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2.
Clin Immunol ; 197: 34-39, 2018 12.
Article in English | MEDLINE | ID: mdl-30170030

ABSTRACT

וֹndoleamine-2,3-dioxygenase (IDO) plays a role in immune regulation. Increased IDO activity was reported in systemic lupus erythematosus (SLE). We investigated the effects of the tolerogenic peptide hCDR1, shown to ameliorate lupus manifestations, on IDO gene expression. mRNA was prepared from splenocytes of hCDR1- treated SLE-afflicted (NZBxNZW)F1 mice, from blood samples of lupus patients, collected before and after their in vivo treatment with hCDR1 and from peripheral blood mononuclear cells (PBMC) of patients incubated with hCDR1. IDO gene expression was determined by real-time RT-PCR. hCDR1 significantly down-regulated IDO expression in SLE-affected mice and in lupus patients (treated in vivo and in vitro). No effects were observed in healthy donors or following treatment with a control peptide. Diminished IDO gene expression was associated with hCDR1 beneficial effects. Our results suggest that the hCDR1-induced FOXP3 expressing regulatory T cells in lupus are not driven by IDO but rather by other hCDR1 regulated pathways.


Subject(s)
Antibodies, Monoclonal/pharmacology , Indoleamine-Pyrrole 2,3,-Dioxygenase/drug effects , Lupus Erythematosus, Systemic/genetics , Peptide Fragments/pharmacology , T-Lymphocytes, Regulatory/drug effects , Adult , Aged , Animals , Antibodies, Monoclonal/therapeutic use , Clinical Trials, Phase II as Topic , Down-Regulation , Female , Forkhead Transcription Factors/metabolism , Humans , Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Lupus Erythematosus, Systemic/drug therapy , Lupus Erythematosus, Systemic/metabolism , Male , Mice , Middle Aged , Peptide Fragments/therapeutic use , Real-Time Polymerase Chain Reaction , Spleen/cytology , T-Lymphocytes, Regulatory/metabolism
3.
Clin Immunol ; 192: 85-91, 2018 07.
Article in English | MEDLINE | ID: mdl-29730433

ABSTRACT

Primary Sjogren's syndrome (pSS) is an autoimmune disease characterized by lymphocytic infiltration of exocrine glands. We investigated whether the tolerogenic peptide, hCDR1, that ameliorates lupus manifestations would have beneficial effects on pSS as well. The in vitro effects of hCDR1 on gene expression of pro-inflammatory cytokines and regulatory molecules were tested in peripheral blood mononuclear cells (PBMC) of 16 pSS patients. hCDR1, but not a control peptide, significantly reduced gene expression of IL-1ß, TNF-α, MX-1 and BlyS and up-regulated immunosuppressive (TGF-ß, FOXP3) molecules in PBMC of pSS patients. hCDR1 did not affect gene expression in patients with rheumatoid arthritis and anti-phospholipid syndrome. Further, hCDR1 up-regulated the expression of Indoleamine 2,3-dioxygenase (IDO) via elevation of TGF-ß. IDO inhibition led to a significant decrease in the expression of FOXP3 which is crucial for the induction of T regulatory cells. Thus, hCDR1 is potential candidate for the specific treatment of pSS patients.


Subject(s)
Cytokines/immunology , Gene Expression Regulation/drug effects , Leukocytes, Mononuclear/drug effects , Peptides/pharmacology , Sjogren's Syndrome/immunology , Adult , Aged , Amino Acid Sequence , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/pharmacology , Cytokines/genetics , Cytokines/metabolism , Female , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/immunology , Forkhead Transcription Factors/metabolism , Gene Expression Regulation/immunology , Humans , Immunologic Factors/pharmacology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Male , Middle Aged , Peptide Fragments/genetics , Peptide Fragments/pharmacology , Peptides/genetics , Sjogren's Syndrome/blood , Sjogren's Syndrome/genetics , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/immunology , Transforming Growth Factor beta/metabolism
4.
J Rheumatol ; 45(3): 310-319, 2018 03.
Article in English | MEDLINE | ID: mdl-29335340

ABSTRACT

OBJECTIVE: Anticitrullinated protein antibodies (ACPA) have major diagnostic significance in rheumatoid arthritis (RA). ACPA are directed against different citrullinated antigens, including filaggrin, fibrinogen, vimentin, and collagen. The presence of ACPA is associated with joint damage and extraarticular manifestations, suggesting that ACPA may have a significant role in the pathogenesis of RA. METHODS: To verify the effect of ACPA on RA-immune cells, peripheral blood mononuclear cells (PBMC) from cyclic citrullinated peptide (CCP)-positive patients with RA and healthy controls were cocultured in vitro with ACPA. ACPA-positive stained cells were analyzed by flow cytometry and the effect of ACPA on mRNA expression levels was evaluated by real-time PCR. We tested whether the stimulatory effects induced by ACPA could be inhibited by the addition of a new multiepitope citrullinated peptide (Cit-ME). RESULTS: We found that ACPA bind specifically to PBMC from CCP-positive patients with RA through the Fab portion. ACPA induce upregulation of pathogenic cytokine expression (4- to 13-fold increase) in PBMC derived from CCP-positive patients with RA. Moreover, ACPA upregulated IL-1ß and IL-6 mRNA expression levels by 10- and 6-fold, respectively, compared to control IgG. Cit-ME, a genuine ligand of ACPA, inhibited the ACPA-induced upregulation of IL-1ß and IL-6 by 30%. CONCLUSION: ACPA bind to a limited percentage of PBMC and upregulate inflammatory cytokine expression, suggesting that ACPA is involved in RA pathogenesis. Targeting ACPA to decrease their pathogenic effects might provide a novel direction in developing therapeutic strategies for RA.


Subject(s)
Arthritis, Rheumatoid/blood , Autoantibodies/blood , Citrulline/immunology , Leukocytes, Mononuclear/metabolism , Peptides, Cyclic/metabolism , Transcriptome/immunology , Antibody Affinity/immunology , Citrulline/chemical synthesis , Filaggrin Proteins , Humans , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin Fc Fragments/metabolism , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Statistics, Nonparametric
5.
J Autoimmun ; 54: 60-71, 2014 Nov.
Article in English | MEDLINE | ID: mdl-24958634

ABSTRACT

Systemic lupus erythematosus (SLE) is a chronic multisystem disease in which various cell types and immunological pathways are dysregulated. Current therapies for SLE are based mainly on the use of non-specific immunosuppressive drugs that cause serious side effects. There is, therefore, an unmet need for novel therapeutic means with improved efficacy and lower toxicity. Based on recent better understanding of the pathogenesis of SLE, targeted biological therapies are under different stages of development. The latter include B-cell targeted treatments, agents directed against the B lymphocyte stimulator (BLyS), inhibitors of T cell activation as well as cytokine blocking means. Out of the latter, Belimumab was the first drug approved by the FDA for the treatment of SLE patients. In addition to the non-antigen specific agents that may affect the normal immune system as well, SLE-specific therapeutic means are under development. These are synthetic peptides (e.g. pConsensus, nucleosomal peptides, P140 and hCDR1) that are sequences of conserved regions of molecules involved in the pathogenesis of lupus. The peptides are tolerogenic T-cell epitopes that immunomodulate only cell types and pathways that play a role in the pathogenesis of SLE without interfering with normal immune functions. Two of the peptides (P140 and hCDR1) were tested in clinical trials and were reported to be safe and well tolerated. Thus, synthetic peptides are attractive potential means for the specific treatment of lupus patients. In this review we discuss the various biological treatments that have been developed for lupus with a special focus on the tolerogenic peptides.


Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , Drug Delivery Systems , Epitopes, T-Lymphocyte/therapeutic use , Immunosuppressive Agents/therapeutic use , Lupus Erythematosus, Systemic , Peptides/therapeutic use , Animals , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Clinical Trials as Topic , Epitopes, T-Lymphocyte/immunology , Humans , Lupus Erythematosus, Systemic/drug therapy , Lupus Erythematosus, Systemic/immunology , Lupus Erythematosus, Systemic/pathology , T-Lymphocytes/immunology , T-Lymphocytes/pathology
6.
PLoS One ; 8(3): e60394, 2013.
Article in English | MEDLINE | ID: mdl-23555966

ABSTRACT

BACKGROUND: The tolerogenic peptide, hCDR1, ameliorated manifestations of systemic lupus erythematosus (SLE) via the immunomodulation of pro-inflammatory and immunosuppressive cytokines and the induction of regulatory T cells. Because type I interferon (IFN-α) has been implicated to play a role in SLE pathogenesis, we investigated the effects of hCDR1 on IFN-α in a murine model of SLE and in human lupus. METHODOLOGY PRINCIPAL FINDINGS: (NZBxNZW)F1 mice with established SLE were treated with hCDR1 (10 weekly injections). Splenocytes were obtained for gene expression studies by real-time RT-PCR. hCDR1 down-regulated significantly IFN-α gene expression (73% inhibition compared to vehicle treated mice, p = 0.002) in association with diminished clinical manifestations. Further, hCDR1 reduced, in vitro, IFN-α gene expression in peripheral blood mononuclear cells (PBMC) of 10 lupus patients (74% inhibition compared to medium, p = 0.002) but had no significant effects on the expression levels of IFN-α in PBMC of primary anti-phospholipid syndrome patients or of healthy controls. Lupus patients were treated for 24 weeks with hCDR1 (5) or placebo (4) by weekly subcutaneous injections. Blood samples collected, before and after treatment, were frozen until mRNA isolation. A significant reduction in IFN-α was determined in hCDR1 treated patients (64.4% inhibition compared to pretreatment expression levels, p = 0.015). No inhibition was observed in the placebo treated patients. In agreement, treatment with hCDR1 resulted in a significant decrease of disease activity. IFN-α appears to play a role in the mechanism of action of hCDR1 since recombinant IFN-α diminished the immunomodulating effects of hCDR1 on IL-1ß, TGFß and FoxP3 gene expression. CONCLUSIONS SIGNIFICANCE: We reported previously that hCDR1 affected various cell types and immune pathways in correlation to disease amelioration. The present studies demonstrate that hCDR1 is also capable of down-regulating significantly (and specifically to lupus) IFN-α gene expression. Thus, hCDR1 has a potential role as a novel, disease specific treatment for lupus.


Subject(s)
Autoantigens/therapeutic use , Down-Regulation/drug effects , Immunologic Factors/therapeutic use , Interferon-alpha/genetics , Lupus Erythematosus, Systemic/drug therapy , Nerve Tissue Proteins/therapeutic use , Peptides/therapeutic use , Amino Acid Sequence , Animals , Autoantigens/chemistry , Cells, Cultured , Female , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Lupus Erythematosus, Systemic/genetics , Male , Mice , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Peptides/chemistry
7.
BMC Med ; 11: 90, 2013 Apr 04.
Article in English | MEDLINE | ID: mdl-23556432

ABSTRACT

BACKGROUND: The 16/6-idiotype (16/6-Id) of the human anti-DNA antibody was found to induce experimental lupus in naïve mice, manifested by production of autoantibodies, leukopenia and elevated inflammatory markers, as well as kidney and brain involvement. We assessed behavior and brain pathology of naive mice injected intra-cerebra-ventricularly (ICV) with the 16/6-Id antibody. METHODS: C3H female mice were injected ICV to the right hemisphere with the human 16/6-Id antibody or commercial human IgG antibodies (control). The mice were tested for depression by the forced swimming test (FST), locomotor and explorative activity by the staircase test, and cognitive functions were examined by the novel object recognition and Y-maze tests. Brain slices were stained for inflammatory processes. RESULTS: 16/6-Id injected mice were cognitively impaired as shown by significant differences in the preference for a new object in the novel object recognition test compared to controls (P = 0.012). Similarly, the preference for spatial novelty in the Y-maze test was significantly higher in the control group compared to the 16/6-Id-injected mice (42% vs. 9%, respectively, P = 0.065). Depression-like behavior and locomotor activity were not significantly different between the16/6-Id-injected and the control mice. Immunohistochemistry analysis revealed an increase in astrocytes and microglial activation in the hippocampus and amygdala, in the 16/6-Id injected group compared to the control. CONCLUSIONS: Passive transfer of 16/6-Id antibodies directly into mice brain resulted in cognitive impairments and histological evidence for brain inflammation. These findings shed additional light on the diverse mosaic pathophysiology of neuropsychiatric lupus.See related Commentary article: http://www.biomedcentral.com/1741-7015/11/91.


Subject(s)
Antibodies/administration & dosage , Antibodies/toxicity , Cognitive Dysfunction/chemically induced , Encephalitis/chemically induced , Animals , Brain/pathology , Female , Histocytochemistry , Humans , Mice , Mice, Inbred C3H
8.
Immunology ; 132(1): 87-95, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20738420

ABSTRACT

Systemic lupus erythematosus (SLE) is an autoimmune disease that involves dysregulation of B and T cells. A tolerogenic peptide, designated hCDR1, ameliorates disease manifestations in SLE-afflicted mice. In the present study, the effect of treatment with hCDR1 on the CD74/macrophage migration inhibitory factor (MIF) pathway was studied. We report here that B lymphocytes from SLE-afflicted mice express relatively elevated levels of CD74, compared with B cells from healthy mice. CD74 is a receptor found in complex with CD44, and it binds the pro-inflammatory cytokine MIF. The latter components were also up-regulated in B cells from the diseased mice, and treatment with hCDR1 resulted in their down-regulation and in reduced B-cell survival. Furthermore, up-regulation of CD74 and CD44 expression was detected in brain hippocampi and kidneys, two target organs in SLE. Treatment with hCDR1 diminished the expression of those molecules to the levels determined for young healthy mice. These results suggest that the CD74/MIF pathway plays an important role in lupus pathology.


Subject(s)
Antigens, Differentiation, B-Lymphocyte/immunology , Autoantigens/immunology , B-Lymphocytes/immunology , Histocompatibility Antigens Class II/immunology , Intramolecular Oxidoreductases/immunology , Lupus Erythematosus, Systemic/immunology , Macrophage Migration-Inhibitory Factors/immunology , Nerve Tissue Proteins/immunology , Peptides/immunology , Animals , Antigens, Differentiation, B-Lymphocyte/biosynthesis , Antigens, Differentiation, B-Lymphocyte/genetics , Apoptosis/drug effects , Apoptosis/immunology , Autoantigens/chemistry , B-Lymphocytes/drug effects , Histocompatibility Antigens Class II/biosynthesis , Histocompatibility Antigens Class II/genetics , Humans , Immunomodulation , Intramolecular Oxidoreductases/biosynthesis , Intramolecular Oxidoreductases/genetics , Lupus Erythematosus, Systemic/pathology , Macrophage Migration-Inhibitory Factors/biosynthesis , Macrophage Migration-Inhibitory Factors/genetics , Mice , Mice, Inbred NZB , Nerve Tissue Proteins/chemistry , Peptides/chemistry , Peptides/pharmacology
9.
J Neuroimmunol ; 232(1-2): 151-7, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21129786

ABSTRACT

To determine the effect of the tolerogenic peptide hCDR1 on hippocampal neurogenesis, we treated SLE-afflicted (NZBxNZW)F1 mice with hCDR1 (once a week for 10weeks). The treatment resulted in the up-regulation of neurogenesis in the dentate gyrus and restored the NeuN immunoreactivity in brain hippocampi of the mice in association with increased gene expression of IGF-1, NGF and BDNF. Furthermore, hCDR1 treatment significantly up-regulated p-ERK and p-Akt that are suggested to be key components in mediating growth factor-induced neurogenesis. The observed effects of hCDR1 on hippocampal-neurogenesis and on associated signaling pathways suggest a potential role for hCDR1 in CNS lupus.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Hippocampus/drug effects , Lupus Vasculitis, Central Nervous System/drug therapy , Neurogenesis/drug effects , Peptide Fragments/therapeutic use , Animals , Blotting, Western , Disease Models, Animal , Enzyme Activation/drug effects , Female , Gene Expression/drug effects , Hippocampus/metabolism , Hippocampus/pathology , Humans , Immunohistochemistry , Lupus Vasculitis, Central Nervous System/metabolism , Lupus Vasculitis, Central Nervous System/pathology , Mice , Nerve Growth Factors/drug effects , Protein Kinases/drug effects , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects
11.
Autoimmun Rev ; 10(1): 22-6, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20659591

ABSTRACT

Treatment with a tolerogenic peptide, hCDR1, designed for the specific treatment of systemic lupus erythematosus (SLE) ameliorated the serological and kidney-related clinical manifestations in murine models of induced and spontaneous lupus. Furthermore, hCDR1 reduced brain pathology and improved behavior parameters in mice with central nervous system manifestations. The beneficial effects were associated with a diminished production of pathogenic cytokines (e.g. IFN-γ, IL-10, and IL-1ß) and with increased production of the immunosuppressive cytokine, TGF-ß. Treatment with hCDR1 up-regulated CD4 and CD8 regulatory T cells (Tregs) that played a key role in the ameliorating effects of hCDR1. Reduction of T cell apoptosis by hCDR1 contributed to the beneficial effects of hCDR. Moreover, treatment with hCDR1 down-regulated B cell maturation and autoreactive B cell survival by diminishing the B cell activating factor (BAFF/BLyS). Finally, hCDR1 suppressed in vivo gene expression of pathogenic cytokines, apoptosis and BLyS and up-regulated immunosuppressive molecules in peripheral blood lymphocytes of SLE patients. The latter was associated with clinical amelioration. Thus, treatment with hCDR1 leads to a cascade of events that culminate in the down-regulation of SLE-associated autoreactive T and B cells and in the clinical amelioration of lupus. hCDR1 is therefore a candidate for the specific treatment of SLE patients.


Subject(s)
Autoantigens/therapeutic use , Immune Tolerance , Lupus Erythematosus, Systemic/therapy , Nerve Tissue Proteins/therapeutic use , Peptides/therapeutic use , Animals , Autoantigens/genetics , Gene Expression Regulation/drug effects , Humans , Immune Tolerance/drug effects , Lupus Erythematosus, Systemic/immunology , Mice , Nerve Tissue Proteins/genetics , Peptides/genetics , Peptides/pharmacology , Swine
12.
J Clin Immunol ; 30(1): 34-44, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19756988

ABSTRACT

INTRODUCTION: Systemic lupus erythematosus (SLE) is characterized by a variety of autoantibodies and systemic clinical manifestations. A tolerogenic peptide, hCDR1, ameliorated lupus manifestations in mice models. The objectives of this study were to induce experimental SLE in pigs and to determine the ability of hCDR1 to immunomodulate the disease manifestations. RESULTS AND DISCUSSION: We report here the successful induction, by a monoclonal anti-DNA antibody, of an SLE-like disease in pigs, manifested by autoantibody production and glomerular immune complex deposits. Treatment of pigs with hCDR1 ameliorated the lupus-related manifestations. Furthermore, the treatment downregulated the gene expression of the pathogenic cytokines, interleukin (IL)-1beta, tumor necrosis factor alpha, interferon gamma, and IL-10, and upregulated the expression of the immunosuppressive cytokine transforming growth factor beta, the antiapoptotic molecule Bcl-xL, and the suppressive master gene, Foxp3, hence restoring the expression of the latter to normal levels. Thus, hCDR1 is capable of ameliorating lupus in large animals and is a potential candidate for the treatment of SLE patients.


Subject(s)
Antibodies, Antinuclear/administration & dosage , Complementarity Determining Regions/administration & dosage , Kidney/metabolism , Lupus Erythematosus, Systemic/immunology , Peptide Fragments/administration & dosage , Animals , Antibodies, Antinuclear/chemistry , Antibodies, Monoclonal/immunology , Cells, Cultured , Complementarity Determining Regions/chemistry , Cytokines/biosynthesis , Cytokines/genetics , Disease Models, Animal , Forkhead Transcription Factors/biosynthesis , Forkhead Transcription Factors/genetics , Gene Expression Regulation/drug effects , Gene Expression Regulation/immunology , Humans , Immune Tolerance , Immunomodulation , Kidney/immunology , Lupus Erythematosus, Systemic/physiopathology , Lupus Erythematosus, Systemic/therapy , Mice , Peptide Fragments/chemistry , Swine/immunology , bcl-X Protein/biosynthesis , bcl-X Protein/genetics
13.
J Autoimmun ; 34(2): 87-95, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19596183

ABSTRACT

Dysregulated expression of Bcl-xL and Bcl-2 may initiate the development of autoimmune diseases including systemic lupus erythematosus (SLE). A tolerogenic peptide designated hCDR1 was shown to ameliorate manifestations of spontaneous and induced murine SLE. Recently, we demonstrated that Bcl-xL plays a critical role in the modulating effects of hCDR1, as manifested by reducing the state of activation of lymphocytes and by down-regulating the secretion of the pathogenic cytokines, IFN-gamma and IL-10. Here we studied the role of Bcl-xL in the development and function of CD4 regulatory T-cells (Treg) from hCDR1-treated, SLE-afflicted (New-Zealand-Black x New-Zealand-White) F1 mice. We report that Bcl-xL was up-regulated in CD4 Treg of tolerized mice, where it played a role in inducing the regulatory/inhibitory molecules Foxp3, CTLA-4, and TGF-beta and in repressing PD-1. Further, Bcl-xL mediated the induction of CTLA-4 and TGF-beta in effector T cells (Teff) by CD4 Treg of the tolerized mice. The induction of Bcl-xL in Teff by Treg was TGF-beta dependent and CTLA-4 independent, leading to inhibition of proliferation and to a decrease in activated Teff. We conclude that Bcl-xL is required for the development and function of CD4 Treg, which ameliorate lupus following treatment with a tolerogenic peptide.


Subject(s)
Antibodies, Monoclonal/administration & dosage , Lupus Erythematosus, Systemic/immunology , Peptide Fragments/administration & dosage , T-Lymphocyte Subsets/metabolism , T-Lymphocytes, Regulatory/metabolism , bcl-X Protein/metabolism , Animals , Antigens, CD/metabolism , Cell Proliferation/drug effects , Cells, Cultured , Female , Forkhead Transcription Factors/biosynthesis , Humans , Immune Tolerance , Interferon-gamma/metabolism , Interleukin-10/metabolism , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/pathology , Lupus Erythematosus, Systemic/therapy , Lymphocyte Activation/drug effects , Mice , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/pathology , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/pathology , Transforming Growth Factor beta/metabolism , Up-Regulation , bcl-X Protein/genetics
14.
Arthritis Rheum ; 60(12): 3744-54, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19950283

ABSTRACT

OBJECTIVE: Central nervous system (CNS) involvement in systemic lupus erythematosus (SLE) is manifested by neurologic deficits and psychiatric disorders. The aim of this study was to examine SLE-associated CNS pathology in lupus-prone (NZBxNZW)F1 (NZB/NZW) mice, and to evaluate the ameliorating effects of treatment with a tolerogenic peptide, hCDR1 (human first complementarity-determining region), on these manifestations. METHODS: Histopathologic analyses of brains from lupus-prone NZB/NZW mice treated with vehicle, hCDR1, or a control scrambled peptide were performed. The messenger RNA expression of SLE-associated cytokines and apoptosis-related molecules from the hippocampi was determined. Anxiety-like behavior was assessed by open-field tests and dark/light transfer tests, and memory deficit was assessed using a novel object recognition test. RESULTS: Infiltration was evident in the hippocampi of the lupus-afflicted mice, and the presence of CD3+ T cells as well as IgG and complement C3 complex deposition was observed. Furthermore, elevated levels of gliosis and loss of neuronal nuclei immunoreactivity were also observed in the hippocampi of the mice with lupus. Treatment with hCDR1 ameliorated the histopathologic changes. Treatment with hCDR1 down-regulated the high expression of interleukin-1beta (IL-1beta), IL-6, IL-10, interferon-gamma, transforming growth factor beta, and the proapoptotic molecule caspase 8 in the hippocampi of the mice with lupus, and up-regulated expression of the antiapoptotic bcl-xL gene. Diseased mice exhibited increased anxiety-like behavior and memory deficit. Treatment with hCDR1 improved these parameters, as assessed by behavior tests. CONCLUSION: Treatment with hCDR1 ameliorated CNS pathology and improved the tested cognitive and mood-related behavior of the mice with lupus. Thus, hCDR1 is a novel candidate for the treatment of CNS lupus.


Subject(s)
Anxiety/immunology , Complementarity Determining Regions/immunology , Hippocampus/drug effects , Lupus Erythematosus, Systemic/drug therapy , Nervous System Diseases/drug therapy , Peptide Fragments/therapeutic use , Animals , Antigen-Antibody Complex/drug effects , Antigen-Antibody Complex/immunology , Anxiety/genetics , Anxiety/metabolism , Apoptosis/drug effects , Behavior, Animal/physiology , CD3 Complex/metabolism , Cytokines/genetics , Cytokines/metabolism , Disease Models, Animal , Female , Gene Expression/drug effects , Hippocampus/metabolism , Hippocampus/pathology , Humans , Immunization , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/immunology , Memory Disorders/drug therapy , Memory Disorders/immunology , Memory Disorders/pathology , Mice , Mice, Inbred NZB , Nervous System Diseases/genetics , Nervous System Diseases/immunology , RNA, Messenger/metabolism , T-Lymphocytes/metabolism , T-Lymphocytes/pathology , bcl-X Protein/genetics , bcl-X Protein/metabolism
15.
PLoS One ; 4(12): e8447, 2009 Dec 24.
Article in English | MEDLINE | ID: mdl-20041142

ABSTRACT

BACKGROUND: Regulatory T cells (Tregs) were shown to be central in maintaining immunological homeostasis and preventing the development of autoimmune diseases. Several subsets of Tregs have been identified to date; however, the dynamics of the interactions between these subsets, and their implications on their regulatory functions are yet to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: We employed a combination of mathematical modeling and frequent in vivo measurements of several T cell subsets. Healthy BALB/c mice received a single injection of either hCDR1--a tolerogenic peptide previously shown to induce Tregs, a control peptide or vehicle alone, and were monitored for 16 days. During this period, splenocytes from the treated mice were analyzed for the levels of CD4, CD25, CD8, CD28 and Foxp3. The collected data were then fitted to mathematical models, in order to test competing hypotheses regarding the interactions between the followed T cell subsets. In all 3 treatment groups, a significant, lasting, non-random perturbation of the immune system could be observed. Our analysis predicted the emergence of functional CD4 Tregs based on inverse oscillations of the latter and CD4(+)CD25(-) cells. Furthermore, CD4 Tregs seemed to require a sufficiently high level of CD8 Tregs in order to become functional, while conversion was unlikely to be their major source. Our results indicated in addition that Foxp3 is not a sufficient marker for regulatory activity. CONCLUSIONS/SIGNIFICANCE: In this work, we unraveled the dynamics of the interplay between CD4, CD8 Tregs and effector T cells, using, for the first time, a mathematical-mechanistic perspective in the analysis of Treg kinetics. Furthermore, the results obtained from this interdisciplinary approach supported the notion that CD4 Tregs need to interact with CD8 Tregs in order to become functional. Finally, we generated predictions regarding the time-dependent function of Tregs, which can be further tested empirically in future work.


Subject(s)
CD4 Antigens/metabolism , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Cell Communication , Interleukin-2 Receptor alpha Subunit/metabolism , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/immunology , Animals , CD28 Antigens/metabolism , CD8 Antigens/metabolism , Female , Flow Cytometry , Forkhead Transcription Factors/metabolism , Kinetics , Lymphocyte Subsets/cytology , Lymphocyte Subsets/immunology , Mice , Mice, Inbred BALB C , Models, Immunological , Spleen/cytology
16.
J Clin Endocrinol Metab ; 94(12): 5117-24, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19890026

ABSTRACT

CONTEXT: Autoimmune Addison's disease is thought to result from T cell mediated autoimmunity. Autoantibodies against the steroidogenic cytochrome P450 enzyme 21-hydroxylase (21OH) are found in most patients, and 21OH is therefore a likely target for antigen-specific T cells. OBJECTIVE: The aim was to study cellular immunity to 21OH and its associations with 21OH autoantibodies and human leukocyte antigen alleles in autoimmune Addison's disease. DESIGN/PATIENTS: Peripheral blood mononuclear cells were collected from 33 patients with autoimmune Addison's disease and 21 controls. Cellular proliferation and production of cytokines in response to stimulation with 21OH or 21OH-derived peptides were tested. RESULTS: Cellular proliferation (P = 0.0009) and secretion of interferon-gamma (P < 0.0001) in response to 21OH was significantly higher in patients compared to healthy controls and associated with the presence of 21OH autoantibodies (P = 0.0052). Furthermore, the 21OH-specific production of interferon-gamma was enhanced in the presence of 21OH autoantibodies. This effect was partially inhibited by antibodies against the Fc receptor for IgG, CD32. Moreover, mature dendritic cells proved superior to the other antigen-presenting cells in invoking cellular responses to 21OH. An association between cellular immunity to 21OH and the high-risk HLA genotype for Addison's disease, DRB1*0301-DQ2/DRB1*0404-DQ8, was observed (P = 0.0089). Finally, a significant association between the DRB1*0404-DQ8 haplotype and cellular responses to a 21OH-derived peptide predicted to bind to DRB1*0404 was detected (P = 0.0055). CONCLUSION: Patients with autoimmune Addison's disease have circulating 21OH-specific T cells, with amino acids 342-361 of 21OH possibly constituting a disease-specific epitope presented by HLA-DRB1*0404.


Subject(s)
Adrenal Insufficiency/enzymology , Autoimmune Diseases/enzymology , Steroid 21-Hydroxylase/metabolism , T-Lymphocytes/enzymology , Adrenal Insufficiency/immunology , Adult , Aged , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Autoantibodies/metabolism , Cell Adhesion/physiology , Cell Proliferation/drug effects , Cytokines/biosynthesis , Dendritic Cells/metabolism , Female , HLA Antigens/genetics , Humans , Interferon-gamma/metabolism , Interleukin-2/metabolism , Male , Middle Aged , Monocytes/metabolism , Receptors, Fc/metabolism , Steroid 21-Hydroxylase/immunology , Young Adult
17.
Immunology ; 128(2): 245-52, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19740381

ABSTRACT

Systemic lupus erythematosus (SLE) is an autoimmune disease mediated by T and B cells. It is characterized by a variety of autoantibodies and systemic clinical manifestations. A tolerogenic peptide, designated hCDR1, ameliorated the serological and clinical manifestations of SLE in both spontaneous and induced models of lupus. In the present study, we evaluated the status of mature B cells in the bone marrow (BM) of SLE-afflicted mice, and determined the effect of treatment with the tolerogenic peptide hCDR1 on these cells. We demonstrate herein that mature B cells of the BM of SLE-afflicted (New Zealand Black x New Zealand White)F(1) mice were largely expanded, and that treatment with hCDR1 down-regulated this population. Moreover, treatment with hCDR1 inhibited the expression of the pathogenic cytokines [interferon-gamma and interleukin (IL)-10], whereas it up-regulated the expression of transforming growth factor-beta in the BM. Treatment with hCDR1 up-regulated the rates of apoptosis of mature B cells. The latter was associated with inhibited expression of the survival Bcl-xL gene and of IL-7 by BM cells. Furthermore, the addition of recombinant IL-7 abrogated the suppressive effects of hCDR1 on Bcl-xL in the BM cells and resulted in elevated levels of apoptosis. Hence, the down-regulated production of IL-7 contributes to the hCDR1-mediated apoptosis of mature B cells in the BM of SLE-afflicted mice.


Subject(s)
Antibodies, Monoclonal/immunology , B-Lymphocyte Subsets/immunology , Bone Marrow Cells/immunology , Interleukin-7/antagonists & inhibitors , Lupus Erythematosus, Systemic/immunology , Peptide Fragments/immunology , Animals , Apoptosis/immunology , Disease Models, Animal , Down-Regulation/immunology , Female , Immune Tolerance/immunology , Interferon-gamma/biosynthesis , Interleukin-10/biosynthesis , Interleukin-7/immunology , Mice , Mice, Inbred Strains , Reverse Transcriptase Polymerase Chain Reaction/methods , Spleen/immunology , Transforming Growth Factor beta/biosynthesis , Up-Regulation/immunology
18.
Clin Immunol ; 133(1): 61-8, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19631585

ABSTRACT

Interferon-gamma (IFN-gamma) plays a pathogenic role in systemic lupus erythematosus (SLE). Uncontrolled IFN-gamma signaling may result from a deficiency in the negative regulator, namely, suppressor of cytokine signaling-1 (SOCS-1). We investigated the activation status of IFN-gamma signaling pathway in SLE-afflicted (New-Zealand-BlackxNew-Zealand-White)F1 mice and determined its responsiveness when treating with a tolerogenic peptide, hCDR1, which ameliorates SLE. SOCS-1 was suppressed and pSTAT1 was enhanced in spleen-derived cells from SLE-affected mice as compared with healthy controls. Treatment with hCDR1 reversed the expression of these two molecules in association with clinical amelioration. In vitro stimulation with IFN-gamma resulted in elevated levels of SOCS-1 in cells from both vehicle and hCDR1-treated mice but this effect reached significance only in cells of the latter group, which also exhibited reduced levels of pSTAT1. Thus, SOCS-1 is diminished in SLE-affected mice, and treatment with hCDR1 results in its up-regulation thereby restoring control of IFN-gamma signaling pathway.


Subject(s)
Autoantigens/therapeutic use , Immunosuppression Therapy , Interferon-gamma/immunology , Lupus Erythematosus, Systemic/therapy , Nerve Tissue Proteins/therapeutic use , STAT1 Transcription Factor/metabolism , Suppressor of Cytokine Signaling Proteins/metabolism , Animals , Disease Models, Animal , Down-Regulation/genetics , Down-Regulation/immunology , Female , Immune Tolerance/drug effects , Immune Tolerance/immunology , Lupus Erythematosus, Systemic/immunology , Mice , Peptides/therapeutic use , STAT1 Transcription Factor/agonists , Signal Transduction/drug effects , Signal Transduction/immunology , Suppressor of Cytokine Signaling 1 Protein , Suppressor of Cytokine Signaling Proteins/agonists , Suppressor of Cytokine Signaling Proteins/antagonists & inhibitors , Up-Regulation/genetics , Up-Regulation/immunology
19.
J Autoimmun ; 33(1): 77-82, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19346102

ABSTRACT

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by dysregulation of cytokines, apoptosis, and B- and T-cell functions. The tolerogenic peptide, hCDR1 (Edratide), ameliorated the clinical manifestations of murine lupus via down-regulation of pro-inflammatory cytokines and apoptosis, up-regulation of the immunosuppressive cytokine TGF-beta, and the induction of regulatory T-cells. In the present study, gene expression was determined in peripheral blood mononuclear cells of 9 lupus patients that were treated for 26 weeks with either hCDR1 (five patients), or placebo (four patients). Disease activity was assessed by SLEDAI-2K and the BILAG scores. Treatment with hCDR1 significantly down-regulated the mRNA expression of the pathogenic cytokines IL-1beta, TNF-alpha, IFN-gamma, and IL-10, of BLyS (B-lymphocyte stimulator) and of the pro-apoptotic molecules caspase-3 and caspase-8. In contrast, the treatment up-regulated in vivo gene expression of both TGF-beta and FoxP3. Furthermore, hCDR1 treatment resulted in a significant decrease in SLEDAI-2K (from 8.0+/-2.45 to 4.4+/-1.67; P=0.02) and BILAG (from 8.2+/-2.7 to 3.6+/-2.9; P=0.03) scores. Thus, the tolerogenic peptide hCDR1, immunomodulates, in vivo, the expression of genes that play a role in SLE, consequently restoring the global immune dysregulation of lupus patients. Hence, hCDR1 has a potential role as a novel disease-specific treatment for lupus patients.


Subject(s)
Antibodies, Monoclonal/administration & dosage , Forkhead Transcription Factors/metabolism , Immunotherapy , Leukocytes, Mononuclear/metabolism , Lupus Erythematosus, Systemic/immunology , Peptide Fragments/administration & dosage , Adult , Apoptosis/drug effects , Caspases/genetics , Caspases/immunology , Caspases/metabolism , Complementarity Determining Regions/immunology , Cytokines/genetics , Cytokines/immunology , Cytokines/metabolism , Disease Progression , Female , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/immunology , Gene Expression Regulation/immunology , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/pathology , Lupus Erythematosus, Systemic/blood , Lupus Erythematosus, Systemic/physiopathology , Lupus Erythematosus, Systemic/therapy , Male , Middle Aged , Self Tolerance/immunology , Severity of Illness Index , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/immunology , Transforming Growth Factor beta/metabolism , Treatment Outcome
20.
J Autoimmun ; 33(1): 58-67, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19329278

ABSTRACT

In primary adrenocortical failure (Addison's disease) caused by autoimmunity, autoantibodies to the steroidogenic cytochrome P450 enzyme 21-hydroxylase (21OH) are detected in the majority of patients. It is currently uncertain whether the autoantibodies themselves participate in the pathogenesis, or if they merely reflect an on-going T cell mediated response. The identification of T cells reactive with 21OH, if any, has been hampered by the lack of a high-quality antigen. In the current study recombinant human 21OH has been expressed in Spodoptera frugiperda insect cells using a baculovirus expression system. Recombinant enzymatically active 21OH was purified to apparent homogeneity by immobilized metal ion affinity chromatography. The purified enzyme was highly immunogenic in immunized SJL/J mice, and immune responses to 21OH-derived peptides assayed as T cell proliferation and interferon gamma production could be invoked after priming with the recombinant protein. Furthermore, purified 21OH was recognized by sera from patients with autoimmune Addison's disease, and it could block the binding of radiolabeled in vitro translated 21OH in a sensitive fluid-phase radioimmunoassay. We conclude that the recombinant preparation of 21OH presented here is of sufficient purity and quality to be used for studies of cellular and humoral immunity in autoimmune Addison's disease.


Subject(s)
Addison Disease/immunology , Autoantigens/metabolism , Peptides/metabolism , Steroid 21-Hydroxylase/metabolism , T-Lymphocytes/metabolism , Addison Disease/blood , Addison Disease/diagnosis , Addison Disease/pathology , Animals , Autoantibodies/blood , Autoantigens/chemistry , Autoantigens/genetics , Autoantigens/immunology , Baculoviridae/genetics , Cell Line , Cell Proliferation , Chromatography, Affinity , Cytochrome P-450 Enzyme System/metabolism , Humans , Immune Sera , Immunization , Interferon-gamma/metabolism , Lymphocyte Activation , Mice , Peptides/chemistry , Peptides/genetics , Peptides/immunology , Spodoptera/enzymology , Spodoptera/genetics , Steroid 21-Hydroxylase/chemistry , Steroid 21-Hydroxylase/genetics , Steroid 21-Hydroxylase/immunology , T-Lymphocytes/immunology , T-Lymphocytes/pathology , Transgenes/immunology
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