Biology subjects

Dimitrov, J. D.

Publications and source records attributed to Dimitrov, J. D..

10 recordsLinked to original sources

bioRxiv2024

An integrative approach for profiling antibody responses in bats to human pathogens

Serological analyses are a fundamental tool for identifying infections by a wide range of pathogens. They offer a current overview of pathogen prevalence and insight into past infections. This is particularly relevant for bats, given their high capacity to tolerate pathogens and their role as reservoirs of zoonotic diseases. At present, serological studies in bats have predominantly employed traditional techniques such as enzyme-linked immunosorbent assay (ELISA). However, these techniques have several limitations, including low throughput and the lack of bat-specific detection antibodies. To address these limitations, we developed an integrative approach for systemic serological analyses based on microarray technology, which enables the simultaneous detection of bat IgG antibodies against >190 human pathogens (viruses, bacteria, protists). The results of our analyses demonstrated an antibody response in bats targeting multiple epitopes from different pathogens, thereby proving the methods high-throughput capability. Furthermore, this approach does not rely on the use of IgG detection reagents, thereby allowing for its application to a diverse range of bat species. This assay offers insights into the infections of bats with pathogens, thereby enhancing our comprehension of zoonotic disease dynamics and facilitating targeted pathogen surveillance.

immunology↗

bioRxiv2024

Spleen tyrosine kinase inhibition mitigates hemin-induced thromboinflammation in the lung and kidney of sickle cell mice

Sickle cell disease (SCD) leads to hemolytic anemia, vaso-occlusive crisis (VOC), hypoperfusion, and progressive organ damage. Hemin, released during hemolysis in SCD, induces platelet activation through CLEC-2, endothelial activation through TLR4, neutrophil adhesion and NETosis, all of which are regulated by spleen tyrosine kinase (Syk). In this study, we assessed neutrophil and platelet recruitment to the pulmonary, renal, splenic, and hepatic microvasculature in control and SCD mice following hemin injection and the effect of Syk inhibition on cell recruitment and organ perfusion. Compared to controls, SCD mice exhibited higher baseline neutrophil and platelet recruitment to the lungs without alterations in lung perfusion as measured by laser speckle contrast imaging. Injection of hemin increased cell recruitment to the pulmonary and renal vasculature with a concomitant reduction in organ perfusion. However, hemin injection did not change cell recruitment or organ perfusion in the spleen and liver, both of which were altered at baseline in SCD mice. Pretreatment of SCD mice with the Syk inhibitor BI-1002494 mitigated baseline and hemin-induced neutrophil and platelet adhesion in the pulmonary and renal microvasculature, with a corresponding normalization of perfusion. Syk regulates vascular integrity in the lung of SCD mice; whilst high concentrations of BI-1002494 increased bleeding, lowering drug concentrations preserved the inhibitory effect on platelet and neutrophil recruitment and lung perfusion and protected from bleeding complications. These data substantiate Syk as a mediator of vascular thrombo-inflammation and hypoperfusion in the lung and kidney of SCD and provide a rationale for pharmacological inhibition as a therapeutic strategy.

pathology↗

bioRxiv2023

Temperature sensitivity of bat antibodies links metabolic state with antigen-recognition diversity

Bat immune system features multiple unique properties such as dampened inflammatory responses and increased tissue protection, explaining their long lifespan and tolerance to viral infections. Here, we demonstrated that body temperature fluctuations corresponding to different physiological states in bats exert a dramatic impact on their antibody repertoires. At elevated temperatures typical for flight, IgG from Myotis myotis and Nyctalus noctula showed elevated antigen binding strength and diversity, recognizing both pathogen-derived antigens and autoantigens. The opposite was observed at temperatures reflecting inactive physiological states. This behavior was not observed for IgG antibodies of human and other mammals, or antibodies of birds. Importantly, diversification of bat antibody specificities resulted in preferential recognition of damaged endothelial and epithelial cells, indicating an anti-inflammatory function. The temperature-sensitivity of bat antibodies was mediated by the variable regions of immunoglobulin molecules. Additionally, we revealed specific molecular features of bat IgG such as low thermodynamic stability and implication of hydrophobic interactions in antigen binding as well as high prevalence of polyreactivity. Overall, our results extend the understanding of bat tolerance to disease and inflammation and highlight the link between metabolism and immunity. This might have important repercussions for human health in the future.

immunology↗

bioRxiv2023

Polyreactivity of antibodies from different B cell subpopulations is determined by distinct sequence patterns of variable region

An antibody molecule that is able to bind to multiple distinct antigens is defined as polyreactive. In the present study we performed statistical analyses to assess sequence correlates of polyreactivity of >600 antibodies cloned from different B cell types of healthy humans. The data reveled a number of sequence patterns of variable regions of heavy and light immunoglobulin chains that determine polyreactivity. The most prominent identified patterns were increased number of basic amino acid residues, reduced frequency of acidic residues, increased number of aromatic and hydrophobic residues, as well as longer length of CDR L1. Importantly, our study revealed that antibodies isolated from different B cell population used distinct sequence patterns (or combinations of them) for polyreactive antigen binding. Furthermore, we combined the data from sequence analyses with molecular modeling of selected polyreactive antibodies, and demonstrate that human antibodies can use multiple pathways for achieving antigen binding promiscuity. These data reconcile some contradictions in literature regarding the determinants of antibody polyreactivity. Moreover, our study demonstrates that mechanism of polyreactivity of antibodies evolves during immune response and might be tailored to specific functional properties of different B cell compartments. Finally, these data can be of use for efforts in development and engineering of therapeutic antibodies.

immunology↗

bioRxiv2023

Post-translational regulation of the Numb/Notch pathway in neurogenesis and cancer by Dlk2

Perturbations in fundamental developmental pathways have a profound influence on tumorigenesis. Numb plays a pivotal role in vertebrate development, including neurogenesis and is a key negative regulator of Notch signaling1, 2. Perturbation of Numb expression affects brain morphology and cell fate3. While extensive research has been conducted on canonical Notch ligands, regulation by vertebrate-specific non-canonical ligands is not understood. Here we identify Delta like non-canonical Notch ligand 2/EGFL9 (Dlk2) as a regulator of zebrafish neurogenesis with mutants exhibiting early increase and subsequent depletion of neural stem cells, decreased radial glial cells density, impaired neuronal cell distribution, and hypersensitivity to stimuli mimicking the embryonic murine Numb/Numblike null phenotype3. Numb function is inactivated by aberrant phosphorylation4, and we show that Dlk2 protein exhibits a high affinity direct interaction with Numb, with loss of Dlk2 in zebrafish telencephalon increasing Numb Ser276 phosphorylation with a concomitant increase in Notch signaling. Patients with tumors exhibiting reduced levels of Dlk2 have a poorer prognosis, while overexpression of Dlk2 in human cancer cell lines reduces cell proliferation. Our findings identify Dlk2 as a key partner of Numb, a gatekeeper of its activity, and an important player in a network of protein interactions regulating both neurogenesis and cancer with potential therapeutic implications.

cell biology↗

bioRxiv2022

Evolutionary trajectory of the physicochemical mechanism of interaction of SARS-CoV-2 spike protein with ACE2

SARS-CoV-2 infects cells by attachment to its receptor - the angiotensin converting enzyme 2 (ACE2). Regardless of the wealth of structural data, little is known about the physicochemical mechanism of interactions of the viral spike (S) protein with ACE2 and how this mechanism has evolved during the pandemic. Here, we applied experimental and computational approaches to characterize the molecular interaction of S proteins from SARS-CoV-2 variants of concern (VOC). Data on kinetics, activation- and equilibrium thermodynamics of binding of the receptor binding domain (RBD) from VOC with ACE2 as well as data from computational protein electrostatics revealed a profound remodeling of the physicochemical characteristics of the interaction during the evolution. Thus, as compared to RBDs from Wuhan strain and other VOC, Omicron RBD presented as a unique protein in terms of conformational dynamics and types of non-covalent forces driving the complex formation with ACE2. Viral evolution resulted in a restriction of the RBD structural dynamics, and a shift to a major role of polar forces for ACE2 binding. Further, we investigated how the reshaping of the physicochemical characteristics of interaction affect the binding specificity of S proteins. Data from various binding assays revealed that SARS-CoV-2 Wuhan and Omicron RBDs manifest capacity for promiscuous recognition of unrelated human proteins, but they harbor distinct reactivity patterns. These findings might contribute for mechanistic understanding of the viral tropism, and capacity to evade immune responses during evolution.

biochemistry↗

bioRxiv2022

Potent Human Broadly SARS-CoV-2 Neutralizing IgA and IgG Antibodies Effective Against Omicron BA.1 and BA.2

Memory B-cell and antibody responses to the SARS-CoV-2 spike protein contribute to long-term immune protection against severe COVID-19, which can also be prevented by antibody-based interventions. Here, wide SARS-CoV-2 immunoprofiling in COVID-19 convalescents combining serological, cellular and monoclonal antibody explorations, revealed humoral immunity coordination. Detailed characterization of a hundred SARS-CoV-2 spike memory B-cell monoclonal antibodies uncovered diversity in their repertoire and antiviral functions. The latter were influenced by the targeted spike region with strong Fc-dependent effectors to the S2 subunit and potent neutralizers to the receptor binding domain. Amongst those, Cv2.1169 and Cv2.3194 antibodies cross-neutralized SARS-CoV-2 variants of concern including Omicron BA.1 and BA.2. Cv2.1169, isolated from a mucosa-derived IgA memory B cell, demonstrated potency boost as IgA dimers and therapeutic efficacy as IgG antibodies in animal models. Structural data provided mechanistic clues to Cv2.1169 potency and breadth. Thus, potent broadly neutralizing IgA antibodies elicited in mucosal tissues can stem SARS-CoV-2 infection, and Cv2.1169 and Cv2.3194 are prime candidates for COVID-19 prevention and treatment.

immunology↗

bioRxiv2021

Induced antigen-binding polyspecificity in human serum IgA

Previous studies have shown that polyreactive antibodies play an important role in the frontline defense against the dissemination of pathogens in the pre-immune host. Interestingly, antigen-binding polyreactivity can not only be inherent, but also acquired post-translationally. The ability of individual monoclonal IgG and IgE antibodies to acquire polyreactivity following contact with various agents that destabilize protein structure (urea, low pH) or have pro-oxidative potential (heme, ferrous ions) has been studied in detail. However, to the best of our knowledge this property of human IgA has previously been described only cursorily. In the present study pooled human serum IgA and two human monoclonal IgA antibodies were exposed to buffers with acidic pH, to free heme or to ferrous ions, and the antigen-binding behavior of the native and modified IgA to viral and bacterial antigens were compared using immunoblot and ELISA. We observed a dose-dependent increase in reactivity to several bacterial extracts and to pure viral antigens. This newly described property of IgA may have therapeutic potential as has already been shown for pooled IgG with induced polyreactivity.

immunology↗

bioRxiv2020

Interaction of clinical-stage antibodies with heme predicts physiochemical and functional liabilities relevant for developability

Immunoglobulin repertoires contain a fraction of antibodies that recognize low molecular weight compounds, including some enzymes cofactors, such as heme. Here, by using a set of 113 samples with variable region sequences matching clinical-stage antibodies, we demonstrated that a considerable number of these antibodies interact with heme. Antibodies that interact with heme possess specific sequence traits of their antigen-binding regions. Moreover they manifest particular physicochemical and functional qualities i.e. increased hydrophobicity, higher propensity of self-binding, higher intrinsic polyreactivity and reduced expression yields. Thus, interaction with heme is a strong predictor of different molecular and functional qualities of antibodies. Notably, these qualities are of high importance for therapeutic antibodies, as their presence was associated with failure of drug candidates to reach clinic. Our study reveled an important facet of information about relationship sequence-function in antibodies. It also offers a convenient tool for detection of liabilities of therapeutic antibodies.

immunology↗

bioRxiv2019

Method for identification of heme-binding proteins and quantification of their interactions

The standard assay for characterization of interaction of heme with proteins is absorbance spectroscopy. However, this approach demands relatively large quantities of proteins and it is difficult to perform in high-throughput manner. Here, we describe an immunosorbent assay based on the covalent in situ conjugation of heme to a pre-coated carrier. Advantage of this assay is that it allows both identification of heme-binding proteins and quantification of their binding avidity, using only minimal amounts of protein (1-10 μg). Importantly, the same approach can be used for covalent linkage of other natural or synthetic compounds and analyzing their interactions with proteins.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗