Anti-integrin Antibodies Research Articles

Vedolizumab Achieved Clinical and Histologic Remission in a Patient with Lung Cancer Who Had a Steroid-Refractory Upper Gastrointestinal Injury Due to Nivolumab Treatment

Abstract Immune checkpoint inhibitors (ICIs) have emerged as a novel therapeutic class for various malignancies. Their immune upregulation promotes significant anti-tumor effect, but simultaneously, can also result in treatment-limiting immune-related adverse events (irAEs). The data on upper gastrointestinal (GI) tract irAEs are sparse. We herein describe a case of steroid-dependent upper GI toxicity with nivolumab (an anti-programmed death [PD] protein-1) that achieved clinical and histological remission with vedolizumab treatment (a GI tract targeted anti-integrin antibody). A 65-year-old male patient with progressive lung cancer was treated with nivolumab and following 16 cycles, developed severe nausea, vomiting, and epigastric abdominal cramps requiring five hospitalizations. His initial esophagogastroduodenoscopy (EGD) showed active inflammation in both the stomach and duodenum. Nivolumab was discontinued, but despite treatment with multiple steroid courses, his symptoms always recurred during prednisone taper. Clinical remission was ultimately achieved with vedolizumab. His last EGD after five infusions of vedolizumab demonstrated resolution of inflammation. His lung cancer has since relapsed and the treatment plan was to resume nivolumab concurrently with vedolizumab. In conclusion, ICIs, such as nivolumab, have emerged as therapy for various malignancies. Their use can be associated with various irAEs including the upper GI adverse events which is uncommon. This case scenario showed that vedolizumab can provide a steroid-sparing therapeutic effect to achieve remission of upper GI irAEs even in cases where multiple steroid courses have failed.

Extracellular Superoxide Dismutase (SOD3) Links Tumor Necrosis Factor‐alpha Receptor 1 to Integrin Signaling

Tumor necrosis factor‐α (TNFα) signaling requires superoxide (O2•−) to be produced extracellularly by NADPH oxidase 1 (Nox1). Extracellular superoxide dismutase (ecSOD) can convert this O2•− to hydrogen peroxide (H2O2). ecSOD is anchored to the cell surface by interaction with either heparin sulfate proteoglycan via a heparin binding domain, or by binding to fibulin‐5 (Fib5), an extracellular matrix protein which interacts with integrin receptors. Fib5 binding does not activate integrins, but blocks interaction with other components of the extracellular matrix. The physiologic significance of ecSOD‐Fib5‐integrin interaction has not been well defined. We hypothesized that ecSOD regulates extracellular redox signaling of TNFα/Nox1 by modulating secondary effects on integrin signaling. We analyzed TNFα‐induced interleukin‐6 (IL‐6) production and NF‐κB activation in cultured vascular smooth muscle cells (VSMC). Knockdown of ecSOD by siRNA blocked TNFα‐mediated IL‐6 (siControl+TNFα 2922±354, si ecSOD+TNFα 952.5±99 pg/mL, p<0.05, n=8–9) and NF‐κB activation (siControl+TNFα 10.8±1.0, si ecSOD+TNFα 4.0±0.3 fold change, p<0.05, n=7). Similar inhibitory effects were observed following knockdown of Fib5 (IL‐6; siControl+TNFα 2372±123, siFib5+TNFα 1686±124 pg/mL, p<0.05, n=4. NF‐κB activation; siControl+TNFα 10.8±1.0, siFib5+TNFα 5.4±0.3 fold change, p<0.05, n=7). We next sought to determine how ecSOD affected integrin signaling. Focal adhesion kinase (FAK) and integrin‐linked kinase (ILK) are critical downstream effectors of integrin activation. TNFα induced phosphorylation of both FAK and Akt, which was used as a surrogate for ILK activation. This approach was validated by the ability of ILK inhibition (Cpd22; 3 μM) to prevent Akt phosphorylation. Both FAK and Akt phosphorylation in response to TNFα were inhibited by an anti‐integrin β1 antibody. Knockdown of ecSOD significantly enhanced TNFα‐induced phosphorylation of both FAK and Akt. FAK inhibition (FAK inhibitor 14; 2 μM) blocked while ILK inhibition (Cpd22; 3 μM) markedly potentiated NF‐κB activation. These results suggest that TNFα transactivates β1 integrins and therefore FAK and ILK in VSMCs. We speculate that ecSOD localizes H2O2 production to the vicinity of integrin receptors via physical association with Fib5 and provides tonic suppression of β1integrin receptor activation. Knockdown of either ecSOD or Fib5 would promote ILK‐dependent inhibition of NF‐κB activation and IL‐6 secretion which occurs via direct TNFR1‐dependent signaling.Support or Funding InformationAmerican Heart Association (16SDG30610002)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Induction and maintenance treatment of inflammatory bowel disease: A comprehensive review

Ulcerative colitis (UC) and Crohn's disease (CD) are the two major types of inflammatory bowel disease (IBD). We conducted a comprehensive review of meta-analyses to summarize the reported effectiveness of different drugs for IBD. We performed a literature search and a total of 110 meta-analyses from 66 articles were summarized and re-analyzed (62 in UC and 48 in CD). In summary, 5-ASA was more effective than placebo in both induction and maintenance treatment of UC, but there were conflicting results on the effect of 5-ASA on the induction treatment or relapse of CD. The use of immunomodulatory agents in the induction or maintenance phase of UC and CD using immunomodulators appeared to be more effective than placebo, but the results were impacted by small number of patients, discordant results with the largest study and risk of biases. Anti-TNF-α and anti-integrin therapeutic antibodies in both, induction and maintenance, showed a better efficacy than placebo in a large proportion of patients analyzed. Other agents, such as probiotics, antibiotics, omega-3, were shown to be more effective than placebo, but the same issues arose as stated above with the use of immunomodulatory agents. In conclusion, we performed a comprehensive review of meta-analysis on comparative efficacy of pharmacotherapy used in the management of IBD. Our review will augment our understanding of the treatment of UC and CD by providing a guideline for interpreting the statistically significant findings and discusses the optimal choice for IBD treatment.

Contribution of sialic acids to integrin α5β1 functioning in melanoma cells

PurposeTo establish the relationship between sialylation of integrin α5β1 and possible alteration in the function of α5β1 receptor in melanoma cells. Materials and methodsIntegrin α5β1 was isolated from primary WM115 (RGP/VGP-like phenotype) and metastatic WM266-4 (lymph node metastasis) cells via affinity chromatography. Integrin α5β1 sialylation and the shift in relative masses of the enzymatically desialylated subunits were confirmed by confocal microscopy and SDS-PAGE, respectively. The ELISA assay was performed to evaluate sialic acid (SA) influence on integrin α5β1 binding to fibronectin (FN). Cell invasion was investigated by the Transwell invasion assay. The effect of neuraminidases treatment on melanoma cells was assessed by flow cytometry using Maackia amurensis and Sambucus nigra lectins. ResultsBoth subunits of integrin α5β1 were found to be more abundantly sialylated in primary than in metastatic cells. The removal of SA had no effect on the purified integrin α5β1 binding to FN. Although metastatic cells underwent more pronounced desialylation than primary cells, invasion of primary WM115 cells was more dependent on the presence of α2-3 linked SA than it was in the case of metastatic WM266-4 cells. In both melanoma cell lines not only integrin α5β1 was involved in invasion, however simultaneous desialylation and usage of anti-integrin α5β1 antibodies resulted in lower invasion abilities of primary WM115 cells. ConclusionsOur data suggest that in primary melanoma cells integrin α5β1 action is more likely dependent on its glycosylation profile, i.e. the presence of SA residues, which influence (decreased) their invasion properties and may facilitate malignant melanoma progression.

P087 ILEAL BIOMARKER OF INNATE IMMUNE ACTIVATION CAN PREDICT CLINICAL RESPONSE TO VEDOLIZUMAB IN CROHN’S DISEASE

Mucosal barrier dysfunction to luminal microbes plays a crucial role in the development of intestinal inflammation in Crohn’s disease (CD). Recently, an inflammatory form of intestinal epithelial cell (IEC) death resulting from innate immune activation, termed pyroptosis, was proposed as a possible cause of this barrier defect. We hypothesize that ileal IEC pyroptosis, as a marker of mucosal barrier dysfunction, may be used to predict clinical response of biologic therapy in CD patients. Vedolizumab is an anti-integrin monoclonal antibody approved for the treatment of CD. The aim of this multi-centered study was to determine the predictive value of pre-treatment ileal IEC pyrotposis for clinical response to vedolizumab in CD. CD patients aged 18 to 78 years from five American IBD centers who had ileal biopsies obtained during colonoscopy before vedolizumab were enrolled. Clinical response, defined as a reduction of Harvey-Bradshaw Index (HBI) of ≥5 points from the pre-treatment baseline, and clinical remission, defined as HBI <5, was determined at ≥6 months after therapy by chart review. Biopsy samples were sectioned and stained for IEC pyroptosis using the Maximus Barrier Assay kit (Maximus Diagnostics LLC). Samples with a minimum of 10 intact villi per patient were analyzed by blinded pathologists for quantitation of ileal IEC pyroptosis. The primary study outcome was clinical response rate to vedolizumab stratified by pre-treatment ileal pyroptosis, using 18 positive cells/1000 IECs as a cut-point. A total of 80 CD patients were enrolled and 5 were excluded from further analysis due to inadequate biopsy samples for IEC pyroptosis quantification. In the remaining 75 patients (40 M, 35 F), the median age was 47 (19, 78) years; 44 (59%) had clinical response to vedolizumab, and 25 (33%) were in clinical remission. There were no significant differences in baseline patient or disease characteristics between responders and non-responders. The clinical response rate in patients with IEC pyroptosis below the critical cut-point of 18 positive cells / 1000 IECs was significantly higher compared to those above: 74% (29/39) vs. 44% (16/36), odds ratio 3.7 (1.4, 10.0), p=0.008. IEC pyroptosis in responders and non-responders were 16.8 ± 11.8 vs. 24.5 ± 11.6 positive cells / 1000 IECs, respectively (p=0.007). The probability of response to vedolizumab was inversely related to the level of ileal IEC pyroptosis, with IEC pyroptosis of > 39 positive cells / 1000 IECs associated with a clinical response rate of 17% (1/6), while a level of <13 positive cells / 1000 IECs was associated with a 90% (18/20) clinical response. Ileal biopsy IEC pyroptosis can predict clinical response to vedolizmab therapy in Crohn’s disease patients. Clinical response stratified by pre-treatment ileal pyroptosis

P087 ILEAL BIOMARKER OF INNATE IMMUNE ACTIVATION CAN PREDICT CLINICAL RESPONSE TO VEDOLIZUMAB IN CROHN’S DISEASE

Mucosal barrier dysfunction to luminal microbes plays a crucial role in the development of intestinal inflammation in Crohn’s disease (CD). Recently, an inflammatory form of intestinal epithelial cell (IEC) death resulting from innate immune activation, termed pyroptosis, was proposed as a possible cause of this barrier defect. We hypothesize that ileal IEC pyroptosis, as a marker of mucosal barrier dysfunction, may be used to predict clinical response of biologic therapy in CD patients. Vedolizumab is an anti-integrin monoclonal antibody approved for the treatment of CD. The aim of this multi-centered study was to determine the predictive value of pre-treatment ileal IEC pyrotposis for clinical response to vedolizumab in CD. CD patients aged 18 to 78 years from five American IBD centers who had ileal biopsies obtained during colonoscopy before vedolizumab were enrolled. Clinical response, defined as a reduction of Harvey-Bradshaw Index (HBI) of ≥5 points from the pre-treatment baseline, and clinical remission, defined as HBI <5, was determined at ≥6 months after therapy by chart review. Biopsy samples were sectioned and stained for IEC pyroptosis using the Maximus Barrier Assay kit (Maximus Diagnostics LLC). Samples with a minimum of 10 intact villi per patient were analyzed by blinded pathologists for quantitation of ileal IEC pyroptosis. The primary study outcome was clinical response rate to vedolizumab stratified by pre-treatment ileal pyroptosis, using 18 positive cells/1000 IECs as a cut-point. A total of 80 CD patients were enrolled and 5 were excluded from further analysis due to inadequate biopsy samples for IEC pyroptosis quantification. In the remaining 75 patients (40 M, 35 F), the median age was 47 (19, 78) years; 44 (59%) had clinical response to vedolizumab, and 25 (33%) were in clinical remission. There were no significant differences in baseline patient or disease characteristics between responders and non-responders. The clinical response rate in patients with IEC pyroptosis below the critical cut-point of 18 positive cells / 1000 IECs was significantly higher compared to those above: 74% (29/39) vs. 44% (16/36), odds ratio 3.7 (1.4, 10.0), p=0.008. IEC pyroptosis in responders and non-responders were 16.8 ± 11.8 vs. 24.5 ± 11.6 positive cells / 1000 IECs, respectively (p=0.007). The probability of response to vedolizumab was inversely related to the level of ileal IEC pyroptosis, with IEC pyroptosis of > 39 positive cells / 1000 IECs associated with a clinical response rate of 17% (1/6), while a level of <13 positive cells / 1000 IECs was associated with a 90% (18/20) clinical response. Ileal biopsy IEC pyroptosis can predict clinical response to vedolizmab therapy in Crohn’s disease patients.

Microfluidic Device for Screening for Target Cell-Specific Binding Molecules by Using Adherent Cells.

This paper proposes a microfluidic device for screening molecules such as aptamers, antibodies, proteins, etc. for target cell-specific binding molecules. The discovery of cancer cell-specific binding molecules was the goal of this study. Its functions include filtering non-target cell-binding molecules, trapping molecules on the surface of target cells, washing away unbound molecules, and collecting target cell-specific binding molecules from target cells. These functions were effectively implemented by using our previously developed micro pillar arrays for cell homogeneous dispersion and pneumatic microvalves for tall microchannels. The device was also equipped with serially connected filter chambers in which non-target cells were cultured to reduce the molecules binding to non-target cells as much as possible. We evaluated the performance of the device using cancer cell lines (N87 cells as target cells and HeLa cells as non-target cells) and two fluorescent dye-labeled antibodies: Anti-human epidermal growth factor receptor 2 (anti-HER2) antibody that binds to target cells and anti-integrin antibody that binds to non-target cells. The results showed that the device could reduce anti-integrin antibodies to the detection limit of fluorescent measurement and collect anti-HER2 antibodies from the target cells.

Abrilumab Found Effective for Moderate-to-Severe Ulcerative Colitis Relative to Placebo

Vedolizumab, an anti-integrin antibody, is FDA-approved to treat ulcerative colitis (UC) and Crohn disease. Abrilumab, a similar anti-integrin, was

Mycoplasma hyopneumoniae resides intracellularly within porcine epithelial cells

Enzootic pneumonia incurs major economic losses to pork production globally. The primary pathogen and causative agent, Mycoplasma hyopneumoniae, colonises ciliated epithelium and disrupts mucociliary function predisposing the upper respiratory tract to secondary pathogens. Alleviation of disease is reliant on antibiotics, vaccination, and sound animal husbandry, but none are effective at eliminating M. hyopneumoniae from large production systems. Sustainable pork production systems strive to lower reliance on antibiotics but lack of a detailed understanding of the pathobiology of M. hyopneumoniae has curtailed efforts to develop effective mitigation strategies. M. hyopneumoniae is considered an extracellular pathogen. Here we show that M. hyopneumoniae associates with integrin β1 on the surface of epithelial cells via interactions with surface-bound fibronectin and initiates signalling events that stimulate pathogen uptake into clathrin-coated vesicles (CCVs) and caveosomes. These early events allow M. hyopneumoniae to exploit an intracellular lifestyle by commandeering the endosomal pathway. Specifically, we show: (i) using a modified gentamicin protection assay that approximately 8% of M. hyopneumoniae cells reside intracellularly; (ii) integrin β1 expression specifically co-localises with the deposition of fibronectin precisely where M. hyopneumoniae cells assemble extracellularly; (iii) anti-integrin β1 antibodies block entry of M. hyopneumoniae into porcine cells; and (iv) M. hyopneumoniae survives phagolysosomal fusion, and resides within recycling endosomes that are trafficked to the cell membrane. Our data creates a paradigm shift by challenging the long-held view that M. hyopneumoniae is a strict extracellular pathogen and calls for in vivo studies to determine if M. hyopneumoniae can traffic to extrapulmonary sites in commercially-reared pigs.

Late breaking abstracts

Late breaking abstracts

RK, the first scorpion peptide with dual disintegrin activity on α1β1 and αvβ3 integrins

Scorpion peptides are well known for their pharmaceutical potential on different targets. These include mainly the ion channels which were found to be highly expressed in many diseases, including cancer, auto-immune pathologies and Alzheimer. So far, however, the disintegrin activity had only been characterized for snake venom molecules. Herein, we present the first short peptide, purified from the venom of Buthus occitanus tunetanus, (termed RK) able to inhibit the cell adhesion of Glioblastoma, Melanoma and Rat pheochromocytoma to different extracellular matrix (ECM) receptors. Anti-integrin antibody assay suggests that RK interacts with both α1β1 and αvβ3 with a more pronounced effect for the former. The examination of the primary structure of RK suggests the involvement of two motifs: KSS, analogue to KTS which was characterized for α1β1 Snake venom disintegrins, and ECD, analogue to RGD which was found to be active on αvβ3. To assess their roles in the disintegrin activity of RK, we conducted a computational analysis. The molecular docking study shows that RK involves mainly two segments to interact with the α1β1 integrin, but the peptide does not implicate the KSS motif in the interaction. The molecular modeling study, suggests the key contribution of the ECD segment in the interaction with αvβ3 integrin.

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease.

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-α4β7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn's disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow. The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field. Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Entyvio lengthen dose-interval study: lengthening vedolizumab dose interval and the risk of clinical relapse in inflammatory bowel disease

Vedolizumab (VDZ), an α4β7 anti-integrin antibody, is efficacious in the induction and maintenance of remission in ulcerative colitis (UC) and Crohn's disease (CD). In the GEMINI long-term safety study, enrolled patients received 4-weekly VDZ. Upon completion, patients were switched to 8-weekly VDZ in Australia. The clinical success rate of treatment de-escalation for patients in remission on VDZ has not been described previously. To determine the proportion of patients who relapsed after switching from 4 to 8-weekly VDZ, the mean time to relapse, and the recapture rate when switching back to 8-weekly dosing. This was a retrospective, observational, multicenter study of patients previously recruited into GEMINI long-term safety in Australia. Data on the demographics and biochemical findings were collected. There were 34 patients [23 men, mean age 49.1 (±13.1) years] and their mean disease duration was 17.6 (±8.5) years. The mean 4-weekly VDZ infusion duration was 286.5 (±48.8) weeks. A total of five (15%) patients relapsed on dose-interval increase (4/17 UC, 1/17 CD) at a median duration from dose interval lengthening to flare of 14 weeks (interquartile range=6-25). Eighty percent (4/5) of patients re-entered remission following dose-interval decrease back to 4-weekly. No clinical predictors of relapse could be determined because of the small cohort size. The risk of patients relapsing when switching from 4 to 8-weekly VDZ ∼15% and is similar between CD and UC. Dose-interval decrease recaptures 80% of patients who relapsed. Therapeutic drug monitoring of VDZ may be of clinical relevance.

Physiological Hypoxia (Physioxia) Impairs the Early Adhesion of Single Lymphoma Cell to Marrow Stromal Cell and Extracellular Matrix. Optical Tweezers Study.

Adhesion is critical for the maintenance of cellular structures as well as intercellular communication, and its dysfunction occurs prevalently during cancer progression. Recently, a growing number of studies indicated the ability of oxygen to regulate adhesion molecules expression, however, the influence of physiological hypoxia (physioxia) on cell adhesion remains elusive. Thus, here we aimed: (i) to develop an optical tweezers based assay to precisely evaluate single diffuse large B-cell lymphoma (DLBCL) cell adhesion to neighbor cells (mesenchymal stromal cells) and extracellular matrix (Matrigel) under normoxia and physioxia; and, (ii) to explore the role of integrins in adhesion of single lymphoma cell. We identified the pronouncedly reduced adhesive properties of lymphoma cell lines and primary lymphocytes B under physioxia to both stromal cells and Matrigel. Corresponding effects were shown in bulk adhesion assays. Then we emphasized that impaired β1, β2 integrins, and cadherin-2 expression, studied by confocal microscopy, account for reduction in lymphocyte adhesion in physioxia. Additionally, the blockade studies conducted with anti-integrin antibodies have revealed the critical role of integrins in lymphoma adhesion. To summarize, the presented approach allows for precise confirmation of the changes in single cell adhesion properties provoked by physiological hypoxia. Thus, our findings reveal an unprecedented role of using physiologically relevant oxygen conditioning and single cell adhesion approaches when investigating tumor adhesion in vitro.

Konvenční a biologická terapie idiopatických střevních zánětů

Crohn's disease and ulcerative colitis are chronic digestive conditions incurable by medication or surgery, which affect mainly young populations in economically developed countries. A medical treatment includes conventional preparations: aminosalicylates, corticosteroids, immunosuppressive drugs, antibiotics and exceptionally also probiotics, while in not responding patients or those at higher risk of an adverse course a biological treatment is indicated. The most frequently administered biologics are antibodies directed against the tumour-necrosis factor α, which are highly effective, but the length of their application is limited by a loss of efficiency and incidence of adverse effects. The new, more selectively acting biologics include anti-integrin antibodies and interleukin 12/23 antibodies. Key words: biological treatment - Crohn's disease - idiopathic bowel inflammation - medication-based treat-ment - ulcerative colitis.

Similar Inhibition of Dynamic Adhesion of Lymphocytes From IBD Patients to MAdCAM-1 by Vedolizumab and Etrolizumab-s.

Although anti-adhesion therapies are a novel mainstay in the treatment of inflammatory bowel diseases (IBDs), the mechanisms controlling integrin-dependent gut homing are poorly elucidated, and the available techniques for translational functional investigations are limited. We used dynamic adhesion assays to study adhesion of CD4+ T cells, CD8+ T cells, CD19+ B cells, and granulocytes to the addressins MAdCAM-1, VCAM-1, and ICAM-1. The effects of vedolizumab, natalizumab, etrolizumab-s, anti-CD11a, and anti-CD18 antibodies were explored. Adhesion of peripheral blood leukocytes from IBD patients and control donors could be validly assessed, and integrin-mediated addressin adhesion could be specifically inhibited by anti-integrin antibodies. Numbers of adhering cells were partly, but not completely, related to integrin expression. Vedolizumab and etrolizumab-s resulted in similar reduction of adhesion to MAdCAM-1, and preliminary data proposed an association of dynamic adhesion to MAdCAM-1 with response to vedolizumab therapy. Dynamic adhesion assays are an easy and broadly applicable method for IBD research that is useful for future translational studies and potentially also for supporting clinical treatment decisions. 10.1093/ibd/izy077_video1izy077_Video_15786486962001.

Pharmacokinetic Modeling and Simulation of Biologicals in Inflammatory Bowel Disease: The Dawning of a New Era for Personalized Treatment.

Anti-tumor necrosis factor-alpha and anti-integrin monoclonal antibodies show great benefits for inducing and maintaining remission, healing the mucosa and restoring the quality of life of patients with inflammatory bowel disease. However, the therapeutic potential of these intrinsically powerful biologicals is abated by a high variability in response. Some patients experience no benefit from these treatments, while others lose response over time. Therapeutic Drug Monitoring (TDM) is a promising tool to further improve therapeutic outcome, substantiated by the finding that highly variable clinical response is correlated with pharmacokinetic (PK) variability. Serum Trough Concentrations (TCs) of the drug are measured and dosage regimens are adapted in order to achieve target TCs that correlate with beneficial therapeutic outcomes. The TC concept is relatively simple but gives only a partial insight in PK. PK profiles should be interpreted in the light of patient specific influences (i.e., covariates) that explain variability. Therefore, the aim of TDM must be to dose the biological in such a way that a personal optimal PK profile is achieved. Furthermore, currently used "treat-to-target" algorithms have proven to increase the therapeutic potential of the drugs, but dosage regimen adaptations are still robust guesswork. A clinical decision support tool for accurately forecasting drug exposure would significantly impact TDM and is suggested to promote successful implementation of individualized predictive treatment in clinical practice. This review provides a clinician-oriented overview of the state-of-the-art, the gaps in current knowledge and future potential of individualized predictive treatment.

Extracellular matrix stiffness controls osteogenic differentiation of mesenchymal stem cells mediated by integrin \u03b15

BackgroundHuman mesenchymal stem cell (hMSC) differentiation into osteoblasts has important clinical significance in treating bone injury, and the stiffness of the extracellular matrix (ECM) has been shown to be an important regulatory factor for hMSC differentiation. The aim of this study was to further delineate how matrix stiffness affects intracellular signaling through integrin α5/β1, FAK, and Wnt signaling, subsequently regulating the osteogenic phenotype of hMSCs.MethodshMSCs were cultured on tunable polyacrylamide hydrogels coated with fibronectin with stiffness corresponding to a Young’s modulus of 13–16 kPa and 62–68 kPa. After hMSCs were cultured on gels for 1 week, gene expression of alpha-1typeIcollagen, BGLAP, and RUNX2 were evaluated by real-time PCR. After hMSCs were cultured on gels for 24 h, signaling molecules relating to integrin α5 (FAK, ERK, p-ERK, Akt, p-Akt, GSK-3β, p-GSK-3β, and β-catenin) were evaluated by western blot analysis.ResultsOsteogenic differentiation was increased on 62–68 kPa ECM, as evidenced by alpha-1 type I collagen, BGLAP, and RUNX2 gene expression, calcium deposition, and ALP staining. In the process of differentiation, gene and protein expression of integrin α5/β1 increased, together with protein expression of the downstream signaling molecules FAK, p-ERK, p-Akt, GSK-3β, p-GSK-3β, and β-catenin, indicating that these molecules can affect the osteogenic differentiation of hMSCs. An antibody blocking integrin α5 suppressed the stiffness-induced expression of all osteoblast markers examined. In particular, alpha-1 type I collagen, RUNX2, and BGLAP were significantly downregulated, indicating that integrin α5 regulates hMSC osteogenic differentiation. Downstream expression of FAK, ERK, p-ERK, and β-catenin protein was unchanged, whereas Akt, p-Akt, GSK-3β, and p-GSK-3β were upregulated. Moreover, expression of Akt and p-Akt was upregulated with anti-integrin α5 antibody, but no difference was observed for FAK, ERK, and p-ERK between the with or without anti-integrin α5 antibody groups. At the same time, expression of GSK-3β and p-GSK-3β was upregulated and β-catenin levels showed no difference between the groups with or without anti-integrin α5 antibody. Since Akt, p-Akt, GSK-3β, and p-GSK-3β displayed the same changes between the groups with or without anti-integrin α5 antibody, we then detected the links among them. Expression of p-Akt and p-GSK-3β was reduced effectively in the presence of the Akt inhibitor Triciribine. However, Akt, GSK-3β, and β-catenin were unchanged. These results suggested that expression of p-GSK-3β was regulated by p-Akt on 62–68 kPa ECM.ConclusionsTaken together, our results provide evidence that matrix stiffness (62–68 kPa) affects the osteogenic outcome of hMSCs through mechanotransduction events that are mediated by integrin α5.

Fibronectin Enhances Cartilage Repair by Activating Progenitor Cells Through Integrin α5β1 Receptor.

This study aimed to determine the effect of fibronectin (FN) on cartilage regeneration through the activation of chondrogenic progenitor cells (CPCs). Cells were isolated from the knee cartilage of mice and cultured in the presence of various concentrations of FN. Proliferation, migration, and chondrogenic differentiation assays were performed in vitro. In some experiments, CPCs were preincubated with anti-integrin α5β1 antibody for 60 min before FN treatment to block the integrin α5β1 receptor. Soluble FN was mixed with Pluronic F-127 and injected into the joint cavity in an early-stage osteoarthritis model. Cartilage repair was evaluated histologically, biochemically, and biomechanically. In vitro, we observed that the isolated CPCs, which exhibited stem cell-relevant markers, proliferated most at a concentration of 20 μg/mL FN (p < 0.05). In addition, FN enhanced the proliferation, migration, and chondrogenic differentiation capacity of CPCs, and the enhancement was significantly decreased by blockade of the integrin α5β1 receptor (p < 0.05). In vivo, FN also significantly promoted cartilage repair along with increased CPC activation and integrin α5β1 expression (p < 0.05). These findings suggest that FN enhances CPC proliferation, migration, and chondrogenic differentiation through the integrin α5β1-dependent signaling pathway. Based on these results, a novel and promising therapy focused on targeted activation of CPCs by FN could be developed for the treatment of cartilage injuries in a clinical setting.

P189 REAL WORLD EVALUATION OF PROLONGED CLINICAL RESPONSE WITH VEDOLIZUMAB IN CROHN’S DISEASE AND ULCERATIVE COLITIS PATIENTS TREATED IN A PRIVATE PRACTICE SETTING

Vedolizumab (VDZ), a gut-selective anti-integrin monoclonal antibody, was approved for the treatment of Crohn’s disease (CD) and ulcerative colitis (UC) in 2014. VDZ is reported to achieve maximal efficacy after several months of therapy. This real-world observational analysis describes effective prolonged treatment of CD and UC patients (pts) with VDZ who achieved initial clinical response. A chart review was performed on all CD and UC pts treated with VDZ in a large gastroenterology private practice. Pts were included that achieved 6 months (mo) of sustained clinical response or remission on VDZ and who had ≥24 mo of available follow-up data. Those were then evaluated at 3 mo intervals for continued VDZ response. Demographics, diagnosis, prior and concurrent therapy, infusions, labs and diagnostics, and assessment of response were compiled. Clinical response was assessed by Kaplan-Meier analysis and associated factors by odds ratio (95% CI). 64 pts (CD-50%; UC-50%) met study criteria. Median age was 42 yrs (range 23–80), 56% female, median disease duration was 9 yrs (range 0.6–42). Treatment prior to VDZ included aminosalicylates in 88% (CD-78%; UC-97%), corticosteroids in 94% (CD-88%; UC-100%), immunomodulators in 73% (CD-81%; UC-66%) and other biologics in 95% (CD-97%; UC-94%). Overall, sustained clinical response was seen in 63%, (n=40) for 24 mo. Pts with CD had a significantly higher sustained response (75%, n=24) versus those with UC (50%, n=16); p=0.026. 3-mo response rates are noted in Figure 1. Response rates were unaffected in either group by prior therapies, treatment interruptions, dose escalations, or disease duration. A majority of pts achieving initial clinical response or remission with VDZ experienced sustained response at 24 mo. More CD pts had sustained response than UC pts. Prolonged clinical response on VDZ was not associated with any identified differences in the population cohort.