonization 8 weeks after the end of treatment. The findings of our pilot study warrant further research in the area of probiotics and intestinal colonization by multidrug-resistant bacteria.

Effect of Low-Dose Aspirin on Soluble FMS-Like Tyrosine Kinase 1/Placental Growth Factor (sFlt-1/PlGF Ratio) in Pregnancies at High Risk for the Development of Preeclampsia.

BACKGROUND: Soluble FMS-like Tyrosine Kinase 1 (sFlt-1) and placental growth factor (PlGF) have been reported to be highly predictive several weeks before the onset of preeclampsia. OBJECTIVE: To investigate longitudinal changes of serum levels sFlt-1 and PlGF in pregnant women at high risk for the development of preeclampsia and to reveal an impact of aspirin on maternal serum concentrations of sFlt-1 and PlGF. METHODS: This was a prospective longitudinal study in 394 women with various risk factors for the development of preeclampsia (chronic hypertension, antiphospholipid syndrome/APS or systemic lupus erythematosus/SLE, thrombophilia, women with a history of preeclampsia, pathologic first trimester screening for preeclampsia) and 68 healthy women. Serum levels of sFlt-1 and PlGF were measured prospectively at 4-week intervals (from gestational weeks 12 until postpartum). RESULTS: The sFlt-1/PlGF ratio was significantly higher in women with an adverse obstetric outcome compared to women with a normal pregnancy, starting between 20 and 24 weeks of gestation. There was no effect of aspirin on sFlt-1/PlGF ratio in women with chronic hypertension, APS/SLE, thrombophilia and controls. The use of aspirin showed a trend towards an improvement of the sFlt-1/PlGF ratio in women with preeclampsia in a previous pregnancy and a significant effect on the sFlt-1/PlGF ratio in women with a pathologic first trimester screening for preeclampsia. CONCLUSIONS: Our findings reveal an impact of aspirin on sFlt-1/PlGF ratio in women with a pathologic first trimester screening for preeclampsia, strongly supporting its prophylactic use.

Cholesterol Deficiency Causes Impaired Osmotic Stability of Cultured Red Blood Cells.

Ex vivo generation of red blood cells (cRBCs) is an attractive tool in basic research and for replacing blood components donated by volunteers. As a prerequisite for the survival of cRBCs during storage as well as in the circulation, the quality of the membrane is of utmost importance. Besides the cytoskeleton and embedded proteins, the lipid bilayer is critical for membrane integrity. Although cRBCs suffer from increased fragility, studies investigating the lipid content of their membrane are still lacking. We investigated the membrane lipid profile of cRBCs from CD34+ human stem and progenitor cells compared to native red blood cells (nRBCs) and native reticulocytes (nRETs). Ex vivo erythropoiesis was performed in a well-established liquid assay. cRBCs showed a maturation grade between nRETs and nRBCs. High-resolution mass spectrometry analysis for cholesterol and the major phospholipid classes, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin and lysophosphatidylcholin, demonstrated severe cholesterol deficiency in cRBCs. Although cRBCs showed normal deformability capacity, they suffered from increased hemolysis due to minimal changes in the osmotic conditions. After additional lipid supplementation, especially cholesterol during culturing, the cholesterol content of cRBCs increased to a subnormal amount. Concurrently, the osmotic resistance recovered completely and became comparable to that of nRETs. Minor differences in the amount of phospholipids in cRBCs compared to native cells could mainly be attributed to the ongoing membrane remodeling process from the reticulocyte to the erythrocyte stage. Obtained results demonstrate severe cholesterol deficiency as a reason for enhanced fragility of cRBCs. Therefore, the supplementation of lipids, especially cholesterol during ex vivo erythropoiesis may overcome this limitation and strengthens the survival of cRBCs ex vivo and in vivo.

Human fetoplacental arterial and venous endothelial cells are differentially programmed by gestational diabetes mellitus, resulting in cell-specific barrier function changes.

AIMS/HYPOTHESIS: An adverse intrauterine environment can result in permanent changes in the physiology of the offspring and predispose to diseases in adulthood. One such exposure, gestational diabetes mellitus (GDM), has been linked to development of metabolic disorders and cardiovascular disease in offspring. Epigenetic variation, including DNA methylation, is recognised as a leading mechanism underpinning fetal programming and we hypothesised that this plays a key role in fetoplacental endothelial dysfunction following exposure to GDM. Thus, we conducted a pilot epigenetic study to analyse concordant DNA methylation and gene expression changes in GDM-exposed fetoplacental endothelial cells. METHODS: Genome-wide methylation analysis of primary fetoplacental arterial endothelial cells (AEC) and venous endothelial cells (VEC) from healthy pregnancies and GDM-complicated pregnancies in parallel with transcriptome analysis identified methylation and expression changes. Most-affected pathways and functions were identified by Ingenuity Pathway Analysis and validated using functional assays. RESULTS: Transcriptome and methylation analyses identified variation in gene expression linked to GDM-associated DNA methylation in 408 genes in AEC and 159 genes in VEC, implying a direct functional link. Pathway analysis found that genes altered by exposure to GDM clustered to functions associated with 'cell morphology' and 'cellular movement' in healthy AEC and VEC. Further functional analysis demonstrated that GDM-exposed cells had altered actin organisation and barrier function. CONCLUSIONS/INTERPRETATION: Our data indicate that exposure to GDM programs atypical morphology and barrier function in fetoplacental endothelial cells by DNA methylation and gene expression change. The effects differ between AEC and VEC, indicating a stringent cell-specific sensitivity to adverse exposures associated with developmental programming in utero. DATA AVAILABILITY: DNA methylation and gene expression datasets generated and analysed during the current study are available at the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database ( http://www.ncbi.nlm.nih.gov/geo ) under accession numbers GSE106099 and GSE103552, respectively.