Cilia Proteins are Biomarkers of Altered Flow in the Vasculature

Cilia, microtubule-based organelles that project from the apical luminal surface of endothelial cells (ECs), are widely regarded as low-flow sensors. Previous reports suggest that upon high shear stress, cilia on the EC surface are lost, and more recent evidence suggests that deciliation—the physical removal of cilia from the cell surface—is a predominant mechanism for cilia loss in mammalian cells. Thus, we hypothesized that EC deciliation facilitated by changes in shear stress would manifest in increased abundance of cilia-related proteins in circulation. To test this hypothesis, we performed shear stress experiments that mimicked flow conditions from low to high shear stress in human primary cells and a zebrafish model system. In the primary cells, we showed that upon shear stress induction, indeed, ciliary fragments were observed in the effluent in vitro, and effluents contained ciliary proteins normally expressed in both endothelial and epithelial cells. In zebrafish, upon shear stress induction, fewer cilia-expressing ECs were observed. To test the translational relevance of these findings, we investigated our hypothesis using patient blood samples from sickle cell disease and found that plasma levels of ciliary proteins were elevated compared with healthy controls. Further, sickled red blood cells demonstrated high levels of ciliary protein (ARL13b) on their surface after adhesion to brain ECs. Brain ECs postinteraction with sickle RBCs showed high reactive oxygen species (ROS) levels. Attenuating ROS levels in brain ECs decreased cilia protein levels on RBCs and rescued ciliary protein levels in brain ECs. Collectively, these data suggest that cilia and ciliary proteins in circulation are detectable under various altered-flow conditions, which could serve as a surrogate biomarker of the damaged endothelium

Evaluation of commercially available fully automated and ELISA-based assays for detecting anti-SARS-CoV-2 neutralizing antibodies

Rapid and accurate measurement of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)-specific neutralizing antibodies (nAbs) is paramount for monitoring immunity in infected and vaccinated subjects. The current gold standard relies on pseudovirus neutralization tests which require sophisticated skills and facilities. Alternatively, recent competitive immunoassays measuring anti-SARS-CoV-2 nAbs are proposed as a quick and commercially available surrogate virus neutralization test (sVNT). Here, we report the performance evaluation of three sVNTs, including two ELISA-based assays and an automated bead-based immunoassay for detecting nAbs against SARS-CoV-2. The performance of three sVNTs, including GenScript cPass, Dynamiker, and Mindray NTAb was assessed in samples collected from SARS-CoV-2 infected patients (n = 160), COVID-19 vaccinated individuals (n = 163), and pre-pandemic controls (n = 70). Samples were collected from infected patients and vaccinated individuals 2–24 weeks after symptoms onset or second dose administration. Correlation analysis with pseudovirus neutralization test (pVNT) and immunoassays detecting anti-SARS-CoV-2 binding antibodies was performed. Receiver operating characteristic (ROC) curve analysis was generated to assess the optimal threshold for detecting nAbs by each assay. All three sVNTs showed an excellent performance in terms of specificity (100%) and sensitivity (100%, 97.0%, and 97.1% for GenScript, Dynamiker, and Mindray, respectively) in samples collected from vaccinated subjects. GenScript demonstrated the strongest correlation with pVNT (r = 0.743, R2 = 0.552), followed by Mindray (r = 0.718, R2 = 0.515) and Dynamiker (r = 0.608, R2 = 0.369). Correlation with anti-SARS-CoV-2 binding antibodies was variable, but the strongest correlations were observed between anti-RBD IgG antibodies and Mindray (r = 0.952, R2 = 0.907). ROC curve analyses demonstrated excellent performance for all three sVNT assays in both groups, with an AUC ranging between 0.99 and 1.0 (p < 0.0001). Also, it was shown that the manufacturer's recommended cutoff values could be modified based on the tested cohort without significantly affecting the sVNT performance. The sVNT provides a rapid, low-cost, and scalable alternative to conventional neutralization assays for measuring and expanding nAbs testing across various research and clinical settings. Also, it could aid in evaluating actual protective immunity at the population level and assessing vaccine effectiveness to lay a foundation for boosters' requirements.Funding was provided by Qatar Foundation (Grant number: UREP28-173-3-057), Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China, Qatar University (Grant number: RRC-2-032)

Assessment of Broadly Reactive Responses in Patients With MERS-CoV Infection and SARS-CoV-2 Vaccination

Importance: In the ongoing COVID-19 pandemic, there remain unanswered questions regarding the nature and importance of the humoral immune response against other coronaviruses. Although coinfection of the Middle East respiratory syndrome coronavirus (MERS-CoV) with the SARS-CoV-2 has not been documented yet, several patients previously infected with MERS-CoV received the COVID-19 vaccine; data describing how preexisting MERS-CoV immunity may shape the response to SARS-CoV-2 following infection or vaccination are lacking. Objective: To characterize the cross-reactive and protective humoral responses in patients exposed to both MERS-CoV infection and SARS-CoV-2 vaccination. Design, Setting, and Participants: This cohort study involved a total of 18 sera samples collected from 14 patients with MERS-CoV infection before (n = 12) and after (n = 6) vaccination with 2 doses of COVID-19 mRNA vaccine (BNT162b2 or mRNA-1273). Of those patients, 4 had prevaccination and postvaccination samples. Antibody responses to SARS-CoV-2 and MERS-CoV were assessed as well as cross-reactive responses to other human coronaviruses. Main Outcomes and Measures: The main outcomes measured were binding antibody responses, neutralizing antibodies, and antibody-dependent cellular cytotoxicity (ADCC) activity. Binding antibodies targeting SARS-CoV-2 main antigens (spike [S], nucleocapsid, and receptor-binding domain) were detected using automated immunoassays. Cross-reactive antibodies with the S1 protein of SARS-CoV, MERS-CoV, and common human coronaviruses were analyzed using a bead-based assay. Neutralizing antibodies (NAbs) against MERS-CoV and SARS-CoV-2 as well as ADCC activity against SARS-CoV-2 were assessed. Results: A total of 18 samples were collected from 14 male patients with MERS-CoV infection (mean [SD] age, 43.8 [14.6] years). Median (IQR) duration between primary COVID-19 vaccination and sample collection was 146 (47-189) days. Prevaccination samples had high levels of anti-MERS S1 immunoglobin M (IgM) and IgG (reactivity index ranging from 0.80 to 54.7 for IgM and from 0.85 to 176.3 for IgG). Cross-reactive antibodies with SARS-CoV and SARS-CoV-2 were also detected in these samples. However, cross-reactivity against other coronaviruses was not detected by the microarray assay. Postvaccination samples showed significantly higher levels of total antibodies, IgG, and IgA targeting SARS-CoV-2 S protein compared with prevaccination samples (eg, mean total antibodies: 8955.0 AU/mL; 95% CI, -5025.0 to 22936.0 arbitrary units/mL; P =.002). In addition, significantly higher anti-SARS S1 IgG levels were detected following vaccination (mean reactivity index, 55.4; 95% CI, -9.1 to 120.0; P =.001), suggesting potential cross-reactivity with these coronaviruses. Also, anti-S NAbs were significantly boosted against SARS-CoV-2 (50.5% neutralization; 95% CI, 17.6% to 83.2% neutralization; P <.001) after vaccination. Furthermore, there was no significant increase in antibody-dependent cellular cytotoxicity against SARS-CoV-2 S protein postvaccination. Conclusions and Relevance: This cohort study found a significant boost in cross-reactive NAbs in some patients exposed to MERS-CoV and SARS-CoV-2 antigens. These findings suggest that isolation of broadly reactive antibodies from these patients may help guide the development of a pancoronavirus vaccine by targeting cross-reactive epitopes between distinct strains of human coronaviruses..This work was supported by internal funds from the Biomedical Research Center of Qatar University. Dr Nasrallah received funding from The WHO Eastern Mediterranean Regional Office (WHO-EMRO) Special Grant for COVID-19 Research

Cilia proteins are biomarkers of altered flow in the vasculature

Cilia, microtubule-based organelles that project from the apical luminal surface of endothelial cells (ECs), are widely regarded as low-flow sensors. Previous reports suggest that upon high shear stress, cilia on the EC surface are lost, and more recent evidence suggests that deciliation- the physical removal of cilia from the cell surface-is a predominant mechanism for cilia loss in mammalian cells. Thus, we hypothesized that EC deciliation facilitated by changes in shear stress would manifest in increased abundance of cilia-related proteins in circulation. To test this hypothesis, we performed shear stress experiments that mimicked flow conditions from low to high shear stress in human primary cells and a zebrafish model system. In the primary cells, we showed that upon shear stress induction, indeed, ciliary fragments were observed in the effluent in vitro, and effluents contained ciliary proteins normally expressed in both endothelial and epithelial cells. In zebrafish, upon shear stress induction, fewer cilia-expressing ECs were observed. To test the translational relevance of these findings, we investigated our hypothesis using patient blood samples from sickle cell disease and found that plasma levels of ciliary proteins were elevated compared with healthy controls. Further, sickled red blood cells demonstrated high levels of ciliary protein (ARL13b) on their surface after adhesion to brain ECs. Brain ECs postinteraction with sickle RBCs showed high reactive oxygen species (ROS) levels. Attenuating ROS levels in brain ECs decreased cilia protein levels on RBCs and rescued ciliary protein levels in brain ECs. Collectively, these data suggest that cilia and ciliary proteins in circulation are detectable under various altered-flow conditions, which could serve as a surrogate biomarker of the damaged endothelium.This work was funded by Qatar National Research Fund, National Priority Research Program (NPRP 10-0123-170222 to HCY). ADS is supported by funds from the Department of Pediatrics, Herma Heart Institute, the National Center for Research Resources, and the National Center for Advancing Translational Sciences, NIH (UL1TR001436)

Comparative analysis of within-host diversity among vaccinated COVID-19 patients infected with different SARS-CoV-2 variants

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a rapidly evolving RNA virus that mutates within hosts and exists as viral quasispecies. Here, we evaluated the within-host diversity among vaccinated and unvaccinated individuals (n = 379) infected with different SARS-CoV-2 Variants of Concern. The majority of samples harbored less than 14 intra-host single-nucleotide variants (iSNVs). A deep analysis revealed a significantly higher intra-host diversity in Omicron samples than in other variants (p value < 0.05). Vaccination status and type had a limited impact on intra-host diversity except for Beta-B.1.315 and Delta-B.1.617.2 vaccinees, who exhibited higher diversity than unvaccinated individuals (p values: <0.0001 and <0.0021, respectively). Three immune-escape mutations were identified: S255F in Delta and R346K and T376A in Omicron-B.1.1.529. The latter 2 mutations were fixed in BA.1 and BA.2 genomes, respectively. Overall, the relatively higher intra-host diversity among vaccinated individuals and the detection of immune-escape mutations, despite being rare, suggest a potential vaccine-induced immune pressure in vaccinated individuals.The authors are grateful for the leadership and assistance provided by the Ministry of Public Health in Qatar, the virology laboratory staff at Hamad Medical Corporation, and Qatar Biobank (QBB) team. This project was funded by Qatar National Research Fund (QNRF; Project number UREP28-164-3-048) and Qatar University (Project number QUCG-BRC-22/23-547). The article processing charges were paid from grant no. QUCG-BRC-2022/23-578

Validation of a Novel Fluorescent Lateral Flow Assay for Rapid Qualitative and Quantitative Assessment of Total Anti-SARS-CoV-2 S-RBD Binding Antibody Units (BAU) from Plasma or Fingerstick Whole-Blood of COVID-19 Vaccinees

Background: Limited commercial LFA assays are available to provide a reliable quantitative measurement of the total binding antibody units (BAU/mL) against the receptor-binding domain of the SARS-CoV-2 spike protein (S-RBD). Aim: This study aimed to evaluate the performance of the fluorescence LFA FinecareTM 2019-nCoV S-RBD test along with its reader (Model No.: FS-113) against the following reference methods: (i) the FDA-approved GenScript surrogate virus-neutralizing assay (sVNT); and (ii) three highly performing automated immunoassays: BioMérieux VIDAS®3, Ortho VITROS®, and Mindray CL-900i®. Methods: Plasma from 488 vaccinees was tested by all aforementioned assays. Fingerstick whole-blood samples from 156 vaccinees were also tested by FinecareTM. Results and conclusions: FinecareTM showed 100% specificity, as none of the pre-pandemic samples tested positive. Equivalent FinecareTM results were observed among the samples taken from fingerstick or plasma (Pearson correlation r = 0.9, p < 0.0001), suggesting that fingerstick samples are sufficient to quantitate the S-RBD BAU/mL. A moderate correlation was observed between FinecareTM and sVNT (r = 0.5, p < 0.0001), indicating that FinecareTM can be used for rapid prediction of the neutralizing antibody (nAb) post-vaccination. FinecareTM BAU results showed strong correlation with VIDAS®3 (r = 0.6, p < 0.0001) and moderate correlation with VITROS® (r = 0.5, p < 0.0001) and CL-900i® (r = 0.4, p < 0.0001), suggesting that FinecareTM can be used as a surrogate for the advanced automated assays to measure S-RBD BAU/mL.This work was made possible by grant number UREP28-173-3-057 from the Qatar National Research Fund (a member of Qatar Foundation). The statements made herein are solely the responsibility of the authors

Effectiveness of mRNA booster doses against the omicron variant

Since its emergence in November, 2021, the omicron (B.1.1.529) variant of SARS-CoV-2 has rapidly spread and quickly overtaken the delta (B.1.617.2) variant as the most common cause of infection.1 Due to numerous spike mutations, the omicron variant showed increased transmissibility and reduced neutralisation by antibodies from prior infections compared with the wild-type virus and other variants of concern. Concurrently, breakthrough infections surged in populations with high vaccination rates, which expedited efforts to scale up booster vaccination

The spectrum of chromosomal translocations in the Arab world: ethnic-specific chromosomal translocations and their relevance to diseases

Chromosomal translocations (CTs) are the most common type of structural chromosomal abnormalities in humans. CTs have been reported in several studies in the Arab world, but the frequency and spectrum of these translocations are not well characterized. The aim of this study is to conduct a systematic review to estimate the frequency and spectrum of CTs in the 22 Arab countries. Four literature databases were searched: PubMed, Science Direct, Scopus, and Web of Science, from the time of inception until July 2021. A combination of broad search terms was used to collect all possible CTs reported in the Arab world. In addition to the literature databases, all captured CTs were searched in three chromosomal rearrangement databases (Mitelman Database, CytoD 1.0 Database, and the Atlas of Genetics and Cytogenetics in Oncology and Hematology), along with PubMed and Google Scholar, to check whether the CTs are unique to the Arabs or shared between Arabs and non-Arabs. A total of 9,053 titles and abstracts were screened, of which 168 studies met our inclusion criteria, and 378 CTs were identified in 15 Arab countries, of which 57 CTs were unique to Arab patients. Approximately 89% of the identified CTs involved autosomal chromosomes. Three CTs, t(9;22), t(13;14), and t(14;18), showed the highest frequency, which were associated with hematological malignancies, recurrent pregnancy loss, and follicular lymphoma, respectively. Complex CTs were commonly reported among Arabs, with a total of 44 CTs, of which 12 were unique to Arabs. This is the first study to focus on the spectrum of CTs in the Arab world and compressively map the ethnic-specific CTs relevant to cancer. It seems that there is a distinctive genotype of Arabs with CTs, of which some manifested with unique clinical phenotypes. Although ethnic-specific CTs are highly relevant to disease mechanism, they are understudied and need to be thoroughly addressed. 2022, The Author(s).Scopu

The spectrum of chromosomal translocations in the Arab world: ethnic-specific chromosomal translocations and their relevance to diseases

Chromosomal translocations (CTs) are the most common type of structural chromosomal abnormalities in humans. CTs have been reported in several studies in the Arab world, but the frequency and spectrum of these translocations are not well characterized. The aim of this study is to conduct a systematic review to estimate the frequency and spectrum of CTs in the 22 Arab countries. Four literature databases were searched: PubMed, Science Direct, Scopus, and Web of Science, from the time of inception until July 2021. A combination of broad search terms was used to collect all possible CTs reported in the Arab world. In addition to the literature databases, all captured CTs were searched in three chromosomal rearrangement databases (Mitelman Database, CytoD 1.0 Database, and the Atlas of Genetics and Cytogenetics in Oncology and Hematology), along with PubMed and Google Scholar, to check whether the CTs are unique to the Arabs or shared between Arabs and non-Arabs. A total of 9,053 titles and abstracts were screened, of which 168 studies met our inclusion criteria, and 378 CTs were identified in 15 Arab countries, of which 57 CTs were unique to Arab patients. Approximately 89% of the identified CTs involved autosomal chromosomes. Three CTs, t(9;22), t(13;14), and t(14;18), showed the highest frequency, which were associated with hematological malignancies, recurrent pregnancy loss, and follicular lymphoma, respectively. Complex CTs were commonly reported among Arabs, with a total of 44 CTs, of which 12 were unique to Arabs. This is the first study to focus on the spectrum of CTs in the Arab world and compressively map the ethnic-specific CTs relevant to cancer. It seems that there is a distinctive genotype of Arabs with CTs, of which some manifested with unique clinical phenotypes. Although ethnic-specific CTs are highly relevant to disease mechanism, they are understudied and need to be thoroughly addressed.Other Information Published in: Chromosoma License: https://creativecommons.org/licenses/by/4.0See article on publisher's website: http://dx.doi.org/10.1007/s00412-022-00775-2</p