Biology subjects

Varghese, P.

Publications and source records attributed to Varghese, P..

5 recordsLinked to original sources

bioRxiv2024

The human complement factor B-C3b complex: Investigation of the interaction using C3b bound to thiol-Sepharose

Factor B, a serine protease proenzyme and part of the complement system, binds to other complement proteins such as C3b and properdin. While it is known to be activated by factor D, other interactions that interfere with the binding between Factor B and C3b are less well-understood. We attached C3b to a thiol Sepharose via its free SH group and conducted a competition assay employing 125I-labelled factor B to study competitive binding. Two anti-C3d monoclonal antibodies (F49b and 4C2) were found to partially inhibit the binding of Factor B to C3b. The inhibitory effects of these monoclonal antibodies were found to be dose-dependent. 4C2 was found to have a maximum inhibition of 50%, while the maximum inhibition by F49b was [~]40%. In contrast, two anti-C3c monoclonal antibodies (F39b and F20b) were able to enhance the formation of the factor B-C3b complex in a dose-dependent manner. Competition binding studies with isolated C3 fragments further supported the involvement of the C3d region in factor B binding, as C3d inhibited the binding of factor B to C3b completely. Furthermore, additional competition binding studies conducted with each of the three domains of factor B (Ba, vWF and SP) demonstrated that each domain independently inhibited the binding of intact factor B to C3b by [~]50%.

immunology↗

bioRxiv2023

Neutrophil-derived complement factor P induces cytotoxicity in basal-like cells via caspase 3/7 activation in pancreatic cancer

Due to profound heterogeneity within stromal immune tumor microenvironment (TME), pancreatic ductal adenocarcinoma (PDAC) remains a hard to treat disease, with the lowest 5-year survival below 10%. Large-scale transcriptomic analysis has revealed two main, clinically relevant PDAC signatures: therapy responsive Classical subtype with better prognosis, and poorly-differentiated Basal-like with poor prognosis. It has also become evident that the cellular and humoral components in the immune TME considerably influence the outcome of tumorigenesis. Complement system, a potent humoral innate immune mechanism, also forms a part of this immune TME. In addition to the regular production of various complement components in the liver, certain infiltrating immune cells such as macrophages, dendritic cells and neutrophils, can produce a few complement components locally at the site of infection and inflammation including TME, and modulate tumorigenic outcomes. Neutrophils are the most prevalent innate immune cells in the PDAC TME; however, its role has been attributed as either pro-tumorigenic or anti-tumorigenic. Neutrophils, when stimulated or under stress, are capable of releasing their secretory granules that also contain the only known up-regulator of the complement alternative pathway, Complement Factor P (CFP) or properdin. Properdin can not only facilitate alternative pathway activation by stabilising the C3 convertase, but also act as a pattern recognition receptor on its own and modify inflammatory response. Here, by combining multicenter transcriptome analysis of PDAC patient tumors, single-cell-RNA-seq analysis, preclinical mouse models and human PDAC specimens, we show that properdin expression and neutrophil surveillance are linked to better prognosis in PDAC patients. Furthermore, properdin expression is substantially higher in well-to-moderately differentiated Classical subtype compared to the highly aggressive basal-like PDAC tumours. Mechanistically, exogenous properdin binds to the cell membrane and activates caspase 3/7 to induce apoptosis in basal-like PDAC cells. Together, these findings suggest that the complement protein, properdin, could be a favorable prognostic factor and exhibit anti-tumorigenic functions in PDAC.

immunology↗

bioRxiv2023

Activation of Complement by Human Fibrin Clots: involvement of C1q and factor H

The classical pathway of the complement system is activated by the binding of C1q in the C1 complex to the target activator including immune complexes. Factor H is regarded as the key downregulatory protein of the alternative pathway of complement. However, C1q and factor H both bind to target surfaces via charge distribution patterns. For few targets, C1q and factor H compete for binding to common or overlapping sites. Factor H, therefore, can effectively regulate the classical pathway activation by such targets, in addition to its previously characterized role in the alternative pathway. Both C1q and factor H are reported to recognize "foreign" or altered-self materials. Clots, formed by the coagulation system, are an example of altered self. Factor H is present abundantly in platelets and is a well-known substrate for FXIIIa. Here, we investigated whether clots activate the classical pathway of complement and whether this is regulated by factor H. We show here that both C1q and factor H bind to fibrin formed in microtitre plates as well as fibrin clots formed under in vitro physiological conditions. Both C1q and factor H become covalently bound to fibrin clots and this is mediated via FXIIIa. We also show that fibrin clots activate the classical pathway of complement, as demonstrated by C4 consumption and membrane attack complex detection assays. Thus, factor H downregulates the classical pathway activation induced by fibrin clots. These results elucidate the intricate molecular mechanisms through which the complement and coagulation pathways intersect and have regulatory consequences.

immunology↗

bioRxiv2023

Mixed IgG Fc immune complexes exhibit blended binding profiles and refine FcR affinity estimates

Immunoglobulin (Ig)G antibodies coordinate immune effector responses by selectively binding to target antigens and then interacting with various effector cells via the Fc{gamma} receptors. The Fc domain of IgG can promote or inhibit distinct effector responses across several different immune cell types through variation based on subclass and Fc domain glycosylation. Extensive characterization of these interactions has revealed how the inclusion of certain Fc subclasses or glycans results in distinct immune responses. During an immune response, however, IgG is produced with mixtures of Fc domain properties, so antigen-IgG immune complexes are likely to almost always be comprised of a combination of Fc forms. Whether and how this mixed composition influences immune effector responses has not been examined. Here, we measured Fc{gamma} receptor binding to immune complexes of mixed Fc domain composition. We found that the binding properties of the mixed-composition immune complexes fell along a continuum between those of the corresponding pure cases. Binding quantitatively matched a mechanistic binding model, except for several low-affinity interactions mostly involving IgG2. We found that the affinities of these interactions are different than previously reported, and that the binding model could be used to provide refined estimates of these affinities. Finally, we demonstrated that the binding model can predict effector-cell elicited platelet depletion in humanized mice, with the model inferring the relevant effector cell populations. Contrary to the previous view in which IgG2 poorly engages with effector populations, we observe appreciable binding through avidity, but insufficient amounts to observe immune effector responses. Overall, this work demonstrates a quantitative framework for reasoning about effector response regulation arising from IgG of mixed Fc composition. Summary pointsO_LIThe binding behavior of mixed Fc immune complexes is a blend of the binding properties for each constituent IgG species. C_LIO_LIAn equilibrium, multivalent binding model can be generalized to incorporate immune complexes of mixed Fc composition. C_LIO_LIParticularly for low-affinity IgG-Fc{gamma} receptor interactions, immune complexes provide better estimates of affinities. C_LIO_LIThe Fc{gamma}R binding model predicts effector-elicited cell clearance in humanized mice. C_LI

immunology↗

bioRxiv2020

A recombinant fragment of Human surfactant protein D binds Spike protein and inhibits infectivity and replication of SARS-CoV-2 in clinical samples

RationaleCOVID-19 is an acute infectious disease caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Human surfactant protein D (SP-D) is known to interact with spike protein of SARS-CoV, but its immune-surveillance against SARS-CoV-2 is not known. ObjectiveThis study aimed to examine the potential of a recombinant fragment of human SP-D (rfhSP-D) as an inhibitor of replication and infection of SARS-CoV-2. MethodsrfhSP-D interaction with spike protein of SARS-CoV-2 and hACE-2 receptor was predicted via docking analysis. The inhibition of interaction between spike protein and ACE-2 by rfhSP-D was confirmed using direct and indirect ELISA. The effect of rfhSP-D on replication and infectivity of SARS-CoV-2 from clinical samples was studied by measuring the expression of RdRp gene of the virus using qPCR. Measurements and Main ResultsIn-silico interaction studies indicated that three amino acid residues in the RBD of spike of SARS-CoV-2 were commonly involved in interacting with rfhSP-D and ACE-2. Studies using clinical samples of SARS-CoV-2 positive cases (asymptomatic, n=7 and symptomatic, n=8 and negative controls n=15) demonstrated that treatment with 5M rfhSP-D inhibited viral replication by ~5.5 fold and was more efficient than Remdesivir (100 M). Approximately, a 2-fold reduction in viral infectivity was also observed after treatment with 5M rfhSP-D. ConclusionsThese results conclusively demonstrate that the calcium independent rfhSP-D mediated inhibition of binding between the receptor binding domain of the S1 subunit of the SARS-CoV-2 spike protein and human ACE-2, its host cell receptor, and a significant reduction in SARS-CoV-2 infection and replication in-vitro.

immunology↗