Induced hepatic stellate cell integrin, α8ß1, enhances cellular contractility and TGFß activity in liver fibrosis.

No effective therapy exists for fatal fibrosis. New therapeutic targets are needed for hepatic fibrosis because the incidence keeps increasing. The activation and differentiation of fibroblasts into myofibroblasts that causes excessive matrix deposition is central to fibrosis. Here, we investigated whether (and which) integrin receptors for matrix proteins activate hepatic stellate cells (HSCs). First, integrin α-subunits were investigated systematically for their expression over the course of HSC activation and their distribution on fibroblasts and other systemic primary cells. The most upregulated in plate culture-activated HSCs and specifically expressed across fibroblast linages was the α8 subunit. An anti-α8 neutralizing mAb was evaluated in three different murine fibrosis models: for cytotoxic (CCl4 treatment), non-alcoholic steatohepatitis-associated and cholestatic fibrosis. In all models, pathology and fibrosis markers (hydroxyproline and α-smooth muscle actin) were improved following the mAb injection. We also CCl4 -treated mice with inducible Itga8-/-; these mice were protected from increased hydroxyproline levels. Furthermore, ITGA8 was upregulated in specimens from 90 patients with liver fibrosis, indicating the relevance of our findings to liver fibrosis in people. Mechanistically, inhibition or ligand engagement of HSC α8 suppressed and enhanced myofibroblast differentiation, respectively, and HSC/fibroblast α8 activated latent TGFß. Finally, integrin α8ß1 potentially fulfils the growing need for anti-fibrotic drugs and is an integrin not to be ignored. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

New Therapeutic Targets for Hepatic Fibrosis in the Integrin Family, α8ß1 and α11ß1, Induced Specifically on Activated Stellate Cells.

A huge effort has been devoted to developing drugs targeting integrins over 30 years, because of the primary roles of integrins in the cell-matrix milieu. Five αv-containing integrins, in the 24 family members, have been a central target of fibrosis. Currently, a small molecule against αvß1 is undergoing a clinical trial for NASH-associated fibrosis as a rare agent aiming at fibrogenesis. Latent TGFß activation, a distinct talent of αv-integrins, has been intriguing as a therapeutic target. None of the αv-integrin inhibitors, however, has been in the clinical market. αv-integrins commonly recognize an Arg-Gly-Asp (RGD) sequence, and thus the pharmacophore of inhibitors for the 5-integrins is based on the same RGD structure. The RGD preference of the integrins, at the same time, dilutes ligand specificity, as the 5-integrins share ligands containing RGD sequence such as fibronectin. With the inherent little specificity in both drugs and targets, "disease specificity" has become less important for the inhibitors than blocking as many αv-integrins. In fact, an almighty inhibitor for αv-integrins, pan-αv, was in a clinical trial. On the contrary, approved integrin inhibitors are all specific to target integrins, which are expressed in a cell-type specific manner: αIIbß3 on platelets, α4ß1, α4ß7 and αLß2 on leukocytes. Herein, "disease specific" integrins would serve as attractive targets. α8ß1 and α11ß1 are selectively expressed in hepatic stellate cells (HSCs) and distinctively induced upon culture activation. The exceptional specificity to activated HSCs reflects a rather "pathology specific" nature of these new integrins. The monoclonal antibodies against α8ß1 and α11ß1 in preclinical examinations may illuminate the road to the first medical agents.

Cross-Talk between Transforming Growth Factor-ß and Periostin Can Be Targeted for Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized as progressive and irreversible fibrosis in the interstitium of lung tissues. There is still an unmet need to develop a novel therapeutic drug for IPF. We have previously demonstrated that periostin, a matricellular protein, plays an important role in the pathogenesis of pulmonary fibrosis. However, the underlying mechanism of how periostin causes pulmonary fibrosis remains unclear. In this study, we sought to learn whether the cross-talk between TGF-ß (transforming growth factor-ß), a central mediator in pulmonary fibrosis, and periostin in lung fibroblasts leads to generation of pulmonary fibrosis and whether inhibitors for integrin αVß3, a periostin receptor, can block pulmonary fibrosis in model mice and the TGF-ß signals in fibroblasts from patients with IPF. We found that cross-talk exists between TGF-ß and periostin signals via αVß3/ß5 converging into Smad3. This cross-talk is necessary for the expression of TGF-ß downstream effector molecules important for pulmonary fibrosis. Moreover, we identified several potent integrin low-molecular-weight inhibitors capable of blocking cross-talk with TGF-ß signaling. One of the compounds, CP4715, attenuated bleomycin-induced pulmonary fibrosis in vivo in mice and the TGF-ß signals in vitro in fibroblasts from patients with IPF. These results suggest that the cross-talk between TGF-ß and periostin can be targeted for pulmonary fibrosis and that CP4715 can be a potential therapeutic agent to block this cross-talk.

Periostin forms a functional complex with IgA in human serum.

BACKGROUND: Periostin is a matricellular protein belonging to the fasciclin family, playing a role for the pathogenesis of allergic diseases by binding to integrins on cell surfaces. Serum periostin is elevated in various allergic diseases reflecting type 2 inflammation and tissue remodeling so that for allergic diseases, periostin is expected to be a novel biomarker for diagnosis, assessing severity or prognosis, and predicting responsiveness to treatments. We have previously shown that most serum periostin exists in the oligomeric form by intermolecular disulfide bonds. METHODS: In this study, we examined how periostin forms a complex in serum, whether the periostin complex in serum is functional, and whether the complex formation interferes with reactivity to anti-periostin Abs. RESULTS: We found that periostin formed a complex with IgA1 at a 1:1 ratio. The periostin in the serum complex contained at least five different isoforms. However, IgA was not essential for the oligomeric formation of periostin in mouse serum or in IgA-lacking serum. The periostin-IgA complex in human serum was functional, sustaining the ability to bind to αVß3 integrin on cell surfaces. Moreover, periostin formed the complex with IgA broadly, which interferes the binding of the Abs recognizing all of the domains except the R4 domain to periostin. CONCLUSIONS: Periostin is a novel member of the IgA-associated molecules. These results are of great potential use to understand the pathological roles of periostin in allergic diseases and, from a practical standpoint, to develop diagnostics or therapeutic agents against periostin.

OPN-a induces muscle inflammation by increasing recruitment and activation of pro-inflammatory macrophages.

NEW FINDINGS: What is the central question of this study? What is the functional relevance of OPN isoform expression in muscle pathology? What is the main finding and its importance? The full-length human OPN-a isoform is the most pro-inflammatory isoform in the muscle microenvironment, acting on macrophages and myoblasts in an RGD-integrin-dependent manner. OPN-a upregulates expression of tenascin-C (TNC), a known Toll-like receptor 4 (TLR4) agonist. Blocking TLR4 signalling inhibits the pro-inflammatory effects of OPN-a, suggesting that a potential mechanism of OPN action is by promoting TNC-TLR4 signalling. Although osteopontin (OPN) is an important mediator of muscle remodelling in health and disease, functional differences in human spliced OPN variants in the muscle microenvironment have not been characterized. We thus sought to define the pro-inflammatory activities of human OPN isoforms (OPN-a, OPN-b and OPN-c) on cells present in regenerating muscle. OPN transcripts were quantified in normal and dystrophic human and dog muscle. Human macrophages and myoblasts were stimulated with recombinant human OPN protein isoforms, and cytokine mRNA and protein induction was assayed. OPN isoforms were greatly increased in dystrophic human (OPN-a > OPN-b > OPN-c) and dog muscle (OPN-a = OPN-c). In healthy human muscle, mechanical loading also upregulated OPN-a expression (eightfold; P < 0.01), but did not significantly upregulate OPN-c expression (twofold; P > 0.05). In vitro, OPN-a displayed the most pronounced pro-inflammatory activity among isoforms, acting on both macrophages and myoblasts. In vitro and in vivo data revealed that OPN-a upregulated tenascin-C (TNC), a known Toll-like receptor 4 (TLR4) agonist. Inhibition of TLR4 signalling attenuated OPN-mediated macrophage cytokine production. In summary, OPN-a is the most abundant and functionally active human spliced isoform in the skeletal muscle microenvironment. Here, OPN-a promotes pro-inflammatory signalling in both macrophages and myoblasts, possibly through induction of TNC-TLR4 signalling. Together, our findings suggest that specific targeting of OPN-a and/or TNC signalling in the damaged muscle microenvironment may be of therapeutic relevance.

Deletion of tetraspanin CD9 diminishes lymphangiogenesis in vivo and in vitro.

Tetraspanins have emerged as key players in malignancy and inflammatory diseases, yet little is known about their roles in angiogenesis, and nothing is known about their involvement in lymphangiogenesis. We found here that tetraspanins are abundantly expressed in human lymphatic endothelial cells (LEC). After intrathoracic tumor implantation, metastasis to lymph nodes was diminished and accompanied by decreased angiogenesis and lymphangiogenesis in tetraspanin CD9-KO mice. Moreover, lymphangiomas induced in CD9-KO mice were less pronounced with decreased lymphangiogenesis compared with those in wild-type mice. Although mouse LEC isolated from CD9-KO mice showed normal adhesion, lymphangiogenesis was markedly impaired in several assays (migration, proliferation, and cable formation) in vitro and in the lymphatic ring assay ex vivo. Consistent with these findings in mouse LEC, knocking down CD9 in human LEC also produced decreased migration, proliferation, and cable formation. Immunoprecipitation analysis demonstrated that deletion of CD9 in LEC diminished formation of functional complexes between VEGF receptor-3 and integrins (α5 and α9). Therefore, knocking down CD9 in LEC attenuated VEGF receptor-3 signaling, as well as downstream signaling such as Erk and p38 upon VEGF-C stimulation. Finally, double deletion of CD9/CD81 in mice caused abnormal development of lymphatic vasculature in the trachea and diaphragm, suggesting that CD9 and a closely related tetraspanin CD81 coordinately play an essential role in physiological lymphangiogenesis. In conclusion, tetraspanin CD9 modulates molecular organization of integrins in LEC, thereby supporting several functions required for lymphangiogenesis.

Genome-Wide Gene Expression Profiling Reveals the Direct Effect of Dienogest on Ovarian Endometriotic Stromal Cells.

Endometriosis affects up to 10% of women of reproductive age, causing dysmenorrhea, chronic pelvic pain, and infertility. The current key drug for endometriosis is dienogest, a progestin with high specificity for the progesterone receptor. To reveal the direct anti-endometriotic effect of dienogest on ovarian endometriotic cells, we investigated the genome-wide gene expression profiles of ovarian endometriotic stromal cells with (Dienogest group) or without dienogest treatment (Control group) and compared the groups' gene expression profiles. We performed a gene ontology (GO) analysis and Ingenuity pathway analysis using these data. To validate the microarray data, we performed real-time RT-PCRs and immunohistochemistry for the differentially expressed genes between the two groups. Of 647 genes differentially expressed between the two groups, 314 genes were upregulated and 333 were downregulated in the Dienogest group versus the Control group. The GO analysis showed that the regulation of macrophage chemotaxis, the collagen catabolic process, and the proteoglycan biosynthetic process are the main biological processes closely associated with the differentially expressed genes. We identified 20 canonical pathways that were most significantly differentially expressed in the Dienogest group versus the Control group. We observed that matrix metalloproteinases (MMPs) are the genes in these pathways that are most closely associated with dienogest treatment. Of components involved in the regulation of macrophage chemotaxis, colony-stimulating factor 1 and macrophage-stimulating 1 are potential upstream regulators of MMPs and were observed herein to be suppressed by dienogest. Our results suggest that dienogest may thus exert its anti-endometriotic effect by directly suppressing MMPs.

Structural basis of spike RBM-specific human antibodies counteracting broad SARS-CoV-2 variants.

The decrease of antibody efficacy to mutated SARS-CoV-2 spike RBD explains the breakthrough infections and reinfections by Omicron variants. Here, we analyzed broadly neutralizing antibodies isolated from long-term hospitalized convalescent patients of early SARS-CoV-2 strains. One of the antibodies named NCV2SG48 is highly potent to broad SARS-CoV-2 variants including Omicron BA.1, BA.2, and BA.4/5. To reveal the mode of action, we determined the sequence and crystal structure of the Fab fragment of NCV2SG48 in a complex with spike RBD from the original, Delta, and Omicron BA.1. NCV2SG48 is from a minor VH but the multiple somatic hypermutations contribute to a markedly extended binding interface and hydrogen bonds to interact with conserved residues at the core receptor-binding motif of RBD, which efficiently neutralizes a broad spectrum of variants. Thus, eliciting the RBD-specific B cells to the longitudinal germinal center reaction confers potent immunity to broad SARS-CoV-2 variants emerging one after another.

Osteopontin undergoes polymerization in vivo and gains chemotactic activity for neutrophils mediated by integrin alpha9beta1.

Osteopontin (OPN) is an integrin-binding inflammatory cytokine that undergoes polymerization catalyzed by transglutaminase 2. We have previously reported that polymeric OPN (polyOPN), but not unpolymerized OPN (OPN*), attracts neutrophils in vitro by presenting an acquired binding site for integrin α9ß1. Among many in vitro substrates for transglutaminase 2, only a few have evidence for in vivo polymerization and concomitant function. Although polyOPN has been identified in bone and aorta, the in vivo functional significance of polyOPN is unknown. To determine whether OPN polymerization contributes to neutrophil recruitment in vivo, we injected OPN* into the peritoneal space of mice. Polymeric OPN was detected by immunoblotting in the peritoneal wash of mice injected with OPN*, and both intraperitoneal and plasma OPN* levels were higher in mice injected with a polymerization-incompetent mutant, confirming that OPN* polymerizes in vivo. OPN* injection induced neutrophil accumulation, which was significantly less following injection of a mutant OPN that was incapable of polymerization. The importance of in vivo polymerization was further confirmed with cystamine, a transglutaminase inhibitor, which blocked the polymerization and attenuated OPN*-mediated neutrophil recruitment. The thrombin-cleaved N-terminal fragment of OPN, another ligand for α9ß1, was not responsible for neutrophil accumulation because a thrombin cleavage-incompetent mutant recruited similar numbers of neutrophils as wild type OPN*. Neutrophil accumulation in response to both wild type and thrombin cleavage-incompetent OPN* was reduced in mice lacking the integrin α9 subunit in leukocytes, indicating that α9ß1 is required for polymerization-induced recruitment. We have illustrated a physiological role of molecular polymerization by demonstrating acquired chemotactic properties for OPN.

Expansion of hedgehog disrupts mesenchymal identity and induces emphysema phenotype.

GWAS have repeatedly mapped susceptibility loci for emphysema to genes that modify hedgehog signaling, but the functional relevance of hedgehog signaling to this morbid disease remains unclear. In the current study, we identified a broad population of mesenchymal cells in the adult murine lung receptive to hedgehog signaling, characterized by higher activation of hedgehog surrounding the proximal airway relative to the distal alveoli. Single-cell RNA-sequencing showed that the hedgehog-receptive mesenchyme is composed of mostly fibroblasts with distinct proximal and distal subsets with discrete identities. Ectopic hedgehog activation in the distal fibroblasts promoted expression of proximal fibroblast markers and loss of distal alveoli and airspace enlargement of over 20% compared with controls. We found that hedgehog suppressed mesenchymal-derived mitogens enriched in distal fibroblasts that regulate alveolar stem cell regeneration and airspace size. Finally, single-cell analysis of the human lung mesenchyme showed that segregated proximal-distal identity with preferential hedgehog activation in the proximal fibroblasts was conserved between mice and humans. In conclusion, we showed that differential hedgehog activation segregates mesenchymal identities of distinct fibroblast subsets and that disruption of fibroblast identity can alter the alveolar stem cell niche, leading to emphysematous changes in the murine lung.

The integrin alpha5beta1 regulates chondrocyte hypertrophic differentiation induced by GTP-bound transglutaminase 2.

Soluble GTP-bound transglutaminase 2 (TG2) induces hypertrophic differentiation in chondrocyte cultures in a beta1 integrin-dependent fashion. beta1 integrin subfamily consists of 12 heterodimers with 12 different alpha subunits and a beta1 subunit. To identify the specific integrin heterodimer(s) responsible for this process, we specifically blocked individual beta1 integrins on the CH-8 immortalized human chondrocytes during hypertrophic differentiation. Blockade of alpha5beta1 inhibited matrix metalloproteinase 13 (MMP-13), type X collagen expression, alkaline phosphatase activity and matrix calcification by 30-50% associated with weak effects of anti-alpha3beta1 and -alpha4beta1. Anti-alpha1beta1, -alpha2beta1 and -alpha6beta1 had no effect. To examine whether the dominant effect of integrin alpha5beta1 was due to a direct interaction with TG2, we incubated the chondrocytic cells on plates coated with GTP-bound TG2. The immobilized GTP-bound TG2 induced hypertrophic differentiation to the same extent as the soluble GTP-bound TG2, which was also inhibited by anti-alpha5beta1. CH-8 cells grown on plates coated with GTP-bound TG2 demonstrated adherence associated with focal adhesion kinase phosphorylation. These properties were inhibited by anti-alpha5beta1. Furthermore, engagement of alpha5beta1 on CH-8 cells via anti-alpha5beta1 antibody did, in fact, induce differentiation. Although CH-8 cells adhered to GTP-free TG2 via integrin alpha5beta1, the cells failed to undergo hypertrophic differentiation. Thus, integrin alpha5beta1 is critical for the chondrocyte hypertrophic differentiation induced by GTP-bound TG2, and this induction is ligand dependent.

Enhanced biological activity of polymeric osteopontin.

Osteopontin is a multifunctional glycoprotein with roles in immunomodulation, inflammatory response, tissue mineralization, and tissue remodeling, which are mediated primarily through integrins. Transglutaminase 2 selectively cross-links proteins by isopeptide bonding. Osteopontin is one of the substrates of this enzyme and undergoes polymerization; however, the biological meaning of this polymerization remains unknown. Using recombinant osteopontin polymerized with purified transglutaminase 2, we examined cell adhesion, spreading, focal contact formation, and migration of SW480 or HUVE cells. All of these cellular behaviors were dramatically enhanced with polymeric osteopontin. These enhancements of cellular functions imply that polymerization might modulate physiological and pathological functions of osteopontin.

Culture time of vitrified/warmed zygotes before microinjection affects the production efficiency of CRISPR-Cas9-mediated knock-in mice.

Robust reproductive engineering techniques are required for the efficient and rapid production of genetically modified mice. We have reported the efficient production of genome-edited mice using reproductive engineering techniques, such as ultra-superovulation, in vitro fertilization (IVF) and vitrification/warming of zygotes. We usually use vitrified/warmed fertilized oocytes created by IVF for microinjection because of work efficiency and flexible scheduling. Here, we investigated whether the culture time of zygotes before microinjection influences the efficiency of producing knock-in mice. Knock-in mice were generated using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system and single-stranded oligodeoxynucleotide (ssODN) or PITCh (Precise Integration into Target Chromosome) system, a method of integrating a donor vector assisted by microhomology-mediated end-joining. The cryopreserved fertilized oocytes were warmed, cultured for several hours and microinjected at different timings. Microinjection was performed with Cas9 protein, guide RNA(s), and an ssODN or PITCh donor plasmid for the ssODN knock-in and the PITCh knock-in, respectively. Different production efficiencies of knock-in mice were observed by changing the timing of microinjection. Our study provides useful information for the CRISPR-Cas9-based generation of knock-in mice.

Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice.

Current advances in producing genetically modified mice using genome-editing technologies have indicated the need for improvement of limiting factors including zygote collection for microinjection and their cryopreservation. Recently, we developed a novel superovulation technique using inhibin antiserum and equine chorionic gonadotropin to promote follicle growth. This method enabled the increased production of fertilized oocytes via in vitro fertilization compared with the conventional superovulation method. Here, we verify that the ultra-superovulation technique can be used for the efficient generation of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout mice by microinjection of plasmid vector or ribonucleoprotein into zygotes. We also investigated whether single-amino-acid-substituted mice and conditional knockout mice could be generated. Founder mice bearing base substitutions were generated more efficiently by co-microinjection of Cas9 protein, a guide RNA and single-stranded oligodeoxynucleotide (ssODN) than by plasmid microinjection with ssODN. The conditional allele was successfully introduced by the one-step insertion of an ssODN designed to carry an exon flanked by two loxP sequences and homology arms using a double-cut CRISPR-Cas9 strategy. Our study presents a useful method for the CRISPR-Cas9-based generation of genetically modified mice from the viewpoints of animal welfare and work efficiency.

Periostin promotes hepatic fibrosis in mice by modulating hepatic stellate cell activation via αv integrin interaction.

BACKGROUND: Periostin is a matricellular protein that serves as a ligand for integrins and is required for tissue remodeling and fibrosis. We investigated the role of periostin in hepatic fibrosis and the mechanisms involved. METHODS: Primary hepatic stellate cells (HSCs) and the HSC-immortalized cell line LX2 were used to study the profibrotic property of periostin and the interaction of periostin with integrins. Wild-type and periostin-deficient (periostin-/-) mice were subjected to two distinct models of liver fibrosis induced by hepatotoxic (carbon tetrachloride or thioacetamide) or cholestatic (3.5-diethoxycarbonyl-1.4-dihydrocollidine) injury. RESULTS: Periostin expression in HSCs and LX2 cells increased in association with their activation. Gene silencing of periostin resulted in a significant reduction in the levels of profibrotic markers. In addition to enhanced cell migration in response to periostin, LX2 cells incubated on periostin showed significant induction of α-smooth muscle actin and collagen, indicating a profibrotic property. An antibody targeting αvß5 and αvß3 integrins suppressed cell attachment to periostin by 60 and 30 % respectively, whereas anti-α5ß1 antibody had no effect. Consistently, αv integrin-silenced LX2 cells exhibited decreased attachment to periostin, with a significant reduction in the levels of profibrotic markers. Moreover, these profibrotic effects of periostin were observed in the mouse models. In contrast to extensive collagen deposition in wild-type mice, periostin-/- mice developed less noticeable hepatic fibrosis induced by hepatotoxic and cholestatic liver injury. Accordingly, the profibrotic markers were significantly reduced in periostin-/- mice. CONCLUSION: Periostin exerts potent profibrotic activity mediated by αv integrin, suggesting the periostin-αv integrin axis as a novel therapeutic target for hepatic fibrosis.

Fibronectin Extra Domain A Promotes Liver Sinusoid Repair following Hepatectomy.

Liver sinusoidal endothelial cells (LSECs) are the main endothelial cells in the liver and are important for maintaining liver homeostasis as well as responding to injury. LSECs express cellular fibronectin containing the alternatively spliced extra domain A (EIIIA-cFN) and increase expression of this isoform after liver injury, although its function is not well understood. Here, we examined the role of EIIIA-cFN in liver regeneration following partial hepatectomy. We carried out two-thirds partial hepatectomies in mice lacking EIIIA-cFN and in their wild type littermates, studied liver endothelial cell adhesion on decellularized, EIIIA-cFN-containing matrices and investigated the role of cellular fibronectins in liver endothelial cell tubulogenesis. We found that liver weight recovery following hepatectomy was significantly delayed and that sinusoidal repair was impaired in EIIIA-cFN null mice, especially females, as was the lipid accumulation typical of the post-hepatectomy liver. In vitro, we found that liver endothelial cells were more adhesive to cell-deposited matrices containing the EIIIA domain and that cellular fibronectin enhanced tubulogenesis and vascular cord formation. The integrin α9ß1, which specifically binds EIIIA-cFN, promoted tubulogenesis and adhesion of liver endothelial cells to EIIIA-cFN. Our findings identify a role for EIIIA-cFN in liver regeneration and tubulogenesis. We suggest that sinusoidal repair is enhanced by increased LSEC adhesion, which is mediated by EIIIA-cFN.

α8ß1 integrin regulates nutrient absorption through an Mfge8-PTEN dependent mechanism.

Coordinated gastrointestinal smooth muscle contraction is critical for proper nutrient absorption and is altered in a number of medical disorders. In this work, we demonstrate a critical role for the RGD-binding integrin α8ß1 in promoting nutrient absorption through regulation of gastrointestinal motility. Smooth muscle-specific deletion and antibody blockade of α8 in mice result in enhanced gastric antral smooth muscle contraction, more rapid gastric emptying, and more rapid transit of food through the small intestine leading to malabsorption of dietary fats and carbohydrates as well as protection from weight gain in a diet-induced model of obesity. Mechanistically, ligation of α8ß1 by the milk protein Mfge8 reduces antral smooth muscle contractile force by preventing RhoA activation through a PTEN-dependent mechanism. Collectively, our results identify a role for α8ß1 in regulating gastrointestinal motility and identify α8 as a potential target for disorders characterized by hypo- or hyper-motility.

Distinct structural requirements for binding of the integrins alphavbeta6, alphavbeta3, alphavbeta5, alpha5beta1 and alpha9beta1 to osteopontin.

The extracellular matrix protein, osteopontin, is a ligand for several members of the integrin family, including alpha5beta1, alphavbeta3, alphavbeta5 and alpha9beta1. Osteopontin is a substrate for a number of extracellular proteases, including thrombin and the metalloproteases MMP-3 and MMP-7, which cleave osteopontin at sites close to or within the mapped integrin binding sites. Using affinity chromatography and cell adhesion assays, we now identify the integrin alphavbeta6 as an additional osteopontin receptor. Utilizing a series of recombinant forms of osteopontin, we compared the structural requirements for alphavbeta6 binding with those for the 4 other osteopontin-binding integrins. Like alpha5beta1, alphavbeta3 and alphavbeta5 (but not alpha9beta1), alphavbeta6 binds to the RGD site in osteopontin, since RGD peptide or mutation of this site to RAA completely inhibits alphavbeta6-mediated cell adhesion. For both alpha9beta1 and alpha5beta1, the N-terminal fragment generated by thrombin cleavage is a much better ligand than full length osteopontin, whereas thrombin-cleavage does not appear to be required for optimal adhesion to alphavbeta3, alphavbeta5 or alphavbeta6. A recombinant fragment predicted to be generated by MMP cleavage no longer supported alpha5beta1 or alpha9beta1-mediated adhesion, but adhesion mediated by alphavbeta5 or alphavbeta6 was unaffected. Finally, adhesion of alphavbeta5 or alphavbeta6 was inhibited by mutation of two aspartic acid residues upstream of the RGD site, whereas adhesion mediated by alphavbeta3, alpha5beta1 or alpha9beta1 was unaffected by these mutations. These results suggest that the hierarchy of integrin interactions with osteopontin can undergo complex regulation at least in part through the action of extracellular proteases.

Epitopes in α8ß1 and other RGD-binding integrins delineate classes of integrin-blocking antibodies and major binding loops in α subunits.

Identification of epitopes for integrin-blocking monoclonal antibodies (mAbs) has aided our understanding of structure-function relationship of integrins. We mapped epitopes of chicken anti-integrin-α8-subunit-blocking mAbs by mutational analyses, examining regions that harboured all mapped epitopes recognized by mAbs against other α-subunits in the RGD-binding-integrin subfamily. Six mAbs exhibited blocking function, and these mAbs recognized residues on the same W2:41-loop on the top-face of the ß-propeller. Loop-tips sufficiently close to W2:41 (<25 Å) contained within a footprint of the mAbs were mutated, and the loop W3:34 on the bottom face was identified as an additional component of the epitope of one antibody, clone YZ5. Binding sequences on the two loops were conserved in virtually all mammals, and that on W3:34 was also conserved in chickens. These indicate 1) YZ5 binds both top and bottom loops, and the binding to W3:34 is by interactions to conserved residues between immunogen and host species, 2) five other blocking mAbs solely bind to W2:41 and 3) the α8 mAbs would cross-react with most mammals. Comparing with the mAbs against the other α-subunits of RGD-integrins, two classes were delineated; those binding to "W3:34 and an top-loop", and "solely W2:41", accounting for 82% of published RGD-integrin-mAbs.

[Repeated tuberculin skin tests in nurse students--observation for 3 years].

In Japan, two-step tuberculin skin test (two-step TST) is recommended for health care workers (HCWs) if the diameter of erythema in the first test is less than 30 mm, and to detect new infection if there is 10 mm or more increase from the base-line diameter. We observed TST in nurse students from the entrance to the graduation for 3 years, and analyzed the change of TST reaction to discuss the usefulness of two-step TST and the criteria for detecting new tuberculous infection among BCG vaccinated HCWs. Mean +/- S.D. (mm) in erythema and induration in each occasion were: in a group with single TST at entrance (T1) and before graduation (TG) (n = 99, group I); T1 = 19.8 +/- 11.3, TG = 23.6 +/- 14.3 for erythema and T1 = 12.5 +/- 5.3, TG = 13.8 +/- 5.0 for induration: in a group with two-step TST at entrance (T1 and T2) (n = 40, group II); T1 = 11.9 +/- 7.8, T2 = 20.4 +/- 10.6, TG = 22.1 +/- 13.5 for erythema and T1 = 8.6 +/- 6.1, T2 = 12.0 +/- 5.1, TG = 13.4 +/- 5.4 for induration: in a group BCG vaccinated after single negative TST (less than 10 mm of erythema and/or less than 5 mm of induration) (n = 12, group I-B); T1 = 4.0 +/- 3.5, TB = 19.8 +/- 5.8, TG = 16.3 +/- 7.6 for erythema and T1 = 0.5 +/- 1.5, TB = 14.1 +/- 3.7, TG = 11.9 +/- 4.4 for induration: in a group BCG vaccinated after two negative TST (n = 10, group II-B); T1 = 3.6 +/- 3.1, T2 = 6.2 +/- 2.4, TB = 23.9 +/- 8.5, TG = 14.1 +/- 6.9 for erythema and T1 = 1.7 +/- 2.7, T2 = 3.2 +/- 1.8, TB = 16.0 +/- 3.2, TG = 7.5 +/- 7.8 for induration. One student in the group II was diagnosed as tuberculosis before the graduation. If we exclude this case form the group II, mean +/- S.D. (mm) of TG in group II were 20.7 +/- 11.2 for erythema and 13.1 +/- 5.0 for induration. Booster phenomenon was significant on two-step TST. There was moderate booster phenomenon even after 34 months from the previous single TST. There was also significant waning of reaction 27 to 31 months after BCG vaccination. But there were no significant waning nor booster after two-step TST. Concerning the difference between the reaction before graduation and at entrance, mean +/- S.D. (mm) in erythema and induration, [TG-T1] in the group I were +3.8 +/- 11.4 and +1.6 +/- 5.8, respectively, [TG-T2 or 1] in the group II were +0.7 +/- 11.4 and +0.4 +/- 5.6, respectively, [TG-TB] in the group I-B and II-B were -6.4 +/- 9.9 and -5.0 +/- 6.5. If we exclude one case who got tuberculosis from the group II, mean +/- S. D. (mm) in erythema and induration of [TG-T2] in group II were -0.6 +/- 8.1 and +0.2 +/- 5.4, respectively. According to the criteria in Japan of more than 10 mm increase in erythema and more than 6 mm increase in induration, recommended by Menzies, significant values of [TG--(T1, T2 or TB)] was observed in 24 (24%) by erythema and 22 (22%) by induration in the group I, while in the group II only in 4 (10%) by erythema and 5 (12.5%) by induration, which included case diagnosed as active tuberculosis. The criterion of 10 mm increase in erythema seems to correspond to that of 6 mm increase in induration. 95% confidential limit in the differences between two tests were 15.6 mm (mean plus 2 standard deviation) in erythema and 11.0 mm in induration in the group II. Considering that these data may include some newly infected persons, appropriate criteria for detecting new tuberculosis infection is estimated to be between 10 to 15 mm increase in erythema and 6 to 11 mm increase in induration from the baseline by two-step TST. Among BCG vaccinated, TST two months after vaccination is useful as the base line. As there is moderate booster phenomenon even after three years from the previous single test and variation is more common, the detection of new tuberculous infection can be made more accurately with the two-step TST in HCWs.