Manni Luthra-Guptasarma

HLA-B27 lacking associated β2-microglobulin rearranges to auto-display or cross-display residues 169–181: a novel molecular mechanism for spondyloarthropathies

Expression of the MHC class I allelle, HLA‐B27, is correlated with autoimmune disease. The misfolding and association of B27 heavy chains through non‐native disulfide bonds has recently been implicated. Here, we propose that β2m‐free, peptide‐free heavy chains support a helix‐coil transition in the segment leading from the α2 domain to the α3 domain, facilitating rotation of backbone angles around residues 167/168, and allowing residues 169–181 (identical to a known B27 ligand) to loop around and occupy the molecules own peptide‐binding cleft. Such ‘auto‐display’, occurring either within B27 molecules, or between B27 molecules, could provoke autoimmune attack.

Fibrotic Remodeling of the Extracellular Matrix through a Novel (Engineered, Dual-Function) Antibody Reactive to a Cryptic Epitope on the N-Terminal 30 kDa Fragment of Fibronectin

Fibrosis is characterized by excessive accumulation of scar tissue as a result of exaggerated deposition of extracellular matrix (ECM), leading to tissue contraction and impaired function of the organ. Fibronectin (Fn) is an essential component of the ECM, and plays an important role in fibrosis. One such fibrotic pathology is that of proliferative vitreoretinopathy (PVR), a sight-threatening complication which develops as a consequence of failure of surgical repair of retinal detachment. Such patients often require repeated surgeries for retinal re-attachment; therefore, a preventive measure for PVR is of utmost importance. The contractile membranes formed in PVR, are composed of various cell types including the retinal pigment epithelial cells (RPE); fibronectin is an important constituent of the ECM surrounding these cells. Together with the vitreous, fibronectin creates microenvironments in which RPE cells proliferate. We have successfully developed a dual-action, fully human, fibronectin-specific single chain variable fragment antibody (scFv) termed Fn52RGDS, which acts in two ways: i) binds to cryptic sites in fibronectin, and thereby prevents its self polymerization/fibrillogenesis, and ii) interacts with the cell surface receptors, ie., integrins (through an attached “RGD” sequence tag), and thereby blocks the downstream cell signaling events. We demonstrate the ability of this antibody to effectively reduce some of the hallmark features of fibrosis - migration, adhesion, fibronectin polymerization, matrix metalloprotease (MMP) expression, as well as reduction of collagen gel contraction (a model of fibrotic tissue remodeling). The data suggests that the antibody can be used as a rational, novel anti-fibrotic candidate.

Targeting the Fibronectin Type III Repeats in Tenascin-C Inhibits Epithelial–Mesenchymal Transition in the Context of Posterior Capsular Opacification

PURPOSE Posterior capsular opacification (PCO) is a common complication following extracapsular surgery, associated with fibrosis, opacification, and contraction of the posterior lens capsule. It is characterized by increased expression of extracellular matrix proteins such as tenascin-C, fibronectin, collagens, and proteoglycans. Tenascin-C is known to be critical for injury-induced epithelial-mesenchymal transition (EMT) in the lens epithelium. We aimed to target fibronectin type III repeats 1-5 within tenascin-C (TNfnIII 1-5) using an scFv (single-chain variable fragment) antibody, and to evaluate its effectiveness in the context of lens epithelial cells. METHODS Phage display library screening was used to generate an antibody against TNfnIII 1-5. Lens epithelial cells were cultured in the presence of the scFv antibodies to evaluate the effects on cell proliferation, migration, fibronectin polymerization and deposition, matrix metalloprotease (MMP) regulation, actin stress fiber distribution, and expression of EMT markers. The effect on SMAD-dependent and SMAD-independent pathways was also examined. RESULTS The scFv TN64 was found to be effective in regulating the proliferation, migration, and expression of MMP-2 and MMP-9, fibronectin polymerization and deposition, and expression of EMT markers. TN64 did not interfere with SMAD3 phosphorylation. Altered localization of β-catenin, as well as downregulation of phosphorylation of mitogen-activated protein (MAP) kinases and focal adhesion kinase (FAK), was involved. CONCLUSIONS Our data suggest that the TNfnIII 1-5 repeats play an important role in PCO pathology. The inhibition of EMT by TN64 is mediated by SMAD-independent, integrin-β-catenin-FAK signaling pathway, and is therefore proposed as a novel antifibrotic therapeutic candidate.

Effect of steroids on the activation status of platelets in patients with Immune thrombocytopenia (ITP)

Abstract The activation status of platelets in Immune Thrombocytopenia (ITP) patients – which is still somewhat controversial – is of potential interest, because activated platelets tend to aggregate (leading to excessive clotting or thromboembolic events) but cannot do so when platelet numbers are low, as in ITP. Although corticosteroids are the first line of therapy in ITP, the effect of steroids on activation of platelets has not been evaluated so far. We examined the status of platelet activation (with and without stimulation with ADP) in ITP patients, at the start of therapy (pre-steroid treatment, naive) and post-steroid treatment (classified on the basis of steroid responsiveness). We used flow cytometry to evaluate the levels of expression of P-selectin, and PAC-1 binding to platelets of 55 ITP patients and a similar number of healthy controls, treated with and without ADP. We found that platelets in ITP patients exist in an activated state. In patients who are responsive to steroids, the treatment reverses this situation. Also, the fold activation of platelets upon treatment with ADP is more in healthy controls than in ITP patients; treatment with steroids causes platelets in steroid-responsive patients to become more responsive to ADP-activation, similar to healthy controls. Thus steroids may cause changes in the ability of platelets to get activated with an agonist like ADP. Our results provide new insights into how, and why, steroid therapy helps in the treatment of ITP.

Phenomenological Perspectives on the Folding of β/α‐Barrel Domains Through the Modular Formation and Assembly of Smaller Structural Elements

The β/α‐barrel motif was once considered to be a single protein domain. In recent years, however, it has been shown to consist of smaller substructures displaying the ability to fold autonomously. Here we review the current status of experimental findings concerning the motifs folding behavior in the light of what is currently known about (a) the relative rates of formation of helices and sheets in proteins, in general, and (b) the peculiarities of topology and architecture of the motif, in particular, to develop a detailed phenomenological understanding of how β/α‐barrels might form through the modular folding and assembly of substructures.

Expression of Granulocyte Colony Stimulating Factor and Its Receptor by Retinal Pigment Epithelial Cells: A Role in Maintaining Differentiation-Competent State

Purpose: Granulocyte colony stimulating factor (GCSF) is a potent hematopoietic factor that stimulates the growth of neutrophil granulocyte precursors, and also regulates the differentiation and survival of neutrophils by inhibiting apoptosis. Incidentally, GCSF is also known to act as an endogenous ligand for brain cells, counteracting acute neuronal degeneration and contributing to long-term plasticity of progenitor cells after cerebral ischemia. Since GCSF was recently reported to be present in retinal ganglions, we examined its expression in retinal pigment epithelial (RPE) cells, which, together with retinal neurons, arise from the same underlying precursor cells. Methods: We used reverse transcriptase polymerase chain reaction (PCR) to assay expression of GCSF and GCSF receptor (GCSFR) genes; immunostaining and flow cytometry to assay the presence of GCSFR on cell surfaces; bromodeoxyuridine (BrdU) incorporation measurement to monitor DNA synthesis; and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to monitor cell proliferation. The effect of GCSF on differentiation of RPE cells was assessed by immunocytochemistry to detect the presence of various marker proteins. Results: The D407 RPE cells, as well as RPE derived from cadaver eyes, were found to express both GCSF and GCSFR. Despite the presence of the GCSF receptor, exogenously added GCSF did not result in any proliferation of these cells. We found that GCSF acts like a de-differentiating factor, maintaining RPE cells in the rounded form, and in a transdifferentiation-competent state. Conclusions: The expression of GCSF and GCSFR by D407 RPE may be an important factor in RPE cell maintenance.

Control of fibrotic changes through the synergistic effects of anti-fibronectin antibody and an RGDS-tagged form of the same antibody.

TGF-β and myofibroblasts play a key role in fibrosis, characterized by aberrant synthesis and deposition of extracellular matrix (ECM) proteins, such as fibronectin (Fn) and collagen type I. There are two major roles played by integrins in the fibrotic pathology: (i) Fn-integrin interaction, coupled with cytokines like TGF-β, facilitates the self-polymerization of Fn and regulates cell–matrix fibrillar adhesions, thereby promoting fibrillogenesis; (ii) Integrin interaction with an RGD (arginine-glycine–aspartic) consensus sequence in the latent TGF-β, resulting in its activation. This study describes an anti-fibrotic strategy using a combination of two antibodies: Fn52 (targeted against the N-terminal 30 kDa region of fibronectin, a major site for Fn self-association), and its engineered form, Fn52RGDS (which binds to integrins). Interestingly, a synergistic effect of the cocktail in causing a decline in fibrotic features was confirmed in the context of fibrotic posterior capsular opacification (PCO), mediated by the lens epithelial cells (left behind after cataract surgery). Inclusion of Fn52RGDS to Fn52 aids in better diffusion of the antibodies; such combination therapies could be useful in the context of pathologies involving extensive remodeling of the fibronectin matrix, where the thick ECM offers a major challenge for efficient drug delivery.

Investigation of the Possible Association between the HLA Antigens and Idiopathic Thrombocytopenic Purpura (ITP)

The etiology of idiopathic thrombocytopenic purpura (ITP), characterized by destruction of platelets, is still poorly understood. Although genetic as well as immunological factors are thought to play a role in the disease pathogenesis, genetic association studies in terms of major histocompatibility complex (MHC) polymorphisms are scarce and discrepant. Results from previous studies suggest that different populations show varying associations with MHC alleles. Since i) there are inconsistencies in HLA associations, and ii) such an association study does not exist for the Indian subcontinent, we carried out sequence specific priming (SSP)-based genotyping of HLA DRB1 alleles in the North Indian population. Data for such studies is available for two East Asian countries, Japan and China, and the association in both cases is different. Further, among the Japanese population too, there are discrepant results. It was therefore important to analyze such an association in the Indian population, belonging to Southern Asia. Our data shows that none of the alleles have any significant association with ITP. Moreover, in contrast to other studies, comparison made between patients who were responsive to steroid therapy against those who were refractory to steroids, also did not show any association of the HLA DRB1 alleles with steroid responsiveness.

Differences in conformational stability of the two alpha domains of the disease-associated and non-disease-associated subtypes of HLA-B27

The MHC Class I molecule, HLA-B27, is strongly linked with development of the inflammatory arthritic disease, ankylosing spondylitis (AS); whereas the B*2705 subtype shows strong association, B*2709 is not associated with disease, even though the two subtypes differ in only a single residue at position 116. Currently, attention is focused on the misfolding propensities of these two subtypes, including studies of disulfide-linked dimers and non-covalently formed high molecular weight (HMW) aggregates. Using mutants retaining only a single cysteine at positions C67 or C164, and using a cysteine-reactive, environment-sensitive, fluorescence probe (acrylodan), we find that within the same overall population of identical single-cysteine HLA-B27 molecules, there exist sub-populations which (a) possess free cysteines which react with acrylodan, (b) form disulfide-linked dimers, and (c) form HMW aggregates. Further, using acrylodan fluorescence, we find (d) that the α1 and α2 domains unfold independently of each other in HMW aggregates, (e) that these two domains of B*2709 are less stable to chemical and thermal denaturation than the corresponding domains of B*2705, suggesting easier clearance of misfolded molecules in the former, and (f) C67 is much more exposed in B*2705 than in B*2709, which could potentially explain how B*2705 more easily forms C67-mediated disulfide-bonded dimers.

Immunodiagnosis of platelet activation in immune thrombocytopenia through scFv antibodies cognate to activated IIb3 integrins

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by low platelet count and presence of IgG autoantibodies to platelet surface glycoproteins, such as αIIbβ3 and GPIb/IX. Our previous work has shown that platelets in ITP patients exist in an activated state. Two different marker-based approaches are used to study the course of platelet activation: (1) binding of PAC-1 antibody, signifying a change in αIIbβ3 conformation, and (2) expression of P-selectin, signifying alpha granule content release from platelets. Here, we describe the development of a new scFv antibody (R38) that, compared with PAC-1, appears to better distinguish between platelets of ITP patients and healthy controls. Notably, R38 was generated using commercially sourced resting-state integrin that was coated on a microtiter plate. Its ability to distinguish between ITP patients and healthy controls thus suggests that inadvertent integrin activation caused by coating involves a conformational change and exposure of a cryptic epitope. This report also describes for the first time the potential use of an scFv antibody in the immunodiagnosis of platelet activation in ITP patients.