VLA-4 Research Articles

P0915 Real-life experience with Upadacitinib in Crohn’s disease (UPARECOL-CD Induction): A Colombian cohort

Abstract Background In Latin America, clinical experience with Upadacitinib for CD in terms of effectiveness and safety is limited. In Colombia, CD is an orphan disease and in many cases is highly refractory to conventional treatments. The objective of this study is to describe the real-life experience in Colombian patients with CD treated with Upadacitinib. Methods Descriptive observational study, patients with CD treated with Upadacitinib in induction phase (45 mg orally once a day for 12 weeks) in different reference centers nationwide. The therapeutic response was evaluated in endoscopic and/or imaging, paraclinical and clinical terms (CDAI). Additionally, the frequency of adverse events (AE), steroid use and extraintestinal manifestations (EIM) were measured. Results 10 patients, 5/10 (50%) female, mean age 43.15 years (SD; 22.7 range 14.2-21.3). Mean age at diagnosis of CD 37.7 (SD 23.15) years (range 6.9-67.5). Mean time between disease onset and start of Upadacitinib 3.33 (SD 8.17) years (range 0.1-15.9). Most patients had moderate to severe CD; 7/10 (70%) patients with ileocolonic disease and 3 (30%) with ileal disease. 5 patients with stricturing disease and 3 patients with active penetrating disease. All patients had previously failed tumor necrosis factor inhibitors, 8/10 (80%) had failed integrin alpha4 beta7 inhibitors (Vedolizumab). Six patients had EIM. Six patients had a history of surgery for CD. Additionally, 4/10 (40%) patients were using a steroid before starting Upadacitinib and 6/10 (60%) patients were using an immunomodulator. During the induction phase, 7/10 (70%) achieved clinical response, 2/10 (20%) clinical remission, and 5/10 (50%) paraclinical remission. 4/6 (66.6%) patients achieved improvement in imaging findings. All patients with EIM showed improvement of EIM. 2/5 (40%) patients showed improvement of perianal disease. Only one patient required surgery for CD. 3/4 (75%) were able to discontinue corticosteroid and 1/4 (25%) achieved steroid dose reduction. One patient required discontinuation of Upadacitinib, due to lack of response and severe sepsis. One patient reported acne after induction. No other AEs were reported. Conclusion Upadacitinib is an effective option for patients with CD with inflammatory, stenosing and penetrating phenotypes refractory to multiple treatments, suggesting a reduced requirement for surgical procedures. Long-term real-life studies are needed to determine whether there is persistence of response in maintenance.

Application of advanced treatment in chronic inflammatory bowel diseases

The treatment options for chronic inflammatory bowel diseases (IBD) have been greatly expanded due to a better understanding of the underlying pathogenesis. A total of five classes of advanced treatment are available. Apractical overview of advanced treatment of IBD. Narrative review. Advanced treatments are indicated for moderate to severe IBD. A timely use is recommended to achieve better response rates and to avoid irreversible bowel damage. Tumor necrosis factor (TNF) inhibitors and Janus kinase (JAK) inhibitors have a broad efficacy, also for extraintestinal disease manifestations. The risk of reactivation of varicella zoster virus is increased with JAK inhibitors. In high-risk patients and an age >65 years there is possibly a moderately elevated cardiovascular risk and neoplastic side effects. The integrin alpha4beta7 inhibitor vedolizumab and the interleukin (IL) 12 and 23 inhibitor ustekinumab have very good safety profiles. Selective IL-23inhibitors are sometimes superior to ustekinumab with comparable safety profiles with respect to efficacy. The sphingosine-1-phosphate receptor modulators ozanimod and etrasimod are approved for oral treatment of ulcerative colitis. The treatment success of the medications remains still limited and aminority of patients will not respond to every individual treatment. Thus, sequential administration of several treatments is often needed. Due to the lack of comparative studies, the personalized choice, sequence and decision for treatments are usually based on personal experience and should take patient preferences, efficacy, safety and individual patient profiles into consideration.

LncRNA MALAT1 silencing represses CXCL12-induced proliferation, invasion, and homing behavior in multiple myeloma by inhibiting CXCR4

ABSTRACT Background: Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been identified in multiple myeloma (MM); its influence on the homing behavior in MM cells is unclear. This study elucidates the impact and mechanism by which MALAT1 affects the homing behavior in MM cells. Methods: Bone marrow was obtained from patients with MM. MALAT1 and C-X-C motif chemokine receptor 4 (CXCR4) expression in cluster of differentiation 138-purified (CD138) plasma cells were detected by qRT-PCR and Western blotting. MALAT1, CXCR4, and C-X-C motif chemokine ligand 12 (CXCL12) influences on MM cell viability, proliferation, and invasion was monitored by CCK-8, 5-Ethynyl-2'-deoxyuridine, and Transwell assays. qRT-PCR, Western blotting, and ELISA were utilized to detect homing effect-related proteins in MM cells, including intercellular adhesion molecule 1 (ICAM-1) and very late antigen-4 (VLA-4). Results: MALAT1 and CXCR4 were overexpressed in CD138-purified plasma cells from bone marrow of MM patients, associating with bone damage. MALAT1 upregulated CXCR4 in MM cells. MALAT1 overexpression enhanced MM cell viability, proliferation, and invasion, whereas CXCR4 silencing reversed them. CXCR4 silencing attenuated MALAT1 induction on ICAM-1 and VLA-4 expression in MM cells. CXCL12 upregulation intensified MM cell proliferation and invasion and ICAM-1 and VLA-4 expression in MM cells. MALAT1 silencing counteracted the impact of CXCL12. Conclusion: MALAT1 silencing may repress CXCL12 induction on MM cell proliferation, invasion, and homing behavior by inhibiting CXCR4. MALAT1 may be a promising target for MM treatment.

Integrin alpha1 beta1 promotes interstitial fibrosis in a mouse model of polycystic kidney disease.

Fibrosis is the cause of end-stage kidney failure in patients with Autosomal Dominant Polycystic Kidney Disease (ADPKD). The molecular and cellular mechanisms involved in fibrosis are complex and anti-fibrotic therapies have so far failed to make an impact on patient welfare. Using unbiased proteomics analysis on the Pkd1 nl/nl mouse, we found that expression of the integrin α1 subunit is increased in this model of ADPKD. In human ADPKD tissue and two single cell RNA kidney disease datasets, ITGA1 was also upregulated. To investigate the functional role of this integrin subunit in ADPKD, we generated a Pkd1 nl/nl Itga1 -/- mouse. We observed a significant reduction in kidney volume and kidney dysfunction in mice lacking the integrin α1 subunit. Kidneys from Pkd1 nl/nl Itga1 -/- mice had smaller cysts and reduced interstitial expansion and tubular atrophy. Picrosirius red staining identified a restriction in collagen staining in the interstitium and the myofibroblast marker α smooth muscle actin was also downregulated. Myofibroblast cell proliferation was reduced in Pkd1 nl/nl Itga1 -/- mice and primary fibroblast cultures demonstrated an abrogated fibrogenic phenotype in integrin α1-depleted fibroblasts. These results highlight a previously unrecognised role for the integrin α1 subunit in kidney fibrosis.

COMPARISON OF THE EFFECTS OF DOCETAXEL and AMYGDALIN TREATMENT ON CELL DEATH, INTEGRIN-α and INTEGRIN-β EXPRESSIONS IN DU145 PROSTATE CANCER CELL LINE

OBJECTIVE: Prostate cancer (PC) ranks second among cancer-related deaths in men, and most deaths are caused by metastasis. Integrins, which are cell surface receptors, play an important role in cancer metastasis. It has been shown that integrin alpha2beta1 expression is effective in cell adhesion, migration, and invasion by increasing binding to collagen I in metastatic PCs. Docetaxel chemotherapy is used in PC, but it is ineffective in advanced stages. Amygdalin is a cyanogenic glycoside commonly found in fruit seeds, there is conflict in the literature regarding its effectiveness in cancer treatment. We aimed to compare the effects of Amygdalin and Docetaxel treatments on the DU145 prostate cancer cell line on integrinalfa2 (ITGA2) and integrinbeta1 (ITGB1) expressions, as well as their effects on cell death, Caspase-3, and Beclin-1. MATERIAL AND METHODS: Propagated DU145 cells were divided into four groups. Amygdalin was given to the first group, Docetaxel was given to the second group, and Amygdalin andDocetaxel were given together to the third group. They were exposed to the active substances for 24 hours. The fourth group (Control) was not given any substance. mRNA levels of ITGA2 and ITGB1 genes were determined by the Real-time PCR method. Caspase-3 and Beclin-1 staining were performed immunocytochemically to evaluate cell death. RESULTS: There was an increase in ITGA2 and ITGB1 expressions in the groups administered by Amygdalin and by Docetaxel (P<0.05). The decrease in ITGB1 expression was significant in the group given Amygdalin+Docetaxel (P<0.001). Caspase-3 (P<0.05) and Beclin-1 (P<0.05) immunoreactivities were observed to increase in all three groups compared to the control group. CONCLUSIONS: It was observed that Docetaxel increased cell death more than Amygdalin in DU145 PC cells, and when Amygdalin and Docetaxel were used together, ITGA2 and ITGB1 expressions were significantly reduced. Our results suggest that dual treatment of Amygdalin and Docetaxel may prevent prostate cancer metastases.

In vitro effects of l-kynurenine and quinolinic acid on adhesion, migration and apoptosis in B16 F10 melanoma cells

IntroductionThe inhibition of melanoma adhesion through adhesion molecules, such as integrins and E-cadherin, may represent a promising strategy for managing melanoma metastasis. Compounds, namely l-kynurenine (L-kyn) and quinolinic acid (Quin), previously displayed anti-cancer effects at half-maximal inhibitory concentration (IC50) against B16 F10 melanoma cells in vitro. However, the role of these compounds in B16 F10 melanoma cell adhesion, migration and apoptosis remain unknown. MethodsPost-exposure to the compounds, flow cytometry was used to analyse the expression of very late antigen-5 (VLA-5), E-cadherin and cleaved caspase-3 in B16 F10 melanoma and RAW 264.7 murine macrophage cells. An adhesion assay was used to quantify the adhesion of both cell lines to vitronectin. A scratch migration assay was used to measure the possible inhibition of cell migration in B16 F10 cells in response to L-kyn and Quin. ResultsIn both B16 F10 and RAW 264.7 cells, neither L-kyn nor Quin induced significant effects on VLA-5 expression or cell adhesion to vitronectin. In B16 F10 cells, both L-kyn and Quin elevated E-cadherin expression and displayed a trend of suppressed migration. However, only L-kyn elevated E-cadherin in RAW 264.7 cells. L-kyn induced apoptosis by elevating cleaved caspase-3 expression in both cell lines. ConclusionL-kyn and Quin demonstrated promising antimetastatic effects in their ability to elevate E-cadherin expression and induce apoptosis in B16 F10 melanoma cells. However, these effects did not occur in response to vitronectin or VLA-5 integrin alterations. Furthermore, it cannot be excluded that L-kyn also induced apoptosis in RAW 264.7 cells. As such, these effects should be confirmed in additional control cell lines and substantiated with in vivo models.

Development of VLA4 and CXCR4 Antagonists for the Mobilization of Hematopoietic Stem and Progenitor Cells.

The treatment of patients diagnosed with hematologic malignancies typically includes hematopoietic stem cell transplantation (HSCT) as part of a therapeutic standard of care. The primary graft source of hematopoietic stem and progenitor cells (HSPCs) for HSCT is mobilized from the bone marrow into the peripheral blood of allogeneic donors or patients. More recently, these mobilized HSPCs have also been the source for gene editing strategies to treat diseases such as sickle-cell anemia. For a HSCT to be successful, it requires the infusion of a sufficient number of HSPCs that are capable of adequate homing to the bone marrow niche and the subsequent regeneration of stable trilineage hematopoiesis in a timely manner. Granulocyte-colony-stimulating factor (G-CSF) is currently the most frequently used agent for HSPC mobilization. However, it requires five or more daily infusions to produce an adequate number of HSPCs and the use of G-CSF alone often results in suboptimal stem cell yields in a significant number of patients. Furthermore, there are several undesirable side effects associated with G-CSF, and it is contraindicated for use in sickle-cell anemia patients, where it has been linked to serious vaso-occlusive and thrombotic events. The chemokine receptor CXCR4 and the cell surface integrin α4β1 (very late antigen 4 (VLA4)) are both involved in the homing and retention of HSPCs within the bone marrow microenvironment. Preclinical and/or clinical studies have shown that targeted disruption of the interaction of the CXCR4 or VLA4 receptors with their endogenous ligands within the bone marrow niche results in the rapid and reversible mobilization of HSPCs into the peripheral circulation and is synergistic when combined with G-CSF. In this review, we discuss the roles CXCR4 and VLA4 play in bone marrow homing and retention and will summarize more recent development of small-molecule CXCR4 and VLA4 inhibitors that, when combined, can synergistically improve the magnitude, quality and convenience of HSPC mobilization for stem cell transplantation and ex vivo gene therapy after the administration of just a single dose. This optimized regimen has the potential to afford a superior alternative to G-CSF for HSPC mobilization.

Albumin-Binding Lutetium-177-Labeled LLP2A Derivatives as Theranostics for Melanoma.

Very late antigen-4 (VLA-4) is a transmembrane integrin protein that is highly expressed in aggressive forms of metastatic melanoma. A small-molecule peptidomimetic, LLP2A, was found to have a low pM affinity binding to VLA-4. Because LLP2A itself does not inhibit cancer cell proliferation and survival, it is an ideal candidate for the imaging and delivery of therapeutic payloads. An analog of [177Lu]Lu-labeled-LLP2A was previously investigated as a therapeutic agent in melanoma tumor-bearing mice, resulting in only a modest improvement in tumor growth inhibition, likely due to rapid clearance of the agent from the tumor. To improve the pharmacokinetic profile, DOTAGA-PEG4-LLP2A with a 4-(p-iodophenyl)butyric acid (pIBA) albumin binding moiety was synthesized. We demonstrate the feasibility of this albumin binding strategy by comparing in vitro cell binding assays and in vivo biodistribution performance of [177Lu]Lu-DOTAGA-PEG4-LLP2A ([177Lu]Lu-1) to the albumin binding [177Lu]Lu-DOTAGA-pIBA-PEG4-LLP2A ([177Lu]Lu-2). In vitro cell binding assay results for [177Lu]Lu-1 and [177Lu]Lu-2 showed Kd values of 0.40 ± 0.07 and 1.75 ± 0.40 nM, with similar Bmax values of 200 ± 6 and 315 ± 15 fmol/mg, respectively. In vivo biodistribution data for both tracers exhibited specific uptake in the tumor, spleen, thymus, and bone due to endogenous expression of VLA-4. Compound [177Lu]Lu-2 exhibited a much longer blood circulation time compared to [177Lu]Lu-1. The tumor uptake for [177Lu]Lu-1 was highest at 1 h (∼15%ID/g) and that for [177Lu]Lu-2 was highest at 4 h (∼23%ID/g). Significant clearance of [177Lu]Lu-1 from the tumor occurs at 24 h (<5%ID/g) while[177Lu]Lu-2 is retained for greater than 96 h (∼10%ID/g). An efficacy study showed that melanoma tumor-bearing mice receiving compound [177Lu]Lu-2 given in two fractions (2 × 14.8 MBq, 14 days apart) had a greater median survival time than mice administered a single 29.6 MBq dose of compound [177Lu]Lu-1, while a single 29.6 MBq dose of [177Lu]Lu-2 imparted hematopoietic toxicity. The in vitro and in vivo data show addition of pIBA to [177Lu]Lu-DOTAGA-PEG4-LLP2A slows blood clearance for a higher tumor uptake, and there is potential of [177Lu]Lu-2 as a theranostic in fractionated administered doses.

Abstract WP295: Blocking the VCAM1-VLA4 Axis Prevents Cognitive Decline in a Mouse Model of Infarct-Induced Neurodegeneration

Introduction: Infarct-induced neurodegeneration occurs chronically after stroke and can be prevented by inhibiting B cell influx. Vascular cell adhesion molecule 1 (VCAM1) facilitates immune cell diapedesis by binding very late antigen 4 (VLA4), and plasma levels are elevated after stroke in humans and mice. We hypothesized that chronic treatment with antibodies anti-VCAM1 or anti-VLA4 would reduce lymphocyte infiltration and prevent cognitive decline. Methods: Aged (10 month old) C57BL/6 male &amp; female mice (N=9-10/group) underwent permanent middle cerebral artery occlusion and were dosed with anti-VCAM1, anti-VLA4, or control IgG every 3 days beginning 4 days after stroke for 10 weeks. Sham mice were also dosed with IgG. Barnes maze and Novel object were performed at -1, 1 and 6 weeks and single-cell RNA sequencing was performed on immune and endothelial cells at 10 weeks. Results: Blocking either VCAM1 or VLA4 reduced B cells 67% or 55%, respectively, compared to IgG treated stroked mice. Stroked mice treated with IgG developed a cognitive deficit in both Barnes maze and novel object by 6 weeks, while both anti-VCAM1 and anti-VLA4 treated mice performed comparably to sham animals in the Barnes maze (p=0.398; p=0.972, respectively). Similarly, sham IgG (p&lt;0.0001), stroke anti-VCAM1 (p&lt;0.0001) and VLA4 (p=0.0059) could all perform the novel object task. Single-cell sequencing of immune and brain endothelial cells demonstrated that most transcriptional changes occur in endothelial cells and not immune cells, and that anti-VCAM1 and anti-VLA4 induce similar transcriptional changes compared to control IgG after stroke. Specifically, both therapeutic antibodies restore heat shock protein expression ( Dnaja1, Hsp90ab1, Hspa8 ) and decrease inflammatory genes CD74 and IL1r1 . Transcriptional changes also reflect increased translation and Sox18 upregulation, consistent with angiogenesis. Conclusion: Blocking either VCAM1 or VLA4 prevents infarct-induced neurodegeneration in aged mice. Changes in endothelial cell transcription and decreased immune cells in the brain may both contribute to therapeutic effects. Together, these findings establish the VCAM1-VLA4 axis as a promising target to prevent infarct-induced neurodegeneration.

Integrated Analysis of DNA Methylation and Gene Expression Profiles in a Rat Model of Osteoarthritis.

Osteoarthritis (OA) is common and affected by several factors, such as age, weight, sex, and genetics. The pathogenesis of OA remains unclear. Therefore, using a rat model of monosodium iodoacetate (MIA)-induced OA, we examined genomic-wide DNA methylation using methyl-seq and characterized the transcriptome using RNA-seq in the articular cartilage tissue from a negative control (NC) and MIA-induced rats. We identified 170 genes (100 hypomethylated and upregulated genes and 70 hypermethylated and downregulated genes) regulated by DNA methylation in OA. DNA methylation-regulated genes were enriched in functions related to focal adhesion, extracellular matrix (ECM)-receptor interaction and the PI3K-Akt and Hippo signaling pathways. Functions related to extracellular matrix organization, extracellular matrix proteoglycans, and collagen formation were involved in OA. A molecular and protein-protein network was constructed using methylated expression-correlated genes. Erk1/2 was a downstream target of OA-induced changes in DNA methylation and RNA expression. We found that the integrin subunit alpha 2 (ITGA2) gene is important in focal adhesion, alpha6-beta4 integrin signaling, and the inflammatory response pathway in OA. Overall, gene expression changes because DNA methylation influences OA pathogenesis. ITGA2, whose gene expression changes are regulated by DNA methylation during OA onset, is a candidate gene. Our findings provide insights into the epigenetic targets of OA processes in rats.

Inhibition of α4β1 Integrin Activity by Small Tellurium Compounds Regulates PD-L1 Expression and Enhances Antitumor Effects.

Various cancer treatment approaches that inhibit the activity of the programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) axis, a key player in tumor immune evasion, have been developed. We show that the immunomodulatory small tellurium complexes AS101 (ammonium trichloro(dioxoethylene-o,o')tellurate) and SAS (octa-O-bis(R,R)-tartarate ditellurane) suppress PD-L1 expression in a variety of human and mouse malignant cells via the modulation of α4β1 very late antigen-4 (VLA-4) integrin activity. Consequently, the expression of pAkt and its downstream effector pNFκB are inhibited. Additionally, SAS promotes the death of mouse malignant cells by activated syngeneic splenocytes or CD8+ T cells, preventing the development of chemoresistance in malignant cells. Moreover, AS101 and SAS may increase, at least in part, chemosensitivity through inhibition of the VLA-4/IL-10/PD-L1 pathway. Additionally, AS101 or SAS treatment of B16/F10 melanoma-bearing mice decreased tumor cell PD-L1 expression, leading to increased CD8+ T-cell infiltration into the tumors and tumor shrinkage. Combination treatment with an αPD-1 antibody and either tellurium compound significantly increased the antitumor efficacy of immunotherapy. Overall, VLA-4 integrin signaling is critical for tumor immune evasion and is a potential target for cancer treatment. Finally, AS101 or SAS, biologically active tellurium compounds, can effectively enhance the therapeutic efficacy of αPD-1-based cancer immunotherapy.

Assessment of Novel Erythroid Signaling Pathways Modulating Hydroxyurea-Mediated Reduction of Vaso-Occlusive Adhesive Interactions in Early Precursors of Sickle Red Blood Cells (RBCs)

Sickle cell disease (SCD) affects ~100,000 Americans and millions worldwide, with healthcare costs in the U.S. exceeding $1 billion annually to treat frequent and unpredictable vaso-occlusive episodes (VOEs). In addition to fetal hemoglobin induction, hydroxyurea (HU) reduces the frequency of VOEs by increasing nitric oxide (NO) bioavailability and decreasing adhesion receptor expression and RBC-endothelial interactions. Very late antigen-4 (VLA-4), the best characterized adhesion receptor in SCD, is highly expressed on circulating reticulocytes (immature RBCs) and white blood cells (WBCs) during VOEs and in SCD patients with severe disease phenotypes and, decreased in response to HU therapy. Like other integrins, VLA-4 is functionally regulated by cell signaling pathways to modulate activity and binding affinity to a wide variety of ligands that are elevated in the SCD micro-environment. There is highly intriguing evidence showing HU modulates adhesion by upregulating a NO/cGMP-dependent pathway to decrease adhesion receptor activity however specific mechanisms remain unclear. The objective of this study was to assess rapid cell signaling pathways involved in HU-mediated reduction in VLA-4 binding to endothelial vascular cell adhesion molecule-1 (VCAM-1). Whole blood (WB) was obtained from patients with SCD at benign clinic visits according to IRB-approved protocols. WB density-gradient centrifugation was used to isolate hematopoietic stem cells (HSCs) and WBCs from the buffy coat layer and, a CD71 monoclonal antibody (Ab) coupled with magnetic beads to isolate reticulocytes from the packed RBC layer. HSCs were matured to erythroblasts (EBs) in appropriate cell culture media and flow cytometric analyses assessed purity of blood samples using CD235a (exclusive WBC marker) and CD45 (exclusive RBC marker) Abs . Blood samples were pretreated with and without a therapeutically relevant hydroxyurea concentration (50mcM) followed by perfusion through micro-fluidic channels coated with VCAM-1 during pulsatile, shear flow (1.67Hz, 1.0 dyne/cm 2). Additionally, EBs were treated with HU combined and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1- one (ODQ; NO-dependent guanylate cyclase inhibitor). Adhered cells were quantified to establish an adhesion index. The non-parametric Wilcoxon matched pairs signed rank test was used to test the statistical differences between groups. Data are presented as mean ± standard error of mean. A p-value &amp;lt; 0.05 was considered statistically significant. There was a significant decrease in WB (p=0.01) and reticulocyte (p&amp;lt;0.0001) adhesion to VCAM-1 in HU-treated samples however, there was no effect on WBC (p&amp;gt;0.99) adhesion suggesting reticulocytes reduce the adhesive burden of blood cells in WB adhesion assays and modulation of VLA-4 binding differs in sickle RBCs when compared to WBCs. More recently, preliminary data from my lab demonstrated that EBs pretreated with HU reduces VLA-4 binding to VCAM-1 (from 126.5 ± 29.42 to 109.5 ± 29.09) by cGMP-dependent mechanisms (HU + ODQ; from 109.5 ± 29.09 to 120.2 ± 42.80). Laboratory-based static and flow adhesion assays have been used for decades to describe adhesion in SCD since earlier studies established that sickle RBCs are more adherent than non-sickle blood cells and, adhesion correlates with SCD severity. For example, the adhesion assay described above has been used to demonstrate that VLA-4 binding can stratify SCD patients based on disease severity and predict impending VOEs. Additionally, reticulocyte and HbF levels strongly correlate with VLA-4 binding to VCAM-1 and, VLA-4-mediated adhesion is increased during patient-reported VOEs and decreased in HU-treated SCD patients. This is the first SCD study demonstrating the role of HU in modulating VLA-4 binding to endothelial VCAM-1 through rapid erythroid signaling pathways. There is also compelling evidence involving downregulation of VLA-4 activity/binding affinity through NO/cGMP dependent mechanisms and enhancement of VLA-4 binding by kinase-dependent pathways. HU has also been shown to modulate adhesion by upregulating a NO/cGMP-dependent pathway to decrease adhesion receptor activity. Collectively, these data suggest that VLA-4 mechanisms may be targeted to therapeutically inhibit vaso-occlusive adhesive events that promote VOEs in SCD.

CD49d Shapes B Cell Receptor Responsiveness and Tissue Distribution in an Aggressive Chronic Lymphocytic Leukemia Murine Model

Background: High expression of the very late antigen-4 (VLA-4) integrin subunit CD49d predicts disease progression in chronic lymphocytic leukemia (CLL). We previously demonstrated that B cell receptor (BCR) signals induce activation of VLA-4 (Tissino et al., J Exp Med. 2018) and characterized Eµ-TCL1-transgenic (TCL1-tg) mice as an adequate model for the human high-risk CD49d-high CLL patient subgroup, with VLA-4 inhibition hampering the disease (Szenes et al., Leukemia 2020). Here, we address the consequences of genetic B cell-specific ablation of CD49d on disease development, engraftment capacity and treatment susceptibility in a novel CLL mouse model. Methods: We crossed ITGA4fl/fl mice, kindly provided by T. Papayannopoulou, University of Washington, with TCL1-tg mice and CD19 Cre mice, subsequently named CD49d∆B TCL1-tg. Tumor development was continuously monitored by regular blood checks and mice were sacrificed at 10 months. Tumor infiltration was quantified in peritoneal cavity (PC), spleen and bone marrow by flow cytometry using anti-CD5/CD19 antibodies. Integrin and chemokine receptor expression was profiled. In vitro responsiveness to BCR stimulation of CD49d-deficient and proficient leukemic cells was assessed using phospho-specific flow cytometry. Furthermore, purified leukemic cells derived from PC or spleen were subjected to RNA-seq analyses. CD49d-deficient or proficient leukemic cells were transplanted into wild-type C57BL/6J recipients in short and long term experiments. Ongoing: CD49d-deficient or proficient leukemic cells were transplanted competitively into C57BL/6J recipients and are receiving continuous Bruton's tyrosine kinase (BTK)-inhibitor (acalabrutinib) treatment upon tumor onset. Results: CD49d∆B TCL1-tg mice showed a B cell-specific integrin loss in all organs. Blood tumor burden was measured monthly. At months seven and eight, leukemic cells of CD49d∆B TCL1-tg mice were reduced by approx. 25% compared to TCL1-tg littermates (Fig. 1A). This trend was reversed at month nine, with approx. 45% increased lymphocytosis in CD49d∆B TCL1-tg mice. CD49d expression in leukemic cells of TCL1-tg mice was unaffected by disease progression. At sacrifice, CD49d∆B TCL1-tg mice showed an 80% ( p=&amp;lt;0.0001) reduction of tumor burden in the bone marrow (Fig. 1B), but comparable leukemic cell numbers in PC and spleen. Leukemic splenocytes derived from CD49d∆B TCL1-tg mice displayed 38% ( p=0.032) higher IgM and 33% higher CD44 expression but 43% ( p=0.015) reduced expression of CXCR4. BCR signaling was modulated by the integrin loss and BCR stimulation with anti-IgM induced 43% ( p=0.022) higher PLCγ2 and 45% ( p=0.022) higher SYK phosphorylation in leukemic cells derived from CD49d∆B TCL1-tg mice compared to littermates, but comparable BTK phosphorylation. RNA-seq analysis revealed strong differences in leukemic cell transcriptomes between PC and spleen, indicating tissue dependent effects on leukemic cells. Particularly, TNF-α signaling via NF-κB, MTORC1, MAPK signaling and JAK-STAT signaling pathways were upregulated in leukemic cells derived from spleen compared to PC irrespective of CD49d expression, while chemokine signaling pathways were downregulated in PC. When comparing CD49d deficient and proficient leukemic cells, the gene set MYC targets v1 was enriched in deficient cells. When injecting CD49d-deficient leukemic splenocytes intravenously, more cells were recovered after 5 days from blood ( p=0.048) and spleen compared to CD49d-proficient cells, but did not engraft in bone marrow or peritoneum. When injected intraperitonally, CD49d-deficient leukemic cells were found in peritoneum in contrast to TCL1-tg littermates ( p=&amp;lt;0.0001). Conclusion: The TCL1 mouse model resembles a human high-risk CLL (e.g. IGHV unmutated, CD49d-expressing), and thus allows to investigate the impact of CD49d expression in this CLL subset. Loss of CD49d expression resulted in an altered redistribution of leukemic cells in tissue sites with a significant reduction of leukemic cells in the bone marrow. Lack of integrin signals resulted in increased antigen-induced BCR signaling and IgM expression, as well as a B cell receptor activation phenotype associated with MYC gene set enrichment, indicating a feedback loop from integrins to the BCR. Currently, the susceptibility of CD49d∆B TCL1-tg mice to BTK inhibitors is under investigation.

Comparison of Various Stem Cell Mobilization Regimens for Multiple Myeloma - a 15-Year Retrospective Institutional Analysis from India

Introduction For multiple myeloma (MM), Cyclophosphamide (Cy) based chemo-mobilization offers advantage in term of higher stem cell yield and can overcome adverse impact of prior lenalidomide therapy on stem cell harvest. However, considering need for hospital stay and risk of febrile neutropenia, steady state mobilization with G-CSF and pre-emptive plerixafor (PEF) is becoming increasingly popular. Until few years back, cost of PEF was prohibitive thereby limiting its use and prompting a search for alternative cheaper drugs. Mouse models have shown that Proteasome Inhibitors aid in hematopoietic stem-cell (HSC) mobilization by down-regulating very-late antigen-4 (VLA-4) on bone marrow stromal cells which interacts with vascular cell adhesion molecule (VCAM-1) on HSC. We previously reported that bortezomib (Bort) in combination with Cy-G-CSF showed a trend towards higher CD34 yield and reduced need for apheresis sessions, without additional toxicity. There is limited data comparing mobilization regimens, especially from low-middle income countries where HSC collection may not be done after 4-6 cycles of first-line therapy. We hereby report our 15-year experience of various mobilization regimens for MM. Methods All patients with MM who underwent HSC mobilization from September 2007 - December 2022 were included in this analysis. Various mobilization regimens [Group 1 - G-CSF + Bort (August 2018 - December 2022); Group 2 - G-CSF + plerixafor (August 2018 - December 2022); Group 3 - Bort-Cy-GCSF (April 2016 - July 2018); Group 4 - Cy + G-CSF (September 2007 - March 2016)] were used as per respective time-periods (Table 1). Our aim was to collect CD34 cell dose of 5million/kg to suffice for 2 transplants. G-CSF (5 mcg/kg subcutaneously BD) was given on days 1-5 (Groups 1 and 2). Bort (1.3mg/m 2 iv / sc) (Group 1) or PEF (Group 2) were given on day 4 twelve hours prior to planned apheresis on day 5. Apheresis was done on day 5, and additionally on day 6, if first harvest was inadequate for 2 transplants. In group 3, Bort (1.3mg/m 2 iv) was given on days-1,4,8,11, with Cy (1gm/m 2 iv) on days 8 and 9, followed by G-CSF from day 11 onwards. Cy (1gm/m 2 iv) was given on day 1 and 2 (Group 4), followed by G-CSF from day 4 onwards. Stem cell harvest was done in groups 3 and 4 once peripheral blood CD34 (PB CD34) was &amp;gt;20/µL. In groups 3 and 4, if PB CD34 was &amp;lt;20/µL for 2 subsequent days, PEF was used according to physician's discretion, if available. Similarly in Group 1, if PB CD34 was between 10-20/µl on day 4, PEF was used according to physician's discretion. PEF was used in any group after the first harvest if the cell dose was deemed inadequate, at physician's discretion. Primary objective was to determine proportion of patients with CD34 + dose ≥5x10 6/kg in first apheresis in various groups. Secondary objectives were to determine total median CD34 + dose (x10 6/kg) collected, median CD34 + (x10 6/kg) in first apheresis, and frequency of mobilization failure. Mobilization failure was defined as total collected CD34 + dose of &amp;lt;2x10 6/kg or abandoned harvest attempt anticipating a poor collection.Analysis was done as per planned regimen (intention to treat analysis). Results Total 205 patients were analyzed. Median age was 48 years (27-65 years) and 143 (70%) were males (Table 1). Median day of first collection in groups 3 and 4 was day10 from the first dose of cyclophosphamide. Percentage of patients who collected ≥5x10 6 CD34 + cells/kg in first apheresis were 26%, 53%, 69% and 63%, respectively in Groups 1-4 (p=0.0001) (Table 1). CD34+ cell-dose in first apheresis and total dose collected are shown in Table 1. In comparison to groups 1 and 2, both chemo-mobilization groups (group 3 and 4) had a significantly higher total cell dose (Figure 1). In comparison to group 1, both Cy chemo-mobilization groups had a significantly higher cell dose in first harvest, but there was no significant difference between group 2 and group 4 (p=0.23) (Figure 1). PEF was required additionally in 47%, 22%, and 7% of patients in groups 1, 3 and 4 respectively (P&amp;lt;0.00001). Amongst transplanted patients, there was no difference in neutrophil or platelet engraftment and the frequency of engraftment syndrome in various groups. Conclusion Cy-based chemo-mobilization regimens have advantage of higher total stem cell yield, while they are equivalent to G-CSF + Plerixafor for harvest in single apheresis. Addition of Bortezomib to Cyclophosphamide may help to increase stem cell yield.

CC16 drives VLA-2-dependent SPLUNC1 expression.

CC16 (Club Cell Secretory Protein) is a protein produced by club cells and other non-ciliated epithelial cells within the lungs. CC16 has been shown to protect against the development of obstructive lung diseases and attenuate pulmonary pathogen burden. Despite recent advances in understanding CC16 effects in circulation, the biological mechanisms of CC16 in pulmonary epithelial responses have not been elucidated. We sought to determine if CC16 deficiency impairs epithelial-driven host responses and identify novel receptors expressed within the pulmonary epithelium through which CC16 imparts activity. We utilized mass spectrometry and quantitative proteomics to investigate how CC16 deficiency impacts apically secreted pulmonary epithelial proteins. Mouse tracheal epithelial cells (MTECS), human nasal epithelial cells (HNECs) and mice were studied in naïve conditions and after Mp challenge. We identified 8 antimicrobial proteins significantly decreased by CC16-/- MTECS, 6 of which were validated by mRNA expression in Severe Asthma Research Program (SARP) cohorts. Short Palate Lung and Nasal Epithelial Clone 1 (SPLUNC1) was the most differentially expressed protein (66-fold) and was the focus of this study. Using a combination of MTECs and HNECs, we found that CC16 enhances pulmonary epithelial-driven SPLUNC1 expression via signaling through the receptor complex Very Late Antigen-2 (VLA-2) and that rCC16 given to mice enhances pulmonary SPLUNC1 production and decreases Mycoplasma pneumoniae (Mp) burden. Likewise, rSPLUNC1 results in decreased Mp burden in mice lacking CC16 mice. The VLA-2 integrin binding site within rCC16 is necessary for induction of SPLUNC1 and the reduction in Mp burden. Our findings demonstrate a novel role for CC16 in epithelial-driven host defense by up-regulating antimicrobials and define a novel epithelial receptor for CC16, VLA-2, through which signaling is necessary for enhanced SPLUNC1 production.

Effects of a gut-selective integrin-targeted therapy in male mice exposed to early immune activation, a model for the study of autism spectrum disorder

To clarify the role of gut mucosal immunity in ASD, we evaluated, in the early-life immune activation (EIA) mouse model, the effects of administration of a monoclonal antibody directed against the integrin alpha4 beta7 (α4β7 mAb), blocking the leukocyte homing into the gut mucosa. EIA is a double-hit variant of the maternal immune-activation (MIA) model, including both prenatal (Poly I:C) and postnatal (LPS) immune challenges.In C57BL6/J EIA male adult offspring mice, IL-1β and IL-17A mRNA colonic tissue content increased when compared with controls. Cytofluorimetric analyses of lymphocytes isolated from mesenteric lymph-nodes (MLN) and spleens of EIA mice show increased percentage of total and CD4+α4β7+, unstimulated and stimulated IL-17A+ and stimulated IFN-γ+ lymphocytes in MLN and CD4+α4β7+ unstimulated and stimulated IL-17A+ and stimulated IFN-γ+ lymphocytes in the spleen. Treatment with anti-α4β7 mAb in EIA male mice was associated with colonic tissue IL-1β, and IL-17A mRNA content and percentage of CD4+ IL-17A+ and IFN-γ+ lymphocytes in MLN and spleens comparable to control mice. The anti-α4β7 mAb treatment rescue social novelty deficit showed in the three-chamber test by EIA male mice. Increased levels of IL-6 and IL-1β and decreased CD68 and TGF-β mRNAs were also observed in hippocampus and prefrontal cortex of EIA male mice together with a reduction of BDNF mRNA levels in all brain regions examined. Anti-α4β7 mAb treatment restored the expression of BDNF, TGF-β and CD68 in hippocampus and prefrontal cortex.Improvement of the gut inflammatory status, obtained by a pharmacological agent acting exclusively at gut level, ameliorates some ASD behavioral features and the neuroinflammatory status. Data provide the first preclinical indication for a therapeutic strategy against gut-immune activation in ASD subjects with peripheral increase of gut-derived (α4β7+) lymphocytes expressing IL-17A.

High-intensity interval training attenuates development of autoimmune encephalomyelitis solely by systemic immunomodulation

The impact of high-intensity interval training (HIIT) on the central nervous system (CNS) in autoimmune neuroinflammation is not known. The aim of this study was to determine the direct effects of HIIT on the CNS and development of experimental autoimmune encephalomyelitis (EAE). Healthy mice were subjected to HIIT by treadmill running and the proteolipid protein (PLP) transfer EAE model was utilized. To examine neuroprotection, PLP-reactive lymph-node cells (LNCs) were transferred to HIIT and sedentary (SED) mice. To examine immunomodulation, PLP-reactive LNCs from HIIT and SED donor mice were transferred to naïve recipients and analyzed in vitro. HIIT in recipient mice did not affect the development of EAE following exposure to PLP-reactive LNCs. HIIT mice exhibited enhanced migration of systemic autoimmune cells into the CNS and increased demyelination. In contrast, EAE severity in recipient mice injected with PLP-reactive LNCs from HIIT donor mice was significantly diminished. The latter positive effect was associated with decreased migration of autoimmune cells into the CNS and inhibition of very late antigen (VLA)-4 expression in LNCs. Thus, the beneficial effect of HIIT on EAE development is attributed solely to systemic immunomodulatory effects, likely because of systemic inhibition of autoreactive cell migration and reduced VLA-4 integrin expression.

Human BBB-on-a-chip reveals barrier disruption, endothelial inflammation, and T cell migration under neuroinflammatory conditions.

The blood-brain barrier (BBB) is a highly selective barrier that ensures a homeostatic environment for the central nervous system (CNS). BBB dysfunction, inflammation, and immune cell infiltration are hallmarks of many CNS disorders, including multiple sclerosis and stroke. Physiologically relevant human in vitro models of the BBB are essential to improve our understanding of its function in health and disease, identify novel drug targets, and assess potential new therapies. We present a BBB-on-a-chip model comprising human brain microvascular endothelial cells (HBMECs) cultured in a microfluidic platform that allows parallel culture of 40 chips. In each chip, a perfused HBMEC vessel was grown against an extracellular matrix gel in a membrane-free manner. BBBs-on-chips were exposed to varying concentrations of pro-inflammatory cytokines tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL-1β) to mimic inflammation. The effect of the inflammatory conditions was studied by assessing the BBBs-on-chips' barrier function, cell morphology, and expression of cell adhesion molecules. Primary human T cells were perfused through the lumen of the BBBs-on-chips to study T cell adhesion, extravasation, and migration. Under inflammatory conditions, the BBBs-on-chips showed decreased trans-endothelial electrical resistance (TEER), increased permeability to sodium fluorescein, and aberrant cell morphology in a concentration-dependent manner. Moreover, we observed increased expression of cell adhesion molecules and concomitant monocyte adhesion. T cells extravasated from the inflamed blood vessels and migrated towards a C-X-C Motif Chemokine Ligand 12 (CXCL12) gradient. T cell adhesion was significantly reduced and a trend towards decreased migration was observed in presence of Natalizumab, an antibody drug that blocks very late antigen-4 (VLA-4) and is used in the treatment of multiple sclerosis. In conclusion, we demonstrate a high-throughput microfluidic model of the human BBB that can be used to model neuroinflammation and assess anti-inflammatory and barrier-restoring interventions to fight neurological disorders.

Targeted imaging of very late antigen-4 for noninvasive assessment of lung inflammation-fibrosis axis

BackgroundThe lack of noninvasive methods for assessment of dysregulated inflammation as a major driver of fibrosis (i.e., inflammation-fibrosis axis) has been a major challenge to precision management of fibrotic lung diseases. Here, we determined the potential of very late antigen-4 (VLA-4)-targeted positron emission tomography (PET) to detect inflammation in a mouse model of bleomycin-induced fibrotic lung injury.MethodSingle time-point and longitudinal VLA-4-targeted PET was performed using a high-affinity peptidomimetic radiotracer, 64Cu-LLP2A, at weeks 1, 2, and 4 after bleomycin-induced (2.5 units/kg) lung injury in C57BL/6J mice. The severity of fibrosis was determined by measuring the hydroxyproline content of the lungs and expression of markers of extracellular matrix remodeling. Flow cytometry and histology was performed to determine VLA-4 expression across different leukocyte subsets and their spatial distribution.ResultsLung uptake of 64Cu-LLP2A was significantly elevated throughout different stages of the progression of bleomycin-induced injury. High lung uptake of 64Cu-LLP2A at week-1 post-bleomycin was a predictor of poor survival over the 4-week follow up, supporting the prognostic potential of 64Cu-LLP2A PET during the early stage of the disease. Additionally, the progressive increase in 64Cu-LLP2A uptake from week-1 to week-4 post-bleomycin correlated with the ultimate extent of lung fibrosis and ECM remodeling. Flow cytometry revealed that LLP2A binding was restricted to leukocytes. A combination of increased expression of VLA-4 by alveolar macrophages and accumulation of VLA-4-expressing interstitial and monocyte-derived macrophages as well as dendritic cells was noted in bleomycin-injured, compared to control, lungs. Histology confirmed the increased expression of VLA-4 in bleomycin-injured lungs, particularly in inflamed and fibrotic regions.ConclusionsVLA-4-targeted PET allows for assessment of the inflammation-fibrosis axis and prediction of disease progression in a murine model. The potential of 64Cu-LLP2A PET for assessment of the inflammation-fibrosis axis in human fibrotic lung diseases needs to be further investigated.

Increased Bone Marrow Uptake and Accumulation of Very-Late Antigen-4 Targeted Lipid Nanoparticles.

Lipid nanoparticles (LNPs) have evolved rapidly as promising delivery systems for oligonucleotides, including siRNAs. However, current clinical LNP formulations show high liver accumulation after systemic administration, which is unfavorable for the treatment of extrahepatic diseases, such as hematological disorders. Here we describe the specific targeting of LNPs to hematopoietic progenitor cells in the bone marrow. Functionalization of the LNPs with a modified Leu-Asp-Val tripeptide, a specific ligand for the very-late antigen 4 resulted in an improved uptake and functional siRNA delivery in patient-derived leukemia cells when compared to their non-targeted counterparts. Moreover, surface-modified LNPs displayed significantly improved bone-marrow accumulation and retention. These were associated with increased LNP uptake by immature hematopoietic progenitor cells, also suggesting similarly improved uptake by leukemic stem cells. In summary, we describe an LNP formulation that successfully targets the bone marrow including leukemic stem cells. Our results thereby support the further development of LNPs for targeted therapeutic interventions for leukemia and other hematological disorders.