Long-term safety and efficacy of daclizumab beta in relapsing-remitting multiple sclerosis: 6-year results from the SELECTED open-label extension study.

OBJECTIVE: SELECTED, an open-label extension study, evaluated daclizumab beta treatment for up to 6 years in participants with relapsing multiple sclerosis who completed the randomized SELECT/SELECTION studies. We report final results of SELECTED. METHODS: Eligible participants who completed 1-2 years of daclizumab beta treatment in SELECT/SELECTION received daclizumab beta 150 mg subcutaneously every 4 weeks for up to 6 years in SELECTED. Safety assessments were evaluated for the SELECTED treatment period; efficacy data were evaluated from first dose of daclizumab beta in SELECT/SELECTION. RESULTS: Ninety percent (410/455) of participants who completed treatment in SELECTION enrolled in SELECTED. Within SELECTED, 69% of participants received daclizumab beta for > 3 years, 39% for > 4 years, and 9% for > 5 years; 87% of participants experienced an adverse event and 26% a serious adverse event (excluding multiple sclerosis relapse). No deaths occurred. Overall, hepatic events were reported in 25% of participants; serious hepatic events in 2%. There were no confirmed cases of immune-mediated encephalitis. Based on weeks from the first daclizumab beta dose in SELECT/SELECTION, adjusted annualized relapse rate (95% confidence interval) for weeks 0-24 was 0.21 (0.16-0.29) and remained low on continued treatment. Overall incidence of 24-week confirmed disability progression was 17.4%. Mean numbers of new/newly enlarging T2 hyperintense lesions remained low; percentage change in whole brain volume decreased over time. CONCLUSIONS: The effects of daclizumab beta on clinical and radiologic outcomes were sustained for up to ~ 8 years of treatment. No new safety concerns were identified in SELECTED. TRIAL REGISTRATION: Clinicaltrials.gov NCT01051349; first registered on January 15, 2010.

Circulating lymphocyte levels and relationship with infection status in patients with relapsing-remitting multiple sclerosis treated with daclizumab beta.

BACKGROUND: Reversible lymphocyte count reductions have occurred following daclizumab beta treatment for relapsing-remitting multiple sclerosis. OBJECTIVE: To analyse total and differential lymphocyte levels and relationship with infection status. METHODS: In DECIDE, blood samples were collected at 12-week intervals from daclizumab beta- ( n = 919) or intramuscular interferon beta-1a-treated ( n = 922) patients. Infections/serious infections were assessed proximate to grade 2/3 lymphopenia or low CD4+/CD8+ T-cell counts. Total safety population (TSP) data were additionally analysed from the entire clinical development programme ( n = 2236). RESULTS: Over 96 weeks in DECIDE, mean absolute lymphocyte count (ALC), CD4+ and CD8+ T-cell counts decreased <10% (7.1% vs 1.6%, 9.7% vs 2.0%, 9.3% vs 5.9%: daclizumab beta vs interferon beta-1a, respectively); shifts to ALC below lower limit of normal occurred in 13% versus 15%, respectively. Grade 3 lymphopenia was uncommon (TSP: <1%) and transient. Lymphocyte changes generally occurred within 24 weeks after treatment initiation and were reversible within 12 weeks of discontinuation. In DECIDE, mean CD4+/CD8+ T-cell counts were similar regardless of infection status. TSP data were consistent with DECIDE. CONCLUSION: When observed, ALC and CD4+/CD8+ T-cell count decreases in daclizumab beta-treated patients were generally mild-to-modest, reversible upon treatment discontinuation and not associated with increased risk of infections, including opportunistic infections.

No evidence of disease activity in patients receiving daclizumab versus intramuscular interferon beta-1a for relapsing-remitting multiple sclerosis in the DECIDE study.

BACKGROUND: No evidence of disease activity (NEDA) is a composite endpoint being increasingly applied as an outcome measure in clinical trials as well as proposed for individual therapeutic decisions in multiple sclerosis (MS). OBJECTIVE: Assess the proportion of patients with relapsing-remitting MS achieving NEDA in the DECIDE study of daclizumab 150 mg subcutaneous versus intramuscular interferon beta-1a 30 µg for 96-144 weeks. METHODS: NEDA was defined as no relapses, no onset of 12-week confirmed disability progression (CDP), no new/newly enlarging T2 hyperintense lesions (NET2), and no gadolinium-enhancing (Gd+) lesions. Logistic regression models adjusted for baseline covariates compared treatment groups for baseline to week 96, weeks 0-24, and weeks 24-96. RESULTS: From baseline to week 96, more daclizumab versus intramuscular interferon beta-1a patients achieved NEDA (24.6% vs 14.2%; odds ratio (OR; 95% confidence interval): 2.059 (1.592-2.661); p < 0.0001). ORs for clinical NEDA (no relapses, no CDP) and magnetic resonance imaging (MRI) NEDA (no NET2, no Gd+ lesions) were 1.651 (1.357-2.007; p < 0.0001) and 2.051 (1.628-2.582; p < 0.0001), respectively. ORs in favor of daclizumab for weeks 24-96 were consistently higher than for weeks 0-24. CONCLUSION: More daclizumab versus intramuscular interferon beta-1a patients achieved NEDA early in DECIDE, with effects increasing over time.

The translational repressor T-cell intracellular antigen-1 (TIA-1) is a key modulator of Th2 and Th17 responses driving pulmonary inflammation induced by exposure to house dust mite.

T-cell intracellular antigen-1 (TIA-1) is a translational repressor that dampens the production of proinflammatory cytokines and enzymes. In this study we investigated the role of TIA-1 in a mouse model of pulmonary inflammation induced by exposure to the allergenic extract (Df) of the house dust mite Dermatophagoides farinae. When intranasally challenged with a low dose of Df, mice lacking TIA-1 protein (Tia-1(-/-)) showed more severe airway and tissue eosinophilia, infiltration of lung bronchovascular bundles, and goblet cell metaplasia than wild-type littermates. Tia-1(-/-) mice also had higher levels of Df-specific IgE and IgG(1) in serum and ex vivo restimulated Tia-1(-/-) lymph node cells and splenocytes transcribed and released more Th2/Th17 cytokines. To evaluate the site of action of TIA-1, we studied the response to Df in bone marrow chimeras. These experiments revealed that TIA-1 acts on both hematopoietic and non-hematopoietic cells to dampen pulmonary inflammation. Our results identify TIA-1 as a negative regulator of allergen-mediated pulmonary inflammation in vivo. Thus, TIA-1 might be an important player in the pathogenesis of bronchial asthma.

The purinergic G protein-coupled receptor 6 inhibits effector T cell activation in allergic pulmonary inflammation.

We show that the P2Y(6) receptor, a purinergic G protein-coupled receptor with a high affinity for the nucleotide uridine diphosphate, is an important endogenous inhibitor of T cell function in allergic pulmonary inflammation. Mice conditionally deficient in P2Y(6) receptors [p2ry6 (flox/flox);cre/+ mice] exhibited severe airway and tissue pathology relative to P2Y(6)-sufficient [p2ry6 (flox/flox)] littermates (+/+ mice) when treated intranasally with an extract of the dust mite Dermatophagoides farinae (Df). P2Y(6) receptors were inducibly expressed by lung, lymph node, and splenic CD4(+) and CD8(+) T cells of Df-treated +/+ mice. Df-restimulated P2Y(6)-deficient lymph node cells produced higher levels of Th1 and Th2 cytokines, and polyclonally stimulated P2Y(6)-deficient CD4(+) T cells proliferated faster than comparably stimulated P2Y(6)-sufficient cells. The absence of P2Y(6) receptors on CD4(+) cells, but not APCs, was sufficient to amplify cytokine generation. Thus, P2Y(6) receptors protect the lung against exuberant allergen-induced pulmonary inflammation by inhibiting the activation of effector T cells.

Group V secretory phospholipase A2 reveals its role in house dust mite-induced allergic pulmonary inflammation by regulation of dendritic cell function.

We have previously shown that group V secretory phospholipase A(2) (sPLA(2)) regulates phagocytosis of zymosan and Candida albicans by a mechanism that depends on fusion of phagosomes with late endosomes in macrophages. In this study, we report that group V sPLA(2) (Pla2g5)-null mice exposed to an extract of house dust mite Dermatophagoides farinae had markedly reduced pulmonary inflammation and goblet cell metaplasia compared with wild-type (WT) mice. Pla2g5-null mice had also impaired Th2-type adaptive immune responses to D. farinae compared with WT mice. Pla2g5-null bone marrow-derived dendritic cells (BMDCs) activated by D. farinae had delayed intracellular processing of allergen and impaired allergen-dependent maturation, a pattern recapitulated by the native lung DCs of D. farinae-challenged mice. Adoptively transferred D. farinae-loaded Pla2g5-null BMDCs were less able than D. farinae-loaded WT BMDCs to induce pulmonary inflammation and Th2 polarization in WT mice. However, Pla2g5-null recipients transferred with WT or Pla2g5-null D. farinae-loaded BMDCs exhibited significantly reduced local inflammatory responses to D. farinae, even though the transfer of WT BMDCs still induced an intact Th2 cytokine response in regional lymph nodes. Thus, the expression of group V sPLA(2) in APCs regulates Ag processing and maturation of DCs and contributes to pulmonary inflammation and immune response against D. farinae. Furthermore, an additional yet to be identified resident cell type is essential for the development of pulmonary inflammation, likely a cell in which group V sPLA(2) is upregulated by D. farinae, and whose function is also regulated by group V sPLA(2).

Fas-activated serine/threonine phosphoprotein promotes immune-mediated pulmonary inflammation.

We generated Fas-activated serine threonine phosphoprotein (FAST)-deficient mice (FAST(-/-)) to study the in vivo role of FAST in immune system function. In a model of house dust mite-induced allergic pulmonary inflammation, wild type mice develop a mixed cellular infiltrate composed of eosinophils, lymphocytes, and neutrophils. FAST(-/-) mice develop airway inflammation that is distinguished by the near absence of neutrophils. Similarly, LPS-induced alveolar neutrophil recruitment is markedly reduced in FAST(-/-) mice compared with wild type controls. This is accompanied by reduced concentrations of cytokines (TNF-alpha and IL-6 and -23) and chemoattractants (MIP-2 and keratinocyte chemoattractant) in bronchoalveolar lavage fluids. Because FAST(-/-) neutrophils exhibit normal chemotaxis and survival, impaired neutrophil recruitment is likely to be due to reduced production of chemoattractants within the pulmonary parenchyma. Studies using bone marrow chimeras implicate lung resident hematopoietic cells (e.g., pulmonary dendritic cells and/or alveolar macrophages) in this process. In conclusion, our results introduce FAST as a proinflammatory factor that modulates the function of lung resident hematopoietic cells to promote neutrophil recruitment and pulmonary inflammation.

Lung mast cells are a source of secreted phospholipases A2.

BACKGROUND: Secreted phospholipases A(2) (sPLA(2)s) are released in plasma and other biologic fluids of patients with inflammatory, autoimmune, and allergic diseases. OBJECTIVE: We sought to evaluate sPLA(2) activity in the bronchoalveolar lavage fluid (BALF) of asthmatic patients and to examine the expression and release of sPLA(2)s from primary human lung mast cells (HLMCs). METHODS: sPLA(2) activity was measured in BALF and supernatants of either unstimulated or anti-IgE-activated HLMCs as hydrolysis of oleic acid from radiolabeled Escherichia coli membranes. Expression of sPLA(2)s was examined by using RT-PCR. The release of cysteinyl leukotriene (LT) C(4) was measured by means of enzyme immunoassay. RESULTS: Phospholipase A(2) (PLA(2)) activity was higher in the BALF of asthmatic patients than in the control group. BALF PLA(2) activity was blocked by the sPLA(2) inhibitors dithiothreitol and Me-Indoxam but not by the cytosolic PLA(2) inhibitor AZ-1. HLMCs spontaneously released a PLA(2) activity that was increased on stimulation with anti-IgE. This PLA(2) activity was blocked by dithiothreitol and Me-Indoxam but not by AZ-1. HLMCs constitutively express mRNA for group IB, IIA, IID, IIE, IIF, III, V, X, XIIA, and XIIB sPLA(2)s. Anti-IgE did not modify the expression of sPLA(2)s. The cell-impermeable inhibitor Me-Indoxam significantly reduced (up to 40%) the production of LTC(4) from anti-IgE-stimulated HLMCs. CONCLUSIONS: sPLA(2) activity is increased in the airways of asthmatic patients. HLMCs express multiple sPLA(2)s and release 1 or more of them when activated by anti-IgE. The sPLA(2)s released by mast cells contribute to LTC(4) production by acting in an autocrine fashion. Mast cells can be a source of sPLA(2)s in the airways of asthmatic patients.

Vascular endothelial growth factors synthesized by human lung mast cells exert angiogenic effects.

BACKGROUND: Angiogenesis and lymphangiogenesis are critical for several allergic, inflammatory, and neoplastic disorders. Mast cells infiltrate the sites of inflammation and tumors. OBJECTIVE: We sought to characterize the expression and functions of vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) in human mast cells. METHODS: VEGF expression was evaluated by means of RT-PCR and Western blotting in primary human lung mast cells and in the mast cell lines LAD-2 and HMC-1. Angiogenic activity of mast cell supernatants was determined by using the chick embryo chorioallantoic membrane assay. VEGFR expression was assessed by means of RT-PCR and flow cytometry. Modified Boyden chambers were used for chemotaxis assay. RESULTS: Human mast cells express VEGF-A, VEGF-B, VEGF-C, and VEGF-D at both the mRNA and protein level. Prostaglandin E(2) (PGE(2)) enhanced the expression of VEGFA, VEGFB, and VEGFC, whereas an adenosine analog (5'-[N-ethylcarboxamido] adenosine [NECA]) increased VEGFA, VEGFC, and VEGFD expression. In addition, PGE(2) and NECA enhanced VEGF-A release, and supernatants of PGE(2)- and NECA-activated human lung mast cells induced angiogenic responses in the chorioallantoic membrane assay that were inhibited by an anti-VEGF-A antibody. Mast cells expressed mRNA for VEGFR1 and VEGFR2. These receptors were present on the mast cell surface. VEGF-A(165), VEGF-B(167), VEGF-C, VEGF-D, and placental growth factor 1 induced mast cell chemotaxis. These chemotactic effects were mediated by the activation of both VEGFR-1 and VEGFR-2. CONCLUSION: Our data indicate that human mast cells are both a source and a target of angiogenic and lymphangiogenic factors and therefore might play a role in inflammatory and neoplastic angiogenesis through the expression of several forms of VEGFs and their receptors.

Expression of phospholipases A2 in primary human lung macrophages: role of cytosolic phospholipase A2-alpha in arachidonic acid release and platelet activating factor synthesis.

Macrophages are a major source of lipid mediators in the human lung. Expression and contribution of cytosolic (cPLA(2)) and secreted phospholipases A(2) (sPLA(2)) to the generation of lipid mediators in human macrophages are unclear. We investigated the expression and role of different PLA(2)s in the production of lipid mediators in primary human lung macrophages. Macrophages express the alpha, but not the zeta isoform of group IV and group VIA cPLA(2) (iPLA(2)). Two structurally-divergent inhibitors of group IV cPLA(2) completely block arachidonic acid release by macrophages in response to non-physiological (Ca(2+) ionophores and phorbol esters) and physiological agonists (lipopolysaccharide and Mycobacterium protein derivative). These inhibitors also reduce by 70% the synthesis of platelet-activating factor by activated macrophages. Among the full set of human sPLA(2)s, macrophages express group IIA, IID, IIE, IIF, V, X and XIIA, but not group IB and III enzymes. Me-Indoxam, a potent and cell impermeable inhibitor of several sPLA(2)s, has no effect on arachidonate release or platelet-activating factor production. Agonist-induced exocytosis is not influenced by cPLA(2) inhibitors at concentrations that block arachidonic acid release. Our results indicate that human macrophages express cPLA(2)-alpha, iPLA(2) and several sPLA(2)s. Cytosolic PLA(2)-alpha is the major enzyme responsible for lipid mediator production in human macrophages.

Lysophospholipid transacetylase in the regulation of PAF levels in human monocytes and macrophages.

The transacetylase (TA), reported to be identical to platelet-activating factor (PAF) acetylhydrolase (II), is a multifunctional enzyme with three catalytic activities: lysophospholipid transacetylase (TA(L)), sphingosine transacetylase (TA(S)), and acetylhydrolase (AH). We report that TA(L) activity participates in the control of PAF levels in monocytes and macrophages and that its regulation differs in these two types of cells. In monocytes, LPS or granulocyte-macrophage colony-stimulating factor (GM-CSF) specifically increased the TA(L) activity. Western blot analysis and enzyme assays on immunoprecipitates revealed that the increased activity can be ascribed to PAF-AH (II) and that both translocation from cytosol to membranes and p38/ERKs-mediated phosphorylation regulate the enzyme activation. Instead, in macrophages differentiated in vitro from monocytes by incubation with FCS, an increase of both TA(L) and AH activities was observed. Moreover, activation of ERKs and p38 MAP kinase was not required for the up-regulation of PAF-AH (II) in differentiated macrophages. The differences observed in macrophages as compared to monocytes can be explained by 1) p38/ERKs-independent phosphorylation of PAF-AH (II) and 2) appearance of plasma PAF-AH in the course of macrophage differentiation.

Secretory phospholipases A2 in inflammatory and allergic diseases: not just enzymes.

Secretory phospholipases A(2) (sPLA(2)s) are molecules released in plasma and biologic fluids of patients with systemic inflammatory, autoimmune, and allergic diseases. Several sPLA(2) isoforms are expressed and released by such human inflammatory cells as neutrophils, eosinophils, basophils, T cells, monocytes, macrophages, and mast cells. Certain sPLA(2)s release arachidonic acid, thereby providing the substrate for the biosynthesis of proinflammatory eicosanoids. However, there are other mechanisms by which sPLA(2)s might participate in the synthesis of lipid mediators. Interestingly, sPLA(2)s activate inflammatory cells through mechanisms unrelated to their enzymatic activity. Several sPLA(2)s induce degranulation of mast cells and eosinophils and activate exocytosis in macrophages. Furthermore, sPLA(2)s promote cytokine and chemokine production from macrophages, neutrophils, eosinophils, monocytes, and endothelial cells. Some of these effects are mediated by the binding of sPLA(2)s to specific receptors expressed on effector cells. Thus sPLA(2)s might play important roles in the initiation and amplification of the inflammatory reaction. Selective inhibitors of sPLA(2)s and specific antagonists of sPLA(2) receptors might prove useful in the treatment of allergic and autoimmune diseases, such as bronchial asthma and rheumatoid arthritis.

Lung involvement in rheumatoid arthritis.

RA is a systemic autoimmune disorder primarily involving the joints. Extra-articular manifestations of RA often include lung involvement with heterogeneous clinical presentation and radiological findings. Autopsy studies reveal that the percentage of RA patients with pathological changes in the lung is significantly higher than that of patients with clinical manifestations. Lung alterations in RA may be primary or secondary to pharmacological treatments and may involve the alveoli, the interstitium, the airways and/or the pleura. These alterations may significantly impair lung function and some of them are potentially life-threatening. Thus, clinical examination and lung function testing should be performed in all patients with RA at the time of diagnosis and during follow-up. Those patients with clinical alterations and/or impaired lung function should undergo a complete radiological study.

Secretory phospholipases A2 as multivalent mediators of inflammatory and allergic disorders.

Phospholipases A(2) (PLA(2)s) are enzymes responsible for mobilization of fatty acids, including arachidonic acid (AA), from phospholipids. These enzymes are classified as high-molecular-weight cytosolic PLA(2)s (cPLA(2)s) and low-molecular-weight secretory PLA(2)s (sPLA(2)s). There is increasing evidence that large quantities of sPLA(2)s are released in the plasma of patients with systemic inflammatory and autoimmune diseases. In addition, high levels of sPLA(2)s can be detected at sites of allergic inflammation including the upper airways of patients with rhinitis and the lower airways of patients with asthma. These extracellular enzymes play an important role in inflammation by releasing AA, which can be subsequently converted to proinflammatory prostaglandins and leukotrienes. Generation of AA mediated by sPLA(2)s occurs through different mechanisms, including (1) the direct hydrolysis of outer cell membrane phospholipids, (2) internalization and transfer of sPLA(2)s to intracellular pools of phospholipids enriched in AA, and (3) activation of cPLA(2)s. In addition, sPLA(2)s induce degranulation and production of cytokines and chemokines from a variety of cells involved in inflammatory and immune responses. These effects are exerted by mechanisms that are independent of the enzymatic activity and are mediated by the interaction of sPLA(2)s with specific or promiscuous membrane receptors. Therefore, sPLA(2)s may have an important role in inflammatory and allergic reactions by activating multiple mechanisms within inflammatory and immune cells, leading to the production of eicosanoids, cytokines and chemokines.