Publications:

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

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

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.

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

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

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

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.
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

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.

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.

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

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

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.
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

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.

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.

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.

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.

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.