A cross-sectional study of COVID-19 vaccination patterns among patients with epilepsy in Hong Kong.

OBJECTIVE: As Hong Kong faced the 5th wave of the COVID-19 pandemic, the facilitators and hurdles toward effective vaccination is important for healthcare professionals to understand the vaccination gap among patients with epilepsy. METHODS: A cross-sectional, pragmatic study of COVID-19 vaccination was performed at a tertiary epilepsy center with regards to patterns of vaccination and any unusually high rate of adverse events. Patients having recent visits at the epilepsy center (4 months) had their anonymized electronic linkage records examined 12 months after the inception of vaccination program for types of vaccines, seizure demographics, and adverse events following immunization (AEFI). RESULTS: A total of 200 patients with epilepsy and their anonymized data were analyzed. The vaccine uptake was approximately 60% of that of the general population. Twice as many patients with epilepsy chose to receive mRNA vaccine as compared with inactivated vaccine. The proportion of patients who kept up-to-date with all available dosing was 7%. Patients with epilepsy with genetic etiology were least likely to receive vaccination (13/38, 34%, P = .02). There was no unreasonably high rate of unacceptable side effects after vaccination among patients with epilepsy. Only 3 patients reported worsening of seizures without meeting the criteria for AEFI. Refractory epilepsy, allergy to antiseizure medications and elder age (≥65) did not confer any significant difference in vaccination patterns or adverse effects. SIGNIFICANCE: A vaccination gap exists among epilepsy patients which calls for actionable strategies for improving vaccine uptake, including education and outreach programs.

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy.

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented in human embryos. Preimplantation genetic testing for aneuploidy (PGT-A) is a genetic test that significantly improves reproductive outcomes by detecting chromosomal abnormalities of embryos. Next-generation sequencing (NGS) provides a high-throughput and cost-effective approach for genetic analysis and has shown clinical applicability in PGT-A. Here, we present a rapid and low-cost semiconductor sequencing-based NGS method for screening of aneuploidy in embryos. The first step of the workflow is whole genome amplification (WGA) of the biopsied embryo specimen, followed by construction of sequencing library, and subsequent sequencing on the semiconductor sequencing system. Generally, for a PGT-A application, 24 samples can be loaded and sequenced on each chip generating 60-80 million reads at an average read length of 150 base pairs. The method provides a refined protocol for performing template amplification and enrichment of sequencing library, making the PGT-A detection reproducible, high-throughput, cost-efficient, and timesaving. The running time of this semiconductor sequencer is only 2-4 hours, shortening the turnaround time from receiving samples to issuing reports into 5 days. All these advantages make this assay an ideal method to detect chromosomal aneuploidies from embryos and thus, facilitate its wide application in PGT-A.

A prospective study of non-invasive preimplantation genetic testing for aneuploidies (NiPGT-A) using next-generation sequencing (NGS) on spent culture media (SCM).

PURPOSE: This study was to evaluate if spent culture media (SCM) of embryos could be used as a non-invasive tool to achieve aneuploidy screening. Ploidy calls, as well as concordance rates between PGT-A results from trophectoderm (TE) and SCM, were compared. Clinical outcomes of single euploid transfers were also evaluated. METHODS: The study was conducted from March 2017 to June 2018 in a university-based ART center. SCM of day 3 to the day(s) of TE biopsy of all biopsied blastocysts were collected for testing. PGT-A results of SCM were compared with the standard results of TE, with clinical relevance and outcomes examined. RESULTS: NiPGT-A using SCM gave a sensitivity of 81.6%, specificity of 48.3%, positive predictive value of 82.6%, and negative predictive value of 46.7% in ploidy calling. The concordance rates for autosomes and sex determination were 62.1% and 82.4%, respectively. There were 14 single embryo transfer cycles of euploids as determined by TE biopsy. Clinical outcomes not only confirmed 3 false positive results from SCM but also reflected the true ploidy status of the transferred embryo in one case. If ploidy calls were dichotomized without mosaic embryos, the sensitivity and NPV would increase to 91.0% and 66.7% (p = 0.60 and p = 0.25), respectively. CONCLUSIONS: Cell-free DNA found in SCM could provide ploidy information of an embryo as in PGT-A from its TE. Given its potential to reflect the comprehensive chromosomal profile of the whole embryo, more research based on clinical outcomes is required to determine if SCM could be a reliable selection tool in PGT-A.

Green tea epigallocatechin-3-gallate inhibits angiogenesis and suppresses vascular endothelial growth factor C/vascular endothelial growth factor receptor 2 expression and signaling in experimental endometriosis in vivo.

OBJECTIVE: To investigate the antiangiogenesis mechanism of epigallocatechin-3-gallate (EGCG) in an endometriosis model in vivo. DESIGN: Animal studies. SETTING: University laboratory. ANIMAL(S): Human endometrium from women with endometriosis (n = 10) was transplanted into immunocompromised mice. INTERVENTION(S): Mice (n = 30) were randomly treated with EGCG, vitamin E (antioxidant control), or vehicle (negative control) for microvessel imaging. MAIN OUTCOME MEASURE(S): Endometriotic implants were collected for angiogenesis microarray and pathway analysis. Differentially expressed angiogenesis molecules were confirmed by quantitative polymerase chain reaction, Western blot, and immunohistochemistry. Effects of EGCG on angiogenesis signal transduction were further characterized in a human endothelial cell line. Microvessel parameters and the angiogenesis signaling pathway in endometriotic implants and endothelial cells were studied. RESULT(S): EGCG, but not vitamin E, inhibited microvessels in endometriotic implants. EGCG selectively suppressed vascular endothelial growth factor C (VEGFC) and tyrosine kinase receptor VEGF receptor 2 (VEGFR2) expression. EGCG down-regulated VEGFC/VEGFR2 signaling through c-JUN, interferon-γ, matrix metalloproteinase 9, and chemokine (C-X-C motif) ligand 3 pathways for endothelial proliferation, inflammatory response, and mobility. EGCG also suppressed VEGFC expression and reduced VEGFR2 and ERK activation in endothelial cells. VEGFC supplementation attenuated the inhibitory effects by EGCG. CONCLUSION(S): EGCG inhibited angiogenesis and suppressed VEGFC/VEGFR2 expression and signaling pathway in experimental endometriosis in vivo and endothelial cells in vitro.

Monoamine oxidase a expression is vital for embryonic brain development by modulating developmental apoptosis.

Monoamine oxidases (MAO-A, MAO-B) metabolize biogenic amines and have been implicated in neuronal apoptosis. Although apoptosis is an important process in embryo development, the role of MAO isoenzymes has not been investigated in detail. We found that expression of MAO-A and MAO-B can be detected early on during embryo development. Expression levels remained constant until around midgestation but then dropped to almost undetectable levels toward birth. Similar expression kinetics were observed in the brain. Isoform-specific expression silencing of MAO-A mediated by siRNA during in vitro embryogenesis induced developmental defects, as indicated by a reduction of the crown rump length and impaired cerebral development. These alterations were paralleled by elevated serotonin levels. Similar abnormalities were observed when embryos were cultured in the presence of the MAO-A inhibitor clorgyline or when the transcriptional inhibitor of MAO-A expression R1 was overexpressed. In contrast, no such alterations were detected when expression of MAO-B was knocked down. To explore the underlying mechanisms for the developmental abnormalities in MAO-A knockdown embryos, we quantified the degree of developmental apoptosis in the developing brain. MAO-A knockdown reduced the number of apoptotic cells in the neuroepithelium, which coincided with impaired activation of caspases 3 and 9. Moreover, we observed reduced cyclin D1 levels as an indicator of impaired cell proliferation in MAO-A knockdown embryos. This data highlights MAO-A as a vital regulator of embryonic brain development.

Association of hemopexin in tear film and conjunctival macrophages with vernal keratoconjunctivitis.

OBJECTIVE: Vernal keratoconjunctivitis (VKC) is a chronic allergic inflammatory disease with unclear etiology and pathogenesis. We investigated the tear film proteome of patients with VKC to understand the pathologic characteristics of VKC. METHODS: Tear samples were collected from healthy volunteers and patients with VKC. Electrophoresis was performed to display the tear proteomic profiles according to VKC severity. The identities of differentially expressed proteins were analyzed by mass spectrometry and quantified by enzyme-linked immunosorbent assay. Impression cytology was performed on VKC conjunctival samples to demonstrate the cellular protein expression. Allergic sensitization was performed in mice to study the pathologic role of these proteins in VKC. RESULTS: Hemopexin, an inflammatory protein, was elevated in the tear film of patients with VKC. The increased hemopexin concentration in VKC tears was significantly associated with disease severity. Impression cytology showed specific high hemopexin expression in dekeratinized conjunctival epithelium and necrotic macrophages in patients with VKC. Immunohistochemical examination of normal lacrimal tissues from mice showed that hemopexin was not expressed in any lacrimal apparatus. Under systemic and topical sensitization and challenge using hemopexin in mice, the affected eye had mild to moderate bead discharge, chemosis, and edema with excessive macrophage infiltration and conjunctival necrosis. CONCLUSION: An association exists between tear hemopexin and the development and pathologic effects of VKC. CLINICAL RELEVANCE: Increased hemopexin may have a role in the development of VKC.

The effect of glucocorticoids on tendon cell viability in human tendon explants.

BACKGROUND AND PURPOSE: Previous studies on the culture of human tenocytes have shown that dexamethasone and triamcinolone reduce cell viability, suppress cell proliferation, and reduce collagen synthesis. However, such cell cultures lack the extracellular matrix and three-dimensional structure of normal tendons, which affects their response to stimuli. We established a human tendon explant culture system and tested the effects of dexamethasone and triamcinolone on cell viability. METHODS: Primary human tendon explant cultures were prepared from healthy hamstring tendons. Tendon strips were harvested from hamstring tendons and cultured in 24-well plates in Dulbecco's modification of Eagle's Medium (DMEM) supplemented with 2% fetal calf serum. The tendon explants were treated with 0 microM (control), 10 microM, or 100 microM dexamethasone sodium phosphate or 0 microM (control), 10 microM, or 100 microM triamcinolone acetonide in DMEM for 96 h. Cell viability was measured by Alamar blue assay before and after glucocorticoid treatment. RESULTS: Incubation with 10 microM and 100 microM dexamethasone reduced cell viability in human tendon explants by 35% and 45%, respectively, as compared to a 6% increase in the controls (p = 0.01, mixed-effects ANOVA). Triamcinolone at 10 microM and 100 microM reduced cell viability by 33% and 36%, respectively, as compared to a 9% increase in the controls (p = 0.07, mixed-effects ANOVA). INTERPRETATION: Human tendon explant cultures can be used to study the effects of glucocorticoids on human tendon. Dexamethasone and triamcinolone suppress the cell viability of human tendon in its natural 3-dimensional environment with matrix anchorage. Human tendon explant cultures provide a species-specific model for further investigation of the effects of glucocorticoids on the metabolism of the extracellular matrix of human tendon, and on its mechanical properties.

Innate immune response by ficolin binding in apoptotic placenta is associated with the clinical syndrome of preeclampsia.

BACKGROUND: Unidentified circulating factors derived from placenta are thought to be responsible for the exaggerated systemic inflammation leading to preeclampsia. Our aim was to identify the circulating factors present in preeclampsia and to investigate their relationship to the underlying systemic immune response responsible for the associated clinical manifestations. METHODS: We obtained blood samples from pregnant women with and without preeclampsia and performed comparative proteomic analyses to identify the abnormal circulating factors by 2-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time of flight for protein separation and identification. In placentas from preeclamptic pregnancies, we evaluated the potential role of the candidate proteins identified by Western and immunohistochemical analysis. We also used proinflammatory cytokine antibody arrays to investigate local and systemic immune responses. RESULTS: We found that ficolins, the pattern-recognition proteins involved in the lectin-complement pathway, were differentially expressed in plasma from preeclamptic pregnancies. Ficolins were present in low concentrations in plasma but at high concentrations in the placenta, particularly in syncytiotrophoblasts undergoing apoptosis. The binding of ficolins in apoptotic trophoblasts induced innate immunity through local and systemic cytokine activation and correlated with the clinical manifestation of preeclampsia. CONCLUSIONS: We identified specific in vivo circulating factors derived from the placenta that are responsible for the local immune recognition and systemic inflammatory response in the development of clinical manifestations of preeclampsia. These findings may have predictive value and also therapeutic applications to lessen adverse clinical outcomes of preeclampsia.