VLA-4 Research Articles

S1426 Identification of Three Upregulated Genes in Tumor Tissues With Poor Prognosis in Gastric Cancer via Bioinformatical Analysis

Introduction: The purpose of the present academic work was to identify genes with worse prognoses of gastric cancer (GC). Methods: GSE54129 & GSE64951 were extracted from GEO database. Differentially expressed genes (DEGs) between GC and normal specimen were identified via GEO2R online tools with logFC > 1 and adjust P value < 0.05. Raw data were checked in Venn software online to detect the common DEGs. The DEGs with logFC > 1 were considered as up-regulated genes. Gene ontology (GO) analysis and KEGG pathway analysis were conducted and visualized via DAVID and Bioinformatics online tool. PPI network was evaluated by STRING and hub genes were selected via CytoHubba app in Cytoscape. Kaplan Meier-plotter was used to conduct survival analysis. GEPIA website was used to analyze the expression. The best discriminate cut-off points between the high and low expression groups were assessed with the Receiver operating characteristic (ROC) curve and area under the curve (AUC) for overexpressed DEGs by using R software with pROC and ggplot2 packages to analyze data from TCGA-STAD with Log2 transformed FPKM. Results: There are 174 GC & 52 normal tissue samples in the two profile datasets, and 605 and 1,860 up-regulated DEGs from GSE64951 and GSE54129, respectively. Venn diagram detected 62 commonly up-regulated DEGs. GO analysis indicated that they were enriched in integrin-mediated signaling pathway, extracellular matrix organization, and collagen fibril organization for biological processes (BP), and enriched in integrin complex, integrin alpha1-beta1 complex, and endoplasmic reticulum lumen for GO cell component (CC), and enriched in cell adhesion molecule binding, collagen-binding involved in cell-matrix adhesion, and transcription corepressor activity for molecular function (MF). KEGG analysis found that they were enriched in Axon guidance, Cell adhesion molecules (CAMs), and Leishmaniasis. Analysis of PPI network complex found top 10 genes as shown in the PPI network figure. Kaplan Meier plotter identified 3core genes (COL5A1, COL12A1, ITGBL1) associated with a worse prognosis (P < 0.05), and GEPIA found a higher expression level of them in GC tissues than normal tissue. ROC curve showed that AUC of COL5A1, COL12A1, ITGBL1 were 0.813, 0.831, and 0.646, respectively, and their cutoff values were 4.494, 2.789, and 0.900. Conclusion: In GC, COL5A1, COL12A1, and ITGBL1 are associated with poor prognoses that could be potential therapeutic targets., COL5A1 and COL12A1 could be potential diagnostic markers.Figure 1.: A) PRISMA flow chart; B) Forest plot of the overall results; C) Funnel Plot.

Eosinophilia during natalizumab treatment: Incidence, risk factors and temporal patterns

Eosinophilia is common during natalizumab treatment for multiple sclerosis but risk factors are unknown. We aimed to identify demographic, clinical and laboratory characteristics predicting eosinophilia. Sustained eosinophilia occurred in 16.8%. Risk factors for sustained eosinophilia included baseline pre-treatment eosinophilia, medical conditions potentially associated with eosinophilia including allergies, and suboptimal compliance. One temporal profile was associated with the highest and most rapidly developing eosinophilia, and was less likely to resolve: in one such case, eosinophilia was symptomatic. Changes in eosinophil and lymphocyte counts were only weakly correlated, suggesting factors other than Very Late Antigen-4 (VLA-4) inhibition drive eosinophilia.

Nintedanib Regulates GRK2 and CXCR2 to Reduce Neutrophil Recruitment in Endotoxin-Induced Lung Injury.

The role of nintedanib, a multiple tyrosine kinase inhibitor, in the treatment of sepsis-induced acute lung injury (ALI) remains unclear. Lipopolysaccharide (LPS), also known as endotoxin, has been used to induce ALI. The goal of this study was to assess the effect of nintedanib in attenuating the histopathological changes of LPS-induced ALI. Nintedanib was administered via oral gavage to male C57BL/6 mice 24 h and 10 min before intratracheal endotoxin instillation. Lung histopathological characteristics, adhesion molecule expression, and the regulatory signaling pathways of neutrophil chemotaxis were analyzed after 24 h. We found that nintedanib significantly reduced histopathological changes and neutrophil recruitment in LPS-induced ALI. The number of neutrophils in bronchoalveolar lavage fluid (BALF) was reduced in nintedanib-treated relative to untreated mice with ALI. Nintedanib mediated the downregulation of the chemotactic response to LPS by reducing the expression of adhesion molecules and the phosphorylated p38:total p38 mitogen-activated protein kinase (MAPK) ratio in the lungs of mice with ALI. Nintedanib also reduced the expression of lymphocyte antigen 6 complex locus G6D (Ly6G) and very late antigen 4 (VLA-4) in BALF neutrophils and mediated the downregulation of chemokine (C-X-C motif) receptor 2 (CXCR2) and upregulation of G protein-coupled receptor kinase 2 (GRK2) activity in peripheral blood neutrophils in mice with LPS-induced ALI. Nintedanib improved the histopathological changes of LPS-induced ALI by reducing neutrophil chemotaxis. These effects were mediated by the inhibition of adhesion molecules via the activation of GRK2 and the inhibition of p38 MAPK and CXCR2.

Generation and Characterization of Novel VLA-4 Inhibitors for Stem Cell Mobilization in Combination with a CXCR2 Agonist

Introduction: The integrin very-late antigen-4 (VLA-4) is comprised of an alpha4 chain (CD49d) that is non-covalently associated with the beta1 integrin chain (CD29). VLA-4 plays an important role in the homing and retention of hematopoietic stem/progenitor cells (HSPCs) in the bone marrow (BM) microenvironment. We previously reported (Karpova et al. Blood 2016;128:659) a novel strategy and pathway for rapid and potent mobilization of HSPCs in mice using a combination of a VLA-4 inhibitor and the truncated isoform of the CXCR2 agonist Gro-beta (tGro-β, SB-251353). These studies were completed with a novel VLA-4 inhibitor, CWHM-823, that exhibited increased potency and/or solubility relative to the previously described comparators BIO5192 (α4β1-specific) and firategrast (α4β1 and α4β7 dual-specific). Here we extended these findings by generating and characterizing additional novel VLA-4 inhibitors.Methods: We designed and synthesized 21 novel VLA-4 inhibitors with the intent of further improving potency toward VLA-4 and achieving a more prolonged mobilization effect in vivo. All novel VLA-4 antagonists were assayed for potency by measuring their inhibition of binding of a soluble VCAM/Fc chimeric protein (sVCAM-1) to VLA-4 expressing human G2 acute lymphoblastic leukemia (ALL) or murine 5TGM1 multiple myeloma cells by flow cytometry. To evaluate the mobilization ofHSPCs, DBA/2 mice were left untreated (nonmobilized) or treated witha VLA-4 inhibitor alone (5 mg/kg, SC) or in combination with tGro-β (2.5 mg/kg, SC). The number of circulating white blood cells (WBCs), colony forming units (CFU), lineage-Sca-1+c-kit+ (LSK) cells and myeloid and lymphoid cell subsets in the peripheral blood were determined at baseline, 0.5h and 2h after treatment.Results: Given the encouraging results with CWHM-823, we designed, synthesized and tested the potency of 21 additional VLA-4 inhibitors to provide further structure-activity relationships. We observed that CWHM-823 (IC50 = 4 nM) was nearly 50-fold more potent than firategrast (IC50 = 198 nM), 7.5-fold more potent than zaurategrast (IC50 = 30.7 nM) and 6-fold less potent than BIO5192 (IC50 = 0.7 nM) at inhibiting the binding of sVCAM-1 to G2 ALL cells. Nine of our 21 novel inhibitors proved to be more potent than CWHM-823 at inhibiting the binding of sVCAM-1 to G2 ALL cells. Encouragingly, 4 of these inhibitors were equipotent to BIO5192 at inhibiting sVCAM-1 binding to G2 ALL or 5TGM1 myeloma cells (IC50 < 1 nM). Initial mobilization studies in mice indicate that single 5 mg/kg injections of these compounds effectively mobilize murine CFUs and LSK cells to levels comparable to that attained with BIO5192. The combination of tGro-β with the best inhibitors resulted in a dramatic increase in circulating CFU and LSK numbers when compared to treatment with single agents (3-5 fold). Further, combination therapy induced an approximately 2-fold increase in total circulating WBCs with pan mobilization of B cells (3.5-fold), monocytes (3.2-fold), plasmacytoid DCs (3-fold), neutrophils (2.5-fold) and T cells (1.4-fold). Similar to BIO5192 and CWHM-823, mobilization with tGro-β and the new compounds was rapid, peaking at 15-30 minutes after injection. However, in contrast to all of the VLA-4 inhibitors that we have tested in vivo to date, one inhibitor (compound 1611) maintained peak levels of circulating CFUs and LSK cells at 2h after injection. This prolonged CFU and LSK cell mobilization exhibited by these new analogues is quite encouraging, and additional analogues based upon these findings are currently being synthesized.Summary: Currently available small molecule VLA-4 antagonists (BIO5192, firategrast, and zaurategrast) have suffered from poor bioavailability and a short half-life. These preliminary data demonstrate that our novel analogs are exceptionally potent ligands for VLA-4, soluble and efficacious in mobilization of stem cells (CFU and LSK cells) in vivo, and thus are suitable leads for optimization to maximize their pharmacodynamic effect in vivo. DisclosuresMeyers:CasterBridge Pharmaceuticals: Consultancy; Indalo Therapeutics: Consultancy; Meyers MedChem Consulting LLC: Equity Ownership. Griggs:Indalo Therapeutics: Consultancy, Equity Ownership, Research Funding. Morrow:Magenta Therapeutics: Employment, Equity Ownership.

Multimerin-1 Binds to Specific Motifs in Vessel Wall Collagens and Contributes to Thrombosis: Novel Insights Regarding the Mechanisms That Support Human and Mouse Platelet Adhesion

Multimerin-1 (MMRN1) is a homopolymeric glycoprotein stored in platelets and vascular endothelial cells for regulated release that enhances platelet adhesion to collagen. We tested Mmrn1 contributions to platelet adhesion ex vivo and in vivo using selective deficient mice and triple-helical collagen mimetic peptides to evaluate a putative Mmrn1-selective binding motif. Selective Mmrn1-deficient (Mmrn1-/-) mice were generated by crossing Mmrn1+/- “knockout-first allele” (Mmrn1flxneo/+ from European Mutant Mouse Archive) with flp deleter mice, and then with transgenic global cre deleter mice. The resultant heterozygous Mmrn1+/- mice were crossed with wild type (Mmrn1+/+) mice to remove the cre and flp transgenes. Inbred, gender-mixed, age-matched Mmrn1+/+ and Mmrn1-/- mice were then used for in vitro and in vivo assessments of platelet function. The following highly conserved GPAGPOGPX sequences in fibrillar collagens (that are distinct from collagen motifs that bind von Willebrand factor [VWF], platelet integrin alpha2beta1 or platelet GPVI) were identified to bind MMRN1: GPAGPOGPI (type I), GPAGPOGPV (type II), and GPAGPOGPQ (type III). In static adhesion assays with washed human platelets, triple-helical GPAGPOGPX peptides enhanced platelet adhesion when co-presented with the alpha2beta1-binding peptide GFOGER, like the VWF-binding peptide derived from type III collagen which was tested as a positive control. Static platelet adhesion to GPAGPOGPX peptides was also enhanced by agonists that induce MMRN1 release (p<0.02 for each comparison, where family-wise α=0.05, k=7), and co-presentation of GPAGPOGPX peptides with both GFOGER and III-23 maximized platelet adhesion (p<0.02 for each comparison, where family-wise α=0.05, k=5), providing insights on how MMRN1 and VWF synergistically enhance platelet adhesion. In high shear (1500 s-1) flow assays of platelet adhesion, platelet adhesion to triple-helical GPAGPOGPX peptides that were co-presented with GFOGER was greater than adhesion to GFOGER alone (p=0.015). Mmrn1-/- mice were viable and fertile, with normal blood cell counts and bleeding times (respective medians [ranges] for Mmrn1+/+ versus Mmrn1-/- mice: 300 [60-900] seconds vs. 330 [90-900] seconds, p=0.07), and confirmed Mmrn1 deficiency by Western blot analyses of platelets. In assays of platelet adhesion at high shear (1500 s-1), platelets from Mmrn1-/- mice showed a significant impairment in adhesion to full-length fibrillar collagen (% surface area covered by platelets ± SEM for Mmrn1+/+ versus Mmrn1-/- mice: 37 ± 3% vs. 18 ± 2%, p<0.001) and in collagen-induced aggregation in assays with washed platelets (20-30% reduction in maximal aggregation for Mmrn1-/- platelets compared to Mmrn1+/+ platelets, p<0.05). In assays of washed platelet adhesion, Mmrn1-/- platelets showed minimal static adhesionto the GPAGPOGPX peptide, unlike Mmrn1+/+ platelets(p<0.0001). Assays of platelet adhesion under conditions of flow indicated that co-presentation of GPAGPOGPX with GFOGER enhanced the adhesion of activated Mmrn1+/+ but not Mmrn1-/- platelets. Intravital microscopyexperiments indicated that Mmrn1-/- mice also had impaired platelet adhesion in arterioles treated with ferric chloride (platelets deposited per minute ± SEM for Mmrn1+/+ versus Mmrn1-/- mice: Mmrn1+/+ : 82 ± 4 vs. Mmrn1-/-: 42 ± 6, p<0.001), and the thrombi that formed in Mmrn1-deficient mice were smaller and less stable, with delayed vessel occlusion times (respective medians [ranges] for Mmrn1+/+ versus Mmrn1-/- mice: 17 [10-35] min vs. 35 [22-40] min, p<0.001). Taken together, our studies illustrate that multimerin-1 has functions in supporting and enhancing platelet adhesive function, in vivo and in vitro, and that fibrillar collagens contain highly conserved GPAGPOGPX motifs that bind Mmrn1 and contribute to platelet adhesion. DisclosuresNo relevant conflicts of interest to declare.

Is the New Approach in Inflammatory Bowel Disease Treat-to-Target?

Inflammatory bowel disease is a chronic inflammatory disease of which the etiology is unknown. Ulcerative colitis and Crohn's disease are the two main entities of inflammatory bowel disease that are challenging clinicians. In addition to tumor necrosis factor blockers, this overview summarizes current and future new drugs, in the treatment of inflammatory bowel disease according to their goals. The infiltration of lymphocytes into the intestinal lining is a target for therapeutic purposes in inflammatory bowel disease. The vascular cell adhesion molecule-1 and the mucosal addressin cell adhesion molecule-1 are a family of integrins for the alpha4 that are specifically expressed in the alimentary canal on vascular endothelial cells. In Crohn's disease, the alpha4beta7 integrin, and its endothelial receptor, the mucosal addressin cell adhesion molecule-1, have proven to be a relevant factor in the development of chronic intestinal inflammation. New biological and chemical drugs are emerging, with additional molecules pending approval.

The epidemiology and biology of pulmonary metastases.

Our goal in this chapter is to explore the complex processes of metastasis and why there is a predisposition for this to occur in the lung. In addition, we aim to describe the incidence of pulmonary metastases in various contexts and based on the origin of the primary tumor. There are unique characteristics of the pulmonary system that make metastases more likely to occur in the lung than anywhere else in the body. Some of these characteristics include receiving the entire cardiac output every minute, having the densest capillary bed in the body, and being the first reservoir of most lymphatic drainage entering the venous system. There are multiple postulated routes of metastasis to the pulmonary system including hematogenous and lymphatic routes with early or late dissemination. The vascularization of pulmonary metastases is variable and complex, often recruiting supply from bronchial and pulmonary origin. There are also many biochemical factors in the tumor microenvironment that play a key role in the development of lung metastases including vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), very late antigen 4 (VLA-4) and intercellular adhesion molecule 1 (ICAM-1). Studies vary widely in reported rates of pulmonary metastases due to differences in clinical study design, however, it is commonly accepted that up to half of autopsies performed on patients who died of malignancy have pulmonary metastases. In a surgical series describing the incidence of primary cancer types with resected pulmonary metastases the most common sites were thyroid, colon, breast, genitourinary tract, skin, liver, breast, and adrenal glands.

Computational evaluation of anticipated PE_PGRS39 protein involvement in host-pathogen interplay and its integration into vaccine development.

Mycobacterium tuberculosis causes more than 1 million deaths every year, which is higher than any other bacterial pathogen. Its success depends on its interaction with the host and its ability to regulate the host’s immune system for its own survival. Mycobacterium tuberculosis H37Rv (Mtb) proteome consists of unique PE_PGRS family proteins, which present a significant role in bacterial pathogenesis over the past years. Earlier evidence suggests that some PE_PGRS proteins display fibronectin-binding activity. In this manuscript, computational characterization of the PE_PGRS39 protein has indicated something peculiar about this protein. Investigation showed that PE_PGRS39 is an extracellular protein that, instead of acting as fibronectin-binding protein, might mimic fibronectin which binds to alpha-5 beta-1 (α5β1) integrin. PE_PGRS39 protein additionally turned into proven pieces of evidence to have motifs such as DXXG and GGXGXD and PXXP that bind with guanosine triphosphate (GTP), calcium, and host Src homology 3 (SH3) domains, respectively, in conjunction with RGD-integrin binding. These interactions designate the direct role of PE_PGRS39 in bacterial pathogenesis via cell adhesion and signaling. Additionally, the analysis showed that PE_PGRS39 is an antigenic protein and epitope prediction provided functional regions of the protein that trigger a cellular immune response facilitated by T or B cells. Further, an experimental analysis could also open up new avenues for developing novel drugs by targeting signaling motifs or novel vaccines using functional epitopes that could evoke an immune response in the host.

Investigating the Effect of a Non-Conservative Mutation (G273D) on Integrin Alpha6-Beta4 Binding Interactions

Integrins are heterodimeric transmembrane receptors which function in cell adhesion and signalling. Mutations on either their alpha or beta subunit result in different debilitating conditions. In particular, a G273D mutation in the Integrin Beta 4 (ITGB4) subunit is known to cause a lethal form of epidermolysis bullosa (EB), a rare disease characterized by very fragile skin and mucosal membranes. To gain a deeper understanding of this disease, this study aimed to elucidate the effects of the G273D mutation on the structure of ITGB4 and its interaction with its partner, Integrin Alpha 6 (ITGA6). In this study, in silico modelling predicted more interfacial contacts, and consequently, stronger binding affinity between ITGA6-ITGB4, than between ITGA6-ITGB4(G273D). The type of ions included in the simulated equilibration medium (i.e. CaCl2 or NaCl) was also observed to affect the binding affinities between the integrin subunits. Steered Molecular Dynamics (SMD) simulations showed significantly longer dissociation times for the CaCl2 equilibrated WT ITGB4 replicates compared to the other setups. Significantly higher pull forces were needed for the dissociation of ITGA6-ITGB4 compared to ITGA6-ITGB4(G273D) when equilibrated with CaCl2. These results corroborate previous findings that Calcium ions are needed to stabilize the binding of the heterodimer albeit in an inactive state. More importantly, these suggest that the G273D mutation disrupts the binding of ITGB4 with ITGA6 even in the presence of calcium ions.

Domains 12 to 16 of tropoelastin promote cell attachment and spreading through interactions with glycosaminoglycan and integrins alphaV and alpha5beta1.

Elastin is an extracellular matrix component with key structural and biological roles in elastic tissues. Interactions between resident cells and tropoelastin, the monomer of elastin, underpin elastin's regulation of cellular processes. However, the nature of tropoelastin-cell interactions and the contributions of individual tropoelastin domains to these interactions are only partly elucidated. In this study, we identified and characterized novel cell-adhesive sites in the tropoelastin N-terminal region between domains 12 and 16. We found that this region interacts with αV and α5β1 integrin receptors, which mediate cell attachment and spreading. A peptide sequence from within this region, spanning domains 14 to mid-domain 16, binds heparan sulfate through electrostatic interactions with peptide lysine residues and induces conformational ordering of the peptide. We propose that domains 14-16 direct initial cell attachment through cell-surface heparan sulfate glycosaminoglycans, followed by αV and α5β1 integrin-promoted attachment and spreading on domains 12-16 of tropoelastin. These findings advance our mechanistic understanding of elastin matrix biology, with the potential to enhance tissue regenerative outcomes of elastin-based materials.

Integrin α6 β4 on Circulating Tumor Cells of Metastatic Breast Cancer Patients.

Background:The detection of circulating tumor cells (CTCs) is prognostic during the disease in women with metastatic breast cancer. Integrins are key role players in nearly every step of cancer progression. In this study, we aimed to analyze integrin alpha6beta4 expression on CTCs isolated from blood samples of patients with advanced breast cancer.Materials and Methods:In this single-center study, peripheral blood samples from 23 breast cancer patients were obtained and analyzed for the presence of CTCs by EasySep™ Direct Human CTC Enrichment Kit combined with subsequent immunocytochemical staining of anti-cytokeratin and anti-epithelial cell adhesion molecules, and β4 integrin on CTCs. Data were correlated with clinicopathological parameters.Results:CTCs were detected in 100% of the patients. The ratio of integrin β4+ CTC was 61% ± 8% of total CTCs. No significant correlation between histopathological parameters and CTC detection was found.Conclusion:Our results demonstrated the importance of α6 β4 integrin expression on CTCs in distant metastasis.

CC16 Binding to α4β1 Integrin Protects against Mycoplasma pneumoniae Infection.

Rationale CC16 (club cell secretory protein) is a pneumoprotein produced predominantly by pulmonary club cells. Circulating CC16 is associated with protection from the inception and progression of the two most common obstructive lung diseases (asthma and chronic obstructive pulmonary disease). Objectives Although exact mechanisms remain elusive, studies consistently suggest a causal role of CC16 in mediating antiinflammatory and antioxidant functions in the lung. We sought to determine any novel receptor systems that could participate in CC16's role in obstructive lung diseases. Methods Protein alignment of CC16 across species led to the discovery of a highly conserved sequence of amino acids, leucine-valine-aspartic acid (LVD), a known integrin-binding motif. Recombinant CC16 was generated with and without the putative integrin-binding site. A Mycoplasma pneumoniae mouse model and a fluorescent cellular adhesion assay were used to determine the impact of the LVD site regarding CC16 function during live infection and on cellular adhesion during inflammatory conditions. Measurements and Main Results CC16 bound to integrin α4β1), also known as the adhesion molecule VLA-4 (very late antigen 4), dependent on the presence of the LVD integrin-binding motif. During infection, recombinant CC16 rescued lung function parameters both when administered to the lung and intravenously but only when the LVD integrin-binding site was intact; likewise, neutrophil recruitment during infection and leukocyte adhesion were both impacted by the loss of the LVD site. Conclusions We discovered a novel receptor for CC16, VLA-4, which has important mechanistic implications for the role of CC16 in circulation as well as in the lung compartment.

The tellurium-based immunomodulator, AS101 ameliorates adjuvant-induced arthritis in rats.

Despite undeniable improvement in the management of rheumatoid arthritis (RA), the discovery of more effective, less toxic and, ideally, less immune suppressive drugs are much needed. In the current study, we set to explore the potential anti-rheumatic activity of the non-toxic, tellurium-based immunomodulator, AS101 in an experimental animal model of RA. The effect of AS101 was assessed on adjuvant-induced arthritis (AIA) rats. Clinical signs of arthritis were assessed. Histopathological examination was used to assess inflammation, synovial changes and tissue lesions. Very late antigen-4 (VLA-4)+ cellular infiltration was detected using immunohistochemical staining. Enzyme-linked immunosorbent assay (ELISA) was used to measure circulating anti-cyclic citrullinated-peptide autoantibody (ACPA) and real-time polymerase chain reaction (PCR) was used to measure the in-vitro effect of AS101 on interleukin (IL)-6 and IL-1β expression in activated primary human fibroblasts. Prophylactic treatment with intraperitoneal AS101 reduced clinical arthritis scores in AIA rats (P<0·01). AS101 abrogated the migration of active chronic inflammatory immune cells, particularly VLA-4+ cells, into joint cartilage and synovium, reduced the extent of joint damage and preserved joint architecture. Compared to phosphate-buffered saline (PBS)-treated AIA rats, histopathological inflammatory scores were significantly reduced (P<0·05). Furthermore, AS101 resulted in a marked reduction of circulating ACPA in comparison to PBS-treated rats (P<0·05). Importantly, AS101 significantly reduced mRNA levels of proinflammatory mediators such as IL-6 (P<0·05) and IL-1β (P<0·01) in activated primary human fibroblasts. Taken together, we report the first demonstration of the anti-rheumatic/inflammatory activity of AS101 in experimental RA model, thereby supporting an alternative early therapeutic intervention and identifying a promising agent for therapeutic intervention.

Partial Sperm beta1 Integrin Subunit Deletion Proves its Involvement in Mouse Gamete Adhesion/Fusion.

We have previously shown, using antibodies, that the sperm alpha6beta1 integrin is involved in mouse gamete fusion in vitro. Here we report the conditional knockdown of the sperm Itgb1 gene. It induced a drastic failure of sperm fusogenic ability with sperm accumulation in the perivitelline space of in vitro inseminated oocytes deleted or not for the Itgb1 gene. These data demonstrate that sperm, but not oocyte, beta1 integrin subunit is involved in gamete adhesion/fusion. Curiously, knockdown males were fertile in vivo probably because of the incomplete Cre-mediated deletion of the sperm Itgb1 floxed gene. Indeed, this was shown by Western blot analysis and confirmed by both the viability and litter size of pups obtained by mating partially sperm Itgb1 deleted males with females producing completely deleted Itgb1 oocytes. Because of the total peri-implantation lethality of Itgb1 deletion in mice, we assume that sperm that escaped the Itgb1 excision seemed to be preferentially used to fertilize in vivo. Here, we showed for the first time that the deletion, even partial, of the sperm Itgb1 gene makes the sperm unable to normally fertilize oocytes. However, to elucidate the question of the essentiality of its role during fertilization, further investigations using a mouse expressing a recombinase more effective in male germ cells are necessary.

Gefitinib induces EGFR and α5β1 integrin co-endocytosis in glioblastoma cells.

Overexpression of EGFR drives glioblastomas (GBM) cell invasion but these tumours remain resistant to EGFR-targeted therapies such as tyrosine kinase inhibitors (TKIs). Endocytosis, an important modulator of EGFR function, is often dysregulated in glioma cells and is associated with therapy resistance. However, the impact of TKIs on EGFR endocytosis has never been examined in GBM cells. In the present study, we showed that gefitinib and other tyrosine kinase inhibitors induced EGFR accumulation in early-endosomes as a result of an increased endocytosis. Moreover, TKIs trigger early-endosome re-localization of another membrane receptor, the fibronectin receptor alpha5beta1 integrin, a promising therapeutic target in GBM that regulates physiological EGFR endocytosis and recycling in cancer cells. Super-resolution dSTORM imaging showed a close-proximity between beta1 integrin and EGFR in intracellular membrane compartments of gefitinib-treated cells, suggesting their potential interaction. Interestingly, integrin depletion delayed gefitinib-mediated EGFR endocytosis. Co-endocytosis of EGFR and alpha5beta1 integrin may alter glioma cell response to gefitinib. Using an in vitro model of glioma cell dissemination from spheroid, we showed that alpha5 integrin-depleted cells were more sensitive to TKIs than alpha5-expressing cells. This work provides evidence for the first time that EGFR TKIs can trigger massive EGFR and alpha5beta1 integrin co-endocytosis, which may modulate glioma cell invasiveness under therapeutic treatment.

Vedolizumab for ulcerative colitis: Real world outcomes from a multicenter observational cohort of Australia and Oxford.

BACKGROUNDVedolizumab (VDZ), a humanised monoclonal antibody that selectively inhibits alpha4-beta7 integrins is approved for use in adult moderate to severe ulcerative colitis (UC) patients.AIMTo assess the efficacy and safety of VDZ in the real-world management of UC in a large multicenter cohort involving two countries and to identify predictors of achieving remission.METHODSA retrospective review of Australian and Oxford, United Kingdom data for UC patients. Clinical response at 3 mo, endoscopic remission at 6 mo and clinical remission at 3, 6 and 12 mo were assessed. Cox regression models and Kaplan Meier curves were performed to assess the time to remission, time to failure and the covariates influencing them. Safety outcomes were recorded.RESULTSThree hundred and three UC patients from 14 centres in Australia and United Kingdom, [60% n = 182, anti-TNF naïve] were included. The clinical response was 79% at 3 mo with more Australian patients achieving clinical response compared to Oxford (83% vs 70% P = 0.01). Clinical remission for all patients was 56%, 62% and 60% at 3, 6 and 12 mo respectively. Anti-TNF naive patients were more likely to achieve remission than exposed patients at all the time points (3 mo 66% vs 40% P < 0.001, 6 mo 73% vs 46% P < 0.001, 12 mo 66% vs 51% P = 0.03). More Australian patients achieved endoscopic remission at 6 mo compared to Oxford (69% vs 43% P = 0.01). On multi-variate analysis, anti-TNF naïve patients were 1.8 (95%CI: 1.3-2.3) times more likely to achieve remission than anti-TNF exposed (P < 0.001). 32 patients (11%) had colectomy by 12 mo.CONCLUSIONVDZ was safe and effective with 60% of UC patients achieving clinical remission at 12 mo and prior anti-TNF exposure influenced this outcome.

Molecular Imaging of Very Late Antigen-4 in Acute Lung Injury.

Inflammation plays a central role in the pathogenesis of acute lung injury (ALI) during both the acute pneumonitis stage and progression into the chronic fibroproliferative phase, leading to pulmonary fibrosis. Currently, there is an unmet clinical and research need for noninvasive ways to monitor lung inflammation through targeting of immunoregulatory pathways contributing to ALI pathogenesis. In this study, we evaluated the role of targeted imaging of very late antigen-4 (VLA-4), as a key integrin mediating the adhesion and recruitment of immune cells to inflamed tissues, in quantifying lung inflammation in a mouse model of lipopolysaccharide-induced ALI. Methods: ALI was induced by a single intratracheal administration of lipopolysaccharide (10, 20, or 40 μg per mouse) in C57BL/6J mice. Control mice were intratracheally instilled with sterile phosphate-buffered saline. VLA-4-targeted PET/CT was performed 24 h after intravenous injection of a 64Cu-labeled high-affinity peptidomimetic ligand referred to as 64Cu-LLP2A, which is conjugated with the chelator (1,4,8,11-tetraazacyclotetradecane-1-(methane phosphonic acid)-8-(methane carboxylic acid) and a polyethylene glycol 4 linker, at day 2 after the induction of ALI. Ex vivo biodistribution of 64Cu-LLP2A was determined by γ-counting of harvested organs. The severity of lung inflammation was assessed histologically and by measuring the expression of inflammatory markers in the lung tissue lysates using reverse transcription quantitative polymerase chain reaction. Results: Intratracheal lipopolysaccharide instillation led to an acute inflammatory response in the lungs, characterized by increased expression of multiple inflammatory markers and infiltration of myeloid cells, along with a significant and specific increase in 64Cu-LLP2A uptake, predominantly in a peribronchial distribution. There was a strong correlation between the lipopolysaccharide dose and 64Cu-LLP2A uptake, as quantified by in vivo PET (R = 0.69, P < 0.01). Expression levels of both subunits of VLA-4, that is, integrins α4 and β1, significantly correlated with the expression of multiple inflammatory markers, including tumor necrosis factor-α, interleukin-1β, and nitric oxide synthase-2, highlighting the potential of VLA-4 as a surrogate marker of acute lung inflammation. Notably, in vivo 64Cu-LLP2A uptake significantly correlated with the expression of multiple inflammatory markers and VLA-4. Conclusion: Our study demonstrates the feasibility of molecular imaging of VLA-4, as a mechanistically relevant target in ALI, and the accuracy of VLA-4-targeted PET in quantification of ongoing lung inflammation in a murine model.

Nintedanib Reduces Neutrophil Chemotaxis via Activating GRK2 in Bleomycin-Induced Pulmonary Fibrosis.

Neutrophils are involved in the alveolitis of idiopathic pulmonary fibrosis (IPF). However, their pathogenic mechanisms are still poorly understood. Nintedanib has antifibrotic and anti-inflammatory activity in IPF. This study aimed to investigate the regulatory mechanism of nintedanib on neutrophil chemotaxis in bleomycin (BLM)-induced pulmonary fibrosis. Nintedanib was administered via oral gavage to male C57BL/6 mice 24 h after a bleomycin intratracheal injection (1.5 U/kg). Lung histopathological findings, the expression of cytokines, and the regulatory signaling pathways of neutrophil chemotaxis were analyzed. The effect of nintedanib was also investigated in a mouse model with adoptive neutrophil transfer in vivo. Nintedanib significantly decreased the histopathological changes and neutrophil recruitment in BLM-induced pulmonary fibrosis. Nintedanib mediated a downregulation of chemokine (C-X-C motif) receptor 2 (CXCR2) and very late antigen 4 (VLA-4) expression, as well as an upregulation of G protein-coupled receptor kinase 2 (GRK2) activity in peripheral blood neutrophils in BLM-induced pulmonary fibrosis. Nintedanib also decreased the activation of endothelial cells by the decreased expression of vascular cell adhesion molecule 1 (VCAM-1). The effect of nintedanib on regulating neutrophil chemotaxis was also confirmed by a mouse model with adoptive neutrophil transfer in vivo. In conclusion, nintedanib reduces neutrophil chemotaxis and endothelial cell activation to regulate the severity of BLM-induced pulmonary fibrosis. These effects are associated with an enhancement of GRK2 activity and a reduction in CXCR2 and VLA-4 expression on neutrophils and a decrease in VCAM-1 expression on endothelial cells.

Short-term culture under pressurized and hypoxic conditions reduces hematopoietic stem cell apoptosis and improves cellular function of transplant products

Background & Aim Hematopoietic stem cell transplantation (HSCT) is a potential curative therapy for numerous diseases. The success of HSCT is dependent upon the viability, homing, and engraftment capabilities of the hematopoietic stem cells (HSCs). Current clinical approaches allow for storage times up to 48 hours at either room temperature (20-24°C) or refrigerated conditions (2-8°C) prior to transplantation, resulting in high levels of cellular apoptosis and reduced functionality, thus compromising the effectiveness of the transplanted cells. To address this, we substituted the current conditions with short-term culturing methods ( Methods, Results & Conclusion Environmental optimization can selectively enhance the fitness and functionality of specific cell types and increase the stemness or differentiation potential of cells in culture. By varying the temperature (4°C, room temperature, 37°C, 43°C), oxygen (20%, 5%, and 1%), and pressure levels (1.00 to 1.35 atm), we optimized the culture conditions to improve the functionality of HSCs. Cell health was measured using assays measuring apoptosis and metabolic function. Significant decreases in cell populations positive for apoptosis markers, annexin V and DilC1(5), were observed after 24 hours of incubation in optimized conditions compared to current clinical approaches. DUSP4, a stress-related gene that prevents apoptosis, was upregulated under optimized environmental conditions; this, along with higher ATP levels, is indicative of functional and healthy cells. Preconditioned cells exhibit characteristics of a product with improved rates of bone marrow homing and immune reconstitution. They show increased surface expression of the receptor for stromal cell-derived factor (CXCR4), the sialomucin receptor for P-selectin glycoprotein ligand-1 (PSGL-1), the β1 integrin very-late antigen (VLA-4), and the tetraspanin CD82, which are all implicated in the homing capacity of HSCs to the bone marrow. Immune reconstitution, as assessed by measuring proliferation and differentiation via a colony forming units in vitro, predicts robust reconstitution capability of HSCs cultured under optimized conditions. As shown, cells for HSCT maintained under optimized temperature, oxygen, and pressure levels result in improved cell viability and function over conventional storage conditions, suggesting a potential clinical benefit in the transplantation setting.

Abstract P2-05-08: Integrin alpha6beta4 signaling switches double strand break repair from homologous recombination to non-homologous end joining to alter TNBC cells response to cisplatin

Abstract Integrin α6β4 is highly expressed in triple negative breast cancer (TNBC) and drives aggressiveness by stimulating proliferation, angiogenesis, cell migration, invasion and metastasis. Signaling from this integrin stimulates DNA repair and apoptosis resistance, suggesting that it could contribute to therapeutic resistance. Upon testing this hypothesis, we found that integrin α6β4 signaling promoted a three-fold greater sensitivity to cisplatin but exhibited no difference in the response to other chemotherapies tested. Mechanistic investigations revealed that integrin α6β4 stimulated quicker and higher amplitude activations of ATM, Chk2, p53, and 53BP1, which required the β4 signaling domain. Genetic manipulation of gene expression demonstrated that mutant p53 cooperated with integrin α6β4 for cisplatin sensitivity and was necessary for downstream phosphorylation of 53BP1 and enhanced ATM activation. Additionally, we discovered that integrin α6β4 preferentially activated DNA-PKc in response to cisplatin, which led to formation of DNA-PKc-p53 complexes and 53BP1 activation. As a result, integrin α6β4 shifted double strand break repair from homologous recombination (HR) to non-homologous end joining (NHEJ), as demonstrated by HR and NHEJ reporter assays, in normal culture which was amplified by cisplatin treatment. Furthermore, DNA-PKc inhibition blocked integrin α6β4 enhanced cisplatin sensitivity. This signaling, against the backdrop of prominent losses and gains of select DNA repair molecules in breast cancer as a whole, may lead to a novel HR-deficiency that characterizes TNBC and their response to select chemotherapies. In summary, we discovered a novel function of integrin α6β4 in switching DSB repair from HR to NHEJ that results in cisplatin sensitivity in TNBC. Citation Format: Min Chen, Brock Marrs, Lei Qi, Teresa Knifley, Stuart G Jarrett, Heidi L Weiss, Rachel L Stewart, John A D'Orazio, Kathleen L O'Connor. Integrin alpha6beta4 signaling switches double strand break repair from homologous recombination to non-homologous end joining to alter TNBC cells response to cisplatin [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-05-08.