Beta1 Integrin Research Articles

Identification of the causative mutation in the ITGB2 gene in a LAD1 patient by whole exome sequencing

Leukocyte adhesion deficiency type 1 (LAD1) is a rare congenital immunodeficiency disease. The cause of disease is determined to be the mutations in the ITGB2 gene that codes for CD18, the beta chain of beta-2 integrins, leads to decreased expression or functioning of CD18. This deficiency leads to severe impairment of leukocyte adhesion to the vascular wall and leukocyte migration to sites of infection and inflammation. LAD1 has also been associated with inhibition of interleukin-23 and interleukin-17 resulting in a hyperinflammatory and chronic inflammation. Patients with LAD1 typically present in early infancy with recurrent, life threatening infections that are frequently fatal before 2 years of age without hematopoietic stem cell transplant (HSCT). However, LAD1 is difficult to diagnose and many LAD1 patients die at a young age despite intensive antibiotic therapy. Accurate diagnosis requires detailed clinical information (delayed umbilical cord loss, severe periodontitis, delayed wound healing and sores, skin abscesses, and recurrent infection), and confirmation the absence of integrins by flow cytometric analysis. A better understanding of the molecular characteristics of this disease is needed to raise awareness and definitive diagnosis infants with LAD1. To definitive diagnosis, whole exome sequencing and Sanger sequencing were performed in an eighteen-month-old boy with severe leukocytosis, recurrent infections, delayed wound healing, and hepatosplenomegaly associated with an acquired cytomegalovirus infection. Two variants: One previously reported mutation (c.533C>T, p.Pro178Leu) and one novel variant (c.59-1G>A), in the ITGB2 gene were detected. These results can be used for definitive genetic diagnosis, genetic counseling, as well as a prenatal diagnosis in LAD1 patients.

Abstract 3146: EGFL6 induces immunosuppressive functions of tumor-associated myeloid cells and mediates resistance to anti-PDL1 therapy

Abstract Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are critical negative regulators of immunity in cancer. Understanding factors which regulate these cells could result in the identification of new approaches to enhance anti-tumor immunotherapy. One such factor is Epidermal growth factor-like 6 (EGFL6). EGFL6 is a secreted factor known to promote cancer stem like cell migration and regulate cancer cell differentiation. Similarly, EGFL6 promotes endothelial cell migration and proliferation. Indicating a potential role for EGFL6 as a myeloid cell regulatory factor, we found that mice which overexpress Egfl6 have an increased numbers of granulocytes and monocytes in both the bone marrow and spleen. In vitro and ex-vivo analysis indicated that EGFL6, via binding with beta integrins and activation of Syk/ERK signaling, (i) acts as a chemotactic factor for human myeloid cells migration and (ii) promotes their differentiation toward a suppressive state. Suggesting an important role in promoting an immunosuppressive tumor microenvironment (TME), using two syngeneic mouse models of ovarian cancer, we found that expression of Egfl6 in tumor cells resulted in increased accumulation of intra-tumoral MDSCs and TAMs and fewer cytotoxic CD8+ T cells. This was associated with increased tumor growth and shortened animal survival. Gene expression profiling and flow cytometry analysis of tumor infiltrating myeloid cells indicated that Egfl6 induced the expression of immunosuppressive factors, including CXCL2, IL-10 and PD-L1. Consistent with Egfl6 driving an immune suppressive TME, EGFL6 expression in an otherwise immune ‘hot’/anti-PD-L1 responsive tumor model completely inhibited response to anti-PD-L-1 therapy. We are currently evaluating the impact of Egfl6 neutralizing antibody on the efficacy of ICI therapy and anti-tumor immunity. Combined our data show that EGFL6 acts as a chemotactic factor to both recruit myeloid cells to the ovarian TME and subsequently promotes their differentiation to an immunosuppressive phenotype. This suggests EGFL6 is a potential novel therapeutic target to ovarian tumor mediated immunosuppression and enhance response to immune therapy in ovarian cancer patients. Citation Format: Sarah Sinno, Shoumei Bai, Claudia Coronnello, Anda Vlad, Ronald J. Buckanovich, Sandra Cascio. EGFL6 induces immunosuppressive functions of tumor-associated myeloid cells and mediates resistance to anti-PDL1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3146.

Tumor specifically internalizing peptide ‘HN-1’: Targeting the putative receptor retinoblastoma-regulated discoidin domain receptor 1 involved in metastasis

BACKGROUNDLess than 0.5% of intravenously injected drugs reach tumors, contributing to side effects. To limit damage to healthy cells, various delivery vectors have been formulated; yet, previously developed vectors suffer from poor penetration into solid tumors. This issue was resolved by the discovery of HN-1 peptide isolated via biopanning a phage-display library. HN-1 targets human head and neck squamous cell carcinoma (HNSCC) (breast, thyroid; potentially lung, cervix, uterine, colon cancer), translocates across the cell membrane, and efficiently infiltrates solid tumors. HN-1 peptide has been conjugated to various anticancer drugs and imaging agents though the identity of its receptor remained enigmatic.AIMTo decipher the clues that pointed to retinoblastoma (Rb)-regulated discoidin-domain receptor 1 as the putative receptor for HN-1 is described.METHODSHN-1 peptide was synthesized and purified using reverse-phase high-performance liquid chromatography and gel electrophoresis. The predicted mass was confirmed by mass spectroscopy. To image the 3-dimensional structure of HN-1 peptide, PyMOL was used. Molecular modeling was also performed with PEP-FOLD3 software via RPBS bioinformatics web portal (INSERM, France). The immunohistochemistry results of discoidin domain receptor 1 (DDR1) protein were obtained from the publicly accessible database in the Human Protein Atlas portal, which contained the images of immunohistochemically labeled human cancers and the corresponding normal tissues.RESULTSThe clues that led to DDR1 involved in metastasis as the putative receptor mediating HN-1 endocytosis are the following: (1) HN-1 is internalized in phosphate-buffered saline and its uptake is competitively inhibited; (2) HN-1 (TSPLNIHNGQKL) exhibits similarity with a stretch of amino acids in alpha5 beta3 integrin (KLLITIHDRKEF). Aside from two identical residues (Ile-His) in the middle, the overall distribution of polar and nonpolar residues throughout the sequences is nearly identical. As HN-1 sequence lacks the Arg-Gly-Asp motif recognized by integrins, HN-1 may interact with an "integrin-like" molecule. The tertiary structure of both peptides showed similarity at the 3-dimensional level; (3) HN-1 is internalized by attached cells but not by suspended cells. As culture plates are typically coated with collagen, collagen-binding receptor (expressed by adherent but not suspended cells) may represent the receptor for HN-1; (4) DDR1 is highly expressed in head and neck cancer (or breast cancer) targeted by HN-1; (5) Upon activation by collagen, DDR1 becomes internalized and compartmentalized in endosomes consistent with the determination of ’energy-dependent clathrin-mediated endocytosis’ as the HN-1 entry route and the identification of HN-1 entrapped vesicles as endosomes; and (6) DDR1 is essential for the development of mammary glands consistent with the common embryonic lineage rationale used to identify breast cancer as an additional target of HN-1. In summary, collagen-activated tyrosine kinase receptor DDR1 overexpressed in HNSCC assumes a critical role in metastasis. Further studies are warranted to assess HN-1 peptide’s interaction with DDR1 and the therapeutic potential of treating metastatic cancer. Additionally, advances in delivery (conformation, endocytic mechanism, repertoire of targeted cancers of HN-1 peptide), tracking (HN-1 conjugated imaging agents), and activity (HN-1 conjugated therapeutic agents) are described.CONCLUSIONThe discovery of DDR1 as HN-1 peptide’s putative receptor represents a significant advance as it enables identification of metastatic cancers or clinical application of previously developed therapeutics to block metastasis.

Recombinant Integrin β1 Signal Peptide Blocks Gliosis Induced by Aβ Oligomers.

Glial cells participate actively in the early cognitive decline in Alzheimer’s disease (AD) pathology. In fact, recent studies have found molecular and functional abnormalities in astrocytes and microglia in both animal models and brains of patients suffering from this pathology. In this regard, reactive gliosis intimately associated with amyloid plaques has become a pathological hallmark of AD. A recent study from our laboratory reports that astrocyte reactivity is caused by a direct interaction between amyloid beta (Aβ) oligomers and integrin β1. Here, we have generated four recombinant peptides including the extracellular domain of integrin β1, and evaluated their capacity both to bind in vitro to Aβ oligomers and to prevent in vivo Aβ oligomer-induced gliosis and endoplasmic reticulum stress. We have identified the minimal region of integrin β1 that binds to Aβ oligomers. This region is called signal peptide and corresponds to the first 20 amino acids of the integrin β1 N-terminal domain. This recombinant integrin β1 signal peptide prevented Aβ oligomer-induced ROS generation in primary astrocyte cultures. Furthermore, we carried out intrahippocampal injection in adult mice of recombinant integrin β1 signal peptide combined with or without Aβ oligomers and we evaluated by immunohistochemistry both astrogliosis and microgliosis as well as endoplasmic reticulum stress. The results show that recombinant integrin β1 signal peptide precluded both astrogliosis and microgliosis and endoplasmic reticulum stress mediated by Aβ oligomers in vivo. We have developed a molecular tool that blocks the activation of the molecular cascade that mediates gliosis via Aβ oligomer/integrin β1 signaling.

Promising role of topical caffeine mesoporous gel in collagen resynthesis and UV protection through proline assessment

BackgroundCaffeine, an alkaloid agent, has been globally used regularly in drinks, for the reduction in skin cancers and wrinkle formation. As a result of the previous, attempts have been carried out to use caffeine in cosmetology due to its antioxidant and UV ray protection effects. Our aim was to evaluate the effect of caffeine on collagen resynthesis via its effect on proline and prolidase biosynthesis on mice, orally and topically as mesoporous silica at three levels, and the influence on UV protection. In skin biopsies of orally and topically treated mice, the following was assessed using ELISA and Western blot techniques, the activity of prolidase, together with the concentrations of proline, beta integrin, insulin growth factor, protein kinases beta, and mitogen-activated protein kinase. Moreover, we loaded the caffeine on mesoporous silica and assessed the aforementioned parameters together with checkpoint kinase 1 and Rad3-related protein.ResultsCaffeine promoted collagen resynthesis in a dose-dependent manner. The mechanism of this process was found at the level of prolidase activity as caffeine significantly increased the enzyme activity. Caffeine also had a protective effect against UV exhibited by the over-expression of beta integrin, insulin growth factor together with the under-expression of protein kinases beta, mitogen-activated protein kinase, checkpoint kinase 1, and Rad3-related protein.ConclusionsOur study revealed the superiority of SYL-C12 (mesoporous silica-loaded caffeine gel), compromising the high level of the three independent factors, in terms of the measured responses in mesoporous silica with caffeine. Moreover, caffeine promoted collagen resynthesis with significant protective effect against UV apoptotic damage.

Current Status of and Global Trends in Platelet Transfusion Refractoriness From 2004 to 2021: A Bibliometric Analysis.

Platelet transfusion refractoriness (PTR) is common in patients with hematology and oncology and is becoming an important barrier in the treatment of thrombocytopenia and hemorrhage. Bibliometrics is an effective method for identifying existing research achievements, important breakthroughs, current research hotspots, and future development trends in any given field. In recent years, research on PTR has received increasing attention, but a bibliometric analysis of this field has not yet been reported. In this study, we applied bibliometrics to analyze the existing literature on PTR research over the past 17 years. On November 1, 2021, we began a publications analysis of PTR research using the Science Citation Index Expanded of the Web of Science Core Collection with collection dates from 2004 to 2021. This research aimed to summarize the state of PTR research using Bibliometrix to identify connections between different elements (i.e., authors, institutions, countries, journals, references, and keywords) using VOS viewer analyses to visualize key topics and trends in PTR research using Cite Space and gCLUTO. The results of all 310 studies showed that the annual number of publications focused on PTR is steadily increasing, with the United States of America and Japan making significant contributions. We noted that the research group led by Dr. Sherrill J. Slichter was prominent in this field, while Estcourt Lise may become the most influential newcomer. Transfusion was the most popular journal, and Blood was the most cited journal. Using various analyses, including co-cited analysis, historiography analysis, citation burst analysis, and factorial analysis, we pointed out and discussed contributing publications. According to occurrence analysis, co-word biclustering analysis, landform map, thematic evolution, and thematic map, we believe that “activation,” “p-selection,” “CD36 deficiency,” “gene-frequencies,” “CD109,” “HPA-1,” and “beta (3) integrin” may become new trends in PTR research. The outcome of our bibliometric analyses has, for the first time, revealed profound insights into the current state and trends in PTR research. The systematic analysis provided by our study clearly demonstrates the field's significant advancements to all researchers who are interested in a quick and comprehensive introduction to the field.

Egfl6 promotes ovarian cancer progression by inducing the immunosuppressive functions of tumor-infiltrating myeloid cells.

Abstract Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are critical negative regulators of immunity in cancer. Understanding factors which regulate these cells could result in the identification of new approaches to enhance anti-tumor immunotherapy. One such factor is epidermal growth factor-like 6 (EGFL6), a secreted factor known to promote cancer stem like cell migration and regulate cancer cell differentiation. We found that mice which overexpress Egfl6 have an increased numbers of granulocytes and monocytes in both the bone marrow and spleen. In vitro and ex-vivo analysis indicated that EGFL6, via binding with beta integrins and activation of SYK and ERK signaling, (i) acts as a chemotactic factor for myeloid cells migration and (ii) promotes their differentiation toward a suppressive state. Suggesting an important role in promoting an immunosuppressive tumor microenvironment (TME), using two syngeneic mouse models of ovarian cancer, we found that expression of Egfl6 in tumor cells resulted in increased accumulation of intra-tumoral MDSCs and TAMs and fewer cytotoxic CD8+ T cells. This was associated with increased tumor growth and shortened animal survival. Gene expression profiling of tumor infiltrating myeloid cells indicated that Egfl6 induced the expression of immunosuppressive factors, including CXCL2, IL-10 and PD-L1. Moreover, in an immune ‘hot’ tumor model, EGFL6 completely inhibited response to a-PD-L-1 therapy. Combined our data show that EGFL6 induces the recruitment of myeloid cells into the ovarian TME and subsequently promotes their immunosuppressive functions. This suggest EGFL6 is a potential novel therapeutic target to enhance response to immune therapy in OvCa patients. Supported by Ovarian Cancer Research Alliance

The Expression of Active CD11b Monocytes in Blood and Disease Progression in Amyotrophic Lateral Sclerosis.

Monocytes expressing the inflammation suppressing active CD11b, a beta2 integrin, may regulate neuroinflammation and modify clinical outcomes in amyotrophic lateral sclerosis (ALS). In this single site, retrospective study, peripheral blood mononuclear cells from 38 individuals living with ALS and 20 non-neurological controls (NNC) were investigated using flow cytometry to study active CD11b integrin classical (CM), intermediate (IM) and non-classical (NCM) monocytes during ALS progression. Seventeen ALS participants were sampled at the baseline (V1) and at two additional time points (V2 and V3) for longitudinal analysis. Active CD11b+ CM frequencies increased steeply between the baseline and V3 (ANOVA repeated measurement, p < 0.001), and the V2/V1 ratio negatively correlated with the disease progression rate, similar to higher frequencies of active CD11b+ NCM at the baseline (R = −0.6567; p = 0.0031 and R = 0.3862; p = 0.0168, respectively). CD11b NCM, clinical covariates and neurofilament light-chain plasma concentration at the baseline predicted shorter survival in a multivariable and univariate analysis (CD11b NCM—HR: 1.05, CI: 1.01–1.11, p = 0.013. Log rank: above median: 43 months and below median: 21.22 months; p = 0.0022). Blood samples with the highest frequencies of active CD11b+ IM and NCM contained the lowest concentrations of soluble CD11b. Our preliminary data suggest that the levels of active CD11b+ monocytes and NCM in the blood predict different clinical outcomes in ALS.

Deletion of STAT3 from Foxd1 cell population protects mice from kidney fibrosis by inhibiting pericytes trans-differentiation and migration.

Signal transduction and activator of transcription 3 (STAT3) is a key transcription factor implicated in the pathogenesis of kidney fibrosis. Although Stat3 deletion in tubular epithelial cells is known to protect mice from fibrosis, vFoxd1 cells remains unclear. Using Foxd1-mediated Stat3 knockout mice, CRISPR, and inhibitors of STAT3, we investigate its function. STAT3 is phosphorylated in tubular epithelial cells in acute kidney injury, whereas it is expanded to interstitial cells in fibrosis in mice and humans. Foxd1-mediated deletion of Stat3 protects mice from folic-acid- and aristolochic-acid-induced kidney fibrosis. Mechanistically, STAT3 upregulates the inflammation and differentiates pericytes into myofibroblasts. STAT3 activation increases migration and profibrotic signaling in genome-edited, pericyte-like cells. Conversely, blocking Stat3 inhibits detachment, migration, and profibrotic signaling. Furthermore, STAT3 binds to the Collagen1a1 promoter in mouse kidneys and cells. Together, our study identifies a previously unknown function of STAT3 that promotes kidney fibrosis and has therapeutic value in fibrosis.

Sulfasalazine decreases astrogliosis-mediated seizure burden.

ObjectivePreviously, we reported that inhibition of the astrocytic cystine/glutamate antiporter system xc‐ (SXC), using sulfasalazine (SAS), decreased evoked excitatory signaling in three distinct hyperexcitability models ex vivo. The current study expands on this work by evaluating the in vivo efficacy of SAS in decreasing astrogliosis‐mediated seizure burden seen in the beta‐1 integrin knockout (B1KO) model.MethodsVideo‐EEG (electroencephalography) monitoring (24/7) was obtained using Biopac EEG acquisition hardware and software. EEG spectral analysis was performed using MATLAB. SAS was used at an equivalence of doses taken by Crohn’s disease patients. Whole‐cell patch‐clamp recordings were made from cortical layer 2/3 pyramidal neurons.ResultsWe report that 100% of B1KO mice that underwent 24/7 video‐EEG monitoring developed spontaneous recurrent seizures and that intraperitoneal administration of SAS significantly reduced seizure frequency in B1KOs compared to B1KOs receiving sham saline. Spectral analysis found an acute reduction in EEG power following SAS treatment in B1KOs; however, this effect was not observed in nonepileptic control mice receiving SAS. Finally, whole‐cell recordings from SXC knockout mice had hyperpolarized neurons and SXC‐B1 double knockouts fired significantly less action potentials in response to current injection compared to B1KOs with SXC.SignificanceTo devise effective strategies in finding relief for one‐in‐three patients with epilepsy who experience drug‐resistant epilepsy we must continue to explore the mechanisms regulating glutamate homeostasis. This study explored the efficacy of targeting an astrocytic glutamate antiporter, SXC, as a novel antiepileptic drug (AED) target and further characterized a unique mouse model in which chronic astrogliosis is sufficient to induce spontaneous seizures and epilepsy. These findings may serve as a foundation to further assess the potential for SAS or inform the development of more potent and specific compounds that target SXC as a novel treatment for epilepsy.

Targeting of the alphav beta3 integrin complex by CAR-T cells leads to rapid regression of diffuse intrinsic pontine glioma and glioblastoma

BackgroundDiffuse intrinsic pontine glioma (DIPG) and glioblastoma (GBM) are two highly aggressive and generally incurable gliomas with little therapeutic advancements made in the past several decades. Despite immense initial success...

P707 Microbial signature of the colon is not associated with response to vedolizumab in Crohn’s disease

Abstract Background Crohn’s disease (CD) is a complex disease where the gut microbiome plays an important role. One of the established treatments in CD is Vedolizumab (VDZ), an alpha(4)beta(7) integrin antibody. Finding biomarkers to predict therapy response is still a clinical unmet need, since this treatment has shown endoscopic remission in only a third of CD patients. Ananthakrishnan et al. demonstrated a strong relationship between microbial metagenomics signature and therapy response to VDZ in CD based on the metagenomics composition of baseline fecal samples. While the majority of studies focus on fecal samples, mucosa-adherent bacterial signature could bring forth stable biomarkers. In this study, we sought to identify the signature of the adherent microbiome in intestinal biopies to differentiate responders (R) from non-responders (NR) to VDZ treatment at baseline. Methods We prospectively collected ileal and colonic biopsies from adult CD patients scheduled to start VDZ treatment during baseline- and follow-up (FU) endoscopies. After median 27 weeks of follow-up, patients were classified as either R or NR based on endoscopic response (≥50% reduction in SES-CD score), steroid-free clinical response (≥3 point drop in HBI or HBI ≤4, no systemic steroids) and/or biochemical response (≥50% reduction in C-reactive protein (CRP) and fecal calprotectin or a basal CRP ≤5 g/mL and fecal calprotectin ≤250 µg/g). Microbiome composition of the biopsies was determined using 16S RNA gene V3V4 amplicon sequencing. We measured alpha and beta diversity using Wilcoxon and Adonis metrics. Results In total, 44 CD patients were included in the baseline cohort (28 R and 16 NR) and 53 CD patients were included in the follow up cohort (37 R and 20 NR), for which 21 patients overlap between baseline and follow up. When comparing alpha-diversity between R and NR, we did not find significant differences in ileal (Wilcoxon, p=0.78) and colonic (Wilcoxon, p=0.70) samples at baseline nor at follow up (ileal Wilcoxon, p=0. 27 and colonic Wilcoxon, p=0. 63). Next, comparing the beta-diversity, we demonstrated no significant differences between R and NR in ileal (baseline p=0.96, follow up p=0.11) and colonic (baseline p=0.11, follow up p=0.4). Microbiome profiles did show high inter-individual variation but were highly similar intra-individually both between body site and over time. Conclusion Here, we investigated the microbial signature of VDZ R and NR and demonstrated mucosa-associated microbiome is mostly stable after resolution/non-resolution of inflammation in CD and does not predict response to VDZ therapy. Further analyses on data of infliximab, adalimumab and ustekinumab treated patients, as part of the EPIC Pioneer study, are ongoing.

Evaluation of Beta-2 Integrin and Platelets Roles in Sickle Cell Disease Pathogenicity in Basrah Governorate Patients

Sickle cell disease (SCD) comprises an inherited blood disorder that is life long and affects many people globally. In spite of the development in treatment, SCA is a considerable cause of mortality and morbidity. The present study tries to assess the role of leukocytes represented by β integrin(CD18) and platelets and their productivity in the pathogenicity of disease during the steady state and crisis in comparison with the healthy as-control group, SCD patients (15) enrolled during crisis and steady state (follow up) showed a significant increase in leukocytes and platelets cells productivity during crisis when compared to the steady state and in the steady state when compared to the healthy control group . In this study, SCD pathophysiology in steady state and crisis affected by the platelets account and leukocyte activation triggered by inflammatory factors and reflex on the adherence and attachment between cells and blood vessel led to vascular occlusion (VOC).

Secretome Profiling of Atlantic Salmon Head Kidney Leukocytes Highlights the Role of Phagocytes in the Immune Response to Soluble β-Glucan

β‐Glucans (BG) are glucose polymers which are produced in bacteria and fungi but not in vertebrate organisms. Being recognized by phagocytic leukocytes including macrophages and neutrophils through receptors such as dectin-1 and Complement receptor 3 (CR3), the BG are perceived by the innate immune system of vertebrates as foreign substances known as Pathogen Associated Molecular Patterns (PAMPs). The yeast-derived BG has been recognized for its potent biological activity and it is used as an immunomodulator in human and veterinary medicine. The goal of the current study was to characterize the immunostimulatory activity of soluble yeast BG in primary cultures of Atlantic salmon (Salmo salar) head kidney leukocytes (HKLs) in which phagocytic cell types including neutrophils and mononuclear phagocytes predominate. The effect of BG on the secretome of HKL cultures, including secretion of extracellular vesicles (EVs) and soluble protein55s was characterized through western blotting and mass spectrometry. The results demonstrate that, along with upregulation of proinflammatory genes, BG induces secretion of ubiquitinated proteins (UbP), MHCII-containing EVs from professional antigen presenting cells as well as proteins derived from granules of polymorphonuclear granulocytes (PMN). Among the most abundant proteins identified in BG-induced EVs were beta-2 integrin subunits, including CD18 and CD11 homologs, which highlights the role of salmon granulocytes and mononuclear phagocytes in the response to soluble BG. Overall, the current work advances the knowledge about the immunostimulatory activity of yeast BG on the salmon immune system by shedding light on the effect of this PAMP on the secretome of salmon leukocytes.

Immunoexpression of Adhesion Molecules During Human Fetal Hair Development

SummaryBackground: Hair follicles are produced in a cyclical manner and the machinery involved in the reproduction of these follicles is present since the fetal stage. Although extensive research has been done on the human hair follicle, very little is known about the importance of adhesion molecules in its development. Methods: We analyzed here, the immunoexpression of beta-1 integrin, p-cadherin, e- cadherin, and beta-catenin in hair follicles from 26 formalin-fixed and paraffin-embedded skin samples from human embryos and fetus between 12-23 weeks of gestational age. Findings: The adhesion molecules beta-1 integrin and e-cadherin/p-cadherin were expressed from 12 weeks and seemed to play a role in regulating epidermis invagination. Beta-catenin immuno stainingwas negative in all cases; down regulation of this protein may be necessary for fetal hair development and thus facilitating hair follicle down growth. Conclusion: Adhesion molecules are essential for hair follicle down growth and proliferation; integrins and cadherins play a major role in this process. More studies are needed to describe hair follicle development.

Pharmacological characterisation of GSK3335103, an oral αvβ6 integrin small molecule RGD-mimetic inhibitor for the treatment of fibrotic disease

Fibrosis is the formation of scar tissue due to injury or long-term inflammation and is a leading cause of morbidity and mortality. Activation of the pro-fibrotic cytokine transforming growth factor-β (TGFβ) via the alpha-V beta-6 (αvβ6) integrin has been identified as playing a key role in the development of fibrosis. Therefore, a drug discovery programme to identify an orally bioavailable small molecule αvβ6 arginyl-glycinyl-aspartic acid (RGD)-mimetic was initiated. As part of a medicinal chemistry programme GSK3335103 was identified and profiled in a range of pre-clinical in vitro and in vivo systems. GSK3335103 was shown to bind to the αvβ6 with high affinity and demonstrated fast binding kinetics. In primary human lung epithelial cells, GSK3335103-induced concentration- and time-dependent internalisation of αvβ6 with a rapid return of integrin to the cell surface observed after washout. Following sustained engagement of the αvβ6 integrin in vitro, lysosomal degradation was induced by GSK3335103. GSK3335103 was shown to engage with the αvβ6 integrin and inhibit the activation of TGFβ in both ex vivo IPF tissue and in a murine model of bleomycin-induced lung fibrosis, as measured by αvβ6 engagement, TGFβ signalling and collagen deposition, with a prolonged duration of action observed in vivo. In summary, GSK3335103 is a potent αvβ6 inhibitor that attenuates TGFβ signalling in vitro and in vivo with a well-defined pharmacokinetic/pharmacodynamic relationship. This translates to a significant reduction of collagen deposition in vivo and therefore GSK3335103 represents a potential novel oral therapy for fibrotic disorders.

Lentiviral Vector-Mediated Gene Transfer Restores CD18 Expression in Long-Term Repopulating Hematopoietic Stem and Progenitor Cells Isolated from a Patient with Leukocyte Adhesion Deficiency Type 1

Leukocyte adhesion deficiency type 1 (LAD-1) is an inherited primary immunodeficiency caused by loss-of-function mutation within the ITGB2 gene, which encodes the beta2 integrin subunit CD18. Individuals with LAD-1 experience significant loss of neutrophil-mediated innate cellular immune function, resulting in delayed wound healing, severe periodontitis, and life-long bouts of bacterial infection. LAD-1 is a prime candidate for lentiviral vector-mediated genetic intervention as i) it is an intractable, potentially life-threatening disease with limited treatment options, ii) it is amenable to current ex vivo gene therapy procedures, and iii) partial phenotypic correction would present a high likelihood of significant clinical benefit. Allogeneic stem cell transplant can be curative, but suffers from matched donor availability and the potential for graft-versus-host disease. Autologous ex vivo gene therapy may provide a viable alternative to allogeneic transplant in LAD-1 patients. We have evaluated the ability of a CD18-expressing lentiviral vector (LV-hCD18) to mediate ex vivo transduction of LAD-1 patient-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) and subsequent long-term LAD-1 HSPC engraftment in immunodeficient NOD-scid IL2Rg null (NSG) mice. An open reading frame encoding human CD18 was placed under the transcriptional control of the MND promoter (a modified retroviral promoter associated with high levels of stable transgene expression) and packaged in VSV-G-pseudotyped lentiviral particles. After 1 day of pre-stimulation, LAD-1 HSPCs were transduced with LV-hCD18 (MOI = 10) in the presence or absence of transduction-enhancing adjuvants, poloxamer 407 (P407) and prostaglandin E2 (PGE 2), for 24 hours. Sublethally irradiated NSG mice (7 mice/group) were transplanted with either mock-transduced LAD-1 HSPCs, LAD-1 HSPCs transduced in the absence of adjuvants, or LAD-1 HSPCs transduced in the presence of P407/PGE 2. Bone marrow was harvested at ~5.5 months post-transplant for flow cytometric analyses of engraftment efficiency, transgene marking, and human blood cell lineage reconstitution. Bone marrow from mice that received mock-transduced LAD-1 HSPCs showed an average total of 6.45 ± 2.54% (mean ± SEM) CD45+ human cells. Mice that received LAD-1 HSPCs transduced in the absence of adjuvants showed 7.99 ± 1.82% CD45+ human cells, whereas mice transplanted with LAD-1 HSPCs transduced in the presence of adjuvants showed 7.33 ± 1.90% CD45+ cells. A Kruskal-Wallis statistical test indicated no significant difference in the level of human cell engraftment among the recipient groups (P=0.72). Consistent with the LAD-1 phenotype, human myeloid cells from mice that received mock-transduced LAD-1 HSPCs displayed only background levels of CD18 marking (0.13 ± 0.06% CD45+CD13+CD18+ cells). Mice that received LAD-1 HSPCs transduced in the absence of adjuvants showed 4.05 ± 0.40% CD18+ human myeloid cells (range 2.19% to 5.50%), whereas mice that received LAD-1 HSPCs transduced in the presence of P407/PGE 2 showed 9.56 ± 0.96% CD18+ human myeloid cells (range 4.63% to 13.10%), thus representing a >2-fold increase in in vivo, vector-mediated transgene marking levels when adjuvant was used. Moreover, vector-mediated expression of CD18 rescued endogenous expression of a major CD18 heterodimerization partner in neutrophils, CD11b. In mock-transduced LAD-1 HSPC recipients, CD13+ human myeloid cells were devoid of cell surface CD11b expression (0.01 ± 0.01% CD45+CD13+CD11b+ cells). In contrast, CD13+ human myeloid cells in mice that received LAD-1 HSPCs transduced in the absence of adjuvant showed detectable levels of CD11b expression (2.62 ± 0.19% of CD18-expressing human myeloid cells), and CD11b levels were increased to 6.90 ± 0.98% in LAD-1 HSPCs transduced in the presence of P407/PGE 2. Multilineage engraftment, as evidenced by the presence of CD3+ T cells and CD20+ B cells, was noted within all groups; however, human myeloid cells represented the most prominent human blood cell compartment observed. Colony-forming-unit assays of transduced cells and non-transduced control cells pre-transplant showed similar clonogenic output and colony diversity. In sum, successful transduction, engraftment, transgene marking, CD11b rescue, and multilineage reconstitution supports further development of lentiviral vector-mediated gene therapy for LAD-1. DisclosuresNo relevant conflicts of interest to declare.

Increased Angiogenesis by Exosomes Secreted by Adipose-Derived Stem Cells upon Lipopolysaccharide Stimulation.

Exosomes secreted by adipose-derived stem cells (ADSCs) enhance angiogenesis and wound healing. However, in clinical settings, wounds may be infected by various bacteria or pathogens. We investigated whether human ADSCs stimulated with lipopolysaccharide (LPS) secrete exosomes (ADSC-LPS-exo) that augment the angiogenesis of human umbilical vein endothelial cells (HUVECs). ExoQuick-TC exosome precipitation solution was used to purify exosomes from human ADSC culture media in the presence or absence of 1 µg/mL LPS treatment for 24 h. The uptake of ADSC-LPS-exo significantly induced the activation of cAMP response element binding protein (CREB), activating protein 1 (AP-1), and nuclear factor-κB (NF-κB) signaling pathways and increased the migration of and tube formation in HUVECs. RNA interference with CREB, AP-1, or NF-κB1 significantly reduced the migration of and tube formation in HUVECs treated with ADSC-LPS-exo. An experiment with an antibody array for 25 angiogenesis-related proteins revealed that only interleukin-8 expression was significantly upregulated in HUVECs treated with ADSC-LPS-exo. In addition, proteomic analysis revealed that eukaryotic translation initiation factor 4E, amyloid beta A4 protein, integrin beta-1, and ras-related C3 botulinum toxin substrate 1 may be potential candidates involved in ADSC-LPS-exo-mediated enhanced angiogenesis.

A novel ITGB2 variant with long survival in patients with leukocyte adhesion defect type-I

Leukocyte adhesion deficiency is an autosomal recessive primary immunodeficiency that has been divided into three types: LAD1 (beta-2 integrin (CD18) family deficiency/defect), LAD2 (absence of fucosylated carbonhydrate ligands for selectins) and LAD3 (defective activation of all beta integrins). However, recently LAD4 has been described in cystic fibrosis patients, with a defect in integrin activation reported in monocytes. LAD-I is the most common type and prevalence of 1 in 1,000,000 live births. Clinical features of LAD patients are recurrent bacterial and fungal infections, omphalitis with delayed umbilical stump separation, significant leukocytosis especially neutrophilia during infection periods, impaired pus formation, and delayed traumatic or surgical wound healing. Flow cytometry is considered a useful tool for rapid diagnosis of the disease. The study of CD18 and CD11 (a, b, c) expression patterns in peripheral blood leukocytes helps to distinguish different phenotypes of LAD-I. In general, patients with ≥ 2% CD18 expression tend to have a less severe infection and often survive until adulthood, whereas < 2% CD18 expression often results in death in infancy. In this case report, three siblings, 10, 15, and 17years old, diagnosed with leukocyte adhesion defect type 1 in adolescence age group, are presented.

Bioinformatics Analysis and Functional Verification of ADAMTS9-AS1/AS2 in Lung Adenocarcinoma.

Long non-coding RNAs (lncRNAs), as competitive endogenous RNAs (ceRNAs), play a critical role in biological processes of cancer. However, the roles of specific lncRNAs in ceRNA network of lung adenocarcinoma (LUAD) remains largely unclear. Herein, we identified the roles of lncRNA ADAMTS9-AS1/AS2 (ADAMTS-AS1/AS2) in lung adenocarcinoma by bioinformatics analyses and functional verification. First, differentially expressed genes ADAMTS9-AS1, ADAMTS9-AS2 and ADAMTS9 were screened out from GSE130779. Then the expression correlation of these three genes was analyzed. The results showed that ADAMTS9-AS1, ADAMTS9-AS2 and ADAMTS9 were down-regulated in LUAD, and were positively correlated with each other. After that, miRcode was used to find miR-150 which binds to ADAMTS9-AS1/ADAMTS9-AS2/ADAMTS9. Next, co-expression analysis and functional enrichment analyses were performed to further analyze differentially expressed genes. The results showed that the differentially expressed genes were mainly enriched in Beta3 integrin cell surface interactions and epithelial-to-mesenchymal transition. Finally, the cell functions of ADAMTS9-AS1 and ADAMTS9-AS2 in A549 and NCI-H1299 cell lines were verified. In vitro cell studies confirmed that ADAMTS9-AS1 and ADAMTS9-AS2 play an inhibitory role in LUAD cells.