Parturition, oxytocin, inflammation, myocyte damage and obesity; A study of myometrium and haematological parameters in human pregnancy and labour at term

The process of parturition resulting in the delivery of a newborn is a fundamental event ensuring survival of the species. In humans, the main clinical problems of parturition include activation of the process too early or too late resulting in the delivery of pre-term and post-term infants, both with their own implications for future health for the mother and baby. Additionally, where parturition systems are not activated correctly, dysfunctional labour with the resulting need for caesarean delivery (CS), in addition to atonic post-partum haemorrhage can also ensue. Overall, in the UK up to 40% of pregnancies are affected by one of these problems. However, the exact processes involved in the initiation and maintenance of parturition in the human are not fully understood. With such an important event, influences are most likely to be multi-factorial, with hormonal, mechanical, inflammatory, biochemical and maternal environmental factors playing a part. The aims of this thesis were to investigate influences on parturition in human pregnancy. Firstly, the myometrial transcriptional effects of long term exposure to the uterotonic oxytocin (OT) were examined. Further investigation of the myometrial and maternal peripheral response to uterine contractions in-vitro and in-vivo was also made with particular reference to the role of inflammation and myocyte damage. Additionally, the influence of maternal factors, particularly obesity, on the myometrial in-vitro contractile function and response to OT was studied. Initially, 150 gene arrays were produced using the Illumina platform. The samples were derived from myometrium taken at pre-labour CS which subsequently underwent functional contractility experiments in an organ bath. Five drug environments were studied, namely OT, acetic acid (OT vehicle), ML7(a tocolytic acting via inhibition of myosin light chain kinase), ML7 & OT and finally DMSO (ML7 vehicle). Additionally, five time-points of 0, 1, 2, 4, and 6 hours after drug addition were used, resulting in 5 samples for each drug and time combination. The results indicated that despite a clear enhancement of myometrial contractile activity by OT, this functional response does not appear to be mediated by cellular transcription. However, there was a clear contraction and time dependent transcriptional wave, with overrepresentation of genes associated with inflammation and cellular damage/apoptosis, and down-regulation of pathways concerning cellular metabolism. These findings were confirmed by QPCR on further myometrial samples undergoing additional in-vitro functional studies. In addition to the temporal and contractile association with the inflammatory response, our data suggest inflammation occurs in response to myocyte cellular damage regardless of mode of damage e.g. contractile or chemically induced. This was demonstrated by inflammatory upregulation in myometrium exposed to the tocolytic agents nifedipine and ritodrine, which is not seen in response to ML7. Additionally, the myometrial inflammatory response was enhanced by the infective agent LPS. However, contrary to other proposals, the enhanced inflammatory response of the myometrium did not alter or promote the in-vitro contractile ability of the myometrium or its response to OT. This myometrial transcriptional data therefore suggests that the inflammatory response of labour is associated with contraction, chemical or infection induced myometrial cellular damage, but would not be considered necessary for a contractile response. Our in-vivo study of peripheral changes in the maternal circulation again supported our in-vitro myometrial data. Data showed that the effect of pregnancy at term was limited to increased white cell count driven by a neutrophilia, with no suggestion of leukocyte priming prior to labour. Additionally, term pregnancy is associated with an increase in CRP, an increase in GCSF (corresponding with the neutrophilia) in addition to suppression of the chemokines CCL11 and CCL22. Subsequently, we found that repeated blood samples taken at 2 hourly intervals during term labour induced dramatic changes in inflammatory cells and inflammatory mediators in the maternal circulation. Importantly, these changes occur in a co-ordinated time and contraction dependent manner, with the degree of inflammation associated with the length of time in labour and the degree of myocyte damage as measured by circulating CK and Mb. Our study of the influence of maternal factors on myometrial contractile ability and response to OT examined in-vitro myometrial contractility of 609 myometrial strips from 85 women. We demonstrated that maternal obesity does not impair spontaneous or OT induced myometrial contractions in-vitro. Furthermore, maternal age, ethnicity, parity, previous caesarean delivery,gestation at delivery and birthweight do not influence in-vitro myometrial spontaneous or OT induced contractile activity. This therefore suggests that the observed implication of these maternal and infant factors on parturition in-vivo (high rates of induction of labour, high rates of intrapartum caesarean delivery and post partum haemorrhage) cannot be explained by an effect on myometrial contraction per se. This therefore merits further investigation as to alternative mechanisms to ultimately promote and effective, uncomplicated and safe labour and vaginal delivery for at risk mothers. In summary, this thesis provides evidence that the myometrial contractions of human labour, whether spontaneous or OT induced are capable of inducing a temporal wave of transcriptional changes associated with the processes of inflammation, cellular damage/apoptosis with inhibition of cellular metabolic processes. In addition, maternal peripheral circulating factors mirror the myometrial transcriptional changes. These changes are highly comparable with those seen in response to exercising skeletal muscle, and in this model have been shown to play an important role in muscle repair and remodelling after exercise. Therefore, we would suggest that the inflammatory reaction typically associated with human labour occurs as a non-specific response to contraction induced cellular damage and may play a role in postpartum repair and remodelling of the uterus

Generation of 3D Skin Equivalents Fully Reconstituted from Human Induced Pluripotent Stem Cells (iPSCs)

Recent generation of patient-specific induced pluripotent stem cells (PS-iPSCs) provides significant advantages for cell- and gene-based therapy. Establishment of iPSC-based therapy for skin diseases requires efficient methodology for differentiating iPSCs into both keratinocytes and fibroblasts, the major cellular components of the skin, as well as the reconstruction of skin structures using these iPSC-derived skin components. We previously reported generation of keratinocytes from human iPSCs for use in the treatment of recessive dystrophic epidermolysis bullosa (RDEB) caused by mutations in the COL7A1 gene. Here, we developed a protocol for differentiating iPSCs into dermal fibroblasts, which also produce type VII collagen and therefore also have the potential to treat RDEB. Moreover, we generated in vitro 3D skin equivalents composed exclusively human iPSC-derived keratinocytes and fibroblasts for disease models and regenerative therapies for skin diseases, first demonstrating that iPSCs can provide the basis for modeling a human organ derived entirely from two different types of iPSC-derived cells

Pharmacologic inhibition of JAK-STAT signaling promotes hair growth

Several forms of hair loss in humans are characterized by the inability of hair follicles to enter the growth phase (anagen) of the hair cycle after being arrested in the resting phase (telogen). Current pharmacologic therapies have been largely unsuccessful in targeting pathways that can be selectively modulated to induce entry into anagen. We show that topical treatment of mouse and human skin with small-molecule inhibitors of the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway results in rapid onset of anagen and subsequent hair growth. We show that JAK inhibition regulates the activation of key hair follicle populations such as the hair germ and improves the inductivity of cultured human dermal papilla cells by controlling a molecular signature enriched in intact, fully inductive dermal papillae. Our findings open new avenues for exploration of JAK-STAT inhibition for promotion of hair growth and highlight the role of this pathway in regulating the activation of hair follicle stem cells

Hair Follicle Dermal Cells Support Expansion of Murine and Human Embryonic and Induced Pluripotent Stem Cells and Promote Haematopoiesis in Mouse Cultures

In the hair follicle, the dermal papilla (DP) and dermal sheath (DS) support and maintain proliferation and differentiation of the epithelial stem cells that produce the hair fibre. In view of their regulatory properties, in this study, we investigated the interaction between hair follicle dermal cells (DP and DS) and embryonic stem cells (ESCs); induced pluripotent stem cells (iPSCs); and haematopoietic stem cells. We found that coculture of follicular dermal cells with ESCs or iPSCs supported their prolonged maintenance in an apparently undifferentiated state as established by differentiation assays, immunocytochemistry, and RT-PCR for markers of undifferentiated ESCs. We further showed that cytokines that are involved in ESC support are also expressed by cultured follicle dermal cells, providing a possible explanation for maintenance of ES cell stemness in cocultures. The same cytokines were expressed within follicles in situ in a pattern more consistent with a role in follicle growth activities than stem cell maintenance. Finally, we show that cultured mouse follicle dermal cells provide good stromal support for haematopoiesis in an established coculture model. Human follicular dermal cells represent an accessible and readily propagated source of feeder cells for pluripotent and haematopoietic cells and have potential for use in clinical applications

Common variation near CDKN1A, POLD3 and SHROOM2 influences colorectal cancer risk

We performed a meta-analysis of five genome-wide association studies to identify common variants influencing colorectal cancer (CRC) risk comprising 8,682 cases and 9,649 controls. Replication analysis was performed in case-control sets totaling 21,096 cases and 19,555 controls. We identified three new CRC risk loci at 6p21 (rs1321311, near CDKN1A; P = 1.14 × 10(-10)), 11q13.4 (rs3824999, intronic to POLD3; P = 3.65 × 10(-10)) and Xp22.2 (rs5934683, near SHROOM2; P = 7.30 × 10(-10)) This brings the number of independent loci associated with CRC risk to 20 and provides further insight into the genetic architecture of inherited susceptibility to CRC.Swedish Research Council et al.Manuscrip

Five endometrial cancer risk loci identified through genome-wide association analysis.

We conducted a meta-analysis of three endometrial cancer genome-wide association studies (GWAS) and two follow-up phases totaling 7,737 endometrial cancer cases and 37,144 controls of European ancestry. Genome-wide imputation and meta-analysis identified five new risk loci of genome-wide significance at likely regulatory regions on chromosomes 13q22.1 (rs11841589, near KLF5), 6q22.31 (rs13328298, in LOC643623 and near HEY2 and NCOA7), 8q24.21 (rs4733613, telomeric to MYC), 15q15.1 (rs937213, in EIF2AK4, near BMF) and 14q32.33 (rs2498796, in AKT1, near SIVA1). We also found a second independent 8q24.21 signal (rs17232730). Functional studies of the 13q22.1 locus showed that rs9600103 (pairwise r(2) = 0.98 with rs11841589) is located in a region of active chromatin that interacts with the KLF5 promoter region. The rs9600103[T] allele that is protective in endometrial cancer suppressed gene expression in vitro, suggesting that regulation of the expression of KLF5, a gene linked to uterine development, is implicated in tumorigenesis. These findings provide enhanced insight into the genetic and biological basis of endometrial cancer.I.T. is supported by Cancer Research UK and the Oxford Comprehensive Biomedical Research Centre. T.H.T.C. is supported by the Rhodes Trust and the Nuffield Department of Medicine. Funding for iCOGS infrastructure came from the European Community's Seventh Framework Programme under grant agreement 223175 (HEALTH-F2-2009-223175) (COGS), Cancer Research UK (C1287/A10118, C1287/A10710, C12292/A11174, C1281/A12014, C5047/A8384, C5047/A15007, C5047/A10692 and C8197/A16565), the US National Institutes of Health (R01 CA128978, U19 CA148537, U19 CA148065 and U19 CA148112), the US Department of Defense (W81XWH-10-1-0341), the Canadian Institutes of Health Research (CIHR) for the CIHR Team in Familial Risks of Breast Cancer, the Susan G. Komen Foundation for the Cure, the Breast Cancer Research Foundation and the Ovarian Cancer Research Fund. SEARCH recruitment was funded by a programme grant from Cancer Research UK (C490/A10124). Stage 1 and stage 2 case genotyping was supported by the NHMRC (552402 and 1031333). Control data were generated by the WTCCC, and a full list of the investigators who contributed to the generation of the data is available from the WTCCC website. We acknowledge use of DNA from the British 1958 Birth Cohort collection, funded by UK Medical Research Council grant G0000934 and Wellcome Trust grant 068545/Z/02; funding for this project was provided by the Wellcome Trust under award 085475. NSECG was supported by the European Union's Framework Programme 7 CHIBCHA grant and Wellcome Trust Centre for Human Genetics Core Grant 090532/Z/09Z, and CORGI was funded by Cancer Research UK. BCAC is funded by Cancer Research UK (C1287/A10118 and C1287/A12014). OCAC is supported by a grant from the Ovarian Cancer Research Fund thanks to donations by the family and friends of Kathryn Sladek Smith (PPD/RPCI.07) and the UK National Institute for Health Research Biomedical Research Centres at the University of Cambridge.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/ng.356

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707