Inhibition of integrin subunit alpha 11 restrains gastric cancer progression through phosphatidylinositol 3-kinase/Akt pathway.

Gastric cancer (GC) is among the most frequent malignancies originating from the digestive system worldwide, while the role and specific mechanism of integrin-subunit alpha 11 (ITGA11) in GC remain unclear. This study probes the expression characteristics and function of ITGA11 in GC. Firstly, the ITGA11 profile in GC tissues and paracancerous non-tumor tissues was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot (WB), and the association between ITGA11 and GC patients' clinicopathological indicators was evaluated. ITGA11 knockdown models were set up in GC cell lines MKN45 and AGS. Cell proliferation was determined by the cell counting kit-8 (CCK-8) assay and colony formation assay. WB was utilized to gauge the expression of apoptosis-related proteins (Bax, Bcl2, Bad, and C-Caspase3) and the PI3K/AKT pathway. We discovered that the ITGA11 expression was boosted in GC tissues and was related to the unfavorable prognosis of GC patients. Additionally, ITGA11 knockdown abated GC cell proliferation, invasion and migration, and enhanced cell apoptosis. In animal experiments, the tumorigenesis of GC cells knocking down ITGA11 was reduced. Mechanically, knocking down ITGA11 notably inactivated the PI3K/AKT axis. The tumor-suppressive effect mediated by ITGA11 knockdown was attenuated after activating the PI3K/AKT pathway with insulin-like growth factor 1 (IGF-1). Overall, this study substantiated that the ITGA11 expression was heightened in GC tissues, which affected GC progression by modulating the PI3K/AKT pathway.

Circular RNA CircITGA7 Promotes Tumorigenesis of Osteosarcoma via miR-370/PIM1 Axis.

Many studies have shown that there are many circular RNA (circRNA) expression abnormalities in osteosarcoma (OS), and this abnormality is related to the development of osteosarcoma. But at present, it is unclear as to what circITGA7 has in the OS and what it does. In this study, qRT-PCR was used to detect the expression of circITGA7, miR-370, and PIM1 mRNA in OS tissues and cells. The CCK-8 assay was used to detect the effect of circITGA7 on cell proliferation. Later, the transwell assay was used to detect cell migration and invasion. The dual-luciferase reporter assay confirmed the existence of the targeting relationship between circITGA7 and miR-370, and miR-370 and PIM1. We found that circITGA7 was upregulated in OS tissues and cell lines. Knockdown of circITGA7 weakened the cell's ability to proliferate and metastasize. Furthermore, we observed that miR-370 was negatively regulated by circITGA7, while PIM1 was positively regulated by it. A functional assay validated that circITGA7 promoted OS progression via suppressing miR-370 and miR-370 affected OS proliferation and migration via PIM6 in OS. In summary, this study shows that circITGA7 promotes OS proliferation and metastasis via miR-370/PIM1.

In situ antibody phage display yields optimal inhibitors of integrin α11/ß1.

Integrins are transmembrane multi-conformation receptors that mediate interactions with the extracellular matrix. In cancer, integrins influence metastasis, proliferation, and survival. Collagen-binding integrin-α11/ß1, a marker of aggressive tumors that is involved in stroma-tumor crosstalk, may be an attractive target for anti-cancer therapeutic antibodies. We performed selections with phage-displayed synthetic antibody libraries for binding to either purified integrin-α11/ß1 or in situ on live cells. The in-situ strategy yielded many diverse antibodies, and strikingly, most of these antibodies did not recognize purified integrin-α11/ß1. Conversely, none of the antibodies selected for binding to purified integrin-α11/ß1 were able to efficiently recognize native cell-surface antigen. Most importantly, only the in-situ selection yielded functional antibodies that were able to compete with collagen-I for binding to cell-surface integrin-α11/ß1, and thus inhibited cell adhesion. In-depth characterization of a subset of in situ-derived clones as full-length immunoglobulins revealed high affinity cellular binding and inhibitory activities in the single-digit nanomolar range. Moreover, the antibodies showed high selectivity for integrin-α11/ß1 with minimal cross-reactivity for close homologs. Taken together, our findings highlight the advantages of in-situ selections for generation of anti-integrin antibodies optimized for recognition and inhibition of native cell-surface proteins, and our work establishes general methods that could be extended to many other membrane proteins.

Integrin α7 correlates with worse clinical features and prognosis, and its knockdown inhibits cell proliferation and stemness in tongue squamous cell carcinoma.

The present study aimed to evaluate the correlation of integrin α7 (ITGA7) with clinicopathological characteristics and overall survival (OS) in patients with tongue squamous cell carcinoma (TSCC), and to investigate the effect of ITGA7 knockdown on proliferation, apoptosis and stemness of TSCC cells in vitro. ITGA7 expression was measured in tumor tissues and paired adjacent normal tissues from 60 patients with TSCC using immunohistochemistry. ITGA7 expression in human TSCC cell lines and normal oral keratinocytes was measured using quantitative PCR and western blotting. Lentiviruses carrying short hairpin (sh) RNA targeting ITGA7 were used to knockdown its expression in CAL­27 and HSC­4 cells, and then proliferation, apoptosis and stemness were measured. In addition, CAL­27 and HSC­4 cancer stem cells (CSCs) were constructed and their ITGA7 expression was measured. The results demonstrated that ITGA7 was upregulated in the tumor tissues compared with the paired adjacent tissues, and its high expression was correlated with worse pathological grade, N stage, TNM stage and OS. In vitro, ITGA7 expression levels were demonstrated to be increased in the TSCC CAL­27, SCC­9, HSC­4 and SCC­25 cell lines compared to the normal HOK cell line. In CAL­27 and HSC­4 cells, ITGA7 knockdown inhibited cell proliferation, promoted apoptosis, increased CD24 expression, decreased CD44 and CD133 expression, reduced drug resistance to cisplatin and attenuated sphere formation efficiency. Finally, ITGA7 expression levels were greatly elevated in CAL­27 and HSC­4 CSCs compared with parental CAL­27 and HSC­4 cells. In conclusion, ITGA7 knockdown inhibited tumor cell proliferation and stemness in TSCC cells. These findings indicated that ITGA7 might serve as a potential marker for CSCs and may correlate with worse clinical features and prognosis in TSCC.

An anti-CD103 antibody-drug conjugate prolongs the survival of pancreatic islet allografts in mice.

CD103 mediates T-cell infiltration and organ allograft rejection, and depletion of CD103-expressing cells is a promising therapeutic strategy for allograft intolerance. Recently, we verified that M290-MC-MMAF, an anti-CD103 antibody-drug conjugate, potently eliminates CD103-positive cells in vivo, with high specificity and minimal toxicity. However, the contribution of M290-MC-MMAF to blocking the CD103/E-cadherin pathway involved in transplant rejection remains unclear. Herein, we examined the impact of systemic administration of M290-MC-MMAF on allografts in an islet transplantation model. M290-MC-MMAF treatment maintained the long-term survival of islet allografts (>60 days) compared to mock injection or unconjugated M290 antibody treatment (<18 days). The change was associated with a decrease in CD103+CD8+ effector T cells and an increase in CD4+CD25+ regulatory T cells. CD103+CD8+ effector T-cell transfer or CD4+CD25+ regulatory T-cell depletion resulted in a rapid loss of allografts in long-surviving islet hosts. Moreover, M290-MC-MMAF treatment reduced IL-4, IL-6, and TNF-α expression levels and increased IL-10 expression in the grafts, which presented an immunosuppressive cytokine profile. In conclusion, targeting CD103 with M290-MC-MMAF induced immunosuppression and prolonged the survival of pancreatic islet allografts in mice, indicating the potential clinical value of M290-MC-MMAF in therapeutic interventions for allograft rejection.

In Vitro Validation of Phosphorodiamidate Morpholino Oligomers.

One of the crucial aspects of screening antisense oligonucleotides destined for therapeutic application is confidence that the antisense oligomer is delivered efficiently into cultured cells. Efficient delivery is particularly vital for antisense phosphorodiamidate morpholino oligomers, which have a neutral backbone, and are known to show poor gymnotic uptake. Here, we report several methods to deliver these oligomers into cultured cells. Although 4D-Nucleofector™ or Neon™ electroporation systems provide efficient delivery and use lower amounts of phosphorodiamidate morpholino oligomer, both systems are costly. We show that some readily available transfection reagents can be used to deliver phosphorodiamidate morpholino oligomers as efficiently as the electroporation systems. Among the transfection reagents tested, we recommend Lipofectamine 3000™ for delivering phosphorodiamidate morpholino oligomers into fibroblasts and Lipofectamine 3000™ or Lipofectamine 2000™ for myoblasts/myotubes. We also provide optimal programs for nucleofection into various cell lines using the P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza), as well as antisense oligomers that redirect expression of ubiquitously expressed genes that may be used as positive treatments for human and murine cell transfections.

Integrin α7 high expression correlates with deteriorative tumor features and worse overall survival, and its knockdown inhibits cell proliferation and invasion but increases apoptosis in breast cancer.

BACKGROUND: This study aimed to investigate the correlation of integrin α7 (ITGA7) expression with clinical/pathological characteristics and overall survival (OS), and its knockdown on inhibiting cell activities in breast cancer. METHODS: A total of 191 breast cancer patients underwent surgery were retrospectively reviewed, and ITGA7 expression in tumor tissues was determined by immunofluorescence and real-time quantitative polymerase chain reaction. Patients' clinical/pathological data were recorded, and OS was calculated. In vitro, control shRNA and ITGA7 shRNA plasmids were transfected into MCF7 cells to evaluate the influence of ITGA7 knockdown on cell proliferation, apoptosis, and invasion. RESULTS: Ninety-two (48.2%) patients presented with ITGA7 high expression, and 99 patients (51.8%) presented with ITGA7 low expression. ITGA7 expression was positively correlated with T stage, tumor-node metastasis (TNM) stage, and pathological grade. Kaplan-Meier curves showed that ITGA7 high expression was associated with shorter OS, and multivariate Cox's proportional hazards regression displayed that ITGA7 high expression was an independent predictive factor for poor OS. Moreover, in vitro experiments disclosed that cell proliferation (by Cell Counting Kit-8 assay) and cell invasion (by Matrigel invasion assay) were reduced, while cell apoptosis rate (by Annexin V/propidium iodide assay) was enhanced by ITGA7 knockdown in MCF-7 cells. CONCLUSION: Integrin α7 high expression correlates with increased T stage, TNM stage, and pathological grade as well as worse OS, and its knockdown enhances cell apoptosis but inhibits cell proliferation and invasion in breast cancer.

ITGA1 is a pre-malignant biomarker that promotes therapy resistance and metastatic potential in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has single-digit 5-year survival rates at <7%. There is a dire need to improve pre-malignant detection methods and identify new therapeutic targets for abrogating PDAC progression. To this end, we mined our previously published pseudopodium-enriched (PDE) protein/phosphoprotein datasets to identify novel PDAC-specific biomarkers and/or therapeutic targets. We discovered that integrin alpha 1 (ITGA1) is frequently upregulated in pancreatic cancers and associated precursor lesions. Expression of ITGA1-specific collagens within the pancreatic cancer microenvironment significantly correlates with indicators of poor patient prognosis, and depleting ITGA1 from PDAC cells revealed that it is required for collagen-induced tumorigenic potential. Notably, collagen/ITGA1 signaling promotes the survival of ALDH1-positive stem-like cells and cooperates with TGFß to drive gemcitabine resistance. Finally, we report that ITGA1 is required for TGFß/collagen-induced EMT and metastasis. Our data suggest that ITGA1 is a new diagnostic biomarker and target that can be leveraged to improve patient outcomes.

Integrin α7 Is a Functional Marker and Potential Therapeutic Target in Glioblastoma.

Functionally relevant markers of glioblastoma stem-like cells (GSCs) have potential for therapeutic targeting to treat this aggressive disease. Here we used generation and screening of thousands of monoclonal antibodies to search for receptors and signaling pathways preferentially enriched in GSCs. We identified integrin α7 (ITGA7) as a major laminin receptor in GSCs and in primary high-grade glioma specimens. Analyses of mRNA profiles in comprehensive datasets revealed that high ITGA7 expression negatively correlated with survival of patients with both low- and high-grade glioma. In vitro and in vivo analyses showed that ITGA7 plays a key functional role in growth and invasiveness of GSCs. We also found that targeting of ITGA7 by RNAi or blocking mAbs impaired laminin-induced signaling, and it led to a significant delay in tumor engraftment plus a strong reduction in tumor size and invasion. Our data, therefore, highlight ITGA7 as a glioblastoma biomarker and candidate therapeutic target.

Integrin Alpha 9 Blockade Suppresses Lymphatic Valve Formation and Promotes Transplant Survival.

PURPOSE: The lymphatic pathway mediates transplant rejection. We recently reported that lymphatic vessels develop luminal valves in the cornea during lymphangiogenesis, and these valves express integrin alpha 9 (Itga-9) and play a critical role in directing lymph flow. In this study, we used an allogeneic corneal transplantation model to investigate whether Itga-9 blockade could suppress valvulogenesis after transplantation, and how this effect would influence the outcomes of the transplants. METHODS: Orthotopic corneal transplantation was performed between fully mismatched C57BL/6 (donor) and BALB/c (recipient) mice. The recipients were randomized to receive subconjunctival injections of either Itga-9 blocking antibody or isotype control twice a week for 8 weeks. Corneal grafts were assessed in vivo by ophthalmic slit-lamp biomicroscopy and analyzed using Kaplan-Meier survival curves. Additionally, whole-mount full-thickness corneas were evaluated ex vivo by immunofluorescent microscopy on both lymphatic vessels and valves. RESULTS: Anti-Itga-9 treatment suppressed lymphatic valvulogenesis after transplantation. Our treatment did not affect lymphatic vessel formation or their nasal polarized distribution in the cornea. More importantly, Itga-9 blockade led to a significant promotion of graft survival. CONCLUSIONS: Lymphatic valvulogenesis is critically involved in transplant rejection. Itga-9 targeting may offer a new and effective strategy to interfere with the immune responses and promote graft survival.

Functional characterization of six aspartate (D) recombinant mojastin mutants (r-Moj): A second aspartate amino acid carboxyl to the RGD in r-Moj-D_ peptides is not sufficient to induce apoptosis of SK-Mel-28 cells.

Disintegrins are small peptides produced in viper venom that act as integrin antagonists. When bound to integrins, disintegrins induce altered cellular behaviors, such as apoptotic induction. Disintegrins with RGDDL or RGDDM motifs induce apoptosis of normal and cancer cells. We hypothesized that a second aspartate (D) carboxyl to the RGD is sufficient to induce apoptosis. Five recombinant mojastin D mutants were produced by site-directed mutagenesis (r-Moj-DA, r-Moj-DG, r-Moj-DL, r-Moj-DN, and r-Moj-DV). Stable αv integrin knockdown and shRNA scrambled control SK-Mel-28 cell lines were produced to test a second hypothesis: r-Moj-D_ peptides bind to αv integrin. Only r-Moj-DL, r-Moj-DM, and r-Moj-DN induced apoptosis of SK-Mel-28 cells (at 29.4%, 25.6%, and 36.2%, respectively). Apoptotic induction was significantly reduced in SK-Mel-28 cells with a stable αv integrin knockdown (to 2%, 17%, and 2%, respectively), but not in SK-Mel-28 cells with a stable scrambled shRNA. All six r-Moj-D_ peptides inhibited cell proliferation; ranging from 49.56% (r-Moj-DN) to 75.6% (r-Moj-DA). Cell proliferation inhibition by r-Moj-D_ peptides was significantly reduced in SK-Mel-28 cells with a stable αv integrin knockdown. All six r-Moj-D_ peptides inhibited SK-Mel-28 cell migration at high levels (69%-100%). As a consequence, rac-1 mRNA expression levels were significantly reduced as early as 1 h after treatment, suggesting that rac-1 is involved in the cell migration activity of SK-Mel-28.

Improvement of Outcome Measures of Dry Eye by a Novel Integrin Antagonist in the Murine Desiccating Stress Model.

PURPOSE: We investigated the effects of GW559090, a novel, competitive, and high-affinity α4 integrin antagonist, in a murine model of dry eye. Through interaction with vascular cell adhesion molecule 1 (VCAM-1) and fibronectin α4ß1 integrin is involved in leukocyte trafficking and activation. METHODS: Female C57BL/6 mice, aged 6 to 8 weeks, were subjected to desiccating stress (DS). Bilateral topical twice daily treatment with GW559090 was compared to vehicle-treated controls. Treatment was initiated at the time of DS induction. Treatment effects were assessed on corneal staining with Oregon Green Dextran (OGD) and expression of inflammatory markers in ocular surface tissues by real time PCR. Dendritic cell activation was measured in draining cervical lymph nodes (CLN) by flow cytometry. Separate groups of mice received GW559090 subcutaneously to evaluate the effects of systemic administration on corneal staining and cells in CLN. RESULTS: Topical GW559090 significantly reduced corneal uptake of OGD compared to vehicle-treated disease controls in a dose-dependent manner (1, 3, 10, and 30 mg/mL) with 30 mg/mL showing the greatest reduction in OGD staining. When administered topically, corneal expression of IL-1α, matrix metalloproteinase (MMP)-9, chemokine ligand 9 (CXCL9), and TGF-ß1 was reduced in GW559090-treated eyes. Topical treatment with GW559090 decreased dendritic cell activation in lymph nodes. The effects on corneal staining and cellular composition in CLN were not reproduced by systemic administration of GW559090, suggestive of a local role for integrin antagonism in the treatment of dry eye. CONCLUSION: The novel α4 integrin antagonist, GW559090, improved outcome measures of corneal staining and ocular surface inflammation in this murine model of dry eye. These results indicate the potential of this novel agent for the treatment of dry eye disease.

Effects of cilengitide in osteoclast maturation and behavior.

Bone metastasis is a common burden in many types of cancer and has a severe impact on the quality of life in patients. Hence, specific therapeutic strategies inhibiting tumor induced osteolysis are urgently needed. In this study, we aimed to interfere with integrin adhesion receptors, which are central players of the bone resorption process. For this purpose, we used cilengitide, a cyclic RGD peptide, which blocks integrin αVß3 and αVß5-ligand binding. Our results revealed that cilengitide blocked osteoclast maturation in a dose-dependent manner. In detail, pre-osteoclasts treated with cilengitide exhibited reduced cell spreading, cell migration and cell adhesion on RGD-containing matrix proteins, which are ligands of integrin αV. The activation of the most upstream signal transduction molecules of the integrin receptor-initiated pathway, FAK and c-Src, were consistently blocked by cilengitide. First evidence suggests that cilengitide might interfere with metastatic bone disease in vivo and this study describes a potential underlying mechanism of the inhibitory effect of cilengitide on αV-integrin expressing pre-osteoclasts by blocking integrin ligand binding and interfering with osteoclast maturation and cell behavior. In conclusion, our findings suggest that cilengitide, which interferes with αV-integrins on osteoclasts, may represent a novel therapeutic strategy in the treatment of malignant bone disease.

Efficient elimination of CD103-expressing cells by anti-CD103 antibody drug conjugates in immunocompetent mice.

CD103 plays an important role in the destruction of islet allografts, and previous studies found that a CD103 immunotoxin (M290-Saporin, or M290-SAP) promoted the long-term survival of pancreatic islet allografts. However, systemic toxicity to the host and the bystander effects of M290-SAP obscure the underlying mechanisms of action and restrict its clinical applications. To overcome these shortcomings, anti-CD103 M290 was conjugated to different cytotoxic agents through cleavable or uncleavable linkages to form three distinct antibody-drug conjugates (ADCs): M290-MC-vc-PAB-MMAE, M290-MC-MMAF, and M290-MCC-DM1. The drug-to-antibody ratio (DAR) and the purity of the ADCs were determined by HIC-HPLC and SEC-HPLC, respectively. The binding characteristics, internalization and cytotoxicity of M290 and the corresponding ADCs were evaluated in vitro. The cell depletion efficacies of the various M290-ADCs against CD103-positive cells were then evaluated in vivo. The M290-ADCs maintained the initial binding affinity for the CD103-positive cell surface antigen and then quickly internalized the CD103-positive cell. Surprisingly, all M290-ADCs potently depleted CD103-positive cells in vivo, with high specificity and reduced toxicity. Our findings show that M290-ADCs have potent and selective depletion effects on CD103-expressing cells in immunocompetent mice. These data indicate that M290-ADCs could potentially serve as a therapeutic intervention to block the CD103/E-cadherin pathway.

Estudio retrospectivo de la eficacia y seguridad del natalizumab en el tratamiento de la esclerosis múltiple en Portugal / The efficacy and safety of natalizumab for the treatment of multiple sclerosis in Portugal: a retrospective study

Introducción. Los estudios han demostrado que el natalizumab constituye un tratamiento eficaz contra la esclerosis múltiple remitente recurrente (EMRR). Hasta la fecha, no había datos de pacientes portugueses. Objetivo. Determinar la eficacia y la seguridad del natalizumab en pacientes con EMRR atendidos en la práctica clínica ordinaria en Portugal. Pacientes y métodos. Los datos clínicos de adultos con EMRR tratados con natalizumab en centros especializados de neurología en Portugal se introdujeron de forma retrospectiva en una base de datos para llevar a cabo un análisis entre octubre de 2010 y febrero de 2012. Se analizó el cambio en la tasa anualizada de brotes (TAB), en las puntuaciones de la escala ampliada de discapacidad (EDSS) y en el estado de discapacidad. Resultados. Se admitió un total de 383 pacientes atendidos en 20 centros. Antes de iniciar el tratamiento con natalizumab, la mediana inicial de la EDSS era de 4,0 y la TAB media, de 1,64. La mayor parte de los pacientes ya había recibido tratamiento contra la esclerosis múltiple (93,0%). La duración media del tratamiento con natalizumab era de 12 meses. El tratamiento propicio reducciones significativas (p < 0,001) de los valores iniciales de la TAB media y de las puntuaciones EDSS en los tratados con el anticuerpo durante ≥ 12 meses (n = 288) y durante ≥ 24 meses (n = 160). El natalizumab resulto mas eficaz en los pacientes que presentaban un menor grado de discapacidad (EDSS < 3,0) y en los que no habían recibido ningún tratamiento modificador de la enfermedad. Se notificaron dos casos de leuco encefalopatía multifocal progresiva. No hubo efectos adversos inesperados. Conclusión. El natalizumab presenta una tolerabilidad satisfactoria y se muestra eficaz en la reducción de las recidivas y la estabilización de la EMRR en el marco de la practica clínica ordinaria en Portugal. Conserva su eficacia con el tratamiento continuado y podría ser eficaz especialmente en los pacientes con menos discapacidad y en aquellos que no han recibido ningún tratamiento modificador de la enfermedad hasta el momento (AU)

Introduction. Studies have shown that natalizumab is an effective treatment for relapsing-remitting multiple sclerosis (RRMS). To date, no data are available in Portuguese patients. Aim. To determine the efficacy and safety of natalizumab in patients with RRMS in routine clinical practice in Portugal. Patients and methods. Clinical data for adult patients with RRMS treated with natalizumab at specialist neurology centres in Portugal were entered retrospectively into a database for analysis between October 2010 and February 2012. Changes in annualized relapse rates (ARR), Expanded Disability Status Scale (EDSS) scores and disability status were analysed. Results. A total of 383 patients from 20 centres were included. Prior to starting natalizumab, the baseline median EDSS score was 4 and the mean ARR was 1.64. Most patients had previously received multiple sclerosis treatment (93.0%). Median natalizumab treatment duration was 12 months. Natalizumab treatment was associated with significant (p < 0.001) reductions from baseline in the mean ARR and EDSS scores in patients treated with natalizumab for ≥ 12 months (n = 288) and for ≥ 24 months (n = 160). Natalizumab was more effective in patients with less disability (EDSS < 3) and in those who had not previously received disease-modifying treatments. Two cases of progressive multifocal leukoencephalopathy were reported. No new unexpected adverse events occurred. Conclusion. Natalizumab is well tolerated, and is effective in reducing relapse rate and stabilising disease in patients with RRMS in the clinical practice setting in Portugal. Its efficacy persists with continued treatment, and it may be particularly effective in patients with less disability and without prior disease modifying therapy (AU)

Highlights of glycosylation and adhesion related genes involved in myogenesis.

BACKGROUND: Myogenesis is initiated by myoblast differentiation and fusion to form myotubes and muscle fibres. A population of myoblasts, known as satellite cells, is responsible for post-natal growth of muscle and for its regeneration. This differentiation requires many changes in cell behaviour and its surrounding environment. These modifications are tightly regulated over time and can be characterized through the study of changes in gene expression associated with this process. During the initial myogenesis steps, using the myoblast cell line C2C12 as a model, Janot et al. (2009) showed significant variations in expression for genes involved in pathways of glycolipid synthesis. In this study we used murine satellite cells (MSC) and their ability to differentiate into myotubes or early fat storage cells to select glycosylation related genes whose variation of expression is myogenesis specific. RESULTS: The comparison of variant genes in both MSC differentiation pathways identified 67 genes associated with myogenesis. Comparison with data obtained for C2C12 revealed that only 14 genes had similar expression profiles in both cell types and that 17 genes were specifically regulated in MSC. Results were validated statistically by without a priori clustering. Classification according to protein function encoded by these 31 genes showed that the main regulated cellular processes during this differentiation were (i) remodeling of the extracellular matrix, particularly, sulfated structures, (ii) down-regulation of O-mannosyl glycan biosynthesis, and (iii) an increase in adhesion protein expression. A functional study was performed on Itga11 and Chst5 encoding two highly up-regulated proteins. The inactivation of Chst5 by specific shRNA delayed the fusion of MSC. By contrast, the inactivation of Itga11 by specific shRNA dramatically decreased the fusion ability of MSC. This result was confirmed by neutralization of Itga11 product by specific antibodies. CONCLUSIONS: Our screening method detected 31 genes specific for myogenic differentiation out of the 383 genes studied. According to their function, interaction networks of the products of these selected genes converged to cell fusion. Functional studies on Itga11 and Chst5 demonstrated the robustness of this screening.

Contribution of the α8 integrin chain to the expression of extracellular matrix components.

In the kidney, the α8 integrin chain (itga8) is expressed in mesenchymal cells and is upregulated in fibrotic disease. We hypothesized that itga8 mediates a profibrotic phenotype of renal cells by promoting extracellular matrix and cytokine expression. Genetic itga8 deficiency caused complex changes in matrix expression patterns in mesangial and smooth-muscle cells, with the only concordant effect in both cell types being a reduction of collagen III expression. Silencing of itga8 with siRNA led to a decline of matrix turnover with repression of matrix metalloproteinases and reduction of matrix production. In contrast, de novo expression of itga8 in tubular epithelial cells resulted in reduced collagen synthesis. Overexpression of itga8 in fibroblasts did not change the expression of matrix molecules or regulators of matrix turnover. Thus, the influence of itga8 on the expression of matrix components was not uniform and celltype dependent. Itga8 seems unlikely to exert overall profibrotic effects in renal cells.

Natalizumab: a review of its use in the management of relapsing-remitting multiple sclerosis.

Natalizumab (Tysabri®) is a humanized monoclonal antibody against the α4 chain of integrins and was the first targeted therapy to be approved for the treatment of relapsing-remitting multiple sclerosis (RRMS). Natalizumab acts as a selective adhesion molecule antagonist, which binds very late antigen (VLA)-4 and inhibits the translocation of activated VLA-4-expressing leukocytes across the blood-brain barrier into the CNS. In a pivotal phase III clinical trial, natalizumab 300 mg intravenously every 4 weeks for 2 years in adults with RRMS significantly reduced the annualized relapse rate and the risk of sustained progression of disability compared with placebo, as well as significantly increasing the proportion of relapse-free patients at 1 and 2 years. Natalizumab also significantly reduced the number of T2-hyperintense, gadolinium-enhancing and T1-hypointense lesions on magnetic resonance imaging, and significantly reduced the volume of T2-hyperintense and T1-hypointense lesions compared with placebo. Natalizumab recipients generally experienced improved health-related quality of life at 1-2 years. Natalizumab was generally well tolerated in pivotal trials. The only adverse events that were more frequent with natalizumab monotherapy than with placebo were fatigue and allergic reactions. The main safety and tolerability issue with natalizumab is the incidence of progressive multifocal leukoencephalopathy (PML). As long as the risk of PML is managed effectively, natalizumab is a valuable therapeutic option for adults with highly active relapsing forms of multiple sclerosis.

Differential regulation of monocyte cytokine release by αV and ß(2) integrins that bind CD23.

The human soluble CD23 (sCD23) protein displays highly pleiotropic cytokine-like activity. Monocytic cells express the sCD23-binding integrins αVß(3), αVß(5), αMß(2) and αXß(2), but it is unclear which of these four integrins most acutely regulates sCD23-driven cytokine release. The hypothesis that ligation of different sCD23-binding integrins promoted release of distinct subsets of cytokines was tested. Lipopolysaccharide (LPS) and sCD23 promoted release of distinct groups of cytokines from the THP-1 model cell line. The sCD23-driven cytokine release signature was characterized by elevated amounts of RANTES (CCL5) and a striking increase in interleukin-8 (IL-8; CXCL8) secretion, but little release of macrophage inflammatory protein 1ß (MIP-1ß; CCL4). Antibodies to αVß(3) or αXß(2) both promoted IL-8 release, consistent with the sCD23-driven pattern, but both also evoked strong MIP-1ß secretion; simultaneous ligation of these two integrins further increased cytokine secretion but did not alter the pattern of cytokine output. In both model cell lines and primary tissue, integrin-mediated cytokine release was more pronounced in immature monocyte cells than in mature cells. The capacity of anti-integrin monoclonal antibodies to elicit a cytokine release response is epitope-dependent and also reflects the differentiation state of the cell. Although a pattern of cytokine release identical to that provoked by sCD23 could not be elicited with any individual anti-integrin monoclonal antibody, αXß(2) and αVß(3) appear to regulate IL-8 release, a hallmark feature of sCD23-driven cytokine secretion, more acutely than αMß(2) or αVß(5).

The role of natalizumab in the treatment of multiple sclerosis.

Natalizumab is an alpha(4)-integrin antagonist, the first in its class for the treatment of multiple sclerosis (MS). Although multiple mechanisms have been proposed for the efficacy of natalizumab in MS, the most likely explanation is that it interferes with the migration of immune cells into the central nervous system. It does this by binding to the alpha(4) subunit of alpha(4)beta(1)-integrin and preventing leukocyte adhesion to endothelial vascular cell adhesion molecule-1. The efficacy of natalizumab in relapsing-remitting MS has been demonstrated in several double-blind, placebo-controlled trials. Natalizumab has been shown to slow the progression of disability in relapsing-remitting MS significantly better than placebo, and to reduce the number of new and enlarging T2 hyperintense and gadolinium-enhanced magnetic resonance imaging lesions. In a post hoc analysis, the proportion of patients with relapsing-remitting MS free of disease activity was significantly greater with natalizumab compared with placebo. Due to the rare risk of progressive multifocal leukoencephalopathy as a complication, natalizumab is primarily recommended in patients who fail, or cannot tolerate, treatment with interferon (IFN) beta or glatiramer acetate (GA). Stratification of those patients most likely to benefit from natalizumab treatment--such as those with highly active disease, severe disease, or extensive functional loss, or those who have failed or cannot tolerate IFN beta or GA therapy--would help define natalizumab's appropriate place in therapy.