Molly Dayan

Cellular Sensitivity to Collagen in Thromboangiitis Obliterans

We studied 39 patients with thromboangiitis obliterans to determine their cellular and humoral immune responses to native human collagen Type I and Type III, which are constituents of blood vessels. Cell-mediated sensitivity to these collagens was measured by an antigen-sensitive thymidine-incorporation assay. The mean stimulation index--the ratio of thymidine incorporation in the presence of antigen to that in its absence--with both Type I and Type III collagens used as antigens was significantly higher in patients with thromboangiitis obliterans than in patients with arteriosclerosis obliterans or in healthy male controls. Lymphocytes from 77 per cent of the patients with thromboangiitis obliterans exhibited cellular sensitivity to human Type I or Type III collagens (or both). Furthermore, in 17 of 39 serum samples from the patients with thromboangiitis obliterans a low but significant level of anticollagen antibody activity was detected, whereas there was no antibody activity in serum samples from controls. These results suggest that there is a distinct etiologic factor in this disease and also raise the possibility of differentiating between thromboangiitis obliterans and arteriosclerosis obliterans by immunologic means.

The beneficial effects of treatment with tamoxifen and anti-oestradiol antibody on experimental systemic lupus erythematosus are associated with cytokine modulations.

In an attempt to elucidate the role of oestrogens in systemic lupus erythematosus (SLE) we investigated the effects of treatment with an oestrogen antagonist – tamoxifen and a monoclonal anti‐oestradiol (anti‐E2) antibody on mice in which experimental systemic lupus erythematosus (SLE) was induced by a human monoclonal anti‐DNA antibody bearing the 16/6 idiotype (16/6 Id). Thus, groups of BALB/c female mice were immunized with the 16/6 Id and 3 weeks following the booster injection, when antibody titres were elevated in the injected mice, treatment protocols with anti‐oestradiol or tamoxifen were initiated. Control groups that were not immunized with the 16/6 Id but were similarly treated with the above agents were included in the study. The treatment with the above agents had no effect on the total autoantibody titres; however, a decrease in the immunoglobulin G (IgG)2a/IgG1 ratio of the anti‐DNA antibodies was determined in the 16/6 Id immunized and treated mice. Further, both the anti‐oestradiol and tamoxifen had beneficial effects on the clinical manifestations (white blood cell counts, levels of protein in the urine and immune complex deposits in the kidneys) of the 16/6 Id immunized and treated mice. We have previously observed a significant elevation in interleukin‐1 (IL‐1) and tumour necrosis factor‐α (TNF‐α) secretion in mice with experimental SLE and a reduction in IL‐2, IL‐4 and interferon‐γ (INF‐γ) levels as compared with the levels detected in healthy controls. Treatment with either the anti‐oestradiol antibody or with tamoxifen restored the levels of all the above cytokines to the normal levels observed in the control mice.These findings suggest that cytokine modulation may be the basis for the therapeutic effects of both anti‐oestrogens in experimental SLE.

Automated long-term tracking and social behavioural phenotyping of animal colonies within a semi-natural environment

Social behaviour has a key role in animal survival across species, ranging from insects to primates and humans. However, the biological mechanisms driving natural interactions between multiple animals, over long-term periods, are poorly studied and remain elusive. Rigorous and objective quantification of behavioural parameters within a group poses a major challenge as it requires simultaneous monitoring of the positions of several individuals and comprehensive consideration of many complex factors. Automatic tracking and phenotyping of interacting animals could thus overcome the limitations of manual tracking methods. Here we report a broadly applicable system that automatically tracks the locations of multiple, uniquely identified animals, such as mice, within a semi-natural setting. The system combines video and radio frequency identified tracking data to obtain detailed behavioural profiles of both individuals and groups. We demonstrate the usefulness of these data in characterizing individual phenotypes, interactions between pairs and the collective social organization of groups.

Activity of matrix metalloproteinase-9 is elevated in sera of patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the increased production of autoantibodies and by systemic clinical manifestations and damage to multiple organs. The aim of the present study was to analyse matrix metalloproteinase (MMP)‐9 activity in sera of patients with active and inactive SLE in order to evaluate its role in the pathogenesis and course of the disease, as well as its diagnostic value. We measured activity levels of MMP‐9 and MMP‐2, using both gel zymography and activity assay kits, in sera of 40 SLE patients and of 25 healthy controls. We found that MMP‐9 activity, but not MMP‐2 activity, is significantly elevated in the sera of SLE patients compared with sera samples of healthy controls. High activity levels of MMP‐9 were determined in sera of 68% of the SLE patients. Elevated levels of MMP‐9 were correlated with the presence of discoid rash, Raynaud phenomenon, pneumonitis, mucosal ulcers and anti‐phospholipid antibodies. Changes in activity levels of MMP‐9, but not of MMP‐2, were observed in sera of the same patient at different periods of the disease course. High levels of MMP‐9 did not correlate with disease activity index (SLEDAI, BILAG) in female patients, but correlated with SLE activity in the group of male patients. The results of the present study suggest that MMP‐9 plays a role in the pathogenesis of SLE.

A peptide based on the complementarity determining region 1 of a human monoclonal autoantibody ameliorates spontaneous and induced lupus manifestations in correlation with cytokine immunomodulation.

A peptide based on the sequence of the complementarity determining region (CDR) 1 of a human monoclonal anti-DNA autoantibody that bears the 16/6 idiotype (16/6Id) was synthesized as a potential candidate for the treatment of SLE patients. The peptide, designated hCDR1, did not induce experimental SLE upon active immunization of mice. The ability of the peptide to treat an already established lupus that was either induced in BALB/c mice or developed spontaneously in (NZB × NZW)F1 mice was tested. Ten weekly injections of hCDR1 (200, 50 μg/mouse) given subcutaneously mitigated disease manifestations (e.g., leukopenia, proteinuria and kidney damage) and resulted in a prominent reduction in the dsDNA specific antibody titers. Furthermore, treatment with hCDR1 resulted in reduced secretion and expression of the “pathogenic” cytokines [i.e., INFγ, IL-1β, TNFα (in the induced model) and IL-10], whereas the immunosuppressive cytokine TGFβ was up-regulated. Thus, the significant ameliorating effects of hCDR1 are manifested at least partially via the immunomodulation of the cytokine profile. These results suggest that hCDR1 is a potential candidate for a novel treatment of SLE patients.

Effect of aging on cytokine production in normal and experimental systemic lupus erythematosus afflicted mice

Aging mice of strains susceptible to the induction of experimental systemic lupus erythematosus (SLE) develop a milder disease than young animals. To find out whether the decrease in susceptibility to disease is due to age-associated changes in cytokine profile, we first examined the secretion of cytokines by healthy mice aged 2-15 months. A gradual age-related decline in the levels of interleukin (IL)-2 and interferon (IFN) gamma, and an increase in IL-4, IL-10, IL-1, and tumor necrosis factor (TNF) alpha were observed. Experimental SLE was induced in 2- and 10-month-old mice by immunization with the monoclonal anti-DNA antibody bearing the 16/6 Id. Early increased production of pro-inflammatory cytokines (TNFalpha and IL-1), followed by a peak of the Th1-type cytokines (IL-2, IFNgamma) were observed in young mice. The Th2-type cytokines (IL-4, IL-10) peaked later. In contrast, only a mild increase in all of the above cytokines was determined in 10-month immunized mice. It thus appears that the decline in susceptibility to SLE induction in older mice may be related to changes in the capacity to produce cytokines.

Treatment of lupus patients with a tolerogenic peptide, hCDR1 (Edratide): immunomodulation of gene expression.

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.

The role of CD8+CD28− regulatory cells in suppressing myasthenia gravis-associated responses by a dual altered peptide ligand

Myasthenia gravis (MG) and experimental autoimmune MG are T cell-dependent antibody-mediated autoimmune diseases. A dual altered peptide ligand (APL), composed of the tandemly arranged two single amino acid analogs of two myasthenogenic peptides, p195-212 and p259-271, down-regulated in vitro and in vivo MG-associated T cell responses. In the present study, we investigated the role of CD8+CD28− regulatory cells in the mechanism of action of the dual APL. We demonstrated that treatment of mice with the dual APL concomitant with immunization with a myasthenogenic peptide resulted in an increased population of CD8+CD28− cells that express forkhead box P3 (Foxp3). The dual APL inhibited the proliferation of lymph node (LN) cells of the Torpedo acetylcholine receptor-immunized WT C57BL/6 mice, whereas the inhibition was abrogated in CD8−/− knockout mice. Moreover, the dual APL did not inhibit the secretion of IFN-γ by LN cells from CD8−/− mice immunized with Torpedo acetylcholine receptor. However, the mRNA expression of IL-10 and TGF-β by LN cells from CD8−/− mice was up-regulated similarly to that of the WT mice. Furthermore, the dual APL elevated the proapoptotic markers caspases 3 and caspase 8, whereas it down-regulated the antiapoptotic marker Bcl-xL in both CD8−/− and WT mice. Finally, the dual APL-induced CD4+CD25+Foxp3+ cells were up-regulated in CD8−/− mice to a similar extent to that observed in the WT mice. Thus, we suggest that CD8+CD28− regulatory cells play a partial role in the mechanism of action by which the dual APL suppresses experimental autoimmune MG-associated T cell responses.

Modulation of autoreactive responses of peripheral blood lymphocytes of patients with systemic lupus erythematosus by peptides based on human and murine anti-DNA autoantibodies

Two peptides, based on the sequences of the complementarity‐determining regions (CDR) 1 and 3 of a pathogenic murine monoclonal anti‐DNA autoatibody that bears the 16/6 idiotype (Id), were shown to either prevent or treat an already established systemic lupus erythematosus (SLE) in two murine models of lupus. Two additional peptides based on the human monoclonal anti‐DNA, 16/6 Id were synthesized. This study was undertaken in order to investigate the ability of the CDR‐based peptides to immunomodulate SLE‐associated responses of peripheral blood lymphocytes (PBL) of SLE patients. PBL of 24 of the 62 SLE patients tested proliferated in vitro following stimulation with the human 16/6 Id. Peptides based on the CDRs of both the human and murine anti‐DNA autoantibodies inhibited efficiently and specifically the 16/6 Id‐induced proliferation and IL‐2 production. The latter inhibitions correlated with an up‐regulated production (by 2·5–3·5‐fold) of the immunosuppressive cytokine, TGF‐β. Overall, the results of our study demonstrate that the CDR‐based peptides are capable of down‐regulating in vitro autoreactive T cell responses of PBL of SLE patients. Thus, these peptides are potential candidates for a novel specific treatment of SLE patients.

Direct binding of a myasthenia gravis related epitope to MHC class II molecules on living murine antigen-presenting cells

MHC gene products present antigenic epitopes to the antigen receptor on T cells. Nevertheless, direct binding of such epitopes to MHC class II proteins on normal living antigen‐presenting cells (APCs) has not yet been demonstrated. We have previously shown a significant difference in the ability of T cells of myasthenia gravis (MG) patients to proliferate in response to the synthetic peptide p195‐212 of the human acetylcholine receptor (AChR) alpha‐subunit in comparison to healthy controls. The observed proliferative responses correlated significantly with HLA‐DR5. Moreover, lymph node cells of various mouse strains that were primed with the T cell epitope, p195‐212, were found to proliferate to different extents. To investigate these observations further, we designed an assay for direct binding of p195‐212 to MHC class II proteins on the surface of freshly prepared splenic adherent cells. Binding of a biotinylated p195‐212 was monitored using phycoerythrin‐avidin by flow cytometry. Fifteen to sixty per cent of the cells were labeled following incubation with the biotinylated peptide. Binding was observed only to splenic adherent cells derived from mouse strains of which T cells were capable of proliferating in response to p195‐212. The binding specificity, in terms of epitope structure and its site of interaction on the cells, was shown by its inhibition with an excess of the unlabeled peptide or with the relevant monoclonal anti‐I‐A antibodies. These results constitute the first direct evidence for the specific binding of a T cell epitope to live APC.