CAGE sequencing reveals CFTR-dependent dysregulation of type I IFN signaling in activated cystic fibrosis macrophages.

An intense, nonresolving airway inflammatory response leads to destructive lung disease in cystic fibrosis (CF). Dysregulation of macrophage immune function may be a key facet governing the progression of CF lung disease, but the underlying mechanisms are not fully understood. We used 5' end centered transcriptome sequencing to profile P. aeruginosa LPS-activated human CF macrophages, showing that CF and non-CF macrophages deploy substantially distinct transcriptional programs at baseline and following activation. This includes a significantly blunted type I IFN signaling response in activated patient cells relative to healthy controls that was reversible upon in vitro treatment with CFTR modulators in patient cells and by CRISPR-Cas9 gene editing to correct the F508del mutation in patient-derived iPSC macrophages. These findings illustrate a previously unidentified immune defect in human CF macrophages that is CFTR dependent and reversible with CFTR modulators, thus providing new avenues in the search for effective anti-inflammatory interventions in CF.

The Effect of Think Aloud on Performance and Brain Oxygenation During Cycling - An Exploratory Study.

In this study, we aimed to investigate the effect of Think Aloud (TA) on performance in trained and untrained participants, using functional Near Infrared Spectroscopy, during incrementally paced cycling. A mixed design was implemented with cycling expertise (10 untrained vs. 9 trained) as the between groups variable and trial stage (5 stages of increasing effort), and condition (silent vs. TA) as within groups independent variables (IVs). Dependent measures were changes in cortical oxygenation (O2Hb) in 12 areas of the prefrontal cortex (PFC) and physiological indicators of percentage heart rate maximum (%HRmax), average power output (APO), peak power output (PPO), rate of perceived exertion (RPE) and blood lactate ([La]b) over time. Trained cyclists had higher APO and significantly higher PPO from stages 2-5, in addition to a greater increase in PPO over the duration of the test (range 168W-480 W vs. 133W-313 W). There were significant main effects of stage on %HRmax, Bla and RPE (p < .001), with effect sizes (ήp2) ranging from .31 to .97. On average, HRmax%, [La]b and RPE were significantly lower after stage 2 onwards within the TA trial than the silent trial, even though similar power outputs were obtained. Thus, the TA trial elicited a better pacing strategy. There was no main effect of group on changes in O2Hb, though O2Hb did change as a function of stage in four areas of the PFC, and as a function of condition in one area. In this first study to assess the effects of TA on performance during self-paced cycling, TA did not disrupt performance outcomes at low through to high levels of physical exertion for either untrained or trained participants.

A gel-free quantitative proteomics analysis of factors released from hypoxic-conditioned placentae.

Characterizing the protein factors released from placentae during pathogenesis remains a key objective toward understanding preeclampsia and related pregnancy disorders. Gel-free proteomics technologies applied to placental explant-conditioned media offers the potential of identifying these factors. Relative quantification mass spectrometry using isobaric tagging for relative and absolute quantification (iTRAQ) labeling was employed to compare the ''secretome'' between healthy term placental tissue cultured under both normoxic and hypoxic oxygen tensions. Of the 499 proteins identified, 45 were differentially expressed (P < .01 level), including interleukin 8 (IL-8) which was significantly upregulated under hypoxia. Global protein level changes are suggestive of decreased extracellular matrix remodeling under the same conditions. A significant enrichment of soluble liberated placental factors is achieved using this model system. Identifying these changes resulting from hypoxic conditioning is hypothesis generating and may provide new mechanistic insights into preeclampsia.

A potential role for free fatty acids in the pathogenesis of preeclampsia.

OBJECTIVE: Pregnant women with the vascular complication of preeclampsia show altered lipid metabolism characterized by elevated circulating triglycerides and nonesterified free fatty acids. We have compared the effect of maternal plasma from women with and without preeclampsia on cultured vascular endothelial cells and determined whether these plasma-induced changes were reproduced with free fatty acid solutions of palmitic, oleic and linoleic acid, representative of circulating levels reported in preeclampsia. METHODS: Lipid accumulation was quantified by oil-red O staining, apoptosis by terminal dUTP nick-end labelling (TUNEL) and the measurement of mitochondrial redox capacity, and membrane potential recorded using MTT reduction and JC-1 accumulation for human umbilical vein endothelial cells (HUVECs) exposed to plasma and free fatty acids. RESULTS: Lipid droplet accumulation was significantly increased in cultured HUVECs conditioned with maternal plasma from pregnancies with preeclampsia compared with normal uncomplicated controls. This increase was replicated following exposure to free fatty acids at the combined concentrations defined in preeclampsia. Plasma from these women also caused a significant decrease in mitochondrial dehydrogenase activity, a marked reduction in mitochondrial membrane potential and an increase in apoptosis compared with normal pregnancy. Again these effects were reproduced using free fatty acids in combination at the levels previously associated with preeclampsia. CONCLUSION: These findings support the concept of a circulating pathogenic factor for preeclampsia and highlight the possibility that this factor is not a single compound but perhaps the combined elevation of the free fatty acids palmitic, oleic and linoleic acid in the maternal circulation.

Maternal celiac disease autoantibodies bind directly to syncytiotrophoblast and inhibit placental tissue transglutaminase activity.

BACKGROUND: Celiac disease (CD) occurs in as many as 1 in 80 pregnant women and is associated with poor pregnancy outcome, but it is not known if this is an effect on maternal nutrient absorption or, alternatively, if the placenta is an autoimmune target. The major autoantigen, tissue transglutaminase (tTG), has previously been shown to be present in the maternal-facing syncytiotrophoblast plasma membrane of the placenta. METHODS: ELISA was used to demonstrate the presence of antibodies to tissue transglutaminase in a panel of CD sera. Immunohistochemistry was used to evaluate the binding of IgA autoantibodies from CD serum to term placenta. In addition, novel direct binding and activity assays were developed to mimic the in vivo exposure of the villous placenta to maternal autoantibody. RESULTS AND DISCUSSION: CD IgA autoantibodies located to the syncytial surface of the placenta significantly more than IgA antibodies in control sera (P < 0.0001). The distribution of antigen was similar to that observed using a monoclonal antibody to tissue transglutaminase. Staining was reduced by pre-absorption of CD serum with recombinant human tissue transglutaminase. In direct binding assays, autoimmune immunoglobulin A (IgA) from the maternal compartment became associated with antigen at the syncytial surface of the placenta, as a result of which transglutaminase activity at this site was inhibited. CONCLUSION: These data indicate that direct immune effects in untreated CD women may compromise placental function.

Oxygen and the liberation of placental factors responsible for vascular compromise.

Maternal endothelial activation in pre-eclampsia is attributed to the release of unknown factors from a hypoperfused placenta. To further characterize these factors, we have used a serum-free placental villous explant culture model and investigated the effect of the liberated soluble factors produced on human endothelial cell cultures. Term placental villous explants from uncomplicated pregnancies were cultured for 4 days in 20, 6 or 1% O2 to mimic placental hyperoxia, normoxia and hypoxia. Medium collected from viable explants was applied to cultured human uterine microvascular endothelial cells. Medium conditioned by hypoxic explants caused a significant decrease in endothelial cell ATP levels and mitochondrial dehydrogenase activity, suggestive of a reduced metabolic rate. An additional reduction in mitochondrial membrane potential and increased endothelial cell death occurred as the oxygen concentration to which explants had been exposed decreased. Effects of the hypoxic explant medium were also seen ex vivo in a wire myography model of myometrial artery function, with increased vasoconstriction and attenuated vasodilation following exposure to hypoxic explant medium. These results suggest that hypoxia (1% O2) may stimulate the release of soluble factors from the placenta, which have an adverse effect on endothelial cell metabolism and mitochondrial integrity in vitro. These potentially pathogenic factors are now being characterized.

A role for tissue transglutaminase in stabilization of membrane-cytoskeletal particles shed from the human placenta.

Tissue transglutaminase (TGM2; also known as TG2 or tTG) localizes to the syncytial microvillous membrane (MVM) of the human placenta, the primary interface between maternal and fetal tissue. To identify TGM2 substrates in the MVM, membrane vesicles were prepared and labeled with biotinylated acyl donor or acceptor probes. Biotinylated species were selected on an avidin affinity matrix and identified by mass spectrometry of tryptic peptides. The most abundant were cytoskeletal (actin, tubulin, and cytokeratin) and membrane-associated (annexins, integrins, and placental alkaline phosphatase) proteins. During pregnancy, apoptotic particulate material, the end product of the trophoblast life cycle, is shed from the MVM into maternal circulation. Shed material was isolated from primary trophoblast cultures in which syncytial-like masses develop by fusion. A substantial fraction of actin in the particles was in the form of covalent polymeric aggregates, in contrast to cellular actin, which dissociated completely into monomer in SDS-PAGE. When cells were cultured in the presence of transglutaminase inhibitors, actin in the shed particles remained exclusively in monomeric form, and a reduction in trophoblast intercellular fusion and differentiation was observed. These findings suggest that transglutaminase-mediated cross-linking stabilizes the particulate material shed from the placenta.

Tissue transglutaminase expression and activity in placenta.

Tissue transglutaminase (tTG) expression, distribution and activity were examined in human placenta and derived cells. Immunochemical techniques and RT-PCR were used to demonstrate tTG protein and mRNA in stromal cells and trophoblast in first trimester and at term, with higher levels later in pregnancy. Decidual cells also produce tTG. The data were confirmed using primary cultures of trophoblast, fibroblasts and decidual stromal cells. Substrate incorporation studies indicated tTG activity in association with fibroblast extracellular matrix and the syncytial microvillous membrane (MVM), where several target polypeptides could be observed. tTG is a major autoantigen in Coeliac disease (CoD) which is associated with poor pregnancy outcome. tTG at the placental MVM is a plausible target of maternal autoantibody action.