Explore peer-reviewed scientific publications from Ayass BioScience, LLC showcasing breakthroughs in genomics, transcriptome analysis, and precision diagnostics.

Publications:

Explore peer-reviewed scientific publications from Ayass BioScience, LLC showcasing breakthroughs in genomics, transcriptome analysis, and precision diagnostics.

Publications:

Aptamer-Enhanced Surface Decontamination: A Novel Approach for Neutralizing Peanut Allergens and Preventing Cell-Degranulation (MDPI)

Published: 8 April 2025

Aptamer-Enhanced Surface Decontamination: A Novel Approach for Neutralizing Peanut Allergens and Preventing Cell-Degranulation

Peanut allergies, driven by sensitization to key allergens Ara h1, Ara h2, and Ara h3, present significant health risks, particularly in food processing and consumer settings where accidental exposure is frequent. To mitigate this risk, we developed AYA22AR321, a novel aptamer with selective, high-affinity binding to these allergens (Kd values: 0.5 nM for Ara h1, 14.5 nM for Ara h2, and 6.6 nM for crude peanut extract). Functional assays using RBL-2H3 (rat basophilic leukemia cell line) cells showed that AYA22AR321 significantly reduces IgE-mediated degranulation, indicating its potential to attenuate allergic responses. To translate these findings into practical use, we formulated an allergen-neutralizing spray, FISTOQ, containing AYA22AR321, which effectively neutralized peanut allergens on peanut-butter-contaminated surfaces. Stability tests confirmed that FISTOQ, comprising eco-friendly surfactant and preservative, maintains its allergen-neutralizing efficacy over time. Comprehensive safety assessments, including immunogenicity, cytotoxicity in human PBMCs, and mutagenicity via the Ames test, demonstrated that AYA22AR321 is non-immunogenic, non-cytotoxic, and non-mutagenic. This study establishes AYA22AR321 as a promising, targeted strategy for allergen control, providing a significant advancement in allergen mitigation and food safety for high-risk environments.

Contributors: Mohamad Ammar Ayass • Trivendra Tripathi • Natalya Griko • Ramya Ramankutty Nair • Tutku Okyay • Jin Zhang • Kevin Zhu  • Victor Pashkov • Lina Abi-Mosleh

AYA22A Aptamers Mitigate Peanut Allergenicity: Insights from Degranulation Assays and Modulating Immune Responses (MDPI)

Published: 6 August 2024

AYA22A Aptamers Mitigate Peanut Allergenicity: Insights from Degranulation Assays and Modulating Immune Responses

Food allergy, particularly peanut allergy (PA), is a growing health concern affecting millions globally. PA can lead to severe reactions, including fatal anaphylaxis. Despite the availability of FDA-approved therapies like Palforzia, a cure remains elusive. Current immunotherapies show promise but lack a definitive cure. This study applies an established computational biology tool to design aptamers targeting Ara h1 and Ara h2. The in silico design aims to streamline the selection process, enabling cost-effective and rapid identification of aptamer candidates. The developed aptamers (AYA22A, including AYA22AR321, AYA22AR211, and AYA22AR524), demonstrated efficacy in inhibiting degranulation of RBL-2H3 cells (rat basophilic leukemia cell line) in vitro. They showed promise in neutralizing peanut allergen-induced immune responses. The selected aptamers inhibited degranulation in RBL-2H3 cells, addressing concerns in raw peanuts. Moreover, these aptamers demonstrated stability and effectiveness in peanut plant seeds and commercial products. Our aptamers exhibited potential in modulating immune responses associated with peanut allergy. They influenced Th1/Th2 balance, indicating a role in cytokine regulation. In vitro studies also showed the aptamers’ impact on immune cell expression and cytokine production, resembling responses observed with established immunotherapies. The findings suggest AYA22A aptamers as a potential therapeutic option for peanut allergy, providing a basis for further in vivo investigations.

Contributors: Mohamad Ammar Ayass • Trivendra Tripathi • Natalya Griko • Ramya Ramankutty Nair • Tutku Okyay • Jin Zhang • Kevin Zhu • Kristen Melendez • Victor Pashkov • Lina Abi-Mosleh

Transcriptome-Based Treatment for Melanoma With Brain Metastasis: A Case Report (Cureus/Part of Springer Nature Group)

Published: 19 March 2024

Transcriptome-Based Treatment for Melanoma With Brain Metastasis: A Case Report

Malignant melanoma with brain metastasis has a high mortality rate. New approaches for diagnosis and treatment are urgently required to improve prognosis. Here we present a 60-year-old male with metastatic melanoma to the brain. Using a transcriptomics pipeline, we analyzed whole blood and resected tumor tissue, identifying enriched gene expression biomarkers and pathways – including seven upregulated ( BRAF, CDK4, EIF1AX, IK, NRAS, PIK3R2, and TP53) and 11 downregulated (CASP8, CDK10, CDKN2A, CTLA4, GNA11, HERC2, IRF4, MC1R, PLA2G6, RREB1, and TPCN2) genes in the blood (across 15 pathways), showing 14% enrichment, and 16 upregulated (CCND1, CDK4, CTLA4, EIF1AX, IK, IRF4, MITF, NRAS, PIK3CB, PIK3R2, PMEL, RREB1, SLC45A2, SOX10, TYR, and TYRP1) and three downregulated ( GNA11, KITLG, and PLA2G6) genes in tissue (across 17 pathways), showing 33% enrichment, with five shared markers and 12 shared pathways. The model connected CDK4 pathway overactivity observed in both samples to inhibitors like ribociclib, abemaciclib, and palbociclib as putative treatments. By enabling objective personalized therapy selection, this approach shows great promise for advancing patient outcomes.

Contributors: Mohamad Ammar Ayass • Kristen Melendez • Natalya Griko • Jin Zhang • Lina Abi-Mosleh.

Dual Checkpoint Aptamer Immunotherapy: Unveiling Tailored Cancer Treatment Targeting CTLA-4 and NKG2A (MDPI/Cancers)

Published: 4 March 2024

Dual Checkpoint Aptamer Immunotherapy: Unveiling Tailored Cancer Treatment Targeting CTLA-4 and NKG2A

Cancer remains a significant health challenge, with persistent annual increases in new cases and mortality rates. Notably, breast, prostate, and uterine cancers are surging. Economically, cancer care bears an overwhelming burden, with costs varying based on cancer phase and type of treatment. Immunotherapy, including T cell immune-checkpoint inhibitors and CAR-directed T lymphocytes, is pivotal in modern oncology, targeting CTLA-4 and PD-1/PD-L1 pathways. Recent research underscores CTLA-4’s diverse role in immune modulation, impacting T cell activation, IDO induction, and T cell proliferation inhibition. Additionally, recognition of the NKG2A axis, interacting with HLA-E on tumor cells, highlights its significance in immune evasion and cancer progression. Current FDA-approved anti-CTLA-4 therapies exhibit promise but face challenges regarding response rates and immune-related adverse events. This study employed computational methods to design aptamers targeting CTLA4 and NKG2A. Aptamer binding specificity was validated through direct and competitive assays using ELISA and flow cytometry. We demonstrated the biological functionality of AYA22T-R2-13, confirming its specific binding to CTLA-4 and NKG2A, and its potential to enhance CD8 T cell and NK cell activity, promoting in vitro tumor cell lysis in human cell lines.

Recent strides in immunotherapy have illuminated the crucial role of CTLA-4 and PD-1/PD-L1 pathways in contemporary oncology, presenting both promises and challenges in response rates and adverse effects. This study employs a computational biology tool (in silico approach) to craft aptamers capable of binding to dual receptors, namely, inhibitory CTLA4 and NKG2A, thereby unleashing both T and NK cells and enhancing CD8+ T and NK cell functions for tumor cell lysis. Computational analysis highlighted AYA22T-R2-13 with HADDOCK scores of −78.2 ± 10.2 (with CTLA4), −60.0 ± 4.2 (with NKG2A), and −77.5 ± 5.6 (with CD94/NKG2A). Confirmation of aptamer binding to targeted proteins was attained via ELISA and flow cytometry methods. In vitro biological functionality was assessed using lactate dehydrogenase (LDH) cytotoxicity assay. Direct and competitive assays using ELISA and flow cytometry demonstrated the selective binding of AYA22T-R2-13 to CTLA4 and NKG2A proteins, as well as to the cell surface receptors of IL-2-stimulated T cells and NK cells. This binding was inhibited in the presence of competition from CTLA4 or NKG2A proteins. Remarkably, the blockade of CTLA4 or NKG2A by AYA22T-R2-13 augmented human CD8 T cell- and NK cell-mediated tumor cell lysis in vitro. Our findings highlight the precise binding specificity of AYA22T-R2-13 for CTLA4-B7-1/B7-2 (CD80/CD86) or CD94/NKG2A-HLA-E interactions, positioning it as a valuable tool for immune checkpoint blockade aptamer research in murine tumor models. These in vitro studies establish a promising foundation for further enhancing binding capacity and establishing efficacy and safety in animal models. Consequently, our results underscore the potential of AYA22T-R2-13 in cancer immunotherapy, offering high specificity, low toxicity, and the potential for cost-effective production.

Contributors: Mohamad Ammar Ayass, Trivendra Tripathi, Natalya Griko, Tutku Okyay, Ramya Ramankutty Nair, Jin Zhang, Kevin Zhu, Kristen Melendez, Victor Pashkov, and Lina Abi-Mosleh.

T Helper (Th) Cell Profiles and Cytokines/Chemokines in Characterization, Treatment, and Monitoring of Autoimmune Diseases (ScienceDirect/Methods)

Published: 13 November 2023

T Helper (Th) Cell Profiles and Cytokines/Chemokines in Characterization, Treatment, and Monitoring of Autoimmune Diseases

T cell subsets play a pivotal role in the pathogenesis of autoimmune diseases (AD). A practical and accurate flow cytometry method to profile T helper (Th) cell phenotypes in the peripheral blood of AD-diagnosed patients facilitates a rapid visual identification of profile irregularities that enhance AD diagnostic power.

Analyzing T cell profiling within the clinical diagnosis of AD offers a fresh perspective on assessing the functionality of naïve to memory Th cell population and their responsiveness to cytokines. This helps determine disease severity, improving our understanding of their influence on immune status, treatment outcomes, effective therapy, and monitoring of AD.

The correlation between the distinct T cell profile and their pathogenic mediators (cytokines/chemokines) can help direct therapy and monitor its effectiveness.

A naïve/memory CD4 T cell ratio below 0.3 is associated with primary immunodeficiency disorders.

Autoimmune diseases (AD) consist of a spectrum of disease entities whose etiologies are very complex and still not well understood. Every individual has the potential for developing AD under appropriate conditions because the body contains lymphocytes that are potentially reactive with self-antigens. The aims of this study are to (1) explore the flow cytometry method to identify the frequency of various circulating CD4+ T helper (Th) cell-subsets, including Th1, Th2, Th9, Th17, Th17.1, and Th22; (2) In parallel, to examine multiplex ELISA method for pathogenic inflammatory cytokines/chemokines, and (3) To assess the correlation of expression of T cell-subsets with serum cytokines/chemokines and understand its clinical importance with available AD treatments. We analyzed Th17, Th17.1, Th22, Th2, Th1, and Th9 Th cell populations and compared the concentrations of 67 cytokines/chemokines in healthy as well as AD-diagnosed patients. We observed that patients with autoimmune markers had significantly elevated percentages of naïve (Th17, Th22, and Th9) as well as memory (Th17 and Th22) Th cell-subsets, along with increased concentrations of cytokines/chemokines (Eotaxin, TNFβ, and FABP4). The percentage of Th cell-subsets correlated positively or negatively with the production of cytokines/chemokines of patients diagnosed with AD. Our study demonstrates that the naïve and memory Th cell-subsets with positive correlations to cytokines/chemokines show new diagnostic markers to predict the patients’ outcome, while the negative correlation of cytokines/chemokines shows the response to autoimmune therapies. Our findings of Th cell-subsets by flow cytometry and cytokines/chemokines by multiplex ELISA suggest that CCR6+ Th cell-subsets (Th17, Th17.1, Th22, and Th9) contribute to our understanding of the pathogenesis of AD and identify the new onset of AD from the autoimmune spectrum. Our findings highlight the importance of CCR6+ as a possible marker in the characterization, treatment, and monitoring of AD.

Contributors: Mohamad Ammar Ayass, Trivendra Tripathi, Kevin Zhu, Ramya Ramankutty Nair, Kristen Melendez, Jin Zhang, Seyedhossein Fatemi, Tutku Okyay, Natalya Griko, Melesse Balcha Ghelan, Victor Pashkov, and Lina Abi-Mosleh

New High-Affinity Thrombin Aptamers for Advancing Coagulation Therapy: Balancing Thrombin Inhibition for Clot Prevention and Effective Bleeding Management with Antidote

Published: 7 September 2023

New High-Affinity Thrombin Aptamers for Advancing Coagulation Therapy: Balancing Thrombin Inhibition for Clot Prevention and Effective Bleeding Management with Antidote

Thrombin is a key enzyme involved in blood clotting, and its dysregulation can lead to thrombotic diseases such as stroke, myocardial infarction, and deep vein thrombosis. Thrombin aptamers have the potential to be used as therapeutic agents to prevent or treat thrombotic diseases. Thrombin DNA aptamers developed in our laboratory exhibit high affinity and specificity to thrombin. In vitro assays have demonstrated their efficacy by significantly decreasing Factor II activity and increasing PT and APTT times in both plasma and whole blood. Aptamers AYA1809002 and AYA1809004, the two most potent aptamers, exhibit high affinity for their target, with affinity constants (Kd) of 10 nM and 13 nM, respectively. Furthermore, the in vitro activity of these aptamers displays dose-dependent behavior, highlighting their efficacy in a concentration-dependent manner. In vitro stability assessments reveal that the aptamers remain stable in plasma and whole blood for up to 24 h. This finding is crucial for their potential application in clinical settings. Importantly, the thrombin inhibitory activity of the aptamers can be reversed by employing reverse complement sequences, providing a mechanism to counteract their anticoagulant effects when necessary to avoid excessive bleeding. These thrombin aptamers have been determined to be safe, with no observed mutagenic or immunogenic effects. Overall, these findings highlight the promising characteristics of these newly developed thrombin DNA aptamers, emphasizing their potential for therapeutic applications in the field of anticoagulation therapy. Moreover, the inclusion of an antidote in the coagulation therapy regimen can improve patient safety, ensure greater therapeutic efficacy, and minimize risk during emergency situations.

Contributors: Mohamad Ammar Ayass, Natalya Griko, Victor Pashkov, Trivendra Tripathi , Jin Zhang, Ramya Ramankutty Nair,
Tutku Okyay, Kevin Zhu and Lina Abi-Mosleh.

Macrophage-Induced Exacerbation of Nasopharyngeal Inflammatory Lymphocytes in COVID-19 Disease (COVID/MDPI)

Published: 13 April 2023

Macrophage-Induced Exacerbation of Nasopharyngeal Inflammatory Lymphocytes in COVID-19 Disease

The nasal microenvironment plays a crucial role in the transmission, modulation, and clinical progression of COVID-19; however, the immune responses at the site of viral entry remain poorly understood. We deciphered the link between nasopharyngeal (NP) immune and inflammatory response that triggers cytokine/chemokine storms in the nasal route of COVID-19-positive patients. We used RT-PCR, multiplex ELISA, flow cytometry, and LC-MS/MS to decipher nasopharyngeal immune perturbations associated with severe COVID-19. In addition, we performed in vitro assays using cultured human monocytes-derived macrophages trained both in the presence and absence of SARS-CoV-2 trimeric spike protein(s) and co-cultured with and without autologous human peripheral blood mononuclear cells (hPBMCs)/total T-cells/CD8 T-cells. In vitro immune perturbations were examined by flow cytometry and LC-MS/MS assays. Our findings confirm that macrophages orchestrate NP immune inflammatory responses and highlight the cytokine/chemokine storms associated with the increased CD8+ T-cells along with Tregs, Th1, and Th17.1 T-helper cells. We observed a correlation between in vitro and nasal findings that trained macrophages, profoundly M2c, differentially promote the inflammatory surfactome on CD8 T-cells, including ITGAM, LGALS3, CD38, TKT, LRPAP1, and SSBP1. The findings of this study conclude that inflammatory lymphocyte perturbations within the nasopharynx of COVID-19 patients may enforce immune homeostasis during SARS-CoV-2-infection and contribute to COVID-19 pathology. This study explored the therapeutic target proteins that could facilitate the development of new medications, which could allow for immediate treatment of possible emerging viral infections.

Contributors: Mohamad Ammar Ayass, Trivendra Tripathi, Natalya Griko, Ramya Ramankutty Nair, Jin Zhang, Kevin Zhu, Wanying Cao, Victor Pashkov, Tutku Okyay, Sharda Kalla Singh, Lina Abi-Mosleh.

High-Affinity Neutralizing DNA Aptamers against SARS-CoV-2 Spike Protein Variants (COVID/MDPI)

Published: 7 April 2023

High-Affinity Neutralizing DNA Aptamers against SARS-CoV-2 Spike Protein Variants

The continuous emergence of new variants of concern for SARS-CoV-2 has created a challenge for existing therapies. To address this, we developed a series of single-stranded DNA aptamers that not only bind specifically to the trimer S protein of SARS-CoV-2 but also block the interaction between the trimer S protein and ACE2 receptors. The systematic evolution of ligands by exponential enrichment (SELEX) was performed to select the aptamers for SARS-CoV-2 trimer S protein. ELISA-based assay and flow cytometry were performed to test the apatmers’ binding and inhibition of trimer S protein in vitro. Binding affinity was measured using surface plasmon resonance. Significance was determined in Prism 9.0 using the one-way ANOVA test (Dunnett’s multiple comparisons test) or two-way ANOVA test (Tukey’s multiple comparisons test) for comparisons. The p values < 0.05 were considered statistically significant. After 12 rounds of SELEX, eight highly enriched aptamers were able to bind to the trimer S protein of the SARS-CoV-2 Wuhan original strain as well as the trimer S proteins of the Delta, Delta plus, Alpha, Lambda, Mu, and Omicron variants, with affinities in the nM range, while also inhibiting their interaction with ACE2 receptors in Vero E6 cells. Modifications to our best aptamer were made by adding forward and reverse primer sequences and truncation. The modified aptamers AYA2012004_L and AYA2012004_L-M1 showed up to 70% inhibition of the binding of virus-like particles (VLPs) expressing S protein to the ACE2 receptor expressed in HEK293T cells. Our findings imply that the selected aptamers can prevent SARS-CoV-2 from entering host cells and hence suppress the viral infection. In addition, the findings suggest that the selected aptamers might be an innovative therapy for the treatment of COVID-19.

Contributors: Mohamad Ammar Ayass, Natalya Griko, Victor Pashkov, Trivendra Tripathi, Wanying Cao, Nazanin Javan, Jun Dai, Jin Zhang, Kevin Zhu, Lina Abi-Mosleh.

Therapeutic Aptamer for COVID-19 Management (Virology Journal)

Published: 30 December 2022

Highly efficacious and safe neutralizing DNA aptamer of SARS-CoV-2 as an emerging therapy for COVID-19 disease

Background

The paucity of SARS-CoV-2-specific virulence factors has greatly hampered the therapeutic management of patients with COVID-19 disease. Although available vaccines and approved therapies have shown tremendous benefits, the continuous emergence of new variants of SARS-CoV-2 and side effects of existing treatments continue to challenge therapy, necessitating the development of a novel effective therapy. We have previously shown that our developed novel single-stranded DNA aptamers not only target the trimer S protein of SARS-CoV-2, but also block the interaction between ACE2 receptors and trimer S protein of Wuhan origin, Delta, Delta plus, Alpha, Lambda, Mu, and Omicron variants of SARS-CoV-2. We herein performed in vivo experiments that administer the aptamer to the lungs by intubation as well as in vitro studies utilizing PBMCs to prove the efficacy and safety of our most effective aptamer, AYA2012004_L.

Methods

In vivo studies were conducted in transgenic mice expressing human ACE2 (K18hACE2), C57BL/6J, and Balb/cJ. Flow cytometry was used to check S-protein expressing pseudo-virus-like particles (VLP) uptake by the lung cells and test the immuogenicity of AYA2012004_L. Ames test was used to assess mutagenicity of AYA2012004_L. RT-PCR and histopathology were used to determine the biodistribution and toxicity of AYA2012004_L in vital organs of mice.

Results

We measured the in vivo uptake of VLPs by lung cells by detecting GFP signal using flow cytometry. AYA2012004_L specifically neutralized VLP uptake and also showed no inflammatory response in mice lungs. In addition, AYA2012004_L did not induce inflammatory response in the lungs of Th1 and Th2 mouse models as well as human PBMCs. AYA2012004_L was detectable in mice lungs and noticeable in insignificant amounts in other vital organs. Accumulation of AYA2012004_L in organs decreased over time. AYA2012004_L did not induce degenerative signs in tissues as seen by histopathology and did not cause changes in the body weight of mice. Ames test also certified that AYA2012004_L is non-mutagenic and proved it to be safe for in vivo studies.

Conclusions

Our aptamer is safe, effective, and can neutralize the uptake of VLPs by lung cells when administered locally suggesting that it can be used as a potential therapeutic agent for COVID-19 management.

Contributors: Mohamad Ammar Ayass, Trivendra Tripathi, Natalya Griko, Victor Pashkov, Jun Dai, Jin Zhang, Fabian C. Herbert, Ramya Ramankutty Nair, Tutku Okyay, Kevin Zhu, Jeremiah J. Gassensmith, Lina Abi-Mosleh.

Prediction of Covid-19 Patient’s Outcome from Nasopharyngeal Samples (PLOS ONE)

Published: September 12, 2022

Identification of Biomarkers in Non-Invasive Nasopharyngeal Samples from SARS-CoV-2 Patients as Predictor of Severity of Covid-19 Disease Outcome

At Ayass BioScience, LLC, we showed that mass spectrometry proteomics analysis of nasopharyngeal swabs can be a powerful and early approach to evaluate patient response to virus infection.

The beauty of the study lies in the use of a non-invasive method of collecting samples from the nasal swabs, allowing us to present the status of proteomics in the nasal microenvironment at the time of diagnosis of COVID-19 virus infection (acute infection phase) without the need for additional laboratory work.

Our study provided an unbiased, hypothesis-free investigation of the nasal mucosal proteome from over 100 patients. Our results indicate that down-regulation of IgG4 correlates with a bad outcome in COVID-19 patients. The decreased level of IgG4 at the point of entry could be useful for early detection of adverse immune response in COVID-19 patients and predict their ultimate outcome. Additionally, a decrease in the levels of coagulation factor 5 with a decrease in fibronectin suggested an increased chance of thrombotic events in COVID-19 patients, a relatively common complication seen in COVID-19 patients.

Other proteins successfully identified in this study could also be promising biomarkers for early diagnosis of COVID-19, disease monitoring, as well as drug targets.

Serum or plasma have been the primary focus of proteomics studies for COVID-19 to identity biomarkers and potential drug targets. The nasal mucosal environment which consists of lipids, mucosal immune cells, and nasal proteome, has been largely neglected but later revealed to have critical role combating SARS-CoV-2 infection. We present a bottom-up proteomics investigation of the host response to SARS-CoV-2 infection in the nasopharyngeal environment, featuring a noninvasive approach using proteins in nasopharyngeal swabs collected from groups of 76 SARS-CoV-2 positive and 76 negative patients. Results showed that 31 significantly down-regulated and 6 up-regulated proteins were identified (p < 0.05, log2 FC > 1.3) in SARS-CoV-2 positive patient samples as compared to the negatives; these proteins carry potential value as markers for the early detection of COVID-19, disease monitoring, as well as be drug targets. The down-regulation of coagulation factor 5 indicates a thrombotic abnormality in COVID-19 patients and the decreased IgG4 suggests an abnormal immune response at the point of entry in human nasopharyngeal environment, which is in consistent with KEGG and GO pathway analysis. Our study also demonstrated that mass spectrometry proteomics analysis of nasopharyngeal swabs can be used as a powerful early approach to evaluate host response to SARS-CoV-2 viral infection.

Noninvasive nasopharyngeal proteomics of COVID-19 patient identify abnormalities related to complement and coagulation cascade and mucosal immune system

Contributors: Mohamad Ammar Ayass, Wanying Cao, Jin Zhang, Jun Dai, Kevin Zhu, Trivendra Tripathi, Natalya Griko, Victor Pashkov, Lina Abi-Mosleh.

Nasopharyngeal inflammatory lymphocytes perturbation in COVID-19 severity prediction (The Journal of Immunology)

Published: 01 May 2022

Nasopharyngeal inflammatory lymphocytes perturbation in COVID-19 severity prediction

SARS-CoV-2 causes severe respiratory infections in some patients due to an uncontrolled immune response. The nasal microenvironment plays a crucial role in the transmission, modulation, and clinical progression of COVID-19, however, the immune-responses at the site of viral entry remain poorly understood. We used flow cytometry to decipher nasopharyngeal immune profiling of COVID-19 positive/negative patients; viral detection and load were performed by RT-PCR. Analysis of viral-induced immune-inflammatory responses, lymphocytes population/activation lymphocytes markers, T cell subtypes and associated inflammatory cytokines. Furthermore, we performed in vitro assays using cultured monocytes-derived-macrophages trained with/without SARS-CoV-2 spike protein and co-cultured with/without autologous T cells. Results showed that positive patients had high inflammatory CD45+ and CD8+ T cells including elevated % of Th1/Th17.1, associated with a worse outcome in a subgroup of COVID-19 infected patients. Positive patients showed increased % of cytokines, IFN-γ+, IL-17+, and IL-10+ T cells suggesting a crucial role of these cytokines in recruiting and activating neutrophils that can migrate to target organs (e.g., lungs) and potentially play an important role in the disease severity. Moreover, upregulated Tregs along with monocytic-induced inflammatory T-cell responses and increased TH1/TH17 cytokines in positive patients as compared to negative, potentially having a nefarious effect on disease progression. Our findings indicate a distinct signature of inflammatory lymphocytes/cytokines responses within the nasopharynx of COVID-19 patients that might predict the clinical outcome/progression of the disease.

This work is supported by Dr. Mohamad Ayass, CEO of Ayass Bioscience, LLC

Contributors: Mohamad Ammar Ayass, Trivendra Tripathi, Natalya Griko, Jin Zhang, Ramya Ramankutty Nair, Jun Dai, Kevin Zhu, Wanying Cao, Victor Pashkov, Sharda Kalla Singh, Lina Abi Mosleh

The Impact of New SARS-CoV-2 Variants on Vaccine Breakthrough: A Pilot Study on Spreading Infection in the Communities (medRxiv)

September 26, 2021

The Impact of New SARS-CoV-2 Variants on Vaccine Breakthrough: A Pilot Study on Spreading Infection in the Communities

Background Coronavirus disease 2019 (COVID-19) vaccines are effective at helping protect against severe disease and death from variants; however, incident of breakthrough infection in vaccinated patients has been increased. Therefore, we aimed to assess the incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) new variants of concern in the communities and investigate vaccine breakthrough cases on our laboratory (Ayass Bioscience LLC) confirmed detection of COVID-19 variants in Dallas-Fort Worth (DFW), Texas.

Methods Epidemiologic study has been performed at our laboratory. We studied the viral whole-genome sequence and genotyping analysis on 166 symptomatic cases of COVID-19 which were randomly selected from nasal swab positive cases assessed from June 1st to August 30th, 2021, by reverse transcription polymerase chain reaction (RT-PCR) cycle threshold (CT) values. COVID-19 variants were identified to be dominated by B.1.617.2 (89.2%) and followed by AY.3 (1.8%), B.1.1.7 (4.8%), a combination of B.1.526.1 and B.1.617.2 (3%), B.1.621 (0.6%), and P.2 (0.6%).

Result The CT values showed significant difference among the three age groups: <30 years, 31-60 years, and >60 years by one-way ANOVA (N1: F (2, 118) =4.96, p=0.009; N2: F (2, 118) =4.95, p=0.009). No significant difference was observed by symptom, status of immunization, or vaccine manufacturer. A two-way ANOVA was performed to examine the effect of gender and variant group (Delta and other variants) on the CT values. The analyses revealed a statistically significant interaction between the effect of gender and variant group (N1, F (1.117) = 3.906, p = 0.05; N2, F (1, 117) = 7.402, p = 0.008).

Conclusion Our study shows that Delta, the dominant variant of COVID-19, is spreading in the communities, and vaccine breakthrough cases occurred in the majority of Delta variant (91%) followed by AY.3 (5%), B.1.1.7 (2%) and 2% of the double variant of B.1.526.1 and B.1.617.2. The incidence of the breakthrough cases was not linked to a specific manufacturer. The CT value is likely to associate with age. This study also supports our laboratory’s ongoing efforts to sequence the SARS-CoV-2 virus from positive patient samples to identify the new viral variants and possible vaccine breakthrough mutations in the community.

Contributors: Mohamad Ammar Ayass, Jin Zhang, Kevin Zhu, Wanying Cao, Natalya Griko, Victor Pashkov, Jun Dai, Trivendra Tripathi, Lina Abi Mosleh

P147 Prevalence of anti-cyclic citrullinated peptide (ACCP) antibodies and their association with rheumatoid factors and other autoimmune parameters (RHEUMATOLOGY)

Published: 20 April 2020

P147 Prevalence of anti-cyclic citrullinated peptide (ACCP) antibodies and their association with rheumatoid factors and other autoimmune parameters

Background

The presence of antibodies against cyclic citrullinated proteins (anti-CCP) precede the development of rheumatoid arthritis (RA) and both anti-CCP antibody and RF positivity indicate more severe disease. This characteristic makes Anti-CCP best biomarker candidate for the prevention of debilitating joint disease. Almost 70% of RA patients are positive for anti-CCP IgG, while only 2% of control subjects are positive for anti-CCP. Understanding the origins of anti-CCP is critical in unraveling the etiology of RA, so we further evaluated the association of the other autoimmune tests/biomarkers with Anti-CCP to describe other comorbid autoimmune diseases/syndrome.

Methods

We conducted a retrospective analysis of patient data from April 18, 2016, to May 29, 2019, in Texas. The study protocol was approved by the ethics review committee and patients consented for sharing their demographics, clinical information and lab values for diagnostic and research purposes. The study population was described by descriptive statistics. Demographic characteristics and laboratory parameters were compared using chi-square tests. We analyzed blood tests/parameters conducted at the clinics and explored their association with the presence of ACCP. logistic regression models were utilized to calculate the odds ratio to delineate the strength of association of different blood parameters with ACCP. IgG anti-CCP was determined according to the manufacturer’s instruction.

Results

Eleven thousand, four hundred fifty-nine tests were performed on 7,200 patients with a mean age of 52 years (range 0-98). Almost half (54%) of study individuals were females, two-third (63%) were White, (29%) Hispanic and (7%) African American. Four hundred and six patients (3.5%) were positive for AntiCCP3.1. Among those who were positive for AntiCCP3.1, (22%) were Rh factor IgA (RhIgA) positive, (11%) RhIgG positive, and (9%) RhIgM. Univariate logistic regression showed that the female gender was positively associated (OR = 3.19) with ACCP presence while patients older than 50 had a positive gradient of ACCP positivity with each age category. Compared to White, African Americans were more likely (OR = 3.19) and Hispanics were less likely (OR = 0.44) to be associated with AntiCCP positivity. After controlling for age, gender and ethnicity the presence of RhIgM and RhIgA antibodies were positively associated with positive ACCP. (OR = 13.3, OR = 5.2). antiphosphatidylserine IgM and anticardiolipin IgM antibodies were positively associated with ACCP (OR = 2.1, OR = 3.4). Similarly, the presence of antimitochondrial M2 antibodies and anti-topoisomerase antibodies (SCL-70) were very significantly associated with ACCP (OR = 16.1, OR = 37.8).

Conclusion

The Anti-CCP antibodies are highly specific for the early diagnosis of RA and with early initiation of treatment, progression to irreversible and destructive diseases can be prevented. Association of Anti-CCP antibodies with other autoimmune antibodies highlights the importance of screening RA patients for primary biliary sclerosis and scleroderma. Similarly, a biomarker’s strength of interaction with another biomarker helps in the prognostic utility of these test for preventing co-existing diseases.

Disclosures

M. Ayass: Other; Physician at Ayass Bioscience. K. Zhu: Other; Employee at Ayass Bioscience. S. Naveed: None. L. Mosleh: Other; Employee at Ayass Bioscience. K. Viswanathan: Other; Employee at Ayass Bioscience. G. Nowshad: Other; Employee at Ayass Bioscience.

Contributors: Mohamad Ammar Ayass, Kevin Zhu, Shazia Naveed, Lina Mosleh, Kasthuribai Viswanathan, Gul Nowshad

P307 PREVALENCE AND COVARIATES OF FOOD ALLERGY IN OUTPATIENT POPULATION: ROLE OF ANTIPHOSPHOLIPID SYNDROME (ScienceDirect)

November 2019

P307 PREVALENCE AND COVARIATES OF FOOD ALLERGY IN OUTPATIENT POPULATION: ROLE OF ANTIPHOSPHOLIPID SYNDROME

Introduction

Antiphospholipid syndrome is an autoimmune disease, genetic predisposition and exaggerated immunological response to viral and bacterial exposures have been postulated as possible etiology. To our knowledge, very few studies have described food-induced allergy association with the antiphospholipid syndrome.

Section snippets

Methods

Retrospective analysis of demographic, clinical and laboratory information for patients from January 2018 to January 2019 was performed after IRB approval. Descriptive statistics were used to describe study population. We analyzed four different categories of Antiphospholipid antibodies; (antibodies against Beta-2-Glycoprotein-1, Cardiolipin , Phosphatidylserine and Phosphatidylserine/Prothrombin complex) .Odds ratios were calculated for the covariates and allergy status using logistic

Result

Four thousand, six hundred eleven patients included in the study with a mean age of 46 years and 56% were females . Majority of patients were white (83%), Hispanic (12%) and African American (5%). Half of the patients (55%) were suffering from some kind of food allergy and most common allergens were white beans (49%), egg whole (41%) and cow milk (26%).
Univariate logistic regression showed that females and Hispanic patients were less likely to be associated with allergy. The presence of

Conclusion

Our findings of Antiphospholipid antibodies association with food allergy may indicate that these daily dose of allergens in food interfere with the immunological responses causing chronic inflammatory and autoimmune diseases.

Contributors: M. Ayass, G. Nowshad, L. Mosleh, O. Alsheikh, K. Zhu, K. Viswanathan

ANTIPHOSPHOLIPID ANTIBODY SYNDROME AS A RISK FACTOR FOR COPD: MULTIPLICATIVE INTERACTION EFFECTS OF POSITIVE ANTIPHOSPHOLIPID ANTIBODIES WITH LOW TESTOSTERONE (CHEST Journal)

April 2019

ANTIPHOSPHOLIPID ANTIBODY SYNDROME AS A RISK FACTOR FOR COPD: MULTIPLICATIVE INTERACTION EFFECTS OF POSITIVE ANTIPHOSPHOLIPID ANTIBODIES WITH LOW TESTOSTERONE

PURPOSE: In this study we investigated the association of Antiphospholipid Antibody Syndrome with Hypogonadism and COPD
METHODS: A retrospective analysis of patients seen at clinics in Texas (January 2017-October 2018). We analyzed four different categories of APAs (antibodies against Cardiolipin, Beta-2-Glycoprotein-1, Phosphatidylserine and Phosphatidylserine/Prothrombin complex) and their interaction with testosterone. Logistic models were employed to determine association between risk factors (testosterone levels, APAs) and COPD.
RESULTS: Almost half (45%) of patient population suffered from COPD, and 54% were females. Patients with COPD were older (68 years) compared to younger (52 years) patients without COPD. Higher level of AntiPhosphatidylserineIgA were positively associated with COPD while for AntiCardiolipin antibodies series, only IgM subtypes were negatively associated with outcome. Multivariate logistic regression analysis, after adjusting for age, gender, showed that older males are more susceptible to disease. Patients with low testosterone were 2 times more likely to develop COPD compared to those with normal levels of testosterone (2.16; 95%CI 1.56:2.98; P<0.00), while having higher levels of testosterone were 60% less susceptible to develop COPD (0.39; 95%CI 0.17-0.91; P = 0.00). Those who have low testosterone and high titer of aPSPTIgM were 12 times more likely to develop COPD compared to those with normal testosterone and normal aPSPTIgM titer. (12.3; 95%CI 1.93:78.15; P<0.00).
CONCLUSIONS: The findings suggest that the presence of APAs has pathologic significance in patients with hypogonadism and COPD.
CLINICAL IMPLICATIONS: Knowledge of biological interplay will help healthcare providers to utilize the available diagnostic resources and provide a personalized care for co-morbid patients.

Contributors: G. Nowshad, M.A. Ayass, L. Abi Mosleh, K. Zhu, K. Viswanathan, K. Wilgers 

0356 Prevalence And Risk Factors For Sleep Disorders In Us Population (SLEEP-Volume 41)

Published: 27 April 2018

0356 Prevalence And Risk Factors For Sleep Disorders In Us Population

Introduction

Sleep disorders (SD) are common medical conditions which are more prevalent and under-diagnosed causing adverse health outcomes. Recently few studies have indicated the role of vitaminD in improving sleep.

It is hypothesized that vitD is controlling sleep and other human diseases such a hypertension, diabetes, depression and multiple sclerosis via brainstem. No optimal serum concentrations vitD for sleep have been established. Higher levels or vitD deficiency may have implications for sleep.

Methods

The purpose of this study is to investigate whether VitD deficiency is a risk factor for SD. To assess the prevalence of SD in noninstitutionalized U.S. population and to examine the relationship of vitD levels with SD, we used demographic, lab and clinical data from the National Health and Nutrition Examination Survey (Years 2011–2012). Sleep disorders were diagnosed by physicians via questionnaire among adult participants. Vitamin D deficiency was defined as serum 25-hydroxyvitaminD <30 nmol/L, insufficiency (30–50), normal (50–125) and hypervitaminosisD as above 125. Logistic regression models were employed to determine association between vitD levels and sleep disorders.

Results

Out of 5,913 study participants, one fourth were diagnosed with SD. Half (51%) were females, mean age 31 years and 35% were White. Individuals suffering from vitD deficiency were 9%, 26% with insufficient levels and 3% had hypervitaminosisD. Univariate analysis exhibited paradoxical association of levels of vitD with occurrence of sleep disorders; those with hypervitaminosisD, have twice odds of developing SD (OR, 1.93; 95% CI, 1.41–2.64), those with insufficient levels were 25% less likely to develop the SD (OR, 0.75; 95% CI, 0.65–0.78) and those with deficient levels were 30% less likely to be diagnosed with SD. (OR, 0.77; 95% CI, 0.55–0.88). Multivariate analysis after controlling for age, gender and ethnicity, only hypervitaminosisD remain positively associated with SD (OR, 1.44; 95% CI, 1.07–2.05). Female gender, older age and being White or Black were also positively associated with SD.

Conclusion

Prevalence of SD was 24% with hypervitaminosisD as a risk factor for SD while paradoxically deficiency and insufficiency were promoting good sleep.

Contributors: M A Ayass, G Nowshad

Hyperhomocysteinemia as a Predictive Biomarker for Impaired Diffusion Capacity of the Lung for Carbon Monoxide (CHEST Journal)

October 28, 2015

Hyperhomocysteinemia as a Predictive Biomarker for Impaired Diffusion Capacity of the Lung for Carbon Monoxide

PURPOSE: Research has suggested that Hyperhomocysteinemia increased the risk of thrombus formation, by irritating the linings of the blood vessels. Although Hyperhomocysteinemia is considered as a cardiovascular predictive biomarker but none of the studies have examined its role as a pulmonary risk factor. The purpose of the study was to describe the association of Hyperhomocysteinemia with pulmonary diffusion capacity.
METHODS: Four hundred study subjects were randomly selected in an outpatient clinic in Texas. Demographic, laboratory and clinical information including co-morbid conditions (OSA, hypertension, diabetes, cardiac diseases) and measurement of the diffusion capacity for carbon monoxide (DCLCo) were obtained for each patient. Standard descriptive and logistic regression techniques were employed with outcome variable as impaired lung diffusion (DCLCo less than 70%) and predictor variable hyperhomocysteinemia (homocysteine >12μmol/L).
RESULTS: 160 patients (41%) with impaired lung diffusion for CO were mostly older (74% older than 65), females, nonsmoker, and obese (62%). Out of these, 14.2% have mild, 10.5 moderate and 17.7% have severely impaired lung diffusion. Unadjusted logistic regression reveals high homocystein levels are associated with impaired lung diffusion capacity (OR 2.54, P=0.01). Multivariate analysis after controlling for age, gender, ethnicity and comorbid conditions indicated that Hyperhomocysteinemia is significantly associated with lung diffusion capacity impairment (OR 2.50, P=0.03). In asthmatic patients, association of Hyperhomocysteinemia and lung diffusion was statistically significant (OR 2.30, P=0.02); in nonasthmatic, the Hyperhomocysteinemia and DCLCo correlation was not significant. (OR 1.21, P=0.71)
CONCLUSIONS: Hyperhomocysteinemia is associated with impaired lung diffusion in adult patients and specifically for those who were asthmatic.
CLINICAL IMPLICATIONS: Risk-identification in specific patient population will improve the quality of customized care and dissemination of relevant information about co-morbid conditions as an additional health risk to the patients, which will result in early prevention and improved patient’s outcomes.
DISCLOSURE: The following authors have nothing to disclose: Gul Nowshad, Mohamad Ayass

Contributors: Gul Nowshad, PhD, Mohamad Ayass, MD

Prevalence and Correlates of Immunoglobulin IgA Deficiency in Adult Outpatient Population (JACI – The Journal of Allergy and Clinical Immunology)

February 2015

Prevalence and Correlates of Immunoglobulin IgA Deficiency in Adult Outpatient Population

Rationale

IgG subclasses are low in 2.5 percent of the general population. Few studies have explored the prevalence and risk factors for IgG deficiency in adult patient population.

Methods

Four hundred patients examined at Ayass Lung Clinic & Sleep Center in 2013 were included in the study to analyzed IgG levels and their association with other diseases and immunoglobulin deficiencies. Demographic, clinical; and laboratory information were obtained from medical records. Logistic regression models were employed to determine association between risk factors and the presence of IgG deficiency.

Results

Seventy three patients (18%) were IgG deficient (< 750mg/dl). Among those who were IgG deficient were 18% IgM deficient and 22% IgA deficient. Three quarter were aged (>65 years), 62% females and 92% were White. Analysis showed that being White, being in age groups >65, suffering from OSA and other immunoglobulin deficiencies were associated with the IgG deficiency. White adult patients compared to non-White patients were eight times more likely to develop IgG deficiency (CI, 02.30-30.0) after controlling for the demographics. Similarly patients with IgA deficiency and IgM deficiency, were 18 times (CI, 6.40-55.4) and 3.4 times (CI, 1.40-8.40) more likely to develop IgG deficiency, respectively. Patients with obstructive sleep apnea were 63% (CI, 0.17-0.78) and older patients were 73% less likely to develop IgG deficiency (CI, 0.14-0.53), respectively.

Conclusions

The prevalence of IgG deficiency in adult outpatient population is Texas was 18%. IgG subclass deficiency is the major phenotype of primary Immunodeficiency and being White, and suffering from OSA were associated with IgG deficiency.

Contributors: Mohamad Ammar Ayass, MD, Ayesha Jabeen, MBBS, Gul Nowshad, MD, MPH, PhD