Gili Hart

Anti-inflammatory effects of an inflammatory chemokine: CCL2 inhibits lymphocyte homing by modulation of CCL21 triggered integrin-mediated adhesions

Our studies focus on the pathways that restrict homing of specific subsets of immune cells, and thereby fine-tune the immune response at specific lymphoid and peripheral tissues. Here, we report that CCL2 (at picomolar [pM] levels) renders both murine and human T cells defective in their ability to develop CCR7-triggered activation of LFA-1- and LFA-1-mediated adhesion strengthening to endothelial ICAM-1 both in vitro and in vivo. CCL2 also attenuated lymphocyte chemotaxis toward lymph node chemokines. Consequently, low-dose CCL2 inhibited lymphocyte homing to peripheral lymph nodes but did not affect lymphocyte trafficking through the spleen. Impaired homing of lymphocytes to peripheral lymph nodes resulted in attenuated progression of both asthma and adjuvant arthritis. Thus, pM levels of circulating CCL2 can exert global suppressive effects on T-cell trafficking and differentiation within peripheral lymph nodes, and may be clinically beneficial as an anti-inflammatory agent.

CCL2 (pM levels) as a therapeutic agent in inflammatory bowel disease models in mice

Background: Chemokines regulate the pathways that restrict homing of specific subsets of immune cells, and thereby fine tune the immune response at specific lymphoid and peripheral tissues. CCL2 is a chemokine that induces migration of monocytes, memory T cells, and dendritic cells. Previously, we demonstrated that pM levels of CCL2 dramatically inhibit migration of T cells. The aim was to test whether subphysiological doses of CCL2 can ameliorate murine colitis and inflammation‐induced colorectal cancer. Methods: TNBS (2,4,6 trinitrobenzene sulfonic acid) colitis and dextran sodium sulfate (DSS) colitis were induced in Balb/c and C57BL/6 mice, respectively. Mice were treated daily with intraperitoneal CCL2 injections. Disease activity was assessed clinically, histologically, and by measuring inflammatory cytokine levels. In addition, an inflammatory cancer model was induced by azoxymethane‐DSS (AOM‐DSS) in Balb/c mice. Mice were treated daily with CCL2 for 11 weeks and then assessed for number of tumors in the colons. Results: Daily administration of CCL2 (60–120 ng) significantly decreased the development of TNBS‐ and DSS‐induced colitis. In a DSS‐AOM model, CCL2‐treated mice developed significantly fewer tumors (P < 0.005) at 11 weeks. Chronic inflammation in the CCL2‐treated mice was significantly less pronounced as compared to phosphate‐buffered saline‐treated mice. Conclusions: Administration of pM levels of CCL2 significantly inhibits migration of T cells in amelioration of TNBS and DSS colitis and inhibits development of colorectal cancer in an AOM‐DSS colitis model in mice. Thus, pM levels of CCL2 may be clinically beneficial as an antiinflammatory agent in IBD. (Inflamm Bowel Dis 2010)

Ly49D Receptor Expressed on Immature B Cells Regulates Their IFN-γ Secretion, Actin Polymerization, and Homing

Low levels of IFN-γ secreted by immature B cells prevent their own migration and homing to the lymph nodes and premature encounter with Ag. In this study we followed the mechanism regulating IFN-γ secretion by immature B cells. We show that the MHC class I receptor, Ly49D, is expressed on immature B cells and is down-regulated during maturation. Activation of this receptor leads to increase in IFN-γ transcription and translation and results in the altered ability of B cells to polymerize actin in response to chemokine stimulation. Moreover, we show that H2-D blockage inhibits the ability of immature B cells to transcribe the IFN-γ gene and results in rescue of cytoskeletal rearrangement. Thus, Ly49D that is expressed on immature B cells recognizes MHC class I on the peripheral tissues, inducing the secretion of low levels of IFN-γ and thereby down-regulating immature B cell homing to the lymph nodes or to sites of inflammation.

Tight Regulation of IFN-γ Transcription and Secretion in Immature and Mature B cells by the Inhibitory MHC Class I Receptor, Ly49G2

To complete their maturation and to participate in the humoral immune response, immature B cells that leave the bone marrow are targeted to specific areas in the spleen, where they differentiate into mature cells. Previously, we showed that immature B cells actively down-regulate their integrin-mediated migration to lymph nodes or sites of inflammation, enabling their targeting to the spleen to allow their final maturation. This inhibition is mediated by IFN-γ, which is transcribed and secreted at low levels by these immature B cells and is down-regulated at the mature stage. The activating MHC class I receptor, Ly49D, which is expressed at high levels on immature B cells, stimulates this IFN-γ secretion. In this study we show that B cells coexpress the inhibitory MHC class I receptor, Ly49G2. In addition, we demonstrate a tight regulation in the expression of the Ly49 family members on B cells that depends on their cell surface levels. High levels of Ly49G2 have a dominant inhibitory effect on Ly49D expressed at low levels on immature bone marrow and mature B cells, resulting in inhibition of IFN-γ secretion. However, low levels of the inhibitory receptor, Ly49G2, coexpressed with high levels of the activating receptor, Ly49D, on the immigrating immature B cells enable the secretion of specific low levels of IFN-γ. This expression pattern insures the inhibitory control of peripheral immature B cell to prevent premature encounter with an Ag while enabling entry to the lymph nodes during the mature stage.

IL-12 and IL-18 down-regulate B cell migration in an Ly49D-dependent manner

In order to complete their maturation and participate in the humoral immune response, immature B cells that leave the bone marrow are targeted to specific areas in the spleen, where they differentiate into mature cells. Previously, we showed that immature B cells actively down‐regulate their integrin‐mediated migration to LN or to sites of inflammation, enabling their targeting to the spleen. This inhibition is mediated by IFN‐γ, which is transcribed and secreted at low levels by these immature B cells; its expression is subsequently down‐regulated following B cell maturation. The activating and inhibitory MHC class I receptors, Ly49D and Ly49G2, regulate IFN‐γ secretion in B cells, preventing their migration to antigen‐enriched sites and their premature encounter with an antigen, while enabling their entry into the LN when mature. In the present study, we elucidate the pathways by which the Ly49 receptors regulate IFN‐γ levels. We show that Ly49D stimulation triggers a signaling cascade that increases transcription of both IL‐12B and IL‐18; these, in turn, can interact with their specific receptors, which are expressed at elevated levels on immature B cells. Ligation of the IL‐12B and IL‐18 receptors induces the secretion of IFN‐γ, thereby regulating their cytoskeleton rearrangement and migration.

In Vitro and in Vivo Characterization of MOD-4023, a Long-Acting Carboxy-Terminal Peptide (CTP)-Modified Human Growth Hormone.

MOD-4023 is a novel long-acting version of human growth hormone (hGH), containing the carboxy-terminal peptide (CTP) of human chorionic gonadotropin (hCG). MOD-4023 is being developed as a treatment for adults and children with growth hormone deficiency (GHD), which would require fewer injections than currently available GH formulations and thus reduce patient discomfort and increase compliance. This study characterizes MOD-4023s binding affinities for the growth hormone receptor, as well as the pharmacokinetic and pharmacodynamics, toxicology, and safety profiles of repeated dosing of MOD-4023 in Sprague-Dawley rats and Rhesus monkeys. Although MOD-4023 exhibited reduced in vitro potency and lower affinity to the GH receptor than recombinant hGH (rhGH), administration of MOD-4023 every 5 days in rats and monkeys resulted in exposure comparable to daily rhGH, and the serum half-life of MOD-4023 was significantly longer. Repeated administration of MOD-4023 led to elevated levels of insulin-like growth factor 1 (IGF-1), and twice-weekly injections of MOD-4023 resulted in larger increase in weight gain with fewer injections and a lower accumulative hGH dose. Thus, the increased half-life of MOD-4023 in comparison to hGH may increase the frequency of protein-receptor interactions and compensate for its decreased in vitro potency. MOD-4023 was found to be well-tolerated in rats and monkeys, with minimal adverse events, suggesting an acceptable safety profile. These results provide a basis for the continued clinical development of MOD-4023 as a novel treatment of GHD in children and adults.

Mad3 Negatively Regulates B Cell Differentiation in the Spleen by Inducing Id2 Expression

Id2 is a negative regulator of B cell differentiation. Its expression was found to depend on Myc–Max–Mad transcriptional complexes. Here, we show that Mad3 expression levels that are elevated in immature B cells are actively involved in inducing Id2 expression by binding to its promoter, resulting in its augmented expression.

CD151 Regulates T-Cell Migration in Health and Inflammatory Bowel Disease.

Abstract:The continuous recirculation of mature lymphocytes and their entry into the peripheral lymph nodes are crucial for the development of an immune response to foreign antigens. Occasionally, the entry and the subsequent response of T lymphocytes in these sites lead to severe inflammation and pathological conditions. Here, we characterized the tetraspanin molecule, CD151, as a regulator of T cell motility in health and in models of inflammatory bowel disease. CD151 formed a cell surface complex with VLA-4 and LFA-1 integrins, and its activation led to enhanced migration of T cells. Picomolar levels of CCL2 that were previously shown to inhibit T-cell migration to lymph nodes suppressed CD151 expression and dissociated CD151-integrin complexes in T lymphocytes, resulting in attenuated migration toward T-cell attractant chemokines. To directly inhibit CD151 function, a truncated CD151 peptide fragment mimicking of the CD151 extracellular loop was designed. CD151 extracellular loop inhibited T-cell migration in vitro and in vivo and attenuated the development of dextrane sulfate sodium-induced colitis. Thus, CD151 is a key orchestrator of T cell motility; interference with its proper function results in attenuated progression of inflammatory bowel disease.

Long-Acting C-Terminal Peptide–Modified hGH (MOD-4023): Results of a Safety and Dose-Finding Study in GHD Children

Context Daily injections are required for growth hormone (GH) replacement therapy, which may cause low compliance as a result of inconvenience and distress in patients. Objective C-terminal peptide-modified human GH (MOD-4023) is developed for once-a-week dosing regimen in GH-deficient (GHD) adults and children. The present trial was a safety and dose-finding study for weekly MOD-4023 in GHD children. Design A multicenter, open-label, randomized, controlled phase 2 study in children with GHD, evaluating the safety, tolerability, pharmacokinetics/pharmacodynamics, and efficacy of three different weekly MOD-4023 doses, compared with daily recombinant human GH (r-hGH). Setting The trial was conducted in 14 endocrinology centers in Europe. Patients Fifty-three prepubertal children with GHD completed 12 months of treatment with either MOD-4023 (N = 42) or r-hGH (N = 11). Interventions C-terminal peptide-modified hGH (MOD-4023) was administered weekly at a dose of either 0.25, 0.48, or 0.66 mg/kg/wk and compared with daily hGH at a dose of 0.24 mg/kg/wk. Results MOD-4023 showed an estimated half-life approximately fivefold to 10-fold longer when compared with daily r-hGH. Insulin-like growth factor (IGF)-I and IGF-binding peptide 3 showed a dose-dependent increase during MOD-4023 treatment. IGF-I standard deviation score for MOD-4023 did not exceed +2. All MOD-4023 cohorts demonstrated adequate catch-up growth. The 0.66 mg/kg/wk dose demonstrated efficacy closest to daily r-hGH. No serious adverse events were observed during MOD-4023 treatment, and its tolerability was consistent with known properties of r-hGH. Conclusions This study confirms the long-acting properties of MOD-4023 and shows a promising safety and tolerability profile. This provides support for initiation of a phase 3 study in GHD children using a single weekly injection of MOD-4023.

Pharmacokinetic and Pharmacodynamic Modeling of MOD-4023, a Long-Acting Human Growth Hormone, in Growth Hormone Deficiency Children

Background/Aims: MOD-4023 is a long-acting human growth hormone (hGH) in clinical trials for the treatment of growth hormone deficiency (GHD). A key goal is maintenance of serum concentrations of insulin-like growth factor (IGF) 1 within normal range throughout GH dosing. The study aimed to develop a pharmacokinetic model for MOD-4023 and a pharmacodynamic model for the effect of MOD-4023 on IGF-1 to allow estimation of peak and mean IGF-1 and to identify the optimal IGF-1 sampling day. Methods: MOD-4023 (0.25, 0.48, or 0.66 mg/kg) was administered weekly for 12 months to 41 GH-naive GHD children (age 3–11 years). The control group (n = 11, age 4–9 years) received daily recombinant human growth hormone (r-hGH; 34 µg/kg). Sparse samples (4/subject) were obtained to determine serum concentrations of MOD-4023 or r-hGH and IGF-1. Results: A 2-compartment pharmacokinetic model with first-order absorption fit MOD-4023 data well; a 1-compartment model was appropriate for r-hGH. For both, weight-normalized systemic parameters were preferred over allometric scaling. For MOD-4023, an indirect model fit IGF-1 SDS data well; baseline IGF-1 increased over time. At steady state, samples obtained 4 days following dose administration predicted mean IGF-1 SDS during the dosing interval well. Conclusion: The IGF-1 profile is consistent with the weekly dosing interval. Sampling 4 days following dose administration allows estimation of mean IGF-1 SDS during the dosing interval in GHD patients.