Expression profiles of 10 circadian clock genes in human peripheral blood mononuclear cells

The circadian clock system regulates daily rhythms of physiology and behavior. The mammalian master clock in the suprachiasmatic nuclei orchestrates these biological rhythms in peripheral tissues. Since blood is the most accessible tissue source, we sought to dissect the human circadian clock system by characterizing clock gene expression in human peripheral blood mononuclear cells (PBMCs) isolated from eight young, healthy subjects. By evaluating the temporal expression profiles of 10 circadian clock genes, we found that Period 1 (Per1), Per2, and Per3 are rhythmically expressed in human blood samples. Our results suggest that evaluating the rhythmic expression of human Per genes could reveal an individual's circadian phenotype.     

Positive and negative strand of hepatitis C virus RNA sequences in peripheral blood mononuclear cells in patients with chronic hepatitis C: No correlation with viral genotypes 1b, 2a,and 2b

Hepatitis C virus (HCV) has many genotypes which are closely associated with the severity of chronic hepatitis and the response to antiviral therapy. Although HCV is essentially hepatotropic, several lines of evidence suggest that this virus can infect peripheral blood mononuclear cells (PBMC) in most patients with chronic HCV infection. However, the methods used previously to detect negative-strand HCV RNA have been questioned, and the PBMC tropism of different HCV genotypes remains unknown. A stringent method was used to investigate the prevalence of positive- and negative-strand HCV RNA in the PBMC of 106 patients with chronic hepatitis C and to analyze the influence of HCV genotype on the tropism of PBMC. HCV type 1b was the predominant strain in the patients. Positive-strand RNA in PBMC was detected in 83 (78%) and 40% had negative-strand RNA. The demographic and clinical features were comparable among different patients grouped by the replication status of HCV in the plasma and PBMC samples. In addition, there was no significant difference of PBMC tropism between type 1b and non-1b HCV. In summary, HCV does indeed infect actively the PBMC of chronic hepatitis C patients and such infection is not correlated to the pathogenesis of liver cell damage. Moreover, the genotype is not associated specifically with PBMC tropism of HCV. J. Med. Virol. 52:270–274, 1997. © 1997 Wiley-Liss, Inc.     

Human tonsillar IgE biosynthesis in vitro. I. Enhancement of IgE and IgG synthesis in the presence of pokeweed mitogen by T-cell irradiation

A study of the events regulating human IgE biosynthesis in vitro was undertaken with tonsillar lymphocytes. IgG synthesis was also studied to evaluate the specificity of our observations. T-cell irradiation significantly enhanced synthesis of IgE by pokeweed mitogen (PWM)-stimulated B cells from 12 of 18 donors and IgG in all 18 donors. This enhancement was the result of de novo immunoglobulin synthesis, since the amount of IgE and IgG spontaneously released from lysed and lysed-and-cultured mononuclear cells was significantly less than that detected in the cell cultures, and the augmentation was completely ablated by the treatment of the cells with cycloheximide or mitomycin C. Enhancement was also dependent on the presence of PWM; T-cell irradiation did not enhance IgE synthesis in unstimulated cultures. Moreover, this enhancement was also observed in the co-cultures of B cells and allogeneic irradiated T cells. These observations suggest that radiosensitive T cells exert a suppressive activity that contributes to regulation of human IgE and IgG synthesis and that the suppressor function as well as the helper function can overcome allogeneic disparities.     

Internal images: human anti-idiotypic Fab antibodies mimic the IgE epitopes of grass pollen allergen Phl p 5a

BACKGROUND: The role of anti-idiotypic antibodies in allergic disease is still poorly understood. According to Jerne, anti-idiotypic antibodies to IgE should represent internal images of an allergen. Our aim was to ultimately prove whether this hypothesis holds true in allergy. Here, we describe the selection of anti-idiotypic antibodies against Phl p 5a-specific IgE directly from the B-cell repertoire of a grass pollen allergic individual. METHODS: Taking Phleum pratense grass pollen allergen Phl p 5 as a model, we selected anti-idiotypic antibodies against allergen-specific IgE directly from the B-cell repertoire of an allergic individual. We screened a combinatorial phage display library of human monovalent antibody heavy and light chain fragments (Fabs) with anti-Phl p 5a-IgE to identify and characterize Fabs with anti-idiotypic specificity. RESULTS: Five different Fab clones with anti-idiotypic specificity for anti-Phl p 5a-IgE were identified. Their hypervariable regions revealed partial sequence homology with solvent accessible antigenic sites of Phl p 5a, which have been identified by our previous mimotope approach. Phagemid DNA derived from the phage clones was used to produce two soluble recombinant anti-idiotypic Fab clones in E. coli. As a proof of molecular mimicry, both Fabs induced anti-Phl p 5a-specific antibodies in immunized BALB/c mice. Molecular modeling of the heavy and light chain hypervariable loops of the anti-idiotypic Fabs illustrated structural similarity with dominant IgE epitopes of Phl p 5a. CONCLUSION: In this straightforward phage technology approach, antibodies with anti-idiotypic specificities could be isolated from a human allergic's repertoire. As predicted by the immune network hypothesis, their hypervariable domains mimic IgE epitopes like internal images and, more importantly, induce allergen-specific immune responses in the absence of the allergen.     

Association of tissue factor with a γ chain homodimer of the IgE receptor type I in cultured human monocytes

Tissue factor (TF) is a high-affinity receptor for coagulation factors VII (F VII) and VIIa. The F VII/VIIa/TF complex is the major cellular initiator of the extrinsic coagulation cascade. We found that the occupancy of TF by its ligand, F VIIa, is involved with signal transduction and that TF is associated with the γ chain homodimer identified as a component of IgE receptor type I (FcϵRI). When 4-day cultured human monocytes were incubated with F VIIa, several polypeptides, especially a 70-kDa polypeptide, were transiently phosphorylated on tyrosine, residues. These phosphorylation events were inhibited by prior binding of anti-TF monoclonal antibody (mAb) HTF-K14, but not anti-TF mAb HTF-K180 to intact cultured monocytes. HTF-K14 blocked the binding of FVII/Vila to cell surface TF, whereas HTF-K180 did not. Anti-TF immunoprecipitates prepared from 1 % digitonin lysates of cultured human monocytes incorporated phosphate in a γ chain homodimer when incubated with [γ-³²P] ATP. The identity of the TF-associated structures as γ chains was established by immunoblot analysis of anti-TF mAb immunoprecipitates with anti-γ chain mAb. In addition, anti-TF immunoblot analysis showed that TF co-precipitated with anti-γ chain mAb. Our data suggest that γ chains may play an important role in signaling via TF in human monocytes/macrophages.     

Identification of a novel immunomodulatory gliadin peptide that causes interleukin-8 release in a chemokine receptor CXCR3-dependent manner only in patients with coeliac disease

The autoimmune enteropathy, coeliac disease (CD), is triggered by ingestion of gluten-containing grains. We recently reported that the chemokine receptor CXCR3 serves as a receptor for specific gliadin peptides that cause zonulin release and subsequent increase in intestinal permeability. To explore the role of CXCR3 in the immune response to gliadin, peripheral blood mononuclear cells from both patients with CD and healthy controls were incubated with either pepsin-trypsin-digested gliadin or 11 α-gliadin synthetic peptides in the presence or absence of a blocking anti-CXCR3 monoclonal antibody. Supernatants were analysed for interleukin-6 (IL-6), IL-8, IL-10, IL-13, IP-10 (CXCL10), tumour necrosis factor-α and interferon-γ. Gliadin broadly induced cytokine production irrespective of the clinical condition. However, IL-8 production occurred only in a subgroup of individuals and cells of the phagocytic lineage were the main source. Induction of IL-8 was reproduced by one of a comprehensive panel of synthetic α-gliadin peptides and was abrogated when CXCR3 was blocked before stimulation with either gliadin or this peptide in the CD group but not in the control group, suggesting that gliadin-induced IL-8 production was CXCR3-dependent gliadin induced IL-8 production only in CD.     

Insensitivity of PI3K/Akt/GSK3 signaling in peripheral blood mononuclear cells of age-related macular degeneration patients

Our recent studies with cultured retinal pigment epithelium cells suggested that overexpression of interleukin 17 receptor C (IL-17RC), a phenomenon observed in peripheral blood and chorioretinal tissues with age-related macular degeneration (AMD), was associated with altered activation of phosphatidylinositide 3-kinase (PI3K), Akt, and glycogen synthase kinase 3 (GSK3). We wondered whether or not altered PI3K, Akt, and GSK3 activities could be detected in peripheral blood mononuclear cells (PBMC) obtained from AMD patients. In the patients' PBMC, absentor reduced serine-phosphorylation of GSK3α or GSK3β was observed, which was accompanied with increased phosphorylation of GSK3 substrates (e.g. CCAAT enhancer binding protein α, insulin receptor substrate 1, and TAU), indicative of enhanced GSK3 activation. In addition, decreased protein mass of PI3K85α and tyrosinephosphorylation of PI3K50α was present in PBMC of the AMD patients, suggesting impaired PI3K activation. Moreover, abnormally lowered molecular weight forms of Akt and GSK3 were detected in PBMC of the AMD patients. These data demonstrate that despite the presence of high levels of IL-17RC, Wnt-3a and vascular endothelial growth factor, the PI3K/Akt/GSK3 signaling pathway is insensitive to these stimuli in PBMC of the AMD patients. Thus, measurement of PI3K/Akt/GSK3 expression and activity in PBMC may serve as a surrogate biomarker for AMD.     

Absence of the OKT4 epitope on blood T cells and thymus cells in a patient with thymoma, hypogammaglobulinemia, and red blood cell aplasia

Human helper/inducer T-lymphocytes that express the T4 antigen are important in the regulation of B and T cell functions. Several epitopes of the T4 molecule have now been recognized; however, the precise role of these molecules in the function of helper/inducer T cells is unclear. We studied a patient with thymoma, hypogammaglobulinemia, and red blood cell aplasia whose blood lymphocytes and thymus cells did not express the epitope recognized by OKT4 monoclonal antibody but did display the T4 epitopes recognized by OKT4A and Leu3A monoclonal antibodies. The absence of the OKT4 epitope on the patient's thymus cells suggested that the abnormality occurred during early T cell differentiation. The patient had intact delayed hypersensitivity to 4/4 antigens, and his blood lymphocytes proliferated normally to phytohemagglutinin, concanavalin A, pokeweed mitogen, tetanus toxoid, and allogeneic cells. The patient's T cells demonstrated augmented suppressor activity that was localized to the OKT8+ population rather than to the unusual T4 subset. Irradiation abrogated suppressor activity and rendered his T cells capable of providing help for polyclonal B cell differentiation. The data emphasize the limitations of OKT4 as the sole reagent for characterizing the subset of human helper/inducer cells and demonstrate that the expression of the T4 epitope recognized by OKT4 monoclonal antibody is not required for certain helper/inducer T cell functions in vitro and in vivo.     

Mincle,the receptor for mycobacterial cord factor,forms a functional receptor complex with MCL and FcεRI‐γ

Upon receptor activation, the myeloid C‐type lectin receptor Mincle signals via the Syk‐CARD9‐Bcl10‐MALT1 pathway. It does so by recruiting the ITAM‐bearing FcεRI‐γ. The related receptor macrophage C‐type Lectin (MCL) has also been shown to be associated with Syk and to be dependent upon this signaling axis. We have previously shown that MCL co‐precipitates with FcεRI‐γ, but were unable to show a direct association, suggesting that MCL associates with FcεRI‐γ via another molecule. Here, we have used rat primary cells and cell lines to investigate this missing link. A combination of flow cytometric and biochemical analysis showed that Mincle and MCL form heteromers on the cell surface. Furthermore, association with MCL and FcεRI‐γ increased Mincle expression and enhanced phagocytosis of Ab‐coated beads. The results presented in this paper suggest that the Mincle/MCL/FcεRI‐γ complex is the functionally optimal form for these C‐type lectin receptors on the surface of myeloid cells.     

Soluble form of the human high-affinity receptor for IgE inhibits recurrent allergic reaction in a novel mouse model of type I allergy

The recombinant soluble form of the human high-affinity receptor for IgE α subunit (sFc?RIα) is able to block the binding of IgE to the cell surface Fc?RI and prevent the activation of mast cells and basophils. To evaluate its anti-allergic effects in vivo, we established a novel model for type I allergy by transplanting antigen-specific IgE-secreting hybridomas into syngeneic mice. The hybridomas continuously produced anti-2,4,6-trinitrophenyl IgE in vivo, and ear swelling responses could be elicited upon applying picryl chloride to the ears of these mice, which reached their maximum 1–2 h after the antigen challenge. The second swelling response, in extent comparable to the first response, was induced by the second antigen challenge several days later. When the sFc?RIα was intravenously administered before the first challenge and periodically betweeen the first and the second challenges, the ear swelling response to the second challenge, but not to the first challenge, was suppressed almost completely. Immunohistochemical examination revealed that treatment with the sFc?RIα blocked the binding of IgE to the cell surface IgE receptors after the first challenge. Thus, our results indicate that the sFc?RIα suppresses recurrent allergic reactions by preventing IgE binding to the cell surface Fc?RI.     

The effects of a monoclonal antibody to interferon-γ on experimental autoimmune thyroiditis (EAT): prevention of disease and decrease of EAT-specific T cells

CBA/J mice immunized with thyreoglobulin (Tg) develop an experimental autoimmune thyroiditis (EAT) with lymphocytic infiltration of the thyroid glands, autoantibodies to Tg and occurrence of EAT-specific T cells. When these mice were treated for 4 weeks after immunization with 1 mg/week of a monoclonal antibody (mAb) that neutralizes the activity of interferon-γ (IFN) a beneficial effect on the onset of EAT was observed. Characteristic features of EAT were significantly reduced, including the lymphocytic infiltrations of the thyroid glands and the serum levels of autoantibodies to Tg. Moreover, in lymphoid organs, mAb to IFN-γ significantly reduced the percentages of Tg-specific CD8⁺ cells, labeled by the anti-clonotypic mAb AG7. These Tg-specific T cells seem responsible for thyroid damages and disease development, since EAT was simultaneously abrogated. These results show that IFN-γ plays an essential role in the pathophysiology of EAT and suggest the possibility to treat autoimmune thyroid diseases with mAb to IFN-γ or drugs able to antagonize the production and/or the action of this cytokine.     

T helper type 2-like cells and therapeutic effects of interferon-γ in combined immunodeficiency with hypereosinophilia (Omenn's syndrome)

We characterized the defects of CD4+ cells in a 17-month-old girl suffering from combined immunodeficiency with hypereosinophilia (Omenn's syndrome). Because the vast majority of peripheral blood CD4⁺ cells expressed the CD45R0 isoform, we purified circulating CD4⁺ CD45R0⁺ cells from the patient and healthy individuals in order to compare their production of cytokines. The patient's CD4⁺ CD45R0⁺ cells spontaneously produced high levels of interleukin-5 (IL-5) in vitro (1600 pg/ml after 24 h of culture) and this was associated with the presence of IL-5 in serum (323 pg/ml). After stimulation with phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187, they produced higher levels of IL-4 (306 vs. 55 ± 4 pg/ml) and IL-5 (2900 vs. 213 ± 72 pg/ml) and lower levels of IL-2 (17 vs. 63 ± 17 IU/ml) and interferon-γ (IFN-γ) (16 vs. 299 ± 70 IU/ml) than controls CD4⁺ CD45R0⁺ cells. This T helper type 2 (Th2) pattern was confirmed by the detection using reverse polymerase chain reaction of IL-4, IL-5 and IL-10 mRNA within peripheral blood mononuclear cells. During a therapeutic trial with human IFN-γ (40 μg/day) which ameliorated the clinical status of the patient, we observed a down-regulation of the in vivo expression of IL-5 and IL-10, a normalization of the eosinophil count and an improvement of the Tcell response to phytohemagglutinin. This observation indicates for the first time that Th2-like cells might be involved in certain forms of congenital immunodeficiency and that IFN-γ might down-regulate their activities in vivo.     

Prominent proliferative response of peripheral blood mononuclear cells to a recombinant non-structural (NS3) protein of hepatitis C virus in patients with chronic hepatitis C.

The proliferative response of peripheral blood mononuclear cells (PBMC) to a recombinant non-structural (NS3) protein of hepatitis C virus (HCV) was studied in 41 patients with chronic hepatitis C. Of them, 28 had chronic persistent hepatitis (CPH) and 13 chronic active hepatitis (CAH). The positive proliferation rate of PBMC to the recombinant NS3 protein, T9Ag, was 66% in the 41 patients (77% in CAH versus 61% in CPH; P > 0.05) when stimulation index (SI) = 4 was set as the cut-off value. However, mean SI of CAH patients was significantly higher than that of CPH patients (8.3 +/- 5.2 versus 5.1 +/- 3.6; P < 0.05). Six other chronic hepatitis patients who were repeatedly negative for anti-HCV antibody but positive for serum HCV RNA also had an SI of > or = 4.0. The frequency of cellular immune response to the T9Ag is among the highest results obtained by using HCV antigens tested so far. Our studies thus indicate that NS3 is an immunologically important region of HCV for T cells. Moreover, the proliferative response to T9Ag may help to establish hepatitis C etiology in chronic hepatitis patients who are seronegative with currently available anti-HCV assays.     

Th2 cytokines,IgE and mast cells play a crucial role in the induction of para-phenylenediamine-induced contact hypersensitivity in mice

We previously reported the establishment of a mouse model system of contact hypersensitivity (CHS) to paraphenylemediamine (PPD). In order to analyse the functional contribution of Th2 cytokines, IL-4 and IL-5, in PPD induced CHS, STAT6 deficient (STAT6-/-) and wild-type control (WT) mice (C57BL/6) were immunized by the topical application of a PPD solution, and then the subsequent skin reactions were examined. Ear swelling was significantly reduced with a delayed peak response in STAT6-/- mice as compared with that of WT mice. A histological analysis showed the infiltration of both eosinophils and neutrophils in the skin of STAT6-/- mice challenged 24 h previously to significantly decrease in comparison with that in the WT mice. The expression of Th2 cytokines (IL-4, IL-5) by ELISA in the PPD-challenged skin tissue specimens as well as the IgE and IgG1 response after challenge were also profoundly reduced in the STAT6-/- mice. The adoptive transfer of the serum obtained from sensitized WT mice for the putative IgE transfer induced a peak response at 3 h and 24 h after challenge. To further investigate the role of mast cells in the induction of PPD-CHS, mast cell deficient W/Wv mice were sensitized with PPD and then were challenged. Maximal ear swelling was detected from 12 to 24 h and another small peak response was observed at 1 h in+/+mice, whereas only a small peak response at 24 h was detected in W/Wv mice. These data indicate that not only Th2 cytokines and IgE but also mast cells play an essential role in the induction of PPD-CHS.     

Manipulation of the superantigen-induced lymphokine response. Selective induction of interleukin-10 or interferon-γ synthesis in small resting CD4+ T cells

The production of several lymphokines by freshly isolated CD4⁺ T cells has been analyzed at the single-cell level, after stimulation with staphylococcal enterotoxin B (SEB). High frequencies of cells producing interleukin-2 (IL-2) and interferon-γ (IFN-γ) were induced, but very low frequencies of CD4⁺ T cells produced IL-4, IL-5 or IL-10 in response to SEB. Exogenously added IL-4 markedly altered the lymphokine profile induced during primary SEB stimulation. IFN-γ production was reduced, while a high fraction of cells contained IL-10 and IL-4 after activation in the presence of IL-4. We further demonstrate that IL-4 and IL-10 or IFN-y production was selectively induced in resting, high-density CD4⁺ T cells during primary stimulation, by SEB + IL-4 or SEB. Under conditions where both IL-10 and IFN-γ were produced, most cells contained only one of the two lymphokines.     

Human IgE mediates stimulus secretion coupling in rat basophilic leukemia cells transfected with the α chain of the human high-affinity receptor

Cell surface expression of the a chain of the human (h) Fee receptor type 1 (FceR1) can be observed following stable transfection of RBL-2H3 cells with the ha chain of the FceR1 complex. Association and dissociation constants for hIgE are similar to those previously reported for the ligand and its receptor. The demonstration of mediator release following the cross-linking of hFc?R1α by hIgE and antigen or anti-hFc?R1α antibody suggests that this system will assist the identification of structural motifs and specific amino acids that are involved in the generation of the signal(s) that will initiate and/or propagate the secretion of mediators from mast cells and basophils.     

Interaction among human leucocyte antigen–peptide–T cell receptor complexes in cow''s milk allergy: the significance of human leucocyte antigen and T cell receptor–complementarity determining region 3 loops

BACKGROUND: Allergic individuals respond to only a few specific antigens, therefore allergic diseases are characterized by antigen specificity. Clarification of the mechanism of antigen specificity will lead to progress in the therapy of allergic diseases. OBJECTIVES: The purpose of this study is to determine the specific association among T cell epitopes, antigen-presenting molecules and T cell receptor (TCR), and to determine the TCR usage in the pathogenesis of allergies using antigen-specific T cell clones (TCCs). The results can clarify the mechanism of the antigen specificity of allergic diseases, and provide new therapeutic possibilities using analogue peptides. METHODS: Short-term T cell clones specific to beta-lactoglobulin (BLG) were established from peripheral blood mononuclear cells (PBMCs) collected from five patients allergic to cow's milk. We then identified the T cell epitopes and antigen-presenting molecules, and examined TCR usage. We also determined the sequence of the TCR-complementarity-determining region 3 (CDR3). RESULTS: Six TCCs established from the five patients recognized three different peptides, and BLGp97-117 was recognized by four of the six TCCs. BLGp101-112 (KYLLFCMENSAE) was the core sequence in the fragment. Sequence analysis of TCR by the RT-PCR method revealed a marked heterogeneity in TCR usage, and similar amino acid sequences were recognized in the CDR3 region. Four of the six TCCs recognized BLG in association with human leucocyte antigen (HLA)-DRB1*0405 as antigen-presenting molecules. CONCLUSION: We proposed the motif of the interaction between the HLA-DRB1*0405 allele and antigen peptide, and suggested that HLA-DRB1*0405 is an immunoregulatory gene product for T cell responses to BLG.     

Harnessing engineered antibodies of the IgE class to combat malignancy: initial assessment of FcɛRI‐mediated basophil activation by a tumour‐specific IgE antibody to evaluate the risk of type I hypersensitivity

Background IgE antibodies, sequestered into tissues and retained locally by the high‐affinity IgE receptor, Fc?RI, on powerful effector cells such as mast cells, macrophages and eosinophils, may offer improvements in the therapy of solid tumours. The chimeric antibody, MOv18 IgE, against the human ovarian carcinoma antigen, folate receptor α (FRα), is more effective than its IgG1 counterpart in xenograft models of ovarian cancer. Although MOv18 IgE binds to a single epitope on FRα and cannot cross‐link IgE receptors on basophils, there remains a risk that components in the circulation of ovarian cancer patients might cross‐link FRα‐MOv18‐IgE‐receptor‐Fc?RI complexes on basophils to cause type I hypersensitivity. Objective To assess the propensity for MOv18 used in a therapeutic setting to cause Fc?RI‐mediated type I hypersensitivity. Methods As validated readouts of the potential for MOv18 to cause Fc?RI‐mediated type I hypersensitivity we measured release of a granule‐stored mediator from a rat basophilic leukaemia cell line RBL SX‐38 stably transfected with human tetrameric (αβγ2) Fc?RI, and induction of CD63 on blood basophils from patients with ovarian carcinoma and healthy controls ex vivo. Results Serum FRα levels were increased in ovarian cancer patients compared with healthy controls. MOv18 IgE alone, or in the presence of its antigen recombinant human FRα, or of healthy volunteer (n=14) or ovarian carcinoma patient (n=32) sera, did not induce RBL SX‐38 cell degranulation. Exposure to FRα‐expressing ovarian tumour cells at target‐to‐effector ratios expected within tumours induced degranulation. MOv18 IgE did not induce expression of CD63 in blood basophils from either healthy volunteers (n=6), or cancer patients, despite detectable levels of circulating FRα (n=5). Conclusion and Clinical Relevance These encouraging data are compatible with the hypothesis that, when ovarian carcinoma patients are treated with MOv18, Fc?RI‐mediated activation of effector cells occurs within the tumour mass but not in the circulation mandating, with due caution, further pre‐clinical studies. Cite this as: S. M. Rudman, D. H. Josephs, H. Cambrook, P. Karagiannis, A. E. Gilbert, T. Dodev, J. Hunt, A. Koers, A. Montes, L. Taams, S. Canevari, M. Figini, P. J. Blower, A. J. Beavil, C. F. Nicodemus, C. Corrigan, S. B. Kaye, F. O. Nestle, H. J. Gould, J. F. Spicer and S. N. Karagiannis, Clinical & Experimental Allergy, 2011 (41) 1400–1413.