Differential impact of COVID-19 on mental health and burnout.

BACKGROUND: There may be differential impact of the COVID-19 pandemic on mental health and burnout rates of healthcare professionals (HCPs) performing different roles. AIMS: To examine mental health and burnout rates, and possible drivers for any disparities between professional roles. METHODS: In this cohort study, online surveys were distributed to HCPs in July-September 2020 (baseline) and re-sent 4 months later (follow-up; December 2020) assessing for probable major depressive disorder (MDD), generalized anxiety disorder (GAD), insomnia, mental well-being and burnout (emotional exhaustion and depersonalization). Separate logistic regression models (at both phases) compared the risk of outcomes between roles: healthcare assistants (HCAs), nurses and midwives (nurses), allied health professionals (AHPs) and doctors (reference group). Separate linear regression models were also developed relating the change in scores to professional role. RESULTS: At baseline (n = 1537), nurses had a 1.9-fold and 2.5-fold increased risk of MDD and insomnia, respectively. AHPs had a 1.7-fold and 1.4-fold increased risk of MDD and emotional exhaustion, respectively. At follow-up (n = 736), the disproportionate risk between doctors and others worsened: nurses and HCAs were at 3.7-fold and 3.6-fold increased risk of insomnia, respectively. Nurses also had a significantly increased risk of MDD, GAD, poor mental well-being and burnout. Nurses also had significantly worsened anxiety, mental well-being and burnout scores over time, relative to doctors. CONCLUSIONS: Nurses and AHPs had excess risk of adverse mental health and burnout during the pandemic, and this difference worsened over time (in nurses especially). Our findings support adoption of targeted strategies accounting for different HCP roles.

Isolation of syncytiotrophoblast microvesicles and exosomes and their characterisation by multicolour flow cytometry and fluorescence Nanoparticle Tracking Analysis.

The human placenta releases multiple types and sizes of syncytiotrophoblast (STB) extracellular vesicles (EV) into the maternal circulation that exhibit diverse biological activities. The placental perfusion technique enables isolation of these STBEV, but conventional flow cytometry can only be used to phenotype EV down to ∼300 nm in size. Fluorescence Nanoparticle Tracking Analysis (fl-NTA) has the potential to phenotype EV down to ∼50 nm, thereby improving current characterisation techniques. The aims of this study were to prepare microvesicle and exosome enriched fractions from human placental perfusate (n=8) and improve fl-NTA STBEV detection. Differential centrifugation and filtration effectively removed contaminating red blood cells from fresh placental perfusates and pelleted a STB microvesicle (STBMV) fraction (10,000×g pellet - 10KP; NTA modal size 395±12 nm), enriched for the STB marker placental alkaline phosphatase (PLAP) and a STB exosome (STBEX) fraction (150,000×g pellet - 150KP; NTA modal size 147±6 nm), enriched for PLAP and exosome markers Alix and CD63. The PLAP positivity of 'standard' 10KP and 150KP pools (four samples/pool), determined by immunobead depletion, was used to optimise fl-NTA camera settings. Individual 10KP and 150KP samples (n=8) were 54.5±5.7% (range 17.8-66.9%) and 30.6±5.6% (range 3.3-51.7%) PLAP positive, respectively. We have developed a reliable method for enriching STBMV and STBEX from placental perfusate. We also standardised fl-NTA settings and improved measurement of PLAP positive EV in STBMV. However, fl-NTA is not as sensitive as anti-PLAP Dynabead capture for STBEX detection, possibly due to STBEX having lower surface expression of PLAP. These important developments will facilitate more detailed studies of the role of STBMV and STBEX in normal and pathological pregnancies.

Review: Does size matter? Placental debris and the pathophysiology of pre-eclampsia.

A variety of 'debris' is shed from the syncytial surface of the human placenta ranging from large deported multinuclear fragments to sub-cellular components. It is increasingly clear that at least some of this material has signalling functions. Many categories of circulating debris are increased in pre-eclampsia, and exhibit proteins that are pro-inflammatory and could contribute to the systemic inflammatory response in normal pregnancy, which is exaggerated in pre-eclampsia. It is now evident that there is a large 'hidden' population of microvesicles and nanovesicles (including exosomes) which are hard to investigate because of their size. We have used a new technology, nanoparticle tracking analysis, to measure the size and concentration of syncytiotrophoblast vesicles prepared by placental perfusion. The vesicles range in size from 50 nm to 1 µm with the majority being <500 nm (which includes both exosomes and microvesicles). We speculate whether changes not only in the numbers, but also in the size (beneficial syncytiotrophoblast exosomes and harmful microvesicles) might be important in the maternal syndrome of pre-eclampsia.

Tryptophan catabolites in mesenteric lymph may contribute to pancreatitis-associated organ failure.

BACKGROUND: Multiple organ failure (MOF) is the key determinant of mortality in acute pancreatitis (AP). Mesenteric lymph cytotoxicity contributes to organ failure in experimental models of systemic inflammation. The aim of this study was to evaluate the mesenteric lymph pathway and the lymph injury proteome in experimental AP-associated MOF, and to test the hypothesis that immunoregulatory tryptophan catabolites contribute to mesenteric lymph cytotoxicity. METHODS: Using an experimental model of AP in rats, the humoral component of mesenteric lymph in AP was compared with that from sham-operated control animals, using in vitro and in vivo cytotoxicity assays, high-throughput proteomics and high-performance liquid chromatography. The experimental findings were corroborated in a cohort of 34 patients with AP. RESULTS: Compared with biologically inactive lymph from sham-operated rats, mesenteric lymph in AP became cytotoxic 3 h after induction. Hierarchical clustering of lymph proteomic mass spectra predicted the biological behaviour of lymph. Levels of the immunoregulatory tryptophan catabolite, 3-hydroxykynurenine, were increased in cytotoxic lymph and re-created cytotoxicity in vitro. In humans with AP, plasma kynurenine concentrations correlated in real time with MOF scores and preceded a requirement for mechanical ventilation and haemodialysis. CONCLUSION: These results support the concept that mesenteric lymph-borne kynurenines may contribute to pancreatitis-associated MOF.

Angiogenesis and pericytes in the initiation of ectopic calcification.

Ectopic calcification of blood vessels, heart valves, and skeletal muscle is a major clinical problem. There is now good evidence that angiogenesis is associated with ectopic calcification in these tissues and that it is necessary, but not sufficient, for calcification to occur. Angiogenesis may regulate ectopic calcification in several ways. First, many angiogenic factors are now known to exert both direct and indirect effects on bone and cartilage formation. Second, cytokines released by endothelial cells can induce the differentiation of osteoprogenitor cells. Third, the new blood vessels provide oxygen and nutrients to support the growing bone. Finally, the new blood vessels can serve as a conduit for osteoprogenitor cells. These osteoprogenitor cells may be derived from the circulation or from pericytes that are present in the neovessels themselves. Indeed, there is now compelling evidence that pericytes can differentiate into osteoblasts and chondrocytes both in vitro and in vivo. Other vascular cells, including adventitial myofibroblasts, calcifying vascular cells, smooth muscle cells, and valvular interstitial cells, have also been shown to exhibit multilineage potential in vitro. Although these cells share many properties with pericytes, the precise relationship between them is not known. Furthermore, it still remains to be determined whether all or some of these cells contribute to the ectopic calcification observed in vivo. A better understanding of the underlying mechanisms that link angiogenesis, pericytes, and ectopic calcification should provide a basis for development of therapeutic strategies to treat or arrest this clinically significant condition.

Curcumin modifies Apc(min) apoptosis resistance and inhibits 2-amino 1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced tumour formation in Apc(min) mice.

Curcumin, the active ingredient of the rhizome of Curcuma longa, promotes apoptosis and may have chemopreventive properties. This study investigates the effects of curcumin on apoptosis and tumorigenesis in male Apc(min) mice treated with the human dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Intestinal epithelial apoptotic index in response to PhIP treatment was approximately twice as great in the wild-type C57BL/6 APC(+/+) strain than in Apc(min) mice (3.7% Apc(+/+) versus 1.9% Apc(min); P < 0.001). PhIP promoted tumour formation in Apc(min) proximal small intestine (4.6 tumours per mouse, PhIP treated versus 2.1 tumours per mouse, control untreated; P < 0.05). Curcumin enhanced PhIP-induced apoptosis (4.0% curcumin + PhIP versus 2.1% PhIP alone; P < 0.01) and inhibited PhIP-induced tumorigenesis in the proximal small intestine of Apc(min) mice (2.2 tumours per mouse, curcumin + PhIP versus 4.6 tumours per mouse PhIP alone; P < 0.05). This study shows that the Apc(min) genotype is associated with resistance to PhIP-induced apoptosis in intestinal epithelium. Curcumin attenuates Apc(min) resistance to PhIP-induced apoptosis and inhibits PhIP-induced tumorigenesis in proximal Apc(min) mouse small intestine.

Peroxisome proliferator-activated receptor alpha is an androgen-responsive gene in human prostate and is highly expressed in prostatic adenocarcinoma.

Peroxisome proliferator-activated receptor (PPAR) alpha is a member of the nuclear receptor superfamily of ligand-activated transcription factors. PPARalpha is activated by peroxisome proliferators and fatty acids and has been shown to be involved in the transcriptional regulation of genes involved in fatty acid metabolism. In rodents, the PPARalpha-mediated change in such genes results in peroxisome proliferation and can lead to the induction of hepatocarcinogenesis. Using the mRNA differential display technique and Northern blot analysis, we have shown that chronic exposure of the prostate cancer epithelial cell line LNCaP to the synthetic androgen mibolerone results in the down-regulation of PPARalpha mRNA. Levels of PPARalpha mRNA are reduced to approximately 40% of control levels in LNCaP cells exposed to 10 nM mibolerone for 96 h. PPARalpha-responsive reporter plasmids derived from human ApoA-II and muscle carnitine palmitoyl-transferase I genes were stimulated by the PPARalpha-activating ligand Wy-14,643 in LNCaP cells. In situ hybridization and immunohistochemical analyses showed that PPARalpha expression in prostate is confined to epithelial cells. In benign prostatic tissue, PPARalpha mRNA was either absent or only weakly expressed in the basal epithelial cells. In 11 of 18 (61%) poorly differentiated (Gleason score, 8-10) prostatic carcinoma specimens, there was strong expression of PPARalpha compared with 4 of 12 Gleason score 7 tumors and 2 of 11 Gleason score 3-6 tumors (P < 0.01). These results suggest that PPARalpha is found and functional in human prostate and is down-regulated by androgens. The role of PPARalpha may be to integrate dietary fatty acid and steroid hormone signaling pathways, and its overexpression in advanced prostate cancer may indicate a role in tumor progression with the potential involvement of dietary factors.

Cell-matrix adhesions differentially regulate fascin phosphorylation.

Cell adhesion to individual macromolecules of the extracellular matrix has dramatic effects on the subcellular localization of the actin-bundling protein fascin and on the ability of cells to form stable fascin microspikes. The actin-binding activity of fascin is down-regulated by phosphorylation, and we used two differentiated cell types, C2C12 skeletal myoblasts and LLC-PK1 kidney epithelial cells, to examine the hypothesis that cell adhesion to the matrix components fibronectin, laminin-1, and thrombospondin-1 differentially regulates fascin phosphorylation. In both cell types, treatment with the PKC activator 12-tetradecanoyl phorbol 13-acetate (TPA) or adhesion to fibronectin led to a diffuse distribution of fascin after 1 h. C2C12 cells contain the PKC family members alpha, gamma, and lambda, and PKCalpha localization was altered upon cell adhesion to fibronectin. Two-dimensional isoelectric focusing/SDS-polyacrylamide gels were used to determine that fascin became phosphorylated in cells adherent to fibronectin and was inhibited by the PKC inhibitors calphostin C and chelerythrine chloride. Phosphorylation of fascin was not detected in cells adherent to thrombospondin-1 or to laminin-1. LLC-PK1 cells expressing green fluorescent protein (GFP)-fascin also displayed similar regulation of fascin phosphorylation. LLC-PK1 cells expressing GFP-fascin S39A, a nonphosphorylatable mutant, did not undergo spreading and focal contact organization on fibronectin, whereas cells expressing a GFP-fascin S39D mutant with constitutive negative charge spread more extensively than wild-type cells. In contrast, C2C12 cells coexpressing S39A fascin with endogenous fascin remained competent to form microspikes on thrombospondin-1, and cells that expressed fascin S39D attached to thrombospondin-1 but did not form microspikes. Blockade of PKCalpha activity by TPA-induced down-regulation led to actin association of wild-type fascin in fibronectin-adherent C2C12 and LLC-PK1 cells but did not alter the distribution of S39A or S39D fascins. The association of fascin with actin in fibronectin-adherent cells was also evident in the presence of an inhibitory antibody to integrin alpha5 subunit. These novel results establish matrix-initiated PKC-dependent regulation of fascin phosphorylation at serine 39 as a mechanism whereby matrix adhesion is coupled to the organization of cytoskeletal structure.

Paracrine regulation of talin mRNA expression by androgen in human prostate.

Androgens are essential for normal prostate physiology and are intimately associated with the growth and progression of prostate cancer. However, few androgen regulated genes in the prostate have been identified. Using the mRNA differential display technique a 164-bp cDNA fragment was identified as being androgen regulated in the human prostate. Nucleotide sequence analysis of this fragment revealed 84% homology with the gene encoding the cytoskeletal protein talin. Confirmation of the androgen regulation of this gene was carried out using Northern analysis. Primary prostatic stromal cells treated with conditioned medium (CM) from androgen-treated primary prostatic epithelial cells showed an approximate 2-fold reduction in talin mRNA levels compared with stromal cells treated with CM from epithelial cells not exposed to androgens. Expression of talin mRNA in human prostatic tissue was confirmed by in situ hybridisation. The highest levels of expression were present in the epithelial cells, with lower levels of expression in the stroma. Thus, androgen regulation of talin expression may play a role in normal and/or aberrant growth and development of the prostate.

Differential distribution of endothelin receptor subtypes in placentae from normal and growth-restricted pregnancies.

The endothelins (ETs) are potent vasoconstrictor peptides that bind to two distinct receptors, ETA and ETB. This study compares the localization of ETA and ETB receptors in placentae complicated by intrauterine growth retardation (IUGR) and abnormal umbilical Doppler waveform, gestationally matched controls, fetuses that were small for gestational age (SGA), and normal term placentae. Quantitative autoradiography was performed using ETA and ETB subtype-selective ligands. Both ETA and ETB receptors were expressed in the human placenta. Gestational and fetal size effects on the receptor density within stem villi were found, but no effect of abnormal placental blood flow could be demonstrated. A distinct spatial distribution of receptor subtypes within the placenta was observed. Smooth muscle cells expressed both receptors with ETA expression predominant in the proximal regions of the villous tree and ETB abundant in the periphery and decidua. Both receptors were also expressed at lower density on paravascular stromal cells in stem villi. Although these data do not demonstrate aberrant localization of ET receptors in IUGR and SGA placentae, the spatially distinct distribution of ET receptors in the human placenta suggests that ETs play a role in modulation of placental blood flow.

Localization of endothelin receptors in human uterus throughout the menstrual cycle.

Quantitative autoradiography employing the ETA selective ligand [125I]PD 151242 and the ETB selective ligand [125I]BQ3020 was used to assess the localization of ETA and ETB receptors in human uterus throughout the menstrual cycle. ETA and ETB receptors were present in endometrium and myometrium across the menstrual cycle. In myometrium, neither ETA nor ETB receptor density showed any detectable change across the menstrual cycle. ETA receptors were expressed in stroma throughout the endometrium and showed an increase in density in proliferative endometrium compared with secretory and menstrual endometrium. Endometrial ETB receptors were expressed at low density in the proliferative phase. In the early secretory phase there was an increase in ETB receptor density in the glandular epithelium of the basal region of the endometrium but not in functional endometrium. In the late secretory phase ETB receptor expression was increased in glandular epithelium throughout the endometrium. The highest density of ETB receptors was seen in menstrual endometrium, where they were present in stromal as well as epithelial cells. These results suggest that ovarian steroid hormones may play a role in the control of expression of ETA and ETB receptors in endometrial stromal and epithelial cells respectively.

Endothelin expression in the uterus.

The endothelins (ETs) comprise a family of 21 amino acid peptides, ET-1, ET-2 and ET-3, first demonstrated as products of vascular endothelium. Subsequent work showed that they are also found in non-endothelial cells from a variety of tissues such as breast, parathyroid and adrenal gland. At first, the ETs were recognized for their pressor effects. However, ET administration in vivo initially caused hypotension at low concentrations by triggering the paracrine release of endothelial-derived vasodilators. The ETs exert powerful contractile actions on myometrium and other types of smooth muscle and are mitogenic, or co-mitogenic for fibroblasts, vascular smooth muscle and other cells. Demonstration of extravascular ET in endometrium has revealed a powerful vasoconstrictor which might act on the spiral arterioles to effect a powerful and sustained contraction of vascular smooth muscle. ETs might also contribute to the process of endometrial repair. In addition, the ETs appear to play a fundamental role in the control of uterine function in pregnancy. Effects on myometrial contractility have been implicated in the mechanisms governing the onset of normal and pre-term labour, and the peptides are likely to be key determinants of placental blood flow by binding to vascular smooth muscle receptors in the placenta.