Identification of the role of DAB2 and CXCL8 in uterine spiral artery remodeling in early-onset preeclampsia.

Aberrant remodeling of uterine spiral arteries (SPA) is strongly associated with the pathogenesis of early-onset preeclampsia (EOPE). However, the complexities of SPA transformation remain inadequately understood. We conducted a single-cell RNA sequencing analysis of whole placental tissues derived from patients with EOPE and their corresponding controls, identified DAB2 as a key gene of interest and explored the mechanism underlying the communication between Extravillous trophoblast cells (EVTs) and decidual vascular smooth muscle cells (dVSMC) through cell models and a placenta-decidua coculture (PDC) model in vitro. DAB2 enhanced the motility and viability of HTR-8/SVneo cells. After exposure to conditioned medium (CM) from HTR-8/SVneoshNC cells, hVSMCs exhibited a rounded morphology, indicative of dedifferentiation, while CM-HTR-8/SVneoshDAB2 cells displayed a spindle-like morphology. Furthermore, the PDC model demonstrated that CM-HTR-8/SVneoshDAB2 was less conducive to vascular remodeling. Further in-depth mechanistic investigations revealed that C-X-C motif chemokine ligand 8 (CXCL8, also known as IL8) is a pivotal regulator governing the dedifferentiation of dVSMC. DAB2 expression in EVTs is critical for orchestrating the phenotypic transition and motility of dVSMC. These processes may be intricately linked to the CXCL8/PI3K/AKT pathway, underscoring its central role in intricate SPA remodeling.

Pregnancy-Specific Beta-1-Glycoprotein 1 Increases HTR-8/SVneo Cell Migration through the Orai1/Akt Signaling Pathway.

The impaired invasion ability of trophoblast cells is related to the occurrence of preeclampsia (PE). We previously found that pregnancy-specific beta-1-glycoprotein 1 (PSG1) levels were decreased in the serum of individuals with early-onset preeclampsia (EOPE). This study investigated the effect of PSG1 on Orai1-mediated store-operated calcium entry (SOCE) and the Akt signaling pathway in human trophoblast cell migration. An enzyme-linked immunosorbent assay (ELISA) was used to determine the level of PSG1 in the serum of pregnant women with EOPE. The effects of PSG1 on trophoblast proliferation and migration were examined using cell counting kit-8 (CCK8) and wound healing experiments, respectively. The expression levels of Orai1, Akt, and phosphorylated Akt (p-Akt) were determined through Western blotting. The results confirmed that the serum PSG1 levels were lower in EOPE women than in healthy pregnant women. The PSG1 treatment upregulated the protein expression of Orai1 and p-Akt. The selective inhibitor of Orai1 (MRS1845) weakened the migration-promoting effect mediated by PSG1 via suppressing the Akt signaling pathway. Our findings revealed one of the mechanisms possibly involved in EOPE pathophysiology, which was that downregulated PSG1 may reduce the Orai1/Akt signaling pathway, thereby inhibiting trophoblast migration. PSG1 may serve as a potential target for the treatment and diagnosis of EOPE.

Evaluation of serum levels of soluble vascular endothelial (sVE)-Cadherin in early- and late-onset preeclampsia serum sVE-Cadherin levels in preeclampsia.

OBJECTIVE: Endothelial dysfunction is a major feature of preeclampsia. sVE-cadherin plays a role in the preservation and regulation of the endothelial barrier. For that reason, to evaluation of sVE-cadherin may help elucidate the disease pathophysiology of preeclampsia. METHODS: The sample size was calculated as a minimum of 46 pregnant women for each group based on serum sVE-Cadherin levels in a pilot study of 10 preeclamptic and 10 control groups. Hundred-twenty pregnancies complicated with early-onset (n = 60) and late-onset (n = 60) preeclampsia were compared with 120 gestational-age (GA)-matched (±1 week) uncomplicated pregnancies. The venous blood sampling was performed upon preeclampsia diagnosis prior to the onset of the labor in the preeclampsia group and the matching (±1 week) pregnancy week in the control group. Demographic and biochemical parameters were evaluated. RESULTS: Mean serum sVE-Cadherin was significantly higher in women with EOPE compared to that of the GA-matched control group (5.86 ± 1.57 ng/mL vs. 2.28 ± 0.80 ng/mL, p < 0.001), in women with LOPE compared to that of the GA-matched control group (3.11 ± 0.97 ng/mL vs. 1.69 ± 0.87 ng/mL, p < 0.001), and in women with EOPE compared to that of LOPE group (5.86 ± 1.57 ng/mL vs. 3.11 ± 0.97 ng/mL, p < 0.001) after correction for GA. Serum sVE-Cadherin positively correlated with systolic and diastolic blood pressure and a negative correlation with gestational age at sampling. CONCLUSION: The serum level of sVE-Cadherin was higher in women with preeclampsia than that of GA-matched healthy pregnant women, in women with EOPE compared to that of LOPE. sVE-Cadherin is an important marker in early-onset pre-eclampsia with severe clinical findings.

Comparison of immune-related gene signatures and immune infiltration features in early- and late-onset preeclampsia.

BACKGROUND: Preeclampsia, a severe pregnancy syndrome, is widely accepted divided into early- and late-onset preeclampsia (EOPE and LOPE) based on the onset time of preeclampsia, with distinct pathophysiological origins. However, the molecular mechanism especially immune-related mechanisms for EOPE and LOPE is currently obscure. In the present study, we focused on placental immune alterations between EOPE and LOPE and search for immune-related biomarkers that could potentially serve as potential therapeutic targets through bioinformatic analysis. METHODS: The gene expression profiling data was obtained from the Gene Expression Omnibus database. ESTIMATE algorithm and Gene Set Enrichment Analysis were employed to evaluate the immune status. The intersection of differentially expressed genes in GSE74341 series and immune-related genes set screened differentially expressed immune-related genes. Protein-protein interaction network and random forest were used to identify hub genes with a validation by a quantitative real-time PCR. Kyoto Encyclopedia of Genes and Genomes pathways, Gene Ontology and gene set variation analysis were utilized to conduct biological function and pathway enrichment analyses. Single-sample gene set enrichment analysis and CIBERSORTx tools were employed to calculate the immune cell infiltration score. Correlation analyses were evaluated by Pearson correlation analysis. Hub genes-miRNA network was performed by the NetworkAnalyst online tool. RESULTS: Immune score and stromal score were all lower in EOPE samples. The immune system-related gene set was significantly downregulated in EOPE compared to LOPE samples. Four hub differentially expressed immune-related genes (IL15, GZMB, IL1B and CXCL12) were identified based on a protein-protein interaction network and random forest. Quantitative real-time polymerase chain reaction validated the lower expression levels of four hub genes in EOPE compared to LOPE samples. Immune cell infiltration analysis found that innate and adaptive immune cells were apparent lacking in EOPE samples compared to LOPE samples. Cytokine-cytokine receptor, para-inflammation, major histocompatibility complex class I and T cell co-stimulation pathways were significantly deficient and highly correlated with hub genes. We constructed a hub genes-miRNA regulatory network, revealing the correlation between hub genes and hsa-miR-374a-5p, hsa-miR-203a-3p, hsa-miR-128-3p, hsa-miR-155-3p, hsa-miR-129-2-3p and hsa-miR-7-5p. CONCLUSIONS: The innate and adaptive immune systems were severely impaired in placentas of EOPE. Four immune-related genes (IL15, GZMB, IL1B and CXCL12) were closely correlated with immune-related pathogenesis of EOPE. The result of our study may provide a new basis for discriminating between EOPE and LOPE and acknowledging the role of the immune landscape in the eventual interference and tailored treatment of EOPE.

Placental progesterone and its receptor in HIV-infected pre-eclamptic women.

Given the high prevalence of HIV infection and pre-eclampsia (PE) in South Africa, this study evaluated and compared the placental immunostaining of progesterone (P) and progesterone receptors (PR) in the synergy of HIV-infected PE compared to normotensive pregnant women using immunohistochemistry interfaced with morphometric image analysis. Progesterone immunostaining was expressed widely across exchange and conducting villi within mesenchymal, endothelial, and trophoblast cells. In contrast, PR was expressed within syncytiotrophoblasts and was absent within endothelial cells. In exchange villi, P and PR immuno-expression was significantly lower in PE compared to the normotensive group (p = < 0.0001 and p = < 0.0001, respectively) and within the early-onset pre-eclampsia (EOPE) compared to the late-onset pre-eclampsia (LOPE) group (p = < 0.0001 and p = < 0.0001, respectively). Progesterone immuno-expression was significantly lower in the HIV+ compared to the HIV- group (p = < 0.0001), whilst PR was non-significant. In conducting villi, P and PR immuno-expression was significantly lower in the EOPE compared to the LOPE group (p = < 0.0001 and p = < 0.0001, respectively) and in the HIV+ compared to the HIV- group (p = < 0.0001 and p = 0.0009, respectively). Progesterone immuno-expression was slightly higher in the PE compared to normotensive group, and PR immuno-expression was non-significant. There was a significant difference between P and PR within exchange versus conducting villi regardless of pregnancy type, with villi type accounting for 34.47% and 15.28% of total variance for P and PR, respectively. Placental P and PR immuno-expression is downregulated in the duality of PE and HIV+ infection. The use of combined antiretroviral therapy (cART) may result in defective P synthesis, which causes insufficient binding to its receptors. Consequently, PI3K/AKT, JAK-STAT, and MAPK signalling pathways are affected, impairing trophoblast invasion and leading to pre-eclampsia development. Notably, the decrease in P and PR immuno-expression in EOPE validates their effect on placentation.

Are Vascular Endothelium and Angiogenesis Effective MicroRNA Biomarkers Associated with the Prediction of Early-Onset Preeclampsia (EOPE) and Adverse Perinatal Outcomes?

MicroRNA is associated with angiogenesis, invasion, proliferation, and vascular endothelial remodeling of various diseases. We aimed to investigate serum MicroRNA (miRNA) levels in preeclampsia (PE) and to determine whether any changes in miRNA levels are useful in predicting early onset preeclampsia (EOPE) and adverse perinatal outcomes. A total of 89 pregnant patients were enrolled in this prospective case-control study (55 PE and 34 healthy controls). miR-17, miR-20a, miR-20b, miR126, miR155, miR-200, miR-222, and miR-210 levels were studied in maternal serum in preeclamptic pregnant women. Multiple logistic regression analyses analyzed the risk factors which are associated with EOPE and adverse maternal outcomes. The Real-time RT-PCR method was used to determine maternal serum miRNA levels. Serum miR-17, miR-20a, miR-20b, miR126, and miR-210 levels were significantly higher in PE than the control group (p < .001, p < .001, p < .001, p < .001 and p = .047 respectively). Increased miR-17, miR-20a, and miR-20b levels were independently associated with PE (OR: 0.642, 95%Cl: 0.486-0.846, p = .002; OR: 0.899, 95%Cl: 0.811-0.996, p = .042 and OR: 0.817, 95%Cl: 0.689-0.970, p = .021). Increased miR-17 and miR-126 levels were negatively correlated with serum EOPE in PE (r = -.313, p = .020), and increased miR-210 levels were significantly positively correlated with EOPE in PE (r = .285, p = .005). Increased expression of serum miR-17, miR-20a, miR-20b, miR126, and miR-210 were found to be associated with PE, also increased expression of miR-17, miR-20a, and miR-20b were to be predicted with PE, also increased maternal serum miR-17 and miR-126 expressions were negatively correlated and increased miR-210 expression was positively correlated with EOPE in PE women.

MicroRNA miR-27a as a possible regulator of anti-inflammatory macrophage phenotype in preeclamptic placenta.

INTRODUCTION: The role of the TGFß signaling pathway, an important cascade responsible for the anti-inflammatory polarization of macrophages, in the development of both early- and late-onset preeclampsia (eoPE and loPE), remains poorly understood. In this study, we examined the components of the TGFß signaling cascade and macrophage markers within placental tissue in normal pregnancy and in PE. METHODS: Patients with eoPE, loPE, and normal pregnancy were enrolled in the study (n = 10 in each group). Following techniques were used for the investigation: immunohistochemistry analysis, western blotting, qRT-PCR, isolation of monocytes by magnetic sorting, transfection, microRNA sequencing, and bioinformatic analysis. RESULTS: We observed a significant decrease in the anti-inflammatory macrophage marker CD206 in the loPE group, alongside with a significant down-regulation of CD206 protein production in both eoPE and loPE groups. The level of CD68-positive cells and relative levels of CD163 and MARCO production were comparable across the groups. However, we identified a significant decrease in the TGFß receptor 2 production and its gene expression in the PE group. Further analysis revealed a link between TGFBR2 and MRC1 (CD206) genes through a single miRNA, hsa-miR-27a-3p. Transfecting CD14-derived macrophages with the hsa-miR-27a-3p mimic significantly changed TGFBR2 production, indicating the potential role of this miRNA in regulating the TGFß signaling pathway. We also revealed the up-regulation of hsa-miR-27a-5p and hsa-miR-27a-3p in the trophoblast BeWo b30 cell line under the severe hypoxia condition and the fact that TGFBR2 3' UTR could serve as a potential target for these miRNAs. DISCUSSION: Our findings uncover a novel potential therapeutic target for managing patients with PE, significantly contributing to a deeper comprehension of the underlying mechanisms involved in the development of this pathology.

The diagnostic protocol for hereditary spherocytosis-2021 update.

BACKGROUND: Hereditary spherocytosis (HS), a commonly encountered hereditary hemolytic disease, is mostly inherited in an autosomal dominant manner. The clinical manifestations in patients with HS show obvious heterogeneity. Moreover, the sensitivity or specificity of some HS diagnostic tests are not ideal and may easily result in misdiagnosis or missed diagnosis in some patients. The objective of this study was to propose a simple and practical diagnostic protocol, which can contribute to the diagnosis of HS and its differential diagnosis with different types of hemolytic anemia such as thalassemia (THAL), autoimmune hemolytic anemia (AIHA), and glucose-6-phosphate dehydrogenase (G6PD) deficiency, thus, to provide an alternative simple and reliable method for better clinical diagnosis of HS. METHODS: Through combing our research with existing experimental technologies and studies, we propose a simple and practical protocol for HS diagnosis, which will help clinicians to improve HS diagnosis. RESULTS: Compared with the existing HS diagnostic protocols, the HS diagnostic protocol we proposed is simpler. In this new protocol, some experimental tests with ideal diagnostic efficiency are added, such as mean reticulocyte volume (MRV), mean sphered cell volume (MSCV), mean corpuscular volume (MCV), in combination with the observation of clinical manifestations, family investigation, routine tests for hemolytic anemia, genetic testing, and other screening tests. CONCLUSION: The HS diagnostic protocol we proposed could improve the clinical practice and efficiency of HS diagnosis.

Exponential Amplification Using Photoredox Autocatalysis.

Exponential molecular amplification such as the polymerase chain reaction is a powerful tool that allows ultrasensitive biodetection. Here, we report a new exponential amplification strategy based on photoredox autocatalysis, where eosin Y, a photocatalyst, amplifies itself by activating a nonfluorescent eosin Y derivative (EYH3-) under green light. The deactivated photocatalyst is stable and rapidly activated under low-intensity light, making the eosin Y amplification suitable for resource-limited settings. Through steady-state kinetic studies and reaction modeling, we found that EYH3- is either oxidized to eosin Y via one-electron oxidation by triplet eosin Y and subsequent 1e-/H+ transfer, or activated by singlet oxygen with the risk of degradation. By reducing the rate of the EYH3- degradation, we successfully improved EYH3--to-eosin Y recovery, achieving efficient autocatalytic eosin Y amplification. Additionally, to demonstrate its flexibility in output signals, we coupled the eosin Y amplification with photoinduced chromogenic polymerization, enabling sensitive visual detection of analytes. Finally, we applied the exponential amplification methods in developing bioassays for detection of biomarkers including SARS-CoV-2 nucleocapsid protein, an antigen used in the diagnosis of COVID-19.

Biochemical, Cellular, and Proteomic Characterization of Hereditary Spherocytosis Among Tunisians.

BACKGROUND/AIMS: Hereditary Spherocytosis (HS) is the most common erythrocyte membrane disorder causing hemolytic anemia. The wide heterogeneity of both clinical and laboratory manifestations of HS contributes to difficulties associated with the diagnosis of this disorder. Although massive data previously reported worldwide, there is yet no data on HS among the Tunisian population. Here we aim to characterize HS in Tunisian patients at biochemical and cellular levels, identify the membrane protein deficiency, and compare the accuracy of the diagnostic tests to identify the most appropriate assay for HS diagnosis. METHODS: We investigated 81 patients with hemolytic anemia and 167 normal controls. The exploration of HS based on clinical and family history, physical examination, and the results of laboratory tests: blood smear, osmotic fragility test (OFT), cryohemolysis test (CT), pink test (PT), eosine-5'-maleimide (EMA) test, and erythrocyte membrane protein electrophoresis. RESULTS: We identified 21 patients with HS, classified as severe (6/21;28.5%), moderate (10/21;47.6%), and mild (5/21;23.8%). The most prevalent protein deficiency was the band 3 protein detected in ten Tunisian HS patients. The EMA test showed a high specificity (97.5%) and sensitivity (94.7%) for HS diagnosis compared to the other screening tests. Interestingly, fourteen among sixteen patients presenting with homozygous sickle cells HbSS showed an increase of EMA fluorescence intensity compared to other anemic patients. CONCLUSION: Our study highlights the efficiency of the EMA dye for the detection of HS whatever the nature of the involved protein deficiency. We report for the first time, the most prevalent protein deficiency among Tunisians with HS. Moreover, we found that the combination of the EMA-binding test with PT or incubated OFT improves the diagnosis sensitivity while maintaining a good specificity.

A clinical and experimental study of adult hereditary spherocytosis in the Chinese population.

Hereditary spherocytosis (HS) is often misdiagnosed due to lack of specific diagnostic methods. Our study summarized clinical characteristics and described the diagnostic workflow for mild and moderate HS in Chinese individuals, using data from 20 adults, 8 of whom presented a familial history for HS. We used scanning electron microscopy (SEM) to diagnose HS. We observed reduced eosin maleimide fluorescence activity (5.50 mean channel fluorescence (MCF) units) in the 10 cases of HS, which differed significantly when compared with 10 normal adults (15.50 units), iron deficiency anemia (15.50 MCF units), and megaloblastic anemia (12.00 MCF units) values (P < .05). Next generation sequencing results revealed that 9 out of 10 patients were found to have mutations in the spectrin alpha chain (SPTB), anchor protein (ANK1), and SLC4A1 genes. These mutations were not reported in the Human Gene Mutation Database (HGMD), 1000 human genome, ExAC, and dbSNP147 databases. Splenectomy proved to be beneficial in alleviating HS symptoms in 10 cases. It was found that for the diagnosis of HS, SEM and next generation gene sequencing method proved to be more ideal than red blood cell membrane protein analysis using sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting.

Flow cytometric osmotic fragility test and eosin-5'-maleimide dye-binding tests are better than conventional osmotic fragility tests for the diagnosis of hereditary spherocytosis.

INTRODUCTION: Hereditary spherocytosis (HS) is the most common inherited hemolytic anemia with heterogeneous clinico-laboratory manifestations. We evaluated the flow-cytometric tests: eosin-5'-maleimide (EMA) and flow-cytometric osmotic fragility test (FOFT) and the conventional osmotic fragility tests (OFT) for the diagnosis of hereditary spherocytosis (HS). METHODS: One hundred two suspected HS patients underwent EMA, FOFT, incubated OFT (IOFT), and room temperature OFT (RT-OFT). In addition, 10 cases of immune hemolytic anemia (IHA) were included, and performance of the above 4 tests was evaluated. For EMA and FOFT, 5 normal controls were assessed together with the patients and cutoffs were calculated using receiver-operator-characteristics curve (ROC) analysis. RESULTS: The best cutoff for %EMA decrease was 12.5%, and for FOFT, %residual red cells (%RRC) was 25.6%. The sensitivity and specificity of RT-OFT was 62.06% and 86.3%, respectively, while that of IOFT was 79.31% and 87.67%, respectively. Both flow cytometric tests performed better. Sensitivity and specificity of EMA was 86.2% and 93.9% respectively, and that of FOFT was 96.6% and 98.63%, respectively. The combination of the FOFT with IOFT or EMA dye-binding test yields a sensitivity of 100%, but with EMA, it had a higher specificity. Hb/MCHC was a predictor of the severity of the disease while %EMA decrease and %RRC did not correlate with severity of the disease. CONCLUSION: Flow-cytometric osmotic fragility test is the best possible single test followed by EMA for diagnosis of HS. A combination of FOFT and EMA can correctly diagnose 100% patients. These tests are likely to replace conventional OFTs in future.

Quirks of dye nomenclature. 10. Eosin Y and its close relatives.

The long history of eosin Y, eosin B and the methyl and ethyl eosins is recounted as well as their synthesis, the variety of their molecular species and some of the myriad applications of these dyes. Chromatographic techniques are described that reveal the purity or lack of it in commercial samples. Toxicological studies are discussed that suggest that the eosins are virtually non toxic, but efforts to remove them from the environment imply that there may be some risk.

[THE CYTOMETRIC TECHNIQUE OF BINDING OF EOSIN-5-MALEIMIDE IN DIAGNOSTIC OF INHERENT SPHEROCYTOSIS].

The laboratory diagnostic of inherent spherocytosis is based on detection of spherocytes in peripheral blood, decreasing of index of sphericity, decreasing of osmotic resistance of erythrocytes. The new test of diagnostic of hereditary spherocytosis build on molecular defect was developed on the basis of binding extracellular fragments of protein of band 3 with eosin-5-maleimide (EMA-test). The study was carried out to implement comparative analysis of sensitivity and specificity of techniques applied to diagnose inherent spherocytosis. The sampling of 94 patients with various forms of anemias was analyzed All patients were applied complex clinical laboratory examination including analysis of osmotic resistance of erythrocytes, erythrocytometry and EMA-test as specific techniques of diagnostic of inherent spherocytosis. In 51 out of 94 patients (54%) decreasing of values of EMA-test was detected and in 47 patients diagnosis of inherent spherocytosis was confirmed. The standard values of EMA-test were established in 43 patients (46%) and 12 patients out of them with established diagnosis of inherent spherocytosis. Therefore, sensitivity of EMA-test made up to 79% and specificity - 80%. The most sensitive techniques of diagnostic remain osmotic resistance of erythrocytes (91%) and index of sphericity (up to 96%). But the highest specificity in this respect has EMA-test (80%). Nowadays, none of implemented techniques of diagnostic of inherent spherocytosis can be applied as a universal one. The implementation of complex examination is needed for proper diagnostic of disease.

Delayed Measurement of Eosin-5'-Maleimide Binding May Affect the Test Results of Highly Hemolyzed Samples In Vivo and In Vitro-A Case Study.

Diagnosis of hereditary spherocytosis (HS) is based on clinical evaluation and eosin-5'-maleimide (EMA) test. A decrease in EMA fluorescence compared with healthy individuals is typical for HS and serves as a basis for HS diagnosis. Sensitivity and specificity of the test is high and false-positive results rarely occur. Studies have shown that anticoagulated blood sample when stored at 4°C for 7 days do not affect the test results. This case study is about an autoimmune hemolytic anemia patient who showed a primary positive result for EMA test (decrease in EMA fluorescence-47% compared with 100% for samples of healthy individual), when the test was performed in the sample stored for 48 hours after venipuncture and before staining. An irrelevant decrease (92.5% compared with 100% for samples of healthy individual) was found when freshly collected sample was analyzed. On the basis of the results obtained, it is recommended that EMA staining should be performed on the same day of blood collection for patients with significant hemolysis.