Predictive biomarkers and initial analysis of maternal immune alterations in postpartum preeclampsia reveal an immune-driven pathology.

Introduction: Postpartum preeclampsia (PPPE) is an under-diagnosed condition, developing within 48 hours to 6 weeks following an uncomplicated pregnancy. The etiology of PPPE is still unknown, leaving patients vulnerable and making the identification and treatment of patients requiring postpartum care an unmet need. We aimed to understand the immune contribution to PPPE at the time of diagnosis, as well as uncover the predictive potential of perinatal biomarkers for the early postnatal identification of high-risk patients. Methods: Placentas were collected at delivery from uncomplicated pregnancies (CTL) and PPPE patients for immunohistochemistry analysis. In this initial study, blood samples in PPPE patients were collected at the time of PPPE diagnosis (48h-25 days postpartum; mean 7.4 days) and compared to CTL blood samples taken 24h after delivery. Single-cell transcriptomics, flow cytometry, intracellular cytokine staining, and the circulating levels of inflammatory mediators were evaluated in the blood. Results: Placental CD163+ cells and 1st trimester blood pressures can be valuable non-invasive and predictive biomarkers of PPPE with strong clinical application prospects. Furthermore, changes in immune cell populations, as well as cytokine production by CD14+, CD4+, and CD8+ cells, suggested a dampened response with an exhausted phenotype including decreased IL1ß, IL12, and IFNγ as well as elevated IL10. Discussion: Understanding maternal immune changes at the time of diagnosis and prenatally within the placenta in our sizable cohort will serve as groundwork for pre-clinical and clinical research, as well as guiding clinical practice for example in the development of immune-targeted therapies, and early postnatal identification of patients who would benefit from more thorough follow-ups and risk education in the weeks following an uncomplicated pregnancy.

Innate and Adaptive Immune Systems in Physiological and Pathological Pregnancy.

The dynamic immunological changes occurring throughout pregnancy are well-orchestrated and important for the success of the pregnancy. One of the key immune adaptations is the maternal immune tolerance towards the semi-allogeneic fetus. In this review, we provide a comprehensive overview of what is known about the innate and adaptive immunological changes in pregnancy and the role(s) of specific immune cells during physiological and pathological pregnancy. Alongside this, we provided details of remaining questions and challenges, as well as future perspectives for this growing field of research. Understanding the immunological changes that occur can inform potential strategies on treatments for the optimal health of the neonate and pregnant individual both during and after pregnancy.

Proinflammatory changes in the maternal circulation, maternal-fetal interface, and placental transcriptome in preterm birth.

BACKGROUND: Preterm birth remains a leading obstetrical complication because of the incomplete understanding of its multifaceted etiology. It is known that immune alterations toward a proinflammatory profile are observed in women with preterm birth, but therapeutic interventions are still lacking because of scarcity of evidence in the integration of maternal and placental interrelated compartments. OBJECTIVE: This study aimed to obtain an integrated view of the maternal and placental contribution to preterm birth compared with normal term pregnancies for an in-depth understanding of the immune/inflammatory involvement, intending to identify novel strategies to mitigate the negative impact of inflammation. STUDY DESIGN: We prospectively recruited 79 women with preterm or term deliveries and collected placentas for RNA sequencing, histologic analyses, and to assess levels of inflammatory mediators. Blood samples were also collected to determine the circulating immune profiles by flow cytometry and to evaluate the circulating levels of inflammatory mediators. RESULTS: Placental transcriptomic analyses revealed 102 differentially expressed genes upregulated in preterm birth, including known and novel targets, which were highly enriched for inflammatory biological processes according to gene ontology analyses. Analysis of maternal immune cells revealed distinct profiles in preterm births vs term births, including an increased percentage of CD3- cells and monocyte subsets and decreased CD3+ cells along with Th17 subsets of CD4+ lymphocytes. Supporting our bioinformatic findings, we found increases in proinflammatory mediators in the plasma, placenta, and fetal membranes (primarily the amnion) of women with preterm birth, such as interleukin-6 and tumor necrosis factor-α. These findings were not distinct between spontaneous and iatrogenic preterm births except at a molecular level where spontaneous preterm birth presented with an elevated inflammatory profile compared with iatrogenic preterm birth. Analysis of placental histology revealed increased structural and inflammatory lesions in preterm vs term births. We found that genes upregulated in placentas with inflammatory lesions have enrichment of proinflammatory pathways. CONCLUSION: This work sheds light on changes within the immune system in preterm birth on multiple levels and compartments to help identify pregnancies at high risk of preterm birth and to discover novel therapeutic targets for preterm birth.

Impact of Exosomes Released by Different Corneal Cell Types on the Wound Healing Properties of Human Corneal Epithelial Cells.

Corneal wound healing involves communication between the different cell types that constitute the three cellular layers of the cornea (epithelium, stroma and endothelium), a process ensured in part by a category of extracellular vesicles called exosomes. In the present study, we isolated exosomes released by primary cultured human corneal epithelial cells (hCECs), corneal fibroblasts (hCFs) and corneal endothelial cells (hCEnCs) and determined whether they have wound healing characteristics of their own and to which point they modify the genetic and proteomic pattern of these cell types. Exosomes released by all three cell types significantly accelerated wound closure of scratch-wounded hCECs in vitro compared to controls (without exosomes). Profiling of activated kinases revealed that exosomes from human corneal cells caused the activation of signal transduction mediators that belong to the HSP27, STAT, ß-catenin, GSK-3ß and p38 pathways. Most of all, data from gene profiling analyses indicated that exosomes, irrespective of their cellular origin, alter a restricted subset of genes that are completely different between each targeted cell type (hCECs, hCFS, hCEnCs). Analysis of the genes specifically differentially regulated for a given cell-type in the microarray data using the Ingenuity Pathway Analysis (IPA) software revealed that the mean gene expression profile of hCECs cultured in the presence of exosomes would likely promote cell proliferation and migration whereas it would reduce differentiation when compared to control cells. Collectively, our findings represent a conceptual advance in understanding the mechanisms of corneal wound repair that may ultimately open new avenues for the development of novel therapeutic approaches to improve closure of corneal wounds.

Influence of the Postmortem/Storage Time of Human Corneas on the Properties of Cultured Limbal Epithelial Cells.

Besides being a powerful model to study the mechanisms of corneal wound healing, tissue-engineered human corneas (hTECs) are sparking interest as suitable substitutes for grafting purposes. To ensure the histological and physiological integrity of hTECs, the primary cultures generated from human cornea (identified as human limbal epithelial cells (hLECs) that are used to produce them must be of the highest possible quality. The goal of the present study consisted in evaluating the impact of the postmortem/storage time (PM/ST) on their properties in culture. hLECs were isolated from the entire cornea comprising the limbus and central cornea. When grown as monolayers, short PM/ST hLECs displayed increased daily doublings and generated more colonies per seeded cells than long PM/ST hLECs. Moreover, hLECs with a short PM/ST exhibited a markedly faster wound closure kinetic both in scratch wound assays and hTECs. Collectively, these results suggest that short PM/ST hLECs have a greater number of highly proliferative stem cells, exhibit a faster and more efficient wound healing response in vitro, and produce hTECs of a higher quality, making them the best candidates to produce biomaterial substitutes for clinical studies.

Diagnostic or Therapeutic Strategies for Pregnancy Complications.

Pregnancy complications including preeclampsia, preterm birth, recurrent pregnancy loss, and fetal growth restriction affect over 12% of all pregnancies worldwide [...].

Pandemic stress and SARS-CoV-2 infection are associated with pathological changes at the maternal-fetal interface.

INTRODUCTION: The reported effects of SARS-CoV-2 on pregnancy outcomes are conflicting; studies frequently overlook the placenta, which is critical for the health of the mother and infant(s). This study aimed to determine the effect of pandemic stress ± SARS CoV-2 infection on placental histopathology. METHODS: Women were recruited in Canada (n = 69); France (n = 21) or in the UK (n = 25), between March and October 2020. Historic controls (N = 20) were also included. Placenta and fetal membrane samples were collected rapidly after delivery and were fixed and stained for histopathological analysis. Maternal demographical data and obstetric outcomes were recorded. RESULTS: Over 80% of the placentas from SARS-CoV-2+ pregnancies had histopathological abnormalities: predominantly structural (71-86%) or inflammatory (9-22%), depending on geographical location. Excessive fibrin was seen in all sites, whereas deciduitis (Canada), calcifications (UK), agglutinations and chorangiosis (France) predominated in different locations. The frequency of abnormalities was significantly higher than in SARS-CoV-2 negative women (50%, p < 0.05). Demographic and obstetric data were similar in the SARS-CoV-2+ women across all sites - characterised by predominantly Black/Middle Eastern women, and women with elevated body mass index. DISCUSSION: Overall, the frequency of placental abnormalities is increased in SARS-CoV-2+ women, but the incidence of placental abnormalities is also higher in SARS-CoV-2- women that gave birth during the pandemic, which highlights the importance of appropriate control groups to ascertain the roles of pandemic stress and SARS-CoV-2 infection on the placenta and pregnancy outcomes.

The Human Tissue-Engineered Cornea (hTEC): Recent Progress.

Each day, about 2000 U.S. workers have a job-related eye injury requiring medical treatment. Corneal diseases are the fifth cause of blindness worldwide. Most of these diseases can be cured using one form or another of corneal transplantation, which is the most successful transplantation in humans. In 2012, it was estimated that 12.7 million people were waiting for a corneal transplantation worldwide. Unfortunately, only 1 in 70 patients received a corneal graft that same year. In order to provide alternatives to the shortage of graftable corneas, considerable progress has been achieved in the development of living corneal substitutes produced by tissue engineering and designed to mimic their in vivo counterpart in terms of cell phenotype and tissue architecture. Most of these substitutes use synthetic biomaterials combined with immortalized cells, which makes them dissimilar from the native cornea. However, studies have emerged that describe the production of tridimensional (3D) tissue-engineered corneas using untransformed human corneal epithelial cells grown on a totally natural stroma synthesized by living corneal fibroblasts, that also show appropriate histology and expression of both extracellular matrix (ECM) components and integrins. This review highlights contributions from laboratories working on the production of human tissue-engineered corneas (hTECs) as future substitutes for grafting purposes. It overviews alternative models to the grafting of cadaveric corneas where cell organization is provided by the substrate, and then focuses on their 3D counterparts that are closer to the native human corneal architecture because of their tissue development and cell arrangement properties. These completely biological hTECs are therefore very promising as models that may help understand many aspects of the molecular and cellular mechanistic response of the cornea toward different types of diseases or wounds, as well as assist in the development of novel drugs that might be promising for therapeutic purposes.

The Self-assembly Approach as a Tool for the Tissue Engineering of a Bi-lamellar Human Cornea.

Tissue engineering is a flourishing field of regenerative medicine that allows the reconstruction of various tissues of our body, including the cornea. In addition to addressing the growing need for organ transplants, such tissue-engineered substitutes may also serve as good in vitro models for fundamental and preclinical studies. Recent progress in the field of corneal tissue engineering has led to the development of new technologies allowing the reconstruction of a human bi-lamellar cornea. One unique feature of this model is the complete absence of exogenous material. Indeed, these human corneal equivalents are exclusively composed of untransformed human corneal fibroblasts (hCFs) entangled in their own extracellular matrix, as well as untransformed human corneal epithelial cells (hCECs), both of which isolated from donor corneas. The reconstructed human bi-lamellar cornea thereby exhibits a well-organized stroma as well as a well-differentiated epithelium. This chapter describes the methods used for the isolation and culture of hCFs, the production and assembly of hCFs stromal sheets, the seeding of hCECs, and the maturation of the tissue-engineered cornea.

Synthesis of Ultrastable Gold Nanoparticles as a New Drug Delivery System.

Nanotechnologies are increasingly being developed for medical purposes. However, these nanomaterials require ultrastability for better control of their pharmacokinetics. The present study describes three types of ultrastable gold nanoparticles stabilized by thiolated polyethylene glycol groups remaining intact when subjected to some of the harshest conditions described thus far in the literature, such as autoclave sterilization, heat and freeze-drying cycles, salts exposure, and ultracentrifugation. Their stability is characterized by transmission electron microscopy, UV-visible spectroscopy, and dynamic light scattering. For comparison purposes, two conventional nanoparticle types were used to assess their colloidal stability under all conditions. The ability of ultrastable gold nanoparticles to encapsulate bimatoprost, a drug for glaucoma treatment, is demonstrated. MTS assays on human corneal epithelial cells is assessed without changing cell viability. The impact of ultrastable gold nanoparticles on wound healing dynamics is assessed on tissue engineered corneas. These results highlight the potential of ultrastable gold nanoparticles as a drug delivery system in ocular therapy.

Contribution of the WNK1 kinase to corneal wound healing using the tissue-engineered human cornea as an in vitro model.

Damage to the corneal epithelium triggers important changes in the extracellular matrix (ECM) to which basal human corneal epithelial cells (hCECs) attach. These changes are perceived by integrin receptors that activate different intracellular signalling pathways, ultimately leading to re-epithelialization of the injured epithelium. In this study, we investigated the impact of pharmacological inhibition of specific signal transduction mediators on corneal wound healing using both monolayers of hCECs and the human tissue-engineered cornea (hTEC) as an in vitro 3D model. RNA and proteins were isolated from the wounded and unwounded hTECs to conduct gene profiling analyses and protein kinase arrays. The impact of WNK1 inhibition was evaluated on the wounded hTECs as well as on hCECs monolayers using a scratch wound assay. Gene profiling and protein kinase arrays revealed that expression and activity of several mediators from the integrin-dependent signaling pathways were altered in response to the ECM changes occurring during corneal wound healing. Phosphorylation of the WNK1 kinase turned out to be the most striking activation event going on during this process. The inhibition of WNK1 by WNK463 reduced the rate of corneal wound closure in both the hTEC and hCECs grown in monolayer compared with their respective negative controls. WNK463 also reduced phosphorylation of the WNK1 downstream targets SPAK/OSR1 in wounded hTECs. These in vitro results allowed for a better understanding of the cellular and molecular mechanisms involved in corneal wound healing and identified WNK1 as a kinase important to ensure proper wound healing of the cornea.

Enhanced wound healing of tissue-engineered human corneas through altered phosphorylation of the CREB and AKT signal transduction pathways.

The cornea is a transparent organ, highly specialized and unique that is continually subjected to abrasive forces and occasional mechanical or chemical trauma because of its anatomical localization. Upon injury, the extracellular matrix (ECM) rapidly changes to promote wound healing through integrin-dependent activation of specific signal transduction mediators whose contribution is to favor faster closure of the wound by altering the adhesive and migratory properties of the cells surrounding the damaged area. In this study, we exploited the human tissue-engineered cornea (hTECs) as a model to study the signal transduction pathways that participate to corneal wound healing. By exploiting both gene profiling and activated kinases arrays, we could demonstrate the occurrence of important alterations in the level of expression and activation of a few mediators from the PI3K/Akt and CREB pathways in response to the ECM remodeling taking place during wound healing of damaged hTECs. Pharmacological inhibition of CREB with C646 considerably accelerated wound closure compared to controls. This process was considerably accelerated further when both C646 and SC79, an Akt agonist, were added together to wounded hTECs. Therefore, our study demonstrate that proper corneal wound healing requires the activation of Akt together with the inhibition of CREB and that wound healing in vitro can be altered by the use of pharmacological inhibitors (such as C646) or agonists (such as SC79) of these mediators. STATEMENT OF SIGNIFICANCE: Corneal wounds account for a large proportion of all visual disabilities in North America. To our knowledge, this is the first time that a tissue-engineered human cornea (hTEC) entirely produced using normal untransformed human cells is used as a biomaterial to study the signal transduction pathways that are critical to corneal wound healing. Through the use of this biomaterial, we demonstrated that human corneal epithelial cells engaged in wound healing reduce phosphorylation of the signal transduction mediator CREB while, in the mean time, they increase that of AKT. By increasing the activation of AKT together with a decrease in CREB activation, we could considerably reduce wound closure time in our punch-damaged hTECs. Considering the increasing interest given to the reconstruction of different types of tissues, we believe these results will have a strong impact on the field of tissue-engineering and biomaterials. Altering the activation status of the Akt and CREB proteins might prove to be a therapeutically interesting avenue and may also find applications in wound healing of other tissues beside the cornea, such as the skin.

The tissue-engineered human cornea as a model to study expression of matrix metalloproteinases during corneal wound healing.

Corneal injuries remain a major cause of consultation in the ophthalmology clinics worldwide. Repair of corneal wounds is a complex mechanism that involves cell death, migration, proliferation, differentiation, and extracellular matrix (ECM) remodeling. In the present study, we used a tissue-engineered, two-layers (epithelium and stroma) human cornea as a biomaterial to study both the cellular and molecular mechanisms of wound healing. Gene profiling on microarrays revealed important alterations in the pattern of genes expressed by tissue-engineered corneas in response to wound healing. Expression of many MMPs-encoding genes was shown by microarray and qPCR analyses to increase in the migrating epithelium of wounded corneas. Many of these enzymes were converted into their enzymatically active form as wound closure proceeded. In addition, expression of MMPs by human corneal epithelial cells (HCECs) was affected both by the stromal fibroblasts and the collagen-enriched ECM they produce. Most of all, results from mass spectrometry analyses provided evidence that a fully stratified epithelium is required for proper synthesis and organization of the ECM on which the epithelial cells adhere. In conclusion, and because of the many characteristics it shares with the native cornea, this human two layers corneal substitute may prove particularly useful to decipher the mechanistic details of corneal wound healing.

Functional Impact of Collagens on the Activity Directed by the Promoter of the α5 Integrin Subunit Gene in Corneal Epithelial Cells.

PURPOSE: The early step of corneal wound healing is characterized by the massive production of fibronectin (FN), whose secretion is progressively replaced by collagens from the basal membrane as wound healing proceeds. Here, we examined whether expression of the gene encoding the α5 subunit from the FN-binding integrin α5ß1 changes as corneal epithelial cells (CECs) are cultured in the presence of collagen type I (CI) or type IV (CIV). METHODS: Responsiveness of the α5 gene toward collagen was determined by transfection of α5 promoter/chloramphenicol acetyltransferase (CAT) plasmids into rabbit and human CECs cultured on BSA or collagens. Electrophoretic mobility shift assays and Western blots were used to monitor the transcription factors required for basal α5 gene transcription in the presence of collagens. Gene profiling on microarrays was used to determine the impact of collagens on the patterns of genes expressed by CECs. RESULTS: All collagen types repressed the full-length α5/CAT promoter activity in confluent CECs. A moderate increase was observed in subconfluent rabbit CECs grown on CIV but not on CI. These collagen-dependent regulatory influences also correlated with alterations in the transcription factors Sp1/Sp3, NFI, and AP-1 that ensure α5 gene basal transcription. Microarray analyses revealed that CI more profoundly altered the pattern of genes expressed by human CECs than CIV. CONCLUSIONS: Collagens considerably suppressed α5 gene expression in CECs, suggesting that during wound healing, they may interfere with the influence FN exerts on CECs by altering their adhesive and migratory properties through a mechanism involving a reduction in α5 gene expression.