Common infectious agents prevalence in antiphospholipid syndrome.

Antiphospholipid syndrome is characterized by thrombosis and pregnancy loss. Infections are generally associated with autoimmune diseases, but in the setting of antiphospholipid syndrome this link has been suggested as having a pathogenic role. In this study, 98 patients with antiphospholipid syndrome were screened for antibodies directed to several infectious agents. The main finding in this study is the significantly higher prevalence of IgM antibodies to toxoplasma and rubella. This novel finding suggests that these infections might be associated with antiphospholipid syndrome. As autoimmune diseases and, in particular, antiphospholipid syndrome are associated with infections, mainly the catastrophic type of the syndrome, this finding implies that a current infection with these agents, i.e. toxoplasma and rubella, might either be related to the pathogenesis of antiphospholipid syndrome or alternatively to its manifestations.

MHC class I expression regulates susceptibility to spontaneous autoimmune disease in (NZBxNZW)F1 mice.

(NZBxNZW)F1 mice spontaneously develop with age an autoimmune disease that resembles the human disease, systemic lupus erythematosus (SLE). Previous studies have demonstrated that susceptibility to experimentally induced SLE depended on the expression of MHC class I molecules: mice deficient in beta2-microglobulin did not express cell surface class I and were resistant to the induction of experimental SLE. Furthermore, the spontaneous SLE-like disease of (NZBxNZW)F1 mice was ameliorated by treatment with an agent that reduces MHC class I expression, methimazole (MMI). In the present study, the role of MHC class I has been examined in (NZBxNZW)F1 mice deficient in beta2-microglobulin expression. Homozygous (NZBxNZW)F1 beta2m-/- mice do not express class I or develop CD8+ T cells. Surprisingly, they show an increased susceptibility to disease. In sharp contrast, heterozygous (NZBxNZW)F1 beta2m+/- express class I, albeit at reduced levels, develop normal levels of CD8+ T cells and are less susceptible to autoimmune disease, relative to their wild-type litter mates. Taken together, these findings suggest that class I expression regulates the development of disease, both positively and negatively. We speculate that MHC class I expression itself confers susceptibility to disease through presentation of self-peptides, while also selecting for a CD8+ suppressor T cell population that mitigates disease.

Autoantibodies to a NR2A peptide of the glutamate/NMDA receptor in sera of patients with systemic lupus erythematosus.

OBJECTIVE: To determine the prevalence of autoantibodies directed against an epitope of the glutamate/N-methyl-D-aspartic acid (NMDA) receptor subunit NR2A (which is highly expressed in human brain) in the sera of lupus patients, and to investigate the possible correlation of these antibodies with clinical and serological manifestations of systemic lupus erythematosus (SLE). METHODS: Sera were obtained from 109 consecutive SLE patients. Controls were 65 patients with myasthenia gravis, 19 with autoimmune polyendocrine syndrome type I (APS I), and 65 healthy donors. A 15 amino acid long peptide based on a sequence within the NR2A subunit of the NMDA/glutamate receptor was synthesised. Antibodies to this peptide were determined by enzyme linked immunosorbent assay. Antibodies against double stranded DNA (dsDNA) were measured by Chrithidia luciliae assay. Disease activity was determined using the SLE disease activity index (SLEDAI). RESULTS: Sera of 34/109 SLE patients (31%) reacted specifically with the NR2A peptide compared with only 4/65 myasthenia gravis patients (6.1%, p<0.001), 1/19 APS I patients (5.3%, p<0.02), and 3/65 healthy controls (4.6%, p<0.001). No correlation was found between the presence of NR2A and dsDNA or anti-cardiolipin specific autoantibodies. In addition, no significant correlation was observed between the presence of NR2A specific antibodies and the SLEDAI score or any lupus related clinical manifestations. CONCLUSIONS: A significant number of SLE patients (31%) have NR2A specific antibodies that do not correlate with anti-dsDNA antibodies. Additional studies of lupus patients with neurological disorders should elucidate the role of NR2A specific antibodies in lupus related CNS manifestations.

Curcumin synergistically potentiates the growth inhibitory and pro-apoptotic effects of celecoxib in pancreatic adenocarcinoma cells.

BACKGROUND AND AIM: Adenocarcinoma of the Pancreas is a leading cause of cancer-related mortality, accounting for an estimated 30,000 deaths per year in the United States. Multiple studies have indicated that specific cyclooxygenase-2 (COX-2) inhibitors may serve in the prevention and treatment of a variety of malignancies including pancreatic adenocarcinoma. Recent studies had shown that the long-term use of high concentration of COX-2 inhibitors is not toxic free and may be limited due to serious gastrointestinal and cardiovascular side effects. The chemopreventive efficacy of the phytochemical, curcumin has been demonstrated in several in vitro and animal models. In this study we investigated whether curcumin potentiates the growth inhibition effect of a COX-2 inhibitor (celecoxib, Pfizer, NY, USA) in human pancreatic cancer cells. METHODS: P-34 (expressing high levels of COX-2), and MIAPaCa (expressing low levels of COX-2) and Panc-1 (no expression of COX-2) evaluated cell lines were exposed to different concentrations of celecoxib (0-40 microM), curcumin (0-20 microM) and their combination. Cell viability was by XTT assay. Apoptosis was assessed by flow cytometry and COX-2 expression was measured by Western blotting analysis. RESULTS: In P-34 cells, curcumin synergistically potentiated the inhibitory effect of celecoxib on cell growth. The growth inhibition was associated with inhibition of proliferation and induction of apoptosis. Western blot analysis showed that COX-2 expression was down-regulated by the combination therapy. CONCLUSION: Curcumin synergistically augments the growth inhibition inserted by celecoxib in pancreatic cancer cells expressing COX-2. The synergistic effect was mediated through inhibition of COX-2. This may enable the use of celecoxib at lower and safer concentrations and may pave the way for a more effective treatment in this devastating disease.

Amelioration of lupus manifestations by a peptide based on the complementarity determining region 1 of an autoantibody in severe combined immunodeficient (SCID) mice engrafted with peripheral blood lymphocytes of systemic lupus erythematosus (SLE) patients.

A peptide based on the complementarity determining region (CDR)1 of a human monoclonal anti-DNA autoantibody (hCDR1) was shown to either prevent or treat an already established murine lupus in systemic lupus erythematosus (SLE)-prone mice or in mice with induced experimental SLE. The present study was undertaken to determine the therapeutic potential of hCDR1 in a model of lupus in severe combined immunodeficient (SCID) mice engrafted with peripheral blood lymphocytes (PBL) of patients with SLE. To this end, PBL obtained from lupus patients were injected intraperitoneally into two equal groups of SCID mice that were treated either with the hCDR1 (50 micro g/mouse) once a week for 8 weeks, or with a control peptide. Mice were tested for human IgG levels, anti-dsDNA autoantibodies, anti-tetanus toxoid antibodies and proteinuria. At sacrifice, the kidneys of the successfully engrafted mice were assessed for human IgG and murine complement C3 deposits. Of the 58 mice transplanted with PBL of SLE patients, 38 (66%) were engrafted successfully. The mice that were treated with the control peptide developed human dsDNA-specific antibodies. Treatment with hCDR1 down-regulated the latter significantly. No significant effect of the treatment on the levels of anti-tetanus toxoid antibodies could be observed. Treatment with hCDR1 resulted in a significant amelioration of the clinical features manifested by proteinuria, human IgG complex deposits as well as deposits of murine complement C3. Thus, the hCDR1 peptide is a potential candidate for a novel specific treatment of SLE patients.

Beneficial effects of the anti-oestrogen tamoxifen on systemic lupus erythematosus of (NZBxNZW)F1 female mice are associated with specific reduction of IgG3 autoantibodies.

BACKGROUND: Sex hormones have been shown to influence the immune system and to modify the course of autoimmune disorders. OBJECTIVE: To examine the effects of the oestrogen antagonist tamoxifen on the course of systemic lupus erythematosus (SLE) in (NZBxNZW)F1 mice. METHODS: Groups of 8 week old (NZBxNZW)F1 female mice were treated with tamoxifen (800 micro g/mouse; twice a week) or with double distilled water for four months. Mice were evaluated monthly for the presence of autoantibodies directed against DNA and nuclear extract (NE) by enzyme linked immunosorbent assay (ELISA). White blood cells and thrombocytes were quantified by a cell counter and proteinuria by combistix kit. At 6 months of age, all mice that did not die spontaneously were killed and evaluated for the presence of glomerular immune deposits by indirect immunofluorescence assay. IgG isotypes of autoantibodies in the mouse sera and glomeruli were determined by gamma chain specific antibodies. RESULTS: Tamoxifen treatment significantly reduced autoantibody production directed against either NE or DNA. The latter reduction was mainly in autoantibodies of the IgG3 isotype. Furthermore, tamoxifen had significant beneficial effects on the course of SLE in (NZBxNZW)F1 mice. At 6 months of age, 40% of the untreated mice died spontaneously, whereas all the tamoxifen treated mice were still alive. All untreated mice showed severe thrombocytopenia and persistent proteinuria, with diffuse glomerular immune deposits of IgG2a and IgG3 isotypes in their kidneys. In contrast, the tamoxifen treated mice had a normal number of thrombocytes and only minimal proteinuria. Moreover, glomerular immune deposits were detected in <40% of the tamoxifen treated mice. The latter were mainly of the IgG2a but not of the IgG3 isotype. CONCLUSION: The results clearly show the remarkable therapeutic effects of tamoxifen on SLE of (NZBxNZW)F1 female mice and suggest that these beneficial effects are related to the specific reduction of IgG3 autoantibodies.

Suppression of experimental systemic lupus erythematosus (SLE) in mice via TNF inhibition by an anti-TNFalpha monoclonal antibody and by pentoxiphylline.

We have previously shown that the clinical manifestations of experimental systemic lupus erythematosus (SLE) correlate with an early increased secretion of TNFalpha and IL-1. In the present study, we examined the efficacy of two therapeutic modalities which lower TNFalpha production or activity, on the clinical manifestations of the disease. Experimental SLE was induced in naive C3H.SW mice by injection of the human anti-DNA monoclonal antibody (mAb) bearing the common idiotype, 16/6 Id. Two weeks after booster injections, treatment with either an anti-TNFalpha mAb, or pentoxiphylline (PTX) was started, for a period of 6 weeks. Production of TNFalpha (by splenocytes) and IL-1 (by peritoneal macrophages) was determined 3 and 7 months after disease induction. The experimental mice were also followed for disease manifestations. Both treatment protocols, with anti-TNFalpha mAb and with PTX, reduced the production of the two pro-inflammatory cytokines. TNFalpha and IL-1, in mice with experimental SLE. Anti-DNA antibodies were significantly lower in the mice treated with either protocol. In addition, a significantly lower rate of leukopenia, proteinuria and immune complex deposition was observed in treated mice. Abrogation of TNFalpha and IL-1 production in the early stages of experimental SLE by an anti-TNFalpha mAb or by PTX improves the clinical status of mice afflicted with this autoimmune disease.

The mechanism by which a peptide based on complementarity-determining region-1 of a pathogenic anti-DNA auto-Ab ameliorates experimental systemic lupus erythematosus.

A peptide based on complementarity-determining region (CDR)-1 of a monoclonal murine anti-DNA Ab that bears the common idiotype, 16/6Id, was synthesized and characterized. The peptide, designated pCDR1, was found to be an immunodominant T-cell epitope in BALB/c mice. The CDR1-based peptide was shown to be capable of inhibiting the in vivo priming of BALB/c mice immunized with the peptide or with the whole anti-DNA 16/6Id(+) mAbs of either mouse or human origin. We show here that administration of pCDR1 (weekly, i.v., 100 microgram/mouse) in aqueous solution for 5 weeks starting at the time of disease induction with the human 16/6Id prevented the development of clinical manifestations of experimental systemic lupus erythematosus (SLE). Further, 10 weekly injections of pCDR1 to BALB/c mice with an established experimental SLE down-regulated clinical manifestations of SLE (e.g., anti-DNA auto-Abs, leukopenia, proteinuria, immune complex deposits in the kidneys) in the treated mice. Prevention of SLE induction was shown to be associated mainly with a decrease in the levels of IL-2, INFgamma, and the proinflammatory cytokine TNFalpha. On the other hand, the secretion of the immunosuppressive cytokine TGFbeta was elevated. Amelioration of the clinical manifestations of an already established experimental SLE correlated with a dramatic decrease in TNFalpha secretion, elevated levels of TGFbeta, and immunomodulation of the Th1 and Th2 type cytokines to levels close to those observed in healthy mice.

A peptide based on the sequence of the CDR3 of a murine anti-DNA mAb is a better modulator of experimental SLE than its single amino acid-substituted analogs.

A peptide based on the complementarity determining region (CDR) 3 of a pathogenic anti-DNA 16/6 Id(+) monoclonal antibody was previously shown to be a dominant T-cell epitope in experimental SLE, and to be capable of inhibiting SLE-associated proliferative responses. Single amino acid-substituted analogs of pCDR3 were designed and analyzed for their ability to stimulate or inhibit the proliferation of a pCDR3-specific T-cell line. Alterations in positions 9 and 10 neutralized the proliferative potential of pCDR3, whereas alterations in positions 6-8 and 11-15 retained the proliferative potential of the peptides. Similar to pCDR3, its analogs Ala11 and Nle13 inhibited efficiently the in vivo priming of lymph node cells either to pCDR3 or to the human monoclonal anti-DNA 16/6 Id(+) antibody. Substituting both positions 11 (Tyr --> Ala) and 13 (Met --> Nle) reduced this inhibitory capacity compared to the single substituted analogs. Also, truncation of pCDR3 at the C- and/or N-terminus obliterated the inhibitory activities of the peptide. Analogs Ala11 and Nle13 immunomodulated serological and clinical smanifestations of experimental SLE. Nevertheless, the original pCDR3 was a more efficient modulator of the disease.

Prevention of systemic lupus erythematosus-like disease in (NZBxNZW)F1 mice by treating with CDR1- and CDR3-based peptides of a pathogenic autoantibody.

Two peptides based on the complementarity-determining regions (CDR) of a pathogenic murine anti-DNA antibody were employed in an attempt to prevent the spontaneous systemic lupus erythematosus (SLE)-like disease of (NZBxNZW)F1 mice. Female mice, at the age of 2 months, were injected with either the CDR1- or the CDR3-based peptides (pCDR1, pCDR3) subcutaneously or intravenously in aqueous solution for a total of 8-10 treatments. A reduction was observed in the total and pathogenic IgG2a and IgG3 anti-DNA antibody titers in the CDR-treated groups. Treatment reduced the number of mice that developed proteinuria and immune complex deposits in their kidneys. The severity of renal pathology was significantly reduced in the pCDR3 (P<0.02) and pCDR1 (P< or = 0.05) treated mice. Thus, both CDR-based peptides administered in aqueous solution were capable of preventing the SLE-like disease in (NZBxNZW)F1 mice, although the beneficial effects of pCDR3 appeared to be more pronounced than those of pCDR1 in the treated mice.

Characterization and role in experimental systemic lupus erythematosus of T-cell lines specific to peptides based on complementarity-determining region-1 and complementarity-determining region-3 of a pathogenic anti-DNA monoclonal antibody.

Peptides based on the complementarity-determining region 1 (CDR1) and CDR3 of an anti-DNA monoclonal antibody (mAb) carrying the 16/6 idiotype (Id) were shown to induce experimental systemic lupus erythematosus (SLE) in susceptible mouse strains. In the present study, T-cell lines specific to the pCDR1 and pCDR3 peptides were established in BALB/c and in SJL mice, respectively. The T-cell lines were characterized and analysed for their pathogenicity upon administration to syngeneic mouse strains. Both T-cell lines expressed the alphabeta T-cell receptor (TCR) and the CD4+ CD8- phenotype. Additionally, both cell lines secreted interleukin (IL)-4 and IL-10 upon stimulation with their specific peptide, thus belonged to the T helper 2 (Th2) subset. Upon immunization, the pCDR3-specific T-cell line induced experimental SLE in SJL mice. The animals produced high levels of autoimmune anti-DNA and antinuclear protein antibodies, as well as anti-16/6 Id antibodies (Abs). Furthermore, the mice developed clinical manifestations, including leukopenia, proteinuria and accumulation of immune complex deposits in their kidneys. The pCDR1-specific T-cell line failed to induce SLE when injected into BALB/c mice. It is thus suggested that pCDR3 is an immunodominant epitope in experimental SLE and that pCDR3-specific T cells initiate autoimmunity, leading to SLE, probably via epitope spreading.

Differing MHC class I requirements for induction and propagation of experimental systemic lupus erythematosus.

Mice deficient in beta2-microglobulin expression are resistant to the induction of experimental systemic lupus erythematosus (SLE). The present studies were designed to identify the beta2-microglobulin-dependent cell surface molecule(s) that confers sensitivity to experimental SLE, and to determine its role in disease development. We report hat mice lacking the transporter associated with antigen presentation (TAP-/-) were also resistant to disease, whereas CD1-/- and CD8-/- mice were susceptible; susceptibility also did not correlate with neonatal Fc receptor or HEPH expression. These data indicate that disease susceptibility is determined by expression of MHC class I. Furthermore, by analyzing both adoptive transfer and radiation bone marrow chimeras, we demonstrate that MHC class I expression is necessary for propagation of disease, but not for induction of pathogenic cells.

DNA levels in immune complexes circulating in mice with induced systemic lupus erythematosus.

The levels of DNA in IgG immune complexes, which appeared in the circulation of mice after the induction of systemic lupus erythematosus (SLE), were measured by an immunochemical quantitative assay using monoclonal anti-dsDNA antibodies. The amount of DNA in immune complexes was already high at 10-12 days following the injection of a human monoclonal anti-DNA antibody bearing the major idiotype designated 16/6 in complete Freund's adjuvant, i.e. long before the appearance of clinical manifestations. The injections of these antibodies in the alum-precipitated form did not induce the formation of DNA:anti-DNA complexes as well as SLE itself. The levels of DNA in circulating immune complexes were in general high throughout the whole experimental period (up to 7 months) decreasing gradually before the first clinical manifestations appeared and thereafter, when the disease was fully developed. Such a decrease could be explained by the retention of immune complexes in kidneys. The levels of DNA in immune complexes circulating in normal mice or in mice receiving injections of complete Freund's adjuvant was very low. Treatment of experimental SLE that affected the clinical manifestations prevented the formation of high levels of DNA containing immune complexes.

Spontaneous autoimmune disease in (NZB x NZW)F1 mice is ameliorated by treatment with methimazole.

(NZB x NZW)F1 mice spontaneously develop with age an autoimmune disease that resembles the human disease, systemic lupus erythematosus (SLE). The present study demonstrates that methimazole (MMI), an agent used in the treatment of autoimmune thyroid disease, is effective in mitigating the development of this SLE-like autoimmune disease in (NZB x NZW)F1 mice. MMI significantly reduces the incidence and severity of proteinuria and deposition of immune complexes in the kidney. Previous studies have demonstrated that development of an experimentally induced SLE, which was prevented by MMI treatment, depended on the expression of MHC class I molecules. We now report that class I levels on both T cells and B cells from old (NZB x NZW)F1 MHC class I are markedly elevated relative to those from young F1 mice. Furthermore, treatment of (NZB x NZW)F1 mice with MMI reduced MHC class I expression on their PBL concomitant with amelioration of disease, raising the possibility that class I molecules may play a role in the generation of spontaneous autoimmune disease in these mice.

Genetic analysis of experimentally induced lupus in mice.

The DBA/2 and C57BL/6 mouse strains, as well as the BXD RI lines derived from these strains, were used to map the genes controlling experimentally induced systemic lupus erythematosus (SLE). SLE was induced using two immunologic approaches: (1) immunization with the human monoclonal anti-DNA antibody expressing the 16/6Id, to which the DBA/2 strain is susceptible (responder) and the C57BL/6 strain is resistant (nonresponder); and (2) induction of autoimmune GVHD in B6D2F1 hosts by inoculation of parental DBA/2 (induces SLE) or C57BL/6 (does not induce SLE) T cells. By both approaches the BXD RI lines could be divided into distinct DBA/2-like and C57BL/6-like categories. Concordance of SLE induced by both methods was observed for susceptibility and resistance in 13/15 BXD lines (P < 0.005). The results suggest that at least two non-H-2 genes control susceptibility and resistance to experimentally induced SLE, one mapping to chromosome 7 and the other mapping to chromosome 14.

Modulation of murine systemic lupus erythematosus with peptides based on complementarity determining regions of a pathogenic anti-DNA monoclonal antibody.

Experimental systemic lupus erythematosus (SLE) can be induced in naive mice by immunization with a murine monoclonal anti-DNA antibody (mAb), 5G12, that bears a major idiotype designated 16/6 Id. Strain-dependent differences were observed in the proliferative responses of lymph node cells of mice immunized with two peptides based on the sequences of the complementarity determining region (CDR) 1 and 3 of mAb 5G12. The capacity of the peptides to bind to major histocompatibility complex class II molecules correlated with the proliferative responses. Immunization of high responder strains with the CDR-based peptides led to production of autoantibodies and clinical manifestations characteristic to experimental SLE. The CDR-based peptides could prevent autoantibody production in neonatal mice that were immunized later either with the peptide or with the pathogenic autoantibody. Furthermore, the peptides inhibited specific proliferation of lymph node cells of mice immunized with the same peptide, with mAb 5G12 or with the human mAb anti-DNA, 16/6 Id. Thus, the CDR-based peptides are potential candidates for therapy of SLE.

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-alpha (TNF-alpha) secretion in mice with experimental SLE and a reduction in IL-2, IL-4 and interferon-gamma (INF-gamma) 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.

Effect of injection of anti-CD4 and anti-CD8 monoclonal antibodies on the development of experimental systemic lupus erythematosus in mice.

Induction of an experimental disease resembling systemic lupus erythematosus (SLE) has been achieved in mice by immunization with a human monoclonal anti-DNA antibody, bearing a common idiotype, designated 16/6 Id. In the present study we used anti-CD4 and anti-CD8 antibodies to modulate the induction and development of the experimental disease. Thus, depletion of CD4+ T cells prior to the immunization with the 16/6 inhibited the induction of experimental SLE. In contrast, injection of anti-CD4 antibodies to mice that were already immunized with the 16/6 Id did not prevent the development of the disease. Furthermore, administration of anti-CD8 antibodies either before or after priming with the 16/6 Id increased the serological and clinical manifestations of the disease. These results demonstrate the pathogenic role of CD4+ and CD8+ T cells in the induction and development of the experimental SLE.