Α5β1 Integrin Research Articles

The novel ECM protein SNED1 mediates cell adhesion via the RGD-binding integrins α5β1 and αvβ3.

The extracellular matrix (ECM) is a complex meshwork comprising over 100 proteins. It serves as an adhesive substrate for cells and, hence, plays critical roles in health and disease. We have recently identified a novel ECM protein, SNED1, and have found that it is required for neural crest cell migration and craniofacial morphogenesis during development and in breast cancer, where it is necessary for the metastatic dissemination of tumor cells. Interestingly, both processes involve the dynamic remodeling of cell-ECM adhesions via cell surface receptors. Sequence analysis revealed that SNED1 contains two amino acid motifs, RGD and LDV, known to bind integrins, the largest class of ECM receptors. We thus sought to investigate the role of SNED1 in cell adhesion. Here, we report that SNED1 mediates breast cancer and neural crest cell adhesion via its RGD motif. We further demonstrate that cell adhesion to SNED1 is mediated by the RGD integrins α5β1 and αvβ3. These findings are a first step toward identifying the signaling pathways activated downstream of the SNED1-integrin interactions guiding craniofacial morphogenesis and breast cancer metastasis.

FNDC1 is a myokine that promotes myogenesis and muscle regeneration.

Myogenesis is essential for skeletal muscle formation and regeneration after injury, yet its regulators are largely unknown. Here we identified fibronectin type III domain containing 1 (FNDC1) as a previously uncharacterized myokine. In vitro studies showed that knockdown of Fndc1 in myoblasts reduces myotube formation, while overexpression of Fndc1 promotes myogenic differentiation. We further generated recombinant truncated mouse FNDC1 (mFNDC1), which retains reliable activity in promoting myoblast differentiation in vitro. Gain- and loss-of-function studies collectively showed that FNDC1 promotes cardiotoxin (CTX)-induced muscle regeneration in adult mice. Furthermore, recombinant FNDC1 treatment ameliorated pathological muscle phenotypes in the mdx mouse model of Duchenne muscular dystrophy. Mechanistically, FNDC1 bound to the integrin α5β1 and activated the downstream FAK/PI3K/AKT/mTOR pathway to promote myogenic differentiation. Pharmacological inhibition of integrin α5β1 or of the downstream FAK/PI3K/AKT/mTOR pathway abolished the pro-myogenic effect of FNDC1. Collectively, these results suggested that myokine FNDC1 might be used as a therapeutic agent to regulate myogenic differentiation and muscle regeneration for the treatment of acute and chronic muscle disease.

An in-silico study reveals that a C-terminal fragment of the adhesion protein Fibulin7 (Fbln7-C) regulates the activation of integrin α5β1 through dynamics of VWA and the hybrid domain in the β1 subunit

A C-terminal fragment of the adhesion protein Fibulin7 (Fbln7-C) binds to monocytes and neutrophils via integrin α5β1, regulating their adhesion and immunological functions through Erk and STAT signaling pathways. It also inhibits cell binding, spreading, and migration on fibronectin. However, the precise structural components of Fbln7-C that interact with various domains of integrin α5β1 and contribute to its regulatory effects are not entirely understood. This study investigated the structural dynamics of Fibulin7 fragments and the mechanisms by which Fbln7-C regulates α5β1 integrin activation using protein modeling, protein-protein docking, molecular dynamics simulation (MDS), and binding free energy calculations. An energy-minimized model of α5β1 integrin, Fibulin7 full length (Fbln7-FL), and Fbln7-C was developed and validated using 100 ns MDS. Additionally, protein-protein docking was used to confirm Fbln7-C’s better integrin binding ability over Fbln7-FL. A 500 ns MDS on the docked Fbln7-C integrin complex revealed the regulatory effects of Fbln7-C on arginine-glycine-aspartic acid (RGD) bound integrin α5β1. The simulation studies showed that Fbln7-C’s attachment to activated α5β1 integrin increased the distance between the RGD and its interacting residues on both integrin subunits, shifting the RGD ligand from its original binding position and inactivating the integrin. Further analysis using free energy landscape (FEL), principal component analysis (PCA), and binding energy calculation validated the alteration in α5β1 integrin’s structural dynamics following Fbln7-C binding. This could relate to obstruction in the outward swing of the integrin’s hybrid domain and result in the low-affinity, inactive headpiece conformation of the α5β1 integrin.

Unraveling the role of integrating signal peptides into natural collagen on modulating cancer cell adhesion

The signal peptides GVMGFO and GFOGER exhibit differential binding affinities towards Michigan Cancer Foundation-7 (MCF-7) breast cancer cells and HT-1080 human fibrosarcoma cells, respectively, which in turn modulate the cell adhesion properties of natural collagen. GVMGFO demonstrates a more potent interaction with discoidin domain receptor 1(DDR1)-expressing MCF-7 cells, whereas GFOGER preferentially binds to the integrin α2β1 present on HT-1080 cells. The integration of GVMGFO into natural collagen through direct doping or crosslinking markedly enhances its association with MCF-7 cells, especially when optimal peptide concentrations and blending ratios are utilized, indicating a synergistic effect. This augmented adhesion is attributed to specific binding at the DDR1-collagen interface, facilitated by a constellation of amino acids within the collagen scaffold engaging with the DDR1 discoidin (DS) domain through polar interactions and hydrogen bonding. Conversely, the incorporation of GFOGER into natural collagen through co-assembling or crosslinking leads to a progressive increase in adherence to HT-1080 cells, as evidenced by the peptide's affinity for integrin α2β1. These findings advance the design of collagen-based biomaterials for targeted cellular interactions in the medical, pharmaceutical, and enhance our understanding of the molecular mechanisms governing peptide-collagen mediated cell adhesion processes.

Integrin Subtypes and Lamellipodia Mediate Spatial Sensing of RGD Ligands during Cell Adhesion.

Understanding how the spatial distribution of adhesive ligands regulates cell behavior is crucial for designing biomaterials. This study investigates how precisely controlled ligand spacing affects cell spreading and integrin subtype engagement. Using engineered polyacrylamide hydrogels with gold nanoparticle arrays, we explored the impact of RGD ligand spacings (30 and 150 nm) on human mesenchymal stromal cells. Cells exhibited distinct morphological behaviors: smaller spacings promoted larger spreading areas, while larger spacings resulted in elongated shapes with reduced spreading. Mechanistically, we found that the α5β1 integrin, not the αvβ3 integrin, played a central role in mediating these responses, alongside lamellipodia formation. Our findings provide critical insights into the spatial sensing of ligands, highlighting the influence of ligand spacing on cellular mechanotransduction and integrin-specific responses. This work advances the understanding of cell-material interactions and offers potential strategies for designing biomaterials to guide cell behavior in tissue engineering.

A Novel P-III Metalloproteinase from Bothrops barnetti Venom Degrades Extracellular Matrix Proteins, Inhibits Platelet Aggregation, and Disrupts Endothelial Cell Adhesion via α5β1 Integrin Receptors to Arginine-Glycine-Aspartic Acid (RGD)-Containing Molecules.

Viperid snake venoms are notably abundant in metalloproteinases (proteins) (SVMPs), which are primarily responsible for inducing hemorrhage and disrupting the hemostatic process and tissue integrity in envenomed victims. In this study, barnettlysin-III (Bar-III), a hemorrhagic P-III SVMP, was purified from the venom of the Peruvian snake Bothrops barnetti. Bar-III has a molecular mass of approximately 50 kDa and is a glycosylation-dependent functional metalloproteinase. Some biochemical properties of Bar-III, including the full amino acid sequence deduced from its cDNA, are reported. Its enzymatic activity is increased by Ca2+ ions and inhibited by an excess of Zn2+. Synthetic metalloproteinase inhibitors and EDTA also inhibit its proteolytic action. Bar-III degrades several plasma and ECM proteins, including fibrin(ogen), fibronectin, laminin, and nidogen. Platelets play a key role in hemostasis and thrombosis and in other biological process, such as inflammation and immunity, and platelet activation is driven by the platelet signaling receptors, glycoprotein (GP)Ib-IX-V, which binds vWF, and GPVI, which binds collagen. Moreover, Bar-III inhibits vWF- and convulxin-induced platelet aggregation in human washed platelets by cleaving the recombinant A1 domain of vWF and GPVI into a soluble ectodomain fraction of ~55 kDa (sGPVI). Bar-III does not reduce the viability of cultured endothelial cells; however, it interferes with the adhesion of these cells to fibronectin, vitronectin, and RGD peptides, as well as their migration profile. Bar-III binds specifically to the surface of these cells, and part of this interaction involves α5β1 integrin receptors. These results contribute to a better comprehension of the pathophysiology of snakebite accidents/incidents and could be used as a tool to explore novel and safer anti-venom therapeutics.

Intercellular junction-driven stromal cell stacking in a confined 3D microcavity.

Understanding the detailed mechanisms driving fibroblast migration within native tissue settings during pathophysiological events presents a critical research challenge. In this study, we elucidate how stromal cells migrate and contribute to the development of three-dimensional (3D) cellular aggregates within confined microcavities. Integrin α5β1 and β-catenin (β-cat) are central in guiding this collective migration and achieving optimal filling of the microcavity. When β-cat is suppressed, cells tend to migrate more sporadically, leading to less efficient cellular organization. Furthermore, we also detail the pivotal roles of Cx43 and N-cadherin (N-cad) in orchestrating collective migration and in shaping efficient cellular stacking. Suppressing gap junctions, especially Cx43, significantly impacts the extracellular matrix expression, integrin α5 and β1, and other elements in the 3D construct, emphasizing the importance of physicochemical cell-cell interactions. The distribution patterns of N-cad and focal adhesion kinase (FAK) further corroborate the essential roles in forming cell-cell junctions and FAK in establishing the foundational layer that underpins the cell stacking within the microcavity. Interestingly, neither Rho-associated protein kinase (ROCK) nor RhoA significantly alter the cell migration pattern toward microcavity. These findings provide fresh perspectives on fibroblast activities in 3D space, enriching our understanding and offering implications for advancements in wound healing and tissue engineering.

Integrin α2β1 deficiency enhances osteogenesis via BMP-2 signaling for accelerated fracture repair

Previous studies have shown that the absence of the collagen-binding integrin α2β1 confers protection against osteoporosis, primarily by enhancing osteoblast-mediated matrix formation, with a particular increase in collagen type I production. This study aimed to elucidate the mechanism underlying this increased matrix production. Our findings demonstrate that osteoblasts lacking integrin α2 secrete a pro-osteogenic factor that activates both TGF-β and BMP signaling pathways. Among these, BMP-2 was identified as the key signaling protein responsible for this effect, as its expression was significantly upregulated during osteoblast differentiation. Moreover, integrin α2 deficiency led to earlier and elevated BMP-2 secretion at the cell surface during osteogenesis, which promoted accelerated osteoblast differentiation. This phenomenon likely contributes to enhanced matrix production in aging animals, providing a protective effect against osteoporosis.To explore the broader implications of this phenotype, we utilized a fracture healing model. In integrin α2-deficient 12 weeks old female mice, elevated serum levels of BMP-2 were detected during the early stages of fracture repair. This upregulation of BMP signaling within the fracture callus accelerated the healing process, resulting in faster formation and mineralization of the cartilaginous callus. Additionally, the elevated BMP-2 levels facilitated earlier differentiation of chondrocytic cells, evidenced by the premature appearance of collagen type II- and type X-positive cells during endochondral ossification. Despite the accelerated healing, the overall biomechanical integrity of the repaired fractures remained uncompromised.Thus, the modulation of integrin α2β1 presents a promising therapeutic target for enhancing fracture repair by regulating BMP-2 signaling in a physiologically relevant manner.

Weekly Intraperitoneal Injection of Tamoxifen in an Inducible In Vivo Model of Junctional Epidermolysis Bullosa Generates Early and Advanced Disease Phenotypes

Junctional epidermolysis bullosa (JEB) caused by loss of function variants in genes encoding the skin basement membrane proteins Laminin 332 (Lam332), type XVII collagen or integrin α6β4, effects patients from birth with severe blistering eventually leading to scarring and early lethality. Here we have optimised a previously published JEB eKO model with weekly tamoxifen intraperitoneal injections resulting in a more controllable and severe model. Due to the titratable dosing this model now recapitulates both early and advanced stages of the human disease, strengthening its use in therapeutic studies. The gradual loss of Lam-α3 in the skin of the mouse through weekly injections lead to generalised blistering and fibrotic dermal changes in multiple skin sites by week 12 post-tamoxifen. Our findings demonstrate the usefulness of optimising tamoxifen induction in Cre-loxP mouse models of extracellular matrix proteins, an approach that could be applicable to other emerging inducible transgenic disease models to improve their ability to mimic the human disease phenotype.

Alterations in the microenvironment of junctional epidermolysis bullosa keratinocytes: a gene expression study

Integrin α6β4 subunits and type XVII collagen are critical transmembrane proteins involved in cell-matrix adhesion in skin, while laminin 332 serves as their ligand in the basement membrane zone (BMZ). Those proteins contribute to the composition of hemidesmosomes (HDs) and pathogenic variants in their corresponding genes cause junctional epidermolysis bullosa (JEB). Although the genotype-phenotype relationships in JEB have been extensively studied, the pathogenetic changes of extracellular matrix (ECM) and cell-matrix adhesion resulting from gene mutations remain unclear. We conducted a global unbiased transcriptome analysis using bulk RNA sequencing (RNA-seq) on selected JEB donor-derived cell lines lacking integrin β4 subunit (ITGB4-), type XVII collagen (COL17-) and laminin β3 chain (LAMB3-), respectively. Additional JEB cell lines and JEB donor skin samples were used for validation of relevant findings. Collectively, the results revealed similar dysregulation patterns of ECM and focal adhesion (FAs) associated genes in ITGB4- and COL17- cell lines, while LAMB3- cells displayed a relatively opposite tendency. Importantly, key nodes in the dysregulated network were associated with ECM proteins involved in wound healing processes. Additionally, a group of inflammatory-associated genes was disclosed to be up-regulated in JEB keratinocytes and could not be normalized by the adhesion rescue. The functional assay further revealed the hierarchy of stable adhesion among mutant cell lines COL17->ITGB4->LAMB3-, which correlates with the severity of their clinical manifestations. Our results indicated a wound healing associated ECM and inflammatory microenvironment established by JEB keratinocytes.

Integrin α6β4 Upregulates PTPRZ1 Through UCHL1-Mediated Hif-1α Nuclear Accumulation to Promote Triple-Negative Breast Cancer Cell Invasive Properties.

Integrin α6β4 drives triple-negative breast cancer (TNBC) aggressiveness through the transcriptional regulation of key genes. Here, we investigated how integrin α6β4 regulates protein tyrosine phosphatase receptor type Z1 (PTPRZ1). Using stable re-expression of integrin β4 (ITGB4) in cells naturally devoid of integrin α6β4 or knockdown or knockout (KO) of ITGB4, we found that integrin α6β4 regulates PTPRZ1 expression. To gain mechanistic insight, we focused on Hif-1α due to the impact of integrin α6β4 on a hypoxia-associated signature. We found that nuclear localization of Hif-1α, but not Hif-2α, was substantially enhanced with integrin α6β4 signaling. Hif-1α knockdown by shRNA or chemical inhibition decreased PTPRZ1 expression, while chemical activation of Hif-1α increased it. Upstream of Hif-1α, integrin α6β4 upregulates UCHL1 to stabilize Hif-1α and ultimately regulate PTPRZ1. Inhibition of UCHL1 and PTPRZ1 dramatically decreases integrin α6β4-mediated cell migration and three-dimensional invasive growth. Finally, public breast cancer database analyses demonstrated that ITGB4 correlates with PTPRZ1 and that high expression of ITGB4, UCHL1, HIF1A, and PTPRZ1 associated with decreased overall survival, distant metastasis free survival, post progression survival, and relapse-free survival. In summary, these findings provide a novel function of integrin α6β4 in promoting tumor invasive phenotypes through UCHL1-Hif-1α-mediated regulation of PTPRZ1.

Machine Learning Classification of Integrin-Expression-Based Magnetic Sorted SW 620 Cells by Simultaneous O-PTIR and SERS.

Immortalized cell lines are commonly used for in vitro studies such as drug efficacy, toxicology, and life cycle due to their cost effectiveness and accessibility; however, subpopulations within a cell line can arise from random mutations or asynchronous cell cycles which may lead to results that make interpretation difficult. A method that could classify these differences and separate unique subpopulations would increase our understanding of heterogeneous cellular responses. In the present work, we explore spectroscopic signals associated with subpopulations of cells magnetically sorted on the basis of α5β1 integrin binding to cyclic-RGDfC which mimics fibronectin in the extracellular matrix. SW620 colon cancer cells were incubated with cyclic-RGDfC functionalized gold-coated, iron core nanoparticles and magnetically sorted. The subpopulations from the sort were imaged (N = 10 positive and N = 10 negative, number of cells) via simultaneous surface-enhanced Raman scattering (SERS) and optical-photothermal infrared spectroscopy (O-PTIR). Pearson correlations of the standard peptide-protein interaction in the SERS channel allowed for visualization of the cyclic RGDfC-integrin α5β1 interaction. Partial least-squares discriminant analysis of the O-PTIR spectra collected from cell maps successfully classified the positively or negatively sorted cells. These results demonstrate that biochemical changes within a single cell line can be sorted via an integrin-activity-based assay using simultaneous SERS and O-PTIR.

Alpha-hemolysin promotes internalization of Staphylococcus aureus into human lung epithelial cells via caveolin-1- and cholesterol-rich lipid rafts

Staphylococcus aureus is a pathogen associated with severe respiratory infections. The ability of S. aureus to internalize into lung epithelial cells complicates the treatment of respiratory infections caused by this bacterium. In the intracellular environment, S. aureus can avoid elimination by the immune system and the action of circulating antibiotics. Consequently, interfering with S. aureus internalization may represent a promising adjunctive therapeutic strategy to enhance the efficacy of conventional treatments. Here, we investigated the host-pathogen molecular interactions involved in S. aureus internalization into human lung epithelial cells. Lipid raft-mediated endocytosis was identified as the main entry mechanism. Thus, bacterial internalization was significantly reduced after the disruption of lipid rafts with methyl-β-cyclodextrin. Confocal microscopy confirmed the colocalization of S. aureus with lipid raft markers such as ganglioside GM1 and caveolin-1. Adhesion of S. aureus to α5β1 integrin on lung epithelial cells via fibronectin-binding proteins (FnBPs) was a prerequisite for bacterial internalization. A mutant S. aureus strain deficient in the expression of alpha-hemolysin (Hla) was significantly impaired in its capacity to enter lung epithelial cells despite retaining its capacity to adhere. This suggests a direct involvement of Hla in the bacterial internalization process. Among the receptors for Hla located in lipid rafts, caveolin-1 was essential for S. aureus internalization, whereas ADAM10 was dispensable for this process. In conclusion, this study supports a significant role of lipid rafts in S. aureus internalization into human lung epithelial cells and highlights the interaction between bacterial Hla and host caveolin-1 as crucial for the internalization process.

Innovative nebulization delivery of lipid nanoparticle-encapsulated siRNA: a therapeutic advance for Staphylococcus aureus-induced pneumonia

BackgroundIntegrin α5β1 plays a crucial role in the invasion of nonphagocytic cells by Staphylococcus aureus (S. aureus), thereby facilitating infection development. Lipid nanoparticles (LNPs) serve as an effective vehicle for delivering small interfering ribonucleic acids (siRNA) that represent a method to knockdown integrin α5β1 in the lungs through nebulization, thereby potentially mitigating the severity of S. aureus pneumonia. The aim of this study was to harness LNP-mediated targeting to precisely knockdown integrin α5β1, thus effectively addressing S. aureus-induced pneumonia.MethodsC57 mice (8 week-old females) infected with S. aureus via an intratracheal nebulizing device were utilized for the experiments. The LNPs were synthesized via microfluidic mixing and characterized by their size, polydispersity index, and encapsulation efficiency. Continuous intratracheal nebulization was employed for consistent siRNA administration, with the pulmonary function metrics affirming biosafety. The therapeutic efficacy of LNP-encapsulated siRNAs against pneumonia was assessed through western blotting, bacterial count measurement, quantitative polymerase chain reaction, and histological analyses.ResultsLNPs, which have an onion-like structure, retained integrity post-nebulization, ensuring prolonged siRNA stability and in vivo safety. Intratracheal nebulization delivery markedly alleviated the severity of S. aureus-induced pneumonia, as indicated by reduced bacterial load and bolstered immune response, thereby localizing the infection to the lungs and averting systemic dissemination.ConclusionsIntratracheal nebulization of LNP-encapsulated siRNAs targeting integrin α5β1 significantly diminished the S. aureus-mediated cellular invasion and disease progression in the lungs, presenting a viable therapeutic approach for respiratory infections.

Behçet's syndrome: one year in review 2024.

The aim of this review is to provide a critical summary of studies published during 2023 that contribute to our understanding of Behçet's syndrome. An increase in the incidence of BS was reported in Northern Spain after 2014.Studies on patient perspectives showed the impact of Behçet's syndrome on quality of life, daily activities, education, work, and relationships. Differences in genetics among Behçet's syndrome patients with different types of organ involvement were reported and an association between HLA-B/MICA and SLCO4A1 polymorphisms and eye involvement and between DDX60L polymorphisms and nervous system involvement were observed. It was suggested that Integrin α9β1 plays a crucial role in the neutrophil-mediated inflammatory pathways underlying this syndrome and the dysfunction of the PDL1/PD-1 pathway may be associated with the hyperactivity of the Th-1/Th-17 axis. Vein wall thickness seems to be increased in patients with Behçet's syndrome, but its pathogenetic and clinical implications are not clear. There is growing evidence regarding the efficacy of TNF inhibitors in different types of organ involvement. A number of small case series point out to the potential role of TCZ and JAK inhibitors.

Fibroblast integrin α11β1 is a collagen assembly receptor in mechanoregulated fibrillar adhesions

Solid epithelial cancers with significant desmoplasia are characterized by an excessive deposition of collagen-based matrix, which often supports tumor progression. However, the mechanism of how collagen receptors mediate collagen fibrillogenesis still remains mostly unclear.We show that the collagen-binding integrin α11β1 can co-localize with tensin-1 and deposited collagen I in human pancreatic ductal adenocarcinoma (PDAC) stroma. In addition to the canonical fibrillar adhesion integrin α5β1 expressed by human PDAC cancer-associated fibroblasts (CAFs), tensin-1-positive fibrillar adhesions contained α11β1 but lacked α1β1 and α2β1. CAFs lacking α5β1 expression displayed mechanoregulated and tensin-1 dependent α11β1 fibrillar adhesions, suggesting independent roles of the two integrins with regards to fibrillar adhesions-based de novo fibrillogenesis. Further, we demonstrate that cell surface-associated collagen I assembly necessitated α11β1, but not α5β1 expression.In summary, α11β1 integrin is a novel component of fibrillar adhesions, which is strategically positioned to mediate de novo collagen fibrillogenesis at the cell surface under pro-fibrotic conditions.

Pepgen-P15 delivery to bone: A novel 3D printed scaffold for enhanced bone regeneration

Pepgen-P15 is a combination of an organic hydroxyapatite matrix derived from bovine sources, combined with a synthetic peptide known as P-15. The interaction between α2β1 integrin and the P15 chain triggers both intracellular and extracellular signaling pathways, resulting in the production of growth factors. Three-dimensional (3D) printing has recently emerged as an innovative strategy for developing personalized therapies in bone tissue regeneration. In this research, various ratios of calcium magnesium silicate (bredigite) nanoparticles were used to modify 3D printed scaffolds made of xanthan gum and polycaprolactone (PCL) via fused deposition modeling (FDM). Scaffolds were subsequently treated with an alkaline solution, covered with graphene oxide, and finally, Pepgen-P15 was applied. the effects of xanthan gum were assessed using swellability and contact angle tests. The results indicated that, the prepared scaffolds exhibited suitable degradation rates, mechanical characteristics, and apatite formation. Alizarin red and alkaline phosphatase assays were also conducted to evaluate the scaffolds’ effectiveness in promoting bone cell differentiation during cell culture. Furthermore, the surface of the scaffold was examined to determine the amount of Pepgen-P15 loaded and released. According to the findings, the scaffold composed of 20 % bredigite and 0.3 % graphene oxide, coated with Pepgen-P15, demonstrate optimal mechanical properties, cell adherence, development, and proliferation. Typically, it is a good candidate for use in bone tissue engineering.

B-266 Therapeutic Drug Monitoring of Natalizumab and Anti-Natalizumab Antibodies

Abstract Background Natalizumab (NTZ) is a monoclonal antibody directed against the α4-integrin subunit of α4β1 and α4β7 integrins. NTZ is used to treat relapsing and remitting forms of multiple sclerosis. Although a majority of patients on standard dosing (intravenous 300 mg, every 4 weeks) reach therapeutic drug concentrations, about 10% of patients will eventually fail NTZ therapy due to the development of anti-NTZ antibodies. NTZ and anti-NTZ antibody concentrations in serum are used together to provide patient-specific pharmacokinetic and immunogenic assessment to aid in patient management. Methods Serum measurements of NTZ and anti-NTZ antibody levels are performed by lab developed electro-chemiluminescent immunoassays where assay design and principle are equivalent to biologic therapeutic drug monitoring described in our previous publication. The natalizumab drug assay is an immunoassay validated to measure free drug (pharmacodynamically active, ADA-unbound) in serum. The anti-natalizumab antibody assay utilizes a solution phase bridging method designed to measure total binding antibodies (including IgM & IgG subtypes). All anti-natalizumab antibody positive samples are confirmed for drug-specificity by an additional signal suppression step. The anti-natalizumab antibody assay is quantitative and drug-tolerant, meaning that circulating drug in patient serum does not interfere with anti-NTZ detection and quantitation. Results Here, we report concomitant measurement of NTZ and anti-NTZ antibody in 160 patient serum samples. Positive anti-NTZ levels from 24 to 235 ng/mL were found in 7.5% (12/160) patients. Anti-NTZ-free samples (148/160) had NTZ drug levels ranging from undetectable (<1.0) to 85 ug/mL. The mean and median drug level in 90 samples (results >1.0) was 10.8 and 2.7, respectively. 61/90 samples had therapeutic drug concentrations (at least 2.0 ug/mL). In the presence of anti-NTZ, 67% (8/12) had undetectable drug level and (4/12) had mean and median drug levels of 1.2 and 1.3, respectively, which is 9- or 2-fold lower than the mean and median drug levels of the anti-NTZ Ab -free group. Conclusions Low serum NTZ and high anti-NTZ are associated with loss of clinical efficacy, while elevated NTZ may increase progressive multifocal leukoencephalopathy risk. Assays to measure biologic drugs and their anti-drug antibodies in patient serum may be useful tools in dose optimization and patient management.

α9β1 integrin & its ligands as new potential biomarkers in FMF.

Background & objectives Familial Mediterranean Fever (FMF) manifests as a hereditary condition characterized by repeated bouts of fever, abdominal, chest, and joint discomfort, and swelling. Colchicine is the most common form of treatment, but it does not eliminate the disease. The underlying causes of the inflammatory mechanism are still not fully known. Methods A total of 20 healthy controls, 16 individuals with FMF in the attack period, and 14 in the remission period participated in the study. ITGA9, ITGB1, OPN, TNC, VEGF, VCAM-1, TGM2, TSP-1, Emilin-1, and vWF levels were measured by ELISA by obtaining serum from blood samples of individuals. In addition, gene expressions of α9β1 (ITGA9, ITGB1) and its best known ligands (TNC, SPP1) were analyzed by quantitative real-time PCR (qPCR). Results The findings of this study showed that serum levels of α9β1 and its ligands were higher in individuals with FMF in the attack period than in the healthy controls and the FMF group in the remission period (P<0.05). The marker levels of the healthy group were also higher than those in the remission period (p<0.05). In addition, when the gene expressions were compared between the healthy controls and FMF group, no significant difference was found for ITGA9, ITGB1, TNC, and SPP1 genes. Interpretation & conclusions The function of α9β1 and its ligands in FMF disease was investigated for the first time in this study as per our knowledge. Serum levels of these biomarkers may help identify potential new targets for FMF disease diagnosis and treatment approaches.

Elucidating the unexpected cell adhesive properties of agarose substrates. The effect of mechanics, fetal bovine serum and specific peptide sequences

2D agarose substrates have recently been surprisingly shown to be permissive for cell adhesion, depending on their mechanics and the use of the adhesive proteins of fetal bovine serum (FBS) in the cell culture medium. Here, we elucidate how the cells exhibit two anchoring mechanisms depending on the amount of FBS. Under low FBS conditions, the cells recognize the surface-coupled adhesive sequences of fibronectin via the binding of the heterodimer α5β1 integrin. Functionality of the actomyosin axis and mechanoactivation of focal adhesion kinase (FAK) are essential for the stretching of the protein, thereby accessing the “synergy” PPSRN site and enhancing cell adhesion in combination with the downstream RGD motif. Under high FBS conditions, the specific peptide sequences are much less relevant as the adsorbed serum proteins conceal the coupled fibronectin and the cells recognize the adhesive protein vitronectin, which is constitutively present in FBS, via the binding of the heterodimer αvβ3 integrin. Similarly, the intracellular tension and FAK activity are decisive, which collectively indicate that the cells stretch the partially cryptic RGD site of vitronectin and thus make it more accessible for integrin binding. Both anchoring mechanisms only work properly if the agarose substrate is mechanically compliant in terms of linear stress-strain response, unraveling a critical balance between the mechanics of the agarose substrate and the presentation of the adhesive peptides. Statement of significanceIn the context of biomaterial design, agarose hydrogels are known to lack intrinsic cell-adhesive peptide motifs and are therefore commonly used for the development of non-permissive 2D substrates. However, we unexpectedly found that agarose hydrogels can become permissive substrates for cell adhesion, depending on a compliant mechanical response of the substrate and the use of fetal bovine serum (FBS) as protein reservoir in the cell culture medium. We describe here two anchoring mechanisms that cells harness to adhere to agarose substrates, depending on the amount of FBS. Our results will have a major impact on the field of mechanobiology and shed light on the central role of FBS as a natural source of adhesive proteins that could promote cell anchoring.