Integrin-positive Cells Research Articles

Integrin αvβ3 and disulfide bonds play important roles in NGR-retargeted adenovirus transduction efficiency

AimsAdenoviruses that have CNGRCVSGCAGRC peptide inserted into fiber (AdFNGR) or hexon (AdHNGR) protein, respectively, showed increased transduction of endothelial cells. In this study we investigated if cysteines within the CNGRCVSGCAGRC sequence inserted into Ad serotype 5 Ad5 fiber or hexon protein form disulfide bond(s) and whether they play a role in retargeting potential of AdFNGR and AdHNGR. MethodsTransduction efficiency of adenoviruses was done by counting infected cells under the microscope. Adenovirus attachment and internalization were measured by qPCR. Flow cytometry was used to evaluate the expression of CD13 and integrins. Gene knockdown was achieved by transfection of small interfering RNA. Mass spectrometry was used for determining disulfide bonds in adenovirus fiber and hexon protein. Molecular modeling was use to predict interaction of CNGRCVSGCAGRC peptide and CD13. Key findingsAdFNGR and AdHNGR attach better to CD13 and/or αvβ3 integrin-positive cells than Adwt. Reducing disulfide bonds using DTT decreased transduction efficiency and attachment of both AdFNGR and AdHNGR. Cysteins from CNGRCVSGCAGRC peptide within AdHNGR do not form disulfide bonds. Knockdown of αvβ3 integrin reduced increased transduction efficiency of both AdFNGR and AdHNGR, while CD13 knockdown had no effect, indicating that retargeting properties of these viruses rely mainly on αvβ3 integrin expression. SignificanceInsertion site of NGR-containing peptides as well as NGR flanking residues are critical for receptor binding affinity/specificity and transduction efficiency of NGR retargeted adenoviral vectors.

BACH1 Binding Links the Genetic Risk for Severe Periodontitis with ST8SIA1

Genome-wide association studies identified various loci associated with periodontal diseases, but assigning causal alleles remains difficult. Likewise, the generation of biological meaning underlying a statistical association has been challenging. Here, we characterized the genetic association at the gene ST8SIA1 that increases the risk for severe periodontitis in smokers. We used CRISPR/dCas9 activation and RNA-sequencing to identify genetic interaction partners of ST8SIA1 and to determine its function in the cell. We used reporter gene assays to identify regulatory elements at the associated single-nucleotide polymorphisms (SNPs) and to determine effect directions and allele-specific changes of enhancer activity. Antibody electrophoretic mobility shift assays proved allele-specific transcription factor binding at the putative causal SNPs. We found the reported periodontitis risk gene ABCA1 as the top upregulated gene following ST8SIA1 activation. Gene set enrichment analysis showed highest effects on integrin cell surface interactions (area under the curve [AUC] = 0.85; q = 4.9 × 10−6) and cell cycle regulation (AUC = 0.89; q = 1.6 × 10−5). We identified 2 associated repressor elements in the introns of ST8SIA1 that bind the transcriptional repressor BACH1. The putative causative variant rs2012722 decreased BACH1 binding by 40%. We also pinpointed ST8SIA1 as the target gene of the association. ST8SIA1 inhibits cell adhesion with extracellular matrix proteins, integrins, and cell cycle, as well as enhances apoptosis. Likewise, tobacco smoke reportedly results in an inhibition of cell adhesion and a decrease in integrin-positive cells and cell growth. We conclude that impaired ST8SIA1 repression, independently caused by reduced BACH1 binding at the effect T allele, as well as by tobacco smoke, contributes to higher ST8SIA1 levels, and in smokers who carry the effect T allele, both factors would be additive with damaging effects on the gingival barrier integrity. The activity of ST8SIA1 is also linked with the periodontitis risk gene ABCA1.

Luteolin-7-glucoside Promotes Human Epidermal Stem Cell Proliferation by Upregulating β-Catenin, c-Myc, and Cyclin Expression.

Skin epidermal stem cells (EpSCs) play critical roles in skin homeostasis and the repair of skin injury. Luteolin-7-glucoside (L7G) has been reported to accelerate skin wound healing through its anti-inflammatory and antioxidative activity. But its effect on EpSCs is not clear. In the present study, we examined the effect of L7G on the proliferation of human EpSCs and explored the mechanisms involved. MTT assay showed that L7G promoted EpSC proliferation in a dose- and time-dependent manner. BrdU incorporation assay and Ki67 immunofluorescence staining confirmed the proproliferative effect of L7G on EpSCs. Cell cycle analysis showed that treatment of EpSCs with L7G decreased the cell number in the G1 phase and increased the cell number in the S phase. In addition, L7G significantly enhanced EpSC migration. Mechanistic studies showed that L7G significantly induced the expression of β-catenin and c-Myc, as well as cyclins D1, A2, and E1 which are critical for G1/S phase transition. L7G stimulated EpSC proliferation through β-catenin and c-Myc. We further examined the effect of L7G on EpSC proliferation in skin tissues by treatment of human skin explants with L7G and examined the number of EpSCs by immunohistochemical stain of EpSC markers α6 integrin and β1 integrin. We found that treatment of human skin tissue explants with L7G significantly increased the thickness of the epidermis and increased the numbers of α6 integrin-positive and β1 integrin-positive cells at the basal layer of the epidermis. Taken together, these results indicate that L7G promotes EpSC proliferation through upregulating β-catenin, c-Myc, and cyclin expression. L7G can be used to expand EpSCs for generating epidermal autografts and engineered skin equivalents.

Failure of active targeting by a cholesterol-anchored ligand and improvement by altering the lipid composition to prevent ligand desorption

Although anti-angiogenic therapy is predicted to be an effective therapy for treating cancer, selectively targeting tumor endothelial cells (TECs), and not normal endothelial cells, remains a major obstacle. Modifying a drug carrier with a targeting ligand is a popular strategy for developing an active-targeting type drug delivery system (DDS). We previously reported that a cyclo(Arg-Gly-Asp-D-Phe-Lys) (cRGD)-equipped liposome that contains encapsulated siRNA (RGD-MEND) achieved an efficient therapeutic outcome in a murine cancer model. To develop a more efficient TEC-targeting DDS, we examined the effect of the length of the polyethylene glycol (PEG) that is used as a peptide-linker on the cholesterol-scaffold, and liposomal composition on the efficiency of delivery of siRNA to cRGD receptor αVβ3 integrin positive cells. An RGD-MEND modified with shorter linker/no-linker, PEG350 or no-PEG, showed a higher cellular uptake in vitro. However, a shorter or no-linker RGD-cholesterol-modified MEND showed no silencing effect despite its high, in vitro silencing efficiency. To examine the possibility that the cholesterol-scaffold ligand was removed from the surface of the RGD-MEND by interactions with serum proteins, the RGD-MEND was incubated in the presence of a 50% serum solution. The cellular uptake of the cholesterol-scaffold ligand was drastically reduced by the incubation in serum. Increasing the cholesterol ratio in the lipid envelope and adding a helper lipid improved the in vivo knockdown efficiency, probably due to an enhanced ligand retention, even in in vivo conditions. The findings reported herein suggest that the lipid composition and the ligand scaffold of the MEND are major factors in successfully developing an efficient active-targeting DDS.

Synthesis and application of biocompatible gold core⿿poly-(l-Lysine) shell nanoparticles

In this work, green synthesis of a kind of fluorescent core-shell nanoparticles via a facile one-pot method was reported. We chose tryptophan, a common amino acid, to serve as the green reducing agent of the gold core. And a biomacromolecule, poly-(l-lysine), was then employed to assemble and cross-link onto gold nanoparticle surfaces serving as the biocompatible shell. Fluorescence enhancement was observed after a cyanine dye was conjugated onto the nanoparticle surfaces. Then, peptides with arginine-glycine-aspartic acid sequence were bound to the shell as targeting ligand. Cell imaging assay demonstrated that the prepared nanoparticles are efficient in target sensing of integrin-positive cells. Moreover, they possess high biocompatibility because of the green synthetic method. Thus, the prepared nanoparticles show great potential in cellular imaging and targeted integrin detection.

Multifunctional Polymeric Nanosystems for Dual-Targeted Combinatorial Chemo/Angiogenesis Therapy of Tumors.

Combination of antiangiogenesis and chemotherapy holds vast promise for effective inhibition of tumor proliferation and invasion. Herein, a multifunctional self-assembled nanosystem consisting of amphiphilic c(RGDyK)-functionalized low-molecular-weight heparin-gambogic acid conjugate (cRHG) is developed, using c(RGDyK) peptide as αv β3 integrin targeting moiety to realize a double-targeted delivery to both tumor cells and angiogenic vasculature. cRHG with a markedly decreased anticoagulant activity can self-assemble into nanosized particles (around 150 nm). cRHG nanoparticles effectively inhibit vascular endothelial growth factor (VEGF)-triggered tube-like formation of HUVEC and neovascularization of subcutaneous Matrigel plugs. More importantly, the targeting ability of cRHG nanoparticles is revealed by their efficient internalization by αv β3 integrin-positive cells (U87MG and HUVEC) in vitro and obvious accumulation of Cy7/cRHG in the tumor site with strong fluorescence signals in both cancer cells and neovasculature. Besides, cRHG maintains the in vitro cytotoxic activity of gambogic acid, while achieving the highest in vivo antitumor activity in U87MG mouse xenograft model and displaying better safety profile than free drugs solutions. Mechanistic investigations reveal the substantial inhibition of hypoxia-inducible factor-1 alpha, VEGF, and CD31 expression by cRHG nanoparticles with remarkable down-regulation of VEGFR2 phosphorylation. These results suggest that cRHG nanoparticles provide a versatile nanoplatform for efficacious combinatorial tumor therapy.

Tumor Angiogenesis Targeted Radiosensitization Therapy Using Gold Nanoprobes Guided by MRI/SPECT Imaging.

Gold nanoparticles (AuNPs) have recently garnered great interest as potential radiosensitizers in tumor therapy. However, major challenges facing their application in this regard are further enhancement of tumor accumulation of the particles in addition to enhanced permeability retention (EPR) effect and an understanding of the optimal particle size and time for applying radiotherapy after the particle administration. In this study, we fabricated novel cyclic c(RGDyC)-peptide-conjugated, Gd- and 99 mTc-labeled AuNPs (RGD@AuNPs-Gd99 mTc) probes with different sizes (29, 51, and 80 nm) and evaluated their potential as radiosensitization therapy both in vitro and in vivo. We found that these probes have a high specificity for αvβ3 integrin positive cells, which resulted in their high cellular uptake and thereby enhanced radiosensitization. Imaging in vivo with MRI and SPECT/CT directly showed that the RGD@AuNPs-Gd99 mTc probes specifically target tumors and exhibit greater accumulation within tumors than the RAD@AuNPs-Gd99 mTc probes. Interestingly, we found that the 80 nm RGD@AuNPs-Gd99 mTc probes exhibit the greatest effects in vitro; however, the 29 nm RGD@AuNPs-Gd99 mTc probes were clearly most efficient in vivo. As a result, radiotherapy of tumors with the 29 nm probe was the most potent. Our study demonstrates that RGD@AuNPs-Gd99 mTc probes are highly useful radiosensitizers capable of guiding and enhancing radiation therapy of tumors.

Identification of β integrin-like- and fibronectin-like proteins in the bivalve mollusk Mytilus trossulus.

Integrins play a key role in the intermediation and coordination between cells and extracellular matrix components. In this study, we first determined the presence of the β integrin-like protein and its presumptive ligand, fibronectin-like protein, during development and in some adult tissues of the bivalve mollusc Mytilus trossulus. We found that β integrin-like protein expression correlated with the development and differentiation of the digestive system in larvae. Besides the presence of β integrin-like protein in the digestive epithelial larval cells, this protein was detected in the hemocytes and some adult tissues of M. trossulus. The fibronectin-like protein was detected firstly at the blastula stage and later, the FN-LP-immunoreactive cells were scattered in the trochophore larvae. The fibronectin-like protein was not expressed in the β integrin-positive cells of either the veliger stage larvae or the adult mussel tissues and the primary hemocyte cell culture. Despite the β integrin- and fibronectin-like proteins being expressed in different cell types of mussel larvae, we do not exclude the possibility of direct interaction between these two proteins during M. trossulus development or in adult tissues.

A Fusion Protein of RGD4C and β-Lactamase Has a Favorable Targeting Effect in Its Use in Antibody Directed Enzyme Prodrug Therapy.

Antibody directed enzyme prodrug therapy (ADEPT) utilizing β-lactamase is a promising treatment strategy to enhance the therapeutic effect and safety of cytotoxic agents. In this method, a conjugate (antibody-β-lactamase fusion protein) is employed to precisely activate nontoxic cephalosporin prodrugs at the tumor site. A major obstacle to the clinical translation of this method, however, is the low catalytic activity and high immunogenicity of the wild-type enzymes. To overcome this challenge, we fused a cyclic decapeptide (RGD4C) targeting to the integrin with a β-lactamase variant with reduced immunogenicity which retains acceptable catalytic activity for prodrug hydrolysis. Here, we made a further investigation on its targeting effect and pharmacokinetic properties, the results demonstrated that the fusion protein retains a targeting effect on integrin positive cells and has acceptable pharmacokinetic characteristics, which benefits its use in ADEPT.

Interleukin-1β-induced autophagy-related gene 5 regulates proliferation of embryonic stem cell-derived odontoblastic cells.

We previously established a method for the differentiation of induced pluripotent stem cells and embryonic stem cells into α2 integrin-positive odontoblast-like cells. We also reported that Wnt5 in response to interleukin (IL)-1β induces matrix metalloproteinase (MMP)-3-regulated cell proliferation in these cells. Our findings suggest that MMP-3 plays a potentially unique physiological role in the generation of odontoblast-like cells under an inflammatory state. Here, we examined whether up-regulation of autophagy-related gene (Atg) 5 by IL-1β was mediated by Wnt5 signaling, thus leading to increased proliferation of odontoblast-like cells. IL-1β increased the mRNA and protein levels of Atg5, microtubule-associated protein 1 light chain (LC3, a mammalian homolog of yeast Atg8) and Atg12. Treatment with siRNAs against Atg5, but not LC3 and Atg12, suppressed the IL-1β-induced increase in MMP-3 expression and cell proliferation. Our siRNA analyses combined with western blot analysis revealed a unique sequential cascade involving Atg5, Wnt5a and MMP-3, which resulted in the potent increase in odontoblastic cell proliferation. These results demonstrate the unique involvement of Atg5 in IL-1β-induced proliferation of embryonic stem cell-derived odontoblast-like cells.

β Integrin-like protein-mediated adhesion and its disturbances during cell cultivation of the mussel Mytilus trossulus.

In this study, we focus on the specific contribution of β integrin-like protein to adhesion-mediated events in molluscan larval cells in culture that could not have been investigated within the whole animal. An analysis of disturbances to cell-substratum adhesion, caused by the integrin receptor inhibiting Arg-Gly-Asp-Ser (RGDS)-peptide, the Ca(2+)/Mg(2+)-chelators and the stress influence of freezing-thawing, reveals that all these factors resulted in the partial destruction of the integrin-extracellular matrix (ECM) interaction in culture and, in particular, changes in the distribution and relative abundance of β integrin-positive cells. The experiments, carried out on selected substrates, found that β integrin-positive cells demonstrate different affinities for the substrates. This finding further supports the assumption that epithelial differentiation in cultivated cells of larval Mytilus may be mediated by β integrin-like proteins via binding to laminin; direct binding to other components of the ECM could not be demonstrated. The mussel β integrin-positive cells are not involved in myogenic or neuronal differentiation on any of the substrates but part of them has tubulin-positive cilia, forming some epithelia-like structures. Our data indicate that β integrin-positive cells are able to proliferate in vitro which suggests that they could participate in renewing the digestive epithelium in larvae. The findings provide evidence that the distribution pattern of β integrin-like protein depends on the cell type and the factors influencing the adhesion.

The involvement of osteopontin and β3 integrin in implantation and endometrial receptivity in an early mouse pregnancy model

ObjectiveTo explore the roles of osteopontin and β3 integrin in successful implantation. Study designIn this study, an early pregnant mouse model was established by peritoneal injection of pregnant mare serum gonadotropin and human chorionic gonadotropin (PMSG+hCG). The expression of osteopontin (OPN) and β3 integrin on the endometrium was measured by immunohistochemistry, RT-PCR, and western blot. The function of OPN and β3 integrin in implantation was investigated by intrauterine injection of OPN and β3 integrin antibody. ResultsWe found that PMSG+hCG injection significantly increased the number of blastocysts during implantation as well as the concentration of estradiol and progesterone in serum and endometrium tissues. OPN and β3 integrin were co-expressed in luminal epithelium and their levels dynamically changed from day 4 to day 8 of pregnancy with peak expression on day 5. The percentages of OPN and β3 integrin positive cells in the luminal epithelium were significantly higher in PMSG+hCG-stimulated mice on day 5 than in control mice. Functional blockade of OPN and β3 integrin significantly inhibited implantation. ConclusionsThis study suggests that co-expression of OPN and β3 integrin is a biological marker for good endometrial receptivity and that both proteins play a crucial role in blastocyst implantation.

Role of Integrins in the Carcinogenesis of Squamous Cell Carcinoma of the Tongue and Lower Lip

This study analyzed the distribution, intensity, and pattern of immunohistochemical expression of α2β1, α3β1, and α5β1 integrins in squamous cell carcinoma (SCC) of the lower lip and tongue to identify biomarkers that reflect the clinical course of this cancer. Immunoexpression was compared considering prognostic parameters such as anatomic site, metastasis, and histologic grade of malignancy. Immunohistochemical analysis at the invasion front showed a predominance of granular cytoplasmic expression of the integrins studied. In most cases, immunopositive cells were diffusely distributed in the tumors, irrespective of their location, except for α3β1 integrin-positive cells which were focally distributed in 53.3% of tongue SCC cases. With respect to staining intensity, positive staining for α2β1 integrin was observed in 80% of lower lip SCCs and in 93.3% of tongue SCCs. Staining for α3β1 integrin was moderately positive in 60% of lower lip and tongue SCCs. The staining intensity of α5β1 integrin was moderately and strongly positive in 53.3% and 46.7% of lower lip SCCs, respectively, and in 46.7% and 53.3% of tongue SCCs. The strong immunoreactivity for integrins α2β1, α3β1, and α5β1 seen in the oral SCC cases studied suggests a significant participation of these proteins in oral carcinogenesis. However, their expression does not reflect the clinical course of this cancer.

Alteration of the actin cytoskeleton and localisation of the α6β1 and α3 integrins during regeneration of the rat submandibular gland

ObjectivesActin filaments, which are regulated by signal transduction via integrins, play important roles in the regulation of cell differentiation and polarity. The aim of this study was to assess alterations in the cytoskeleton and the localisation of integrins during regeneration of the rat submandibular gland. DesignAfter obstruction for 7 days, the regenerating glands were collected at days 0, 1, 3, 7, 14 after duct release for analysis of regeneration. Alterations in the actin filaments were examined using phalloidin, which specifically binds to filamentous actin (F-actin), and the distributions of the α6β1 and α3 integrins were examined immunohistochemically. ResultsF-actin was strongly localised at the apical region in the intercalated ducts of normal and day-14 glands and in duct-like structures during the regenerative process. Thereafter, actin accumulated at the basement membrane in mature acinar cells. A temporo-spatial correlation was found between the apical distribution of F-actin and α3 integrin staining. Diffuse α6β1 integrin staining, which occurred at a distal site in α3 integrin-positive cells, was observed in immature cells at day 3. At day 14, α6β1 integrin was detected at the basement membrane in terminal differentiated acinar cells. ConclusionThese findings suggest that duct-like structures have the same properties as intercalated ducts, that alterations in α3 to α6β1 integrins regulate the generation of acinar cells from duct-like structures, and that the α6β1 integrin is involved in the differentiation of acinar cells during regeneration of the rat submandibular gland.

Cellular Origin of Testis-Derived Pluripotent Stem Cells: A Case for Very Small Embryonic-Like Stem Cells

It has been suggested that testicular germ stem cells represent the only adult body stem cells that dedifferentiate and reprogram into a pluripotent state without any genetic modification. Emerging debate about the authenticity of embryonic stem cell (ES)-like cells derived from adult testicular tissue has prompted us to put forth this letter. We wish to reinforce our findings that pluripotent very small embryonic-like stem cells (VSELs) exist as a small population in adult mammalian testis and may result in ES-like colonies. Because of their small size, it is felt that VSELs could be contaminating the initial cells used for seeding, although efforts were made to place a single germ cell per well in a 96-well plate for clonal expansion, or magnetic activated cell sorting (MACS)-sorted α6 integrin positive cells were used. On a similar note, it is felt that the presence of VSELs in various tissues along with mesenchymal stem cells (MSCs) may provide an alternative explanation to the transdifferentiation potential of MSCs. We conclude that like Oct-4 biology, presence of VSELs in adult body tissues has somewhat surprised stem cell biologists.

β4 Integrin Marks Interstitial Myogenic Progenitor Cells in Adult Murine Skeletal Muscle

Skeletal muscle growth and its regeneration following injury rely on myogenic progenitor cells, a heterogeneous population that includes the satellite cells and other interstitial progenitors. The present study demonstrates that surface expression of β4 integrin marks a population of vessel-associated interstitial muscle progenitor cells. Muscle β4 integrin-positive cells do not express myogenic markers upon isolation. However, they are capable of undergoing myogenic specification in vitro and in vivo: β4 integrin cells differentiate into multinucleated myotubes in culture dishes and contribute to muscle regeneration upon delivery into diseased mice. Subfractionation of β4 integrin-expressing cells based on CD31 expression does not further enrich for myogenic precursors. These findings support the expression of β4 integrin in interstitial, vessel-associated cells with myogenic activity within adult skeletal muscle.

Neisseria meningitidis Adhesin NadA Targets β1 Integrins: FUNCTIONAL SIMILARITY TO YERSINIA INVASIN

Meningococci are facultative-pathogenic bacteria endowed with a set of adhesins allowing colonization of the human upper respiratory tract, leading to fulminant meningitis and septicemia. The Neisseria adhesin NadA was identified in about 50% of N. meningitidis isolates and is closely related to the Yersinia adhesin YadA, the prototype of the oligomeric coiled-coil adhesin (Oca) family. NadA is known to be involved in cell adhesion, invasion, and induction of proinflammatory cytokines. Because of the enormous diversity of neisserial cell adhesins the analysis of the specific contribution of NadA in meningococcal host interactions is limited. Therefore, we used a non-invasive Y. enterocolitica mutant as carrier to study the role of NadA in host cell interaction. NadA was shown to be efficiently produced and localized in its oligomeric form on the bacterial surface of Y. enterocolitica. Additionally, NadA mediated a β1 integrin-dependent adherence with subsequent internalization of yersiniae by a β1 integrin-positive cell line. Using recombinant NadA(24-210) protein and human and murine β1 integrin-expressing cell lines we could demonstrate the role of the β1 integrin subunit as putative receptor for NadA. Subsequent inhibition assays revealed specific interaction of NadA(24-210) with the human β1 integrin subunit. Cumulatively, these results indicate that Y. enterocolitica is a suitable toolbox system for analysis of the adhesive properties of NadA, revealing strong evidence that β1 integrins are important receptors for NadA. Thus, this study demonstrated for the first time a direct interaction between the Oca-family member NadA and human β1 integrins.

Embryonic Vasculogenesis in Nodular Melanomas and Tumour Differentiation

The relationship of vasculogenic mimicry to pigment in nodular vertical growth phase [VGP] cutaneous melanomas is assessed in this study. 10 nodules each from 27 tumors, 15 pigmented and 12 amelanotic were sampled in proportion to the pigment level. Serial frozen and paraffin sections subjected to HE, Reticulin, PAS to assess the vascular pattern; Dopa Oxidase and Immunopositivity for HMB45, LN5 [laminin 5] & integrin[α(5)β(1)], and EM [electron microscopy] to identify Weibel-Palade bodies within endothelial cells. The vascular pattern, pigment and the immunopositivity was mapped to assess the percentage VM [vasculogenic sinusoids] vs INC [incorporated microvasculature]. In pigmented melanomas, INC from pre-existing stromal vessels is predominant. Amelanotic melanomas show embryonic vasculogenic mimicry, a self-propagating system of spaces within the sheets of tumors cells. Both INC and VM co-exist in tumors with both amelanotic and melanotic nodules. In areas with VM, loci of LN5 and α(5)β(1) integrin positive cells appear within the proliferating columns, positivity in these cells suggesting a switch to a more aggressive form. Irregular spaces appear lined by tumor cells, with initial hemopoeitic activity, coalesce and interlink into tubular networks. Spaces lined by tumor cells extend into an intricate network which then connects with the angiogenetic system. The tumor cells lining the vasculogenic spaces are positive for LN5, α(5)β(1) integrin. Statistically, INC is significantly higher in pigmented melanomas, whereas amelanotic melanomas show significantly higher VM. Pigmentation is correlated positively with INC and negatively with VM. INC and VM are negatively correlated with each other.

The Effect of Anti-GPIIIa49-66 Antibody on Megakaryocyte Differentiation.

AbstractAbstract 1434We previously reported that patients with early-onset HIV-1 ITP develop a unique anti-platelet integrin GPIIIa antibody against the GPIIIa49-66 epitope. Anti-GPIIIa49-66 antibody-induced platelet fragmentation requires sequential activation of the platelet 12-lipoxygenase (12-LO) and NADPH oxidase to release reactive oxygen species (ROS). 12-LO is upstream of the NADPH oxidase pathway and 12(S)-HETE, the product of 12-LO, induces the same oxidative platelet fragmentation as anti-GPIIIa49-66. Since the megakaryocyte (MK) is the progenitor cells for platelets and may contain similar signal pathways, we have investigated the effect of anti-GPIIIa49-66 on MK differentiation and, in particular, the potential role of anti-GPIIIa49-66 induced ROS in this process. We first show that polyclonal anti-GPIIIa49-66 antibody isolated from HIV-1 ITP patients inhibits MK proliferation 2.5 fold in in vitro culture of mouse bone marrow Lin-/- cells driven by thrombopoietin (TPO). We also observed a 3 fold decrease in the number of MK colony-forming units in the presence of a human monoclonal anti-GPIIIa49-66 antibody we generated. However, we could not detect ROS release in DCFH-loaded MEG-01 cells treated with anti-GPIIIa49-66 antibody. In addition, 12(S)-HETE does not inhibit the in vitro differentiation of MK cell line L8057 induced by TPO. In fact, we found a dose dependent increasing of the percentage of αIIb integrin positive cells (from 17.1% to 48.7%) in in vitro culture of L8057 treated by various concentration of H2O2 (from 5 to 20μM). Thus, our data suggests that ROS is not involved in the inhibition of MK differentiation induced by anti-GPIIIa49-66, in contrast to the effect that this antibody has on mature platelets. We therefore conclude that the anti-GPIIIa49-66 antibody dysregulates ROS independent β3 integrin signaling to inhibit MK differentiation. Disclosures:No relevant conflicts of interest to declare.

Changes in integrin-positive cells and T cell subpopulations in the peripheral blood and intestine of calves fed soya protein

Changes in integrin-positive cells and T cell subpopulations in the peripheral blood and intestine of calves fed soya protein