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Corneal wound healing requires epithelial reorganization and stromal extracellular matrix (ECM) remodeling, with ECM proteins such as Tenascin C (TnC) regulating and maintaining corneal homeostasis. The N-terminal globular domain and C-terminal fibrinogen-related domains of TnC are separated by epidermal growth factor (EGF)-like repeats, and upto fifteen fibronectin type III domains (Tn fn). Overexpression of Tn fn 1-5 and its splice variants occurs in varied pathologies. We have previously used Tn64 (a single chain variable fragment antibody cognate to Tn fn 1-5) to establish roles of Tn fn 1-5 in fibrotic pathologies such as rheumatoid arthritis and posterior capsular opacification. Here, we show that Tn64 binds to Tn fn repeats 3-5 (which constitute the major site for binding of soluble fibronectin within TnC). Unlike other Tn fn domains, Tn fn 3-5 displays no inhibition of fibronectin matrix assembly. Rather, the Tn fn 3-5 construct is pro-fibrotic and elicits increased expression of fibronectin. We examined corneal epithelial as well as stromal wound healing through Tn64 binding to Tn fn 3-5, using a human corneal epithelial cell (HCEC) line, primary cultures of human corneal fibroblasts (HCFs), and an ex-vivo corneal organ culture model. Tn64 enhanced proliferation and adhesion of corneal epithelial cells, while inhibiting the migration of corneal fibroblasts and myofibroblasts. Tn64 appears to attenuate inflammation through downregulation of TNF-α, prevent corneal fibrosis by limiting fibronectin polymerization, and promote regeneration of corneal epithelia and stroma, suggesting that it could be developed as a therapeutic agent for effective anti-fibrotic corneal wound healing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Limb Girdle Muscular Dystrophy R1 (LGMDR1) is an autosomal recessive neuromuscular disease caused by mutations in the calpain-3 (CAPN3) gene. As clinical and pathological features may overlap with other types of LGMD, therefore definite molecular diagnosis is required to understand the progression of this debilitating disease. This study aims to identify novel variants of CAPN3 gene in LGMDR1 patients., Results: Thirty-four patients with clinical and histopathological features suggestive of LGMD were studied. The muscle biopsy samples were evaluated using Enzyme histochemistry, Immunohistochemistry, followed by Western Blotting and Sanger sequencing. Out of 34 LGMD cases, 13 patients were diagnosed as LGMDR1 by immunoblot analysis, demonstrating reduced or absent calpain-3 protein as compared to controls. Variants of CAPN3 gene were also found and pathogenicity was predicted using in-silico prediction tools. The CAPN3 gene variants found in this study, included, two missense variants [CAPN3: c.1189T > C, CAPN3: c.2338G > C], one insertion-deletion [c.1688delinsTC], one splice site variant [c.2051-1G > T], and one nonsense variant [c.1939G > T; p.Glu647Ter]., Conclusions: We confirmed 6 patients as LGMDR1 (with CAPN3 variants) from our cohort and calpain-3 protein expression was significantly reduced by immunoblot analysis as compared to control. Besides the previously known variants, our study found two novel variants in CAPN3 gene by Sanger sequencing-based approach indicating that genetic variants in LGMDR1 patients may help to understand the etiology of the disease and future prognostication., (© 2024. The Author(s).)

Background: Corneal disease is a major cause of blindness. Transplantation of cadaver-derived corneas (keratoplasty) is still the current therapy of choice; however, the global shortage of donor corneas continues to drive a search for alternatives. To this end, biosynthetic corneal substitutes have recently begun to gain importance. Here, we present a novel method for the generation of a cornea-like tissue (CLT), using corneo-scleral rims discarded after keratoplasty., Methods and Results: Type I collagen was polymerized within the corneo-scleral rim, which functioned as a 'host' mould, directing the 'guest' collagen to polymerize into disc-shaped cornea-like material (CLM), displaying the shape, curvature, thickness, and transparency of normal cornea. This polymerization of collagen appears to derive from some morphogenetic influence exerted by the corneo-scleral rim. Once the CLM had formed naturally, we used collagen crosslinking to fortify it, and then introduced cells to generate a stratified epithelial layer to create cornea-like tissue (CLT) displaying characteristics of native cornea. Through the excision and reuse of rims, each rim turned out to be useful for the generation of multiple cornea-shaped CLTs., Conclusions: The approach effectively helps to shorten the gap between demand and supply of CLMs/CLTs for transplantation. We are exploring the surgical transplantation of this CLT into animal eyes, as keratoprostheses, as a precursor to future applications involving human eyes. It is possible to use either the CLM or CLT, for patients with varying corneal blinding diseases., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: The opioid receptors in the central nervous system and immune system contribute to its reinforcing effect. Xenobiotics-associated molecular pattern of opioids interacts with Toll-like receptor-4 (TLR-4) on the glial cell surface and increases dopaminergic activity in the nucleus accumbens in preclinical studies. We wanted to examine whether treatment with buprenorphine-naloxone (BNX) might be associated with changes in immunological markers in individuals with opioid dependence (OD)., Methods: We recruited 30 individuals with OD on buprenorphine and 30 age- and sex-matched healthy controls (HCs). We measured the neutrophil (N), lymphocyte (L), CD-4, and CD-8 T-cell count and estimated plasma TLR-4 level in the HC group once. We measured the immunological markers, craving, pain, and perceived stress in the OD group at the treatment initiation (baseline) and after 4 weeks (±2 weeks) of treatment with BNX., Results: The mean severity score on the OD questionnaire was 72.8 (SD 5.4). At baseline, OD had a higher N: L ratio and lower lymphocyte percentage than HC. Plasma TLR-4 concentration increased significantly after 1 month of treatment ( t = -3.09, P = 0.004). Craving, pain, and perceived stress correlated with absolute neutrophil count, N: L ratio, and CD-8 T-cell count, although lost significance after corrections for multiple comparisons., Conclusion: The increase in TLR-4 after treatment with BNX may indicate the rescue from nonprescription opioid-induced immunosuppression or the introduction of a novel xenobiotics-associated molecular pattern of BNX., Competing Interests: There are no conflicts of interest., (Copyright: © 2023 Indian Journal of Psychiatry.)

Objective: To compare CYP1B1 and MYOC variants in a cohort of neonatal-onset (NO) and infantile-onset (IO) primary congenital glaucoma (PCG)., Methods: This prospective observational study included 43 infants with PCG (14 NO and 29 IO) presenting between January 2017 and January 2019 with a minimum 1-year follow-up. CYP1B1 and MYOC genes were screened using Sanger sequencing with in-silico analysis of the variants using Polymorphism Phenotyping v.2 and Protein Variation Effect Analyser platforms. Allelic frequency was estimated using Genome Aggregation Database (gnomAd). Disease presentation and outcome were correlated to the genetic variants in both groups., Results: Babies with CYP1B1 mutations had more severe disease at presentation and worse outcomes. Six of 14 (42.8%) NO glaucoma and 5 of 29 (17.2%) IO harboured CYP1B1 mutations. Five of six babies in the NO group and three of five in the IO group harboured the variant c.1169G>A, [p.R390H]. They required more surgeries and had a poorer outcome. On in-silico analysis c.1169G>A, [p.R390H] scored very likely pathogenic. Two patients in the IO group who had the c.1294C>G, [p.L432V] variant had a good outcome. Five of 14 NO-PCG and 8 of 29 IO-PCG harboured the variant c.227G>A, [p.R76K] in the MYOC gene, which was scored benign by in-silico analysis, and was also found in 2 of 15 normal controls., Conclusions: Patients with CYP1B1 pathogenic variants had a poorer outcome than those without. We found more NO PCG babies with CYP1B1 mutations compared with IO PCG. This may be one of the reasons for NO PCG having a poorer prognosis compared with IO PCG., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Several hypotheses have been proposed to explain the high rate of disease association of HLA-B27 with ankylosing spondylitis (AS), including formation of disulfide-bonded dimers and misfolding of the heavy chain (HC), involving formation of high molecular weight (HMW) multimers. Recently, we have shown that the HMW entities of non-disease associated (non-DA) subtypes cause activation of endosomal-lysosomal pathways, while disease-associated (DA) subtypes of HLA-B27 cause activation of autophagy and unfolded protein response (UPR) pathways. In this paper, we seek an explanation for the failure of these pathways to degrade the HMW entities of DA subtypes of HLA-B27, using a combination of in vitro assays, using extracellular domains of heavy chains (EDHC), as well as in vivo assays, using stable transfectants of the full lengths of heavy chains (FLHC) of DA and non-DA subtypes. Our data shows that both DA and non-DA subtypes form HMW entities. However, non-DA HMW entities display far greater levels of degradation than DA HMW species. Non-DA EDHC display greater loss of structure at lysosomal pH in vitro. This was confirmed by experiments showing that (i) DA FLHCs co-localize with LAMP1, and (ii) induction of autophagy by rapamycin causes significant decrease in levels of non-DA HMW entities, but not that of DA HMW entities. These results point towards lack of facile lysosomal clearance of FLHCs of DA subtypes, suggesting that disease association of HLA-B27 subtypes is correlated with higher persistence of HMW entities in the low pH of lysosomes, with higher potential to trigger immune response., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Purpose: Triple-negative breast cancer (TNBC), a highly aggressive cancer with poor outcome and lacking specific diagnostic, prognostic, or targeted therapeutic strategies, constitutes roughly 20% of all breast cancer cases. TNBC cells lack receptors for estrogen, progesterone, and human epidermal growth factor. The effort continues to find a suitable correlate that could serve as a TNBC biomarker, or as therapeutic target, or both., Materials and Methods: A retrospective study was performed with 88 TNBC and 74 non-TNBC patients who had undergone mastectomy/lumpectomy with axillary clearance for carcinoma breast. Immunohistochemical staining was carried out for levels of proteinase-activated receptor 2 (PAR2), encoded by F2RL1 gene, and staining scores were calculated, based on intensity and percentage positivity., Results: PAR2 levels were markedly upregulated in TNBC patients, compared with patients with other breast cancer subtypes. Amongst different non-TNBC subtypes, higher expression was noted in luminal B (88.8%) and HER2+ (100%), compared with luminal A (52.5%). PAR2 levels were significantly high in TNBC patients with age more than 40 years than corresponding patients of non-TNBC group (P=0.0017). Furthermore, there was a statistically significant increase in levels of PAR2 expression in lymph node negative (P=0.0096) and early stage (P=0.005) of TNBC versus non-TNBC patients. PAR2 staining of ductal carcinoma in situ and invasive ductal carcinoma revealed lower expression in invasive component., Conclusions: Our data suggest that PAR2 levels constitute a correlate of concern for TNBC, tying in with a recent report that higher levels of F2RL1 gene expression correlate with poorer disease-free, as well as overall survival in TNBCs., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Ankylosing spondylitis (AS) belongs to a group of diseases, called spondyloarthropathies (SpA), that are strongly associated with the genetic marker HLA-B27. AS is characterized by inflammation of joints and primarily affects the spine. Over 160 subtypes of HLA-B27 are known, owing to high polymorphism. Some are strongly associated with disease (e.g., B*2704), whereas others are not (e.g., B*2709). Misfolding of HLA-B27 molecules [as dimers, or as high-molecular-weight (HMW) oligomers] is one of several hypotheses proposed to explain the link between HLA-B27 and AS. Our group has previously established the existence of HMW species of HLA-B27 in AS patients. Still, very little is known about the mechanisms underlying differences in pathogenic outcomes of different HLA-B27 subtypes. We conducted a proteomics-based evaluation of the differential disease association of HLA B*2704 and B*2709, using stable transfectants of genes encoding the two proteins. A clear difference was observed in protein clearance mechanisms: whereas unfolded protein response (UPR), autophagy, and aggresomes were involved in the degradation of B*2704, the endosome-lysosome machinery was primarily involved in B*2709 degradation. These differences offer insights into the differential disease association of B*2704 and B*2709., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Thakur and Luthra-Guptasarma.)

We propose that hyper-inflammation (HYPi) is a ''runaway'' consequence of acute inflammation (ACUi) that arises more easily (and also abates less easily) in those who host a pre-existing chronic inflammation (CHRi), because (i) most factors involved in generating an ACUi to limit viral proliferation are already present when there is an underlying CHRi, and also because (ii) anti-inflammatory (AI) mechanisms for the abatement of ACUi (following containment of viral proliferation) are suppressed and desensitized where there is an underlying CHRi, with this causing the ACUi to spiral into a HYPi. Stress, pollution, diet, and gut microbiomes (alterable in weeks through dietary changes) have an intimate and bidirectional cause-effect relationship with CHRi. We propose that avoidance of CHRi-promoting foods and adoption of CHRi-suppressing foods could reduce susceptibility to HYPi, in Covid-19 and in other viral diseases, such as influenza, which are characterized by episodic and unpredictable HYPi., (© 2021 Wiley Periodicals LLC.)

Vibrio mimicus collagenase (VMC), a Class II Vibrio metalloprotease, contains an HEXXH motif in a zinc-binding catalytic domain, and two FAXWXXT motifs in its C-terminal domain, which is its collagen binding domain (CBD). To understand the functional role of the individual CBD motifs in the activity of VMC, if any, we created and characterized a series of VMC variants: i) VMA, with 51 amino acids deleted from the C-terminal end of full-length VMC; ii) VMT1, a form of VMA mutated in the first CBD motif; iii) VMT2, a form of VMA mutated in the second CBD motif; iv) DM, a form of VMA with both CBD motifs mutated; v) CT, a truncated form of VMA, lacking the entire CBD region; and vi) CBD, a construct containing the collagen binding domain alone. The activity of each variant was assessed by multiple means, in relation to VMA. We report that VMT1 and VMT2 show 1.6-fold and 10-fold reduced activity, respectively. The reduced activity of VMT2 correlates with reduced binding to insoluble collagen as well as an inability to cause structural perturbation of collagen. VMC appears to cause unwinding and structural alteration of the collagen triple helix prior to hydrolysis of the substrate (using both motifs for collagen binding), like Clostridium collagenases. In the absence of a known structure for VMC, our findings suggest that Vibrio collagenase, functions like Clostridium collagenases, although the two show very little sequence similarity. Also, VMC shows reduced activity with respect to Clostridium collagenases, making it an ideal enzyme for therapeutic applications., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose: Collagen is a key player contributing to vitreoelasticity and vitreoretinal adhesions. Molecular reorganization causes spontaneous weakening of these adhesions with age, resulting in the separation of the posterior hyaloid membrane (PHM) from the retina in what is called complete posterior vitreous detachment (PVD). Incomplete separation of the posterior hyaloid or tight adherence or both can lead to retinal detachment, vitreomacular traction syndrome, or epiretinal membrane formation, which requires surgical intervention. Pharmacological vitrectomy has the potential of avoiding surgical vitrectomy; it is also useful as an adjunct during retinal surgery to induce PVD. Previously studied enzymatic reagents, such as collagenase derived from Clostridium histolyticum, are nonspecific and potentially toxic. We studied a novel collagenase from Vibrio mimicus (VMC) which remains active (VMA), even after deletion of 51 C-terminal amino acids. To limit the activity of VMA to the vitreous cavity, a fusion construct (inhibitor of hyaluronic acid-VMA [iHA-VMA]) was made in which a 12-mer peptide (iHA, which binds to HA) was fused to the N-terminus of VMA. The construct was evaluated in the context of PVD., Methods: VMA and iHA-VMA were expressed in Escherichia coli, purified, and characterized with gelatin zymography, collagen degradation assay, fluorescamine-based assay, and cell-based assays. Two sets of experiments were performed in New Zealand albino rabbits. Group A (n = 10) received iHA-VMA, while group B (n = 5) received the equivalent dose of VMA. In both groups, saline was injected as a control in the contralateral eyes. Animals were monitored with indirect ophthalmoscopy, optical coherence tomography (OCT), and B-scan ultrasonography. Retinal toxicity was assessed with hematoxylin and eosin (H&E) staining of retinal tissue., Results: The activity of iHA-VMA and VMA was comparable and 65-fold lower than that of C. histolyticum collagenase Type IV. In the iHA-VMA group, all the rabbits (n = 10) developed PVD, with complete PVD seen in six animals. No statistically significant histomorphological changes were seen. In the VMA group, four of the five rabbits developed complete PVD; however, retinal morphological changes were seen in two animals., Conclusions: iHA-VMA displays targeted action confined to the vitreous and shows potential for safe pharmacologic vitreolysis., (Copyright © 2021 Molecular Vision.)

Induction of posterior vitreous detachment (PVD) by pharmacologic vitreolysis has been largely attempted through the use of enzymatic reagents. Ocriplasmin has been the only FDA-approved clinical reagent so far. Several adverse effects of ocriplasmin have emerged, however, and the search for alternative PVD-inducing reagents continues. Since i) collagen forms an important structural component of the vitreous, and ii) strong vitreo-retinal adhesions exist between the cortical vitreous and the internal limiting membrane (ILM) of the retina, an effective PVD-inducing reagent would require both, vitreous liquefaction, and concurrent dehiscence of vitreoretinal adhesion, without being toxic to retinal cells. We designed a combination of two reagents to achieve these two objectives; a triple helix-destabilizing collagen binding domain (CBD), and a fusion of RGD (integrin-binding) tripeptide with CBD (RCBD) to facilitate separation of posterior cortical vitreous from retinal surface. Based on in vitro, ex-vivo, and in vivo experiments, we show that a combination of CBD and RCBD displays potential for safe pharmacologic vitreolysis. Our findings assume significance in light of the fact that synthetic RGD-containing peptides have already been used for inhibition of tumor cell invasion. Proteins such as variants of collagen binding domains could have extended therapeutic uses in the future.

Tenascin-C (TN-C) levels are elevated in the synovial tissue and fluid, as well as cartilage of rheumatoid arthritis (RA) patients. In addition, the presence of TN-C fragments has also been documented in arthritic cartilage. We have previously shown that a single chain variable fragment antibody (TN64), directed against the fibronectin type III repeats 1-5 (TNfnIII 1-5) of TN-C, effectively inhibits fibrotic pathology. Given that fibrosis results from chronic inflammation, and the fact that increased levels of TN-C in the synovial fluid of patients with RA contributes to synovial inflammation and joint destruction, we aimed to investigate the role of TNfnIII 1-5 region of TN-C in RA pathogenesis. Using either the wild type or variants of the two integrin-binding motifs (RGD and AEIDGIEL) present within the TNfnIII 1-5 polypeptide, we demonstrate that the adhesion and migration of synovial fibroblasts is RGD-dependent. The antibody TN64 is effective in inhibiting migration of cells in response to TnfnIII 1-5, and prevents fibroblast-mediated destruction of cartilage. The TN64 antibody was further tested in collagen antibody induced arthritic (CAIA) mice. Our data shows the efficacy of TN64 in preventing induction of arthritis, with significant downregulation of RA-associated cytokines. This suggests that components of the extracellular matrix such as the TNfnIII 1-5 region of TN-C could be exploited to develop therapies to suppress inflammation seen in RA. The TN64 antibody is one such promising candidate in the development of novel treatments for RA., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Elevated levels of a thrombin-cleaved fragment of osteopontin (OPNT) are seen in synovial fluid (SF) and tissues of rheumatoid arthritis (RA) patients. OPNT binds to integrins on cell surfaces, inducing adhesion, migration and survival of inflammatory cells in the synovial joints, where OPNT binds to fibronectin to link fibroblast-like synoviocytes (FLS) with B cells, stimulating the latter to produce inflammatory cytokines. Our aim was to block OPNT-fibronectin interactions and examine whether this reduces inflammation. A human antibody (phage displayed) library was used to select scFv antibodies cognate to OPNT, and a particular scFv antibody (scFv 31) was evaluated. Adhesion, migration and fibronectin polymerization of FLS cells derived from RA patients were monitored, in cultures incorporating scFv 31. Also, scFv 31 was used in mice with CAIA (collagen antibody-induced arthritis), subjected to clinical and histological assessment, analysis of fibronectin and cartilage damage and induction of pro-inflammatory cytokines. The scFv antibody, scFv 31, appeared to cause significantly reduced migration of synovial fibroblasts, altered cell morphology, changes in actin stress fiber arrangement, and marked reduction in fibronectin. In CAIA mice, scFv 31 appeared to prevent arthritic changes through inhibition of synovial hypertrophy and loss of articular cartilage, decrease in fibronectin polymerization and expression of pro-inflammatory cytokines implicated in arthritis. Osteopontin-fibronectin interaction(s) appear to play a role in the expression of key inflammatory molecules by B cells infiltrating the synovial joint. The scFv antibody, scFv 31, provides a potential therapeutic lead for inhibition of some processes implicated in rheumatoid arthritis., (Copyright © 2017. Published by Elsevier B.V.)

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., (Copyright © 2016. Published by Elsevier B.V.)

A self-derived-peptide with the same amino acid sequence (N-RRYLENGKETLQR-C) as residues 169-181 of the human leukocyte antigen (HLA) B27 heavy chain is known to bind to MHC Class I complexes containing the HLA-B27 heavy chain. This observation has been invoked previously in at least two different (but related) molecular explanations for the disease-association of the HLA-B27 allele. Here, we use a combination of fluorescence polarization, competitive inhibition and gel filtration chromatographic studies to show that a fluorescently-labeled peptide of the above sequence binds to two disease-associated subtypes of HLA-B27 (namely HLA-B*27:04 and HLA-B*27:05) but not to non-disease-associated subtypes (HLA-B*27:06 or HLA-B*27:09). This differential binding behavior is seen both in (a) peptide binding to complexes of heavy chain (HLA-B27) and light chain (β 2 microglobulin), and in (b) peptide binding to β 2 microglobulin-free heavy chains in the aggregated state. Such subtype-specific differences are not seen with two other control peptides known to bind to HLA-B27. Our results support the likelihood of differential peptide binding holding at least one of the keys to HLA-B27's disease association.

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.

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.

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by low platelet count and presence of IgG autoantibodies to platelet surface glycoproteins, such as α IIbβ3 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.

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., (Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.)

Background: It is well understood that epithelial mesenchymal transformation occurs when retinal pigment epithelial cells, sourced from either a cell line or cadaver eye, are cultured in the presence of cadaver-derived vitreous. We sought to study the changes in retinal pigment epithelial cells when cell line-derived retinal pigment epithelial cells are cultured in the presence of pathological vitreous., Design: Prospective study., Samples: 42 patients with rhegmatogenous retinal detachments., Methods: D407 retinal pigment epithelial cells were cultured in the presence of cadaver-derived vitreous or vitreous/subretinal fluid derived from patients undergoing retinal reattachment surgeries. Besides the changes in phenotypic characteristics, the viability, proliferation, migration, mesenchymal marker expression and changes in the extracellular matrix components were also evaluated., Main Outcome Measures: Fibrotic phenotype in cell culture., Results: Our study clearly demonstrates that cell line-derived retinal pigment epithelial cells (unlike donor-derived retinal pigment epithelial cells) cultured in the presence of patient-derived vitreous/subretinal fluid, exhibit characteristic features of proliferative vitreoretinopathy., Conclusions: We propose that it is the synergistic effect of the combined use of (i) pathological vitreous, rather than cadaver-derived vitreous (since rhegmatogenous retinal detachment-derived pathological vitreous and subretinal fluid contain exaggerated amounts of growth factors, which could predispose to proliferative vitreoretinopathy development) and (ii) cells from an immortal cell culture (cell line), rather than from primary cell cultures (since cells subjected to continuous serial passaging acquire some mesenchymal characteristics), which together result in not only a unique phenotype, but also prime these cells towards display of features associated with proliferative vitreoretinopathy., (© 2014 Royal Australian and New Zealand College of Ophthalmologists.)

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.

Retinal pigment epithelia (RPE) and retinal neurons (RN) arise from the same underlying precursor cell population. It is worthwhile to explore the transdifferentiation potential of RPE cells, given the benefits that could accrue from turning RPE into neural retina containing all its normal cellular subpopulations. Factors such as retinoic acid (RA, acting as an inducer of transdifferentiation) and epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) (E+F, acting as stem cell maintenance and predifferentiating factors) have been known to stimulate the conversion of RPE into observably neuron-like cells. However, since such treatment does not actually transform RPE into neurons, there is a need to rationally explore the efficacy of other factors as well. In this paper, we have compared the effects of RA, a known transdifferentiating factor in optical and other tissues, with the effects of cadaver vitreous on D407 RPE cells, using a combination of light microscopy, immunofluorescent microimaging and flow cytometry. We show that cadaver-derived vitreous (CV) is comparable to, or better than, RA as a transdifferentiation factor in an EGF + bFGF maintenance background. The vitreous humor could thus be an important stimulator of the differentiation of dedifferentiated RPE cells into neurons. This finding assumes significance in light of the fact that vitreous humor is a factor naturally present in the environment of the developing retina.

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.

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.

We describe and demonstrate a technique for detection of cell surface antigens, with potential use in tissue antigen research. Briefly, a small volume of wetted chromatographic beads (specifically, 100 μL of Nickel-nitriloacetic acid (NTA) agarose) was bound to small quantities (specifically, ∼0.1-0.2 μg) of a single-chain Fv antibody recognizing the Class I MHC heavy chain antigen. The beads were then used to capture and detect cells bearing this antigen, through SDS polyacrylamide gel electrophoresis of boiled bead-cell complexes. A key feature of this method is its use of "signal amplification." Although the antibody-mediated binding and immobilization of intact cells involves relatively small numbers of antibodies, and cells, what is being detected is simply the presence of cells. Each bound cell contains a number of abundant proteins that are present in millions of copies per cell, and the abundance of these proteins ensures that they are detectable on SDS-PAGE, signaling cell-binding. As few as 10(10) -10(12) scFv antibodies immobilized on 100 μL of Ni-NTA beads are thus enough for the trapping of enough cells to allow visualization of their abundant proteins. Conceptually, this method could be easily developed and applied to detection of cells bearing any other cell specific antigen., (Copyright © 2010 American Institute of Chemical Engineers (AIChE).)

It is widely accepted that protein aggregates tend to be pathologic, although little is known about why they are pathologic. Here, we summarize published findings about protein aggregates which have implications for pathology, but which have not yet been covered in any review or hypothesis on the subject, to the best of our knowledge. These findings suggest that protein aggregates can: (i) act as proteases, using exposed surface serines, (ii) function as immunogens, using novel conformational epitopes, (iii) behave as photosensitization-aids, using a novel peptide-based fluorescence, and (iv) act as electrical conductors, using electrons tunneling through hydrogen-bonded networks of peptide bonds. The potential pathological consequences of each finding are speculated upon., ((c) 2010 Elsevier Ltd. All rights reserved.)

Background: The twin phenomena of aggregation and degradation are classically associated with protein storage. However, although aggregation has been thought to be a possible consequence of protein degradation, it has never before been proposed to be a cause of degradation., Methods: Proteins stored under physiological conditions and electrophoresed on SDS-PAGE were examined zymographically for the presence of detergent-resistant high molecular weight (HMW) forms, and association of such HMW forms with time-correlated, seeding-dependent gelatinolytic activity, under various conditions., Results: Eight different proteins aggregate naturally during storage at near-neutral pH, with concomitant development of 'gelatinolytic' activity diminished greatly by storage at low temperatures, extremes of pH, arginine, imidazole, BSA, azide, EDTA, DTT, PMSF (but not AEBSF), and diisopropyl fluorophosphate (DFP), suggesting involvement of surface serine residues in a novel aggregate-borne proteolytic activity., Conclusions: Naturally-formed aggregates of proteins appear to use surface serines to perform peptide bond hydrolysis, explaining degradation of proteins during storage, and indicating why aggregates are cytotoxic., General Significance: The study suggests that a bi-directional cause-effect relationship operates between protein aggregation, and protein degradation, providing clues to the design of better conditions for long-term protein storage.

The N-terminal 'unstructured' region of the human prion protein [PrP((90-231))] is believed to play a role in its aggregation because mutations in this region are associated with seeding-independent deposition disorders like Gerstmann-Straussler-Scheinker disease (GSS). One way of examining the effects of such mutations is to search combinatorially derived libraries for sequence variants showing a propensity to aggregate and/or the ability to interact with prion molecules folded into a beta-sheet-based conformation (i.e., beta-PrP or PrP(Sc)). We created a library of 1.8x10(7) variants randomized between positions 101 and 112, displayed it on filamentous bacteriophage, and 'spiked' it with a approximately 25% population of phages-bearing wild-type prion (wt-PrP). Screening was performed through four rounds of biopanning and amplification against immobilized beta-PrP, and yielded three beta-PrP-binding populations: wt-PrP (26% representation) and two non-wt-PrP variants ( approximately 10% and approximately 64% representation, respectively). The remarkable enrichment of one non-wt-PrP variant (MutPrP) incorporating residues KPSKPKTNMKHM in place of KGVLTWFSPLWQ, despite its initial representation at a 5 million-fold lower level than wt-PrP, caused us to produce it and discover: (i) that it readily aggregates into thioflavin-T-binding amyloids between pH 6.0 and 9.0, (ii) that it adopts a soluble beta-sheet based monomeric structure at pH 10.0, (iii) that it is less thermally stable and more compact than wt-PrP, and (iv) that it displays significantly greater resistance to proteolysis than wt-PrP. Our results suggest that sequence variations in the 101-112 region can indeed predispose the prion for aggregation.

Refolding of the heavy chain of the Class I HLA molecule, HLA-B27, in the absence of beta(2)m, yields soluble high molecular weight (HMW) oligomers reminiscent of the oligomeric forms of beta(2)m-free heavy chains (FHCs) of class I HLA antigens observed on cell surfaces. Here we examine the structural characteristics of HMW B27 in respect of features potentially relevant to autoimmunity, such as: (a) retention of native-like structure, since this could facilitate non-canonical interactions with T-cell receptors even in the absence of bound beta(2)m and peptide, or (b) presence of non-native structure, since this could yield novel (non-self) antigenic conformational epitopes that could elicit immune attack. We report that HMW B27 is characterized by high secondary structural content, structural stability, stability to proteolysis by trypsin, and structural features that are both partly native-like, and partly non-native-like, as assessed through the binding of conformationally-distinguishing and cross-reacting scFv antibodies specifically selected against HMW B27. We also present cell ELISA data with conformation-specific scFv antibodies that distinguish between lymphocytes from individuals who are healthy and B27 positive, and those who are B27 positive but suffering from ankylosing spondylitis.

Expression of the MHC class I allele, 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 beta2m-free, peptide-free heavy chains support a helix-coil transition in the segment leading from the alpha2 domain to the alpha3 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 molecule's own peptide-binding cleft. Such 'auto-display', occurring either within B27 molecules, or between B27 molecules, could provoke autoimmune attack.

The beta/alpha-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 motif's 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 beta/alpha-barrels might form through the modular folding and assembly of substructures.