The potential of oral treatment with FWGE (FWGE ), a new benzoquinone-containing fermentation product of wheat germ, on features of experimental systemic lupus erythematosus (SLE) in naive mice, induced by idiotypic manipulation, was studied. We assessed the effect of FWGE on the profile of autoantibody production and the response of Th1=Th2 related cytokines as well as the clinical picture of experimental SLE in the SLE-induced mice. When the product was given in the pre-immunization period, down-regulation of autoantibody production (anti-dsDNA, mouse 16=6 Id, and anti-histones) following treatment with FWGE  was noted (eg anti-dsDNA decreased from 0.898 0.097 OD at 405 nm to 0.519 0.103 OD following treatment). This effect was sustained for at least 4 weeks after discontinuation of the therapy. Serological manifestations associated with a delay in Th2 response (IL-4 and IL-10) were recorded (eg IL-4 decreased from 91.7 8.11 to 59.55 7.78 ng=ml in splenocyte condition media). The mice showed normal ESR, WBC and less than 100 mg=dl of protein in the urine in comparison to > 300 mg=dl protein in the SLE non-treated mice. In conclusion, oral intake of FWGE can ameliorate the clinical manifestations of experimental SLE, via affecting the Th1=Th2 network inhibiting Th2 response. Lupus (2001) 10, 622–627.

Animal models of systemic lupus erythematosus (SLE) and of antiphospholipid syndrome (APS), are of a great value in assessing novel therapies and approaches for the treatment of these conditions, due to the obvious limitations of evaluating new treatment modalities on patients. A few years ago, we and others introduced a novel method for the induction of experimental autoimmune conditions1–8 including SLE and APS, in naive mice, based on the idiotypic immunization. Thus immunization of naive mice with a specific autoantibody (Ab1; eg anti-dsDNA, anti-cardiolipin, or anti-proteinase-3 Abs), led to the generation of Ab2 (anti-Id), namely an anti-autoantibody, and 2 – 3 months later to the generation of mouse Ab3 (antianti-autoantibody), which simulates the original auto antibody (human or mouse origin) in its binding properties. This antibody network in naive mice leads to the production of specific pathogenic autoantibodies (eg Ab3), followed by emergence of a spectrum of serological, immunohistochemical and clinical manifestations of the respective autoimmune disease (eg immunization with anti-dsDNA led to experimental SLE, immunization with anti-cardiolipin resulted in experimental APS). The idiotypically induced model showed an upregulation of Th1 cytokine (IL-2, IFNg) production at the early stage of the disease, while the increase in the Th2 cytokine (IL-4, IL-10) expression was ascendant as the autoimmune status progress.9

 Amelioration of the clinical manifestation of the disease was achieved by immunomodulation of the cytokine profile via reversing the Th2 response to Th1.10 These idiotypically induced experimental models of autoimmune diseases enabled a series of studies evaluating novel therapeutic modalities.11 – 14 The studied treatments entailed hormonal manipulation (androgens, anti-prolactin agents), intravenous immune globulins (IVIG), Id-specific suppressor T cells, anti-idiotypic antibodies, and cyclosporin-A (CsA). The recently introduced novel approaches include anti-CD4þ antibodies, oral tolerance, bone marrow transplantation, methimazole, tamoxifen, ciprofloxacin, IL-3 and dietary manipulation. Diet enriched with long-chain polyunsaturated fats has a suppressing effect on the expression of experimental SLE in BALB=c mice,15 and linseed oil (enriched with omega-3 unsaturated fatty acid) prevented the appearance of clinical manifestations of experimental antiphospholipid syndrome.16 Obviously, the main advantage of dietary manipulation is the lack of undesired side effects, normally associated with various immunosuppressive treatment modalities in SLE. Wheat is one of the staple foods of mankind. Its kernel also contains the germ, which has been considered as an animal feed so far. Only a small portion of this milling by-product has been marketed for human consumption. Remarkable non-nutrients of wheat germ include the methoxy-substituted benzoquinones, which are present as glycosides of the corresponding methoxyhydroquinones.

Three weeks later a booster injection of the immunoglobulin (10 mg) in phosphate buffered saline (PBS) was given at the same site, as previously detailed.2,3 The mice were bled every 2 weeks, and the levels of autoantibody titers were determined in the sera by ELISA for mouse 16=6 Id, anti-ssDNA, anit-dsDNA, anti-histones, anti-cardiolipin (aCL), and for anti-phosphatidylcholine (aPC) as a negative control. Established parameters of experimental SLE, as described previously (increased erythrocyte sedimentation rate, leukopenia and proteinuria),1–5 were evaluated. Treatment of experimental SLE with FWGE FWGE was introduced daily per os, 7 days before disease induction and during a period of 9 weeks, to the BALB=c mice (5 mg=mouse) at a rate of 0.1 ml=mouse by biomedical needle (Thomas Scientific, Popper & Sons Inc, NY, USA). The groups of mice that were included in the study were: mice induced by anti-ssDNA mAb to develop lupus like disease with or without FWGE supplementation, and non-immunized mice with or without FWGE feeding. Spot ELISA to determine autoantibody secreting cells Mouse splenocytes (1107 cells=ml) were assayed for their ability to secrete in vitro mouse 16=6, antissDNA and aCL antibodies.10 The preparation of splenocytes was done by teasing the spleen and passing the splenocytes through 0.45 m nylon-mesh. The erythrocytes were lysed with 0.08 M Tris-buffered ammonium chloride. The cells were seeded in RPMI 1640, into 24-well tissue culture plates (Nunclon, Dalta, Denmark) precoated with dsDNA, cardiolipin or anti-16=6, phosphatidylcholine (using the same methods as coating ELISA plates), and blocked for 2 h with 3% BSA. The cells were incubated overnight.

The titers of anti-dsDNA decreased from OD 1.0175 0.162 at 405 nm to OD of 0.5966 0.106 (P < 0.02). The Pvalues between the treated and non-treated groups of mice, for all the tested autoantibodies were between 0.01 and 0.02. Comparison of the titers of all autoantibodies tested, in the treated vs the untreated group revealed decreased titers of autoantibodies (42 – 56% less) in the FWGE -treated group of mice as compared to the non-treated lupus group of mice. Table 1 demonstrates the antibody-forming cell activity (AFC) of splenocytes derived from the experimentally induced SLE mice following treatment with FWGE . The data shows a marked and significant inhibition in the number of anti-dsDNA and anti16=6 Id AFC activity following treatment with AVEMAR (eg the number of anti-dsDNA AFC dropped from 73.7 4.2 to 32.3 4.2 upon treatment with AVEMAR). Moderate inhibition of the number of aCL AFC was documented, but was non-significant (P > 0.05), when compared to anti-phosphatidylcholine AFC activity (Table 1). The long duration of the downregulatory effect of AVEMAR on autoantibody titers is demonstrated in Figure 2. Anti-dsDNA titers dropped from an OD of 0.898 0.097 at 405 nm to 0.596 0.106 OD and remained decreased 0.591 0.103 OD, 4 weeks after the end of treatment. Non-significant changes in the titers of the tested autoantibodies in the plasma of thedifferent groups of mice were observed during the tested time points (P > 0.05). Th1=Th2 in vitro cytokine production In vitro production of IL-2, IFNg, IL-4 and IL-10 by splenocytes derived from experimentally induced SLE mice treated with AVEMAR is shown in Figure 3. 

A non-significant change in the level of IL-2 and IFNg production (P > 0.05 for both groups), and a pronounced decrease in the production of IL-4 (P < 0.01) and IL-10 (P < 0.01), upon exposure of lupus mice to FWGE , can be clearly seen. The concentration of IL-4 in the splenocyte condition media decreased from 91.7 8.11 to 59.55 7.78 ng=ml, while IL-10 dropped from 91.65 5.8 to 63.95 5.8 ng=ml, upon FWGE administration to the lupus mice. The ESR (mm=6 h), the WBC count (cells=mm3 ) and the amount of proteinuria (mg=dl) are shown in Table 2. FWGE -treated lupus mice showed a normal ESR, normal WBC (as compared to the leukopenia observed in the non-treated group of lupus mice), and developed only a very moderate proteinuria ( < 100 vs > 300 mg=dl in the untreated group of lupus mice or less than 30 mg=dl in the nonimmunized mice). Discussion Nutritional status is now recognized as having a significant impact upon normal immunocompetence. Among the many therapeutic modalities which are currently being tested in experimental as well as human SLE are dietary immunomodulations.10 – 16,22 – 28 Dietary manipulation may provide the most cost-effective and least toxic therapy for patients with SLE, saving the patient from the undesired side effects normally associated with various immunosuppressive treatments. AVEMAR, is an orally applied fermentation product of wheat germ containing 0.04% substituted benzoquinone (AVEMAR). Preliminary studies have shown that the product has antimetastatic effects as well as immunomodulating properties.

18 – 20 It has also been recently shown that FWGE significantly enhanced the degree of blastic transformation of mice splenic lymphocytes caused by Concanavalin A.20 Oral supplementation of FWGE was also found to have an immune-restoring effect in a skin graft system by shortening the survival time of skin grafts in a co-isogenic mouse skin transplantation model.20 In the current study, the material was found to possess immunosuppressive capabilities. Compared to control mice which were not fed with FWGE and developed experimental SLE, the mice which were exposed to the treatment, showed a significantly lower humoral response (approximately 50%), as shown by production of autoantibodies (eg anti-ssDNA, anti-dsDNA, mouse 16=6 Id, anti-histones). The antibody response was associated with normal values of the ESR, WBC, and by moderate proteinuria when compared to mice which did not receive FWGE and developed high titers of autoantibodies in the sera, increased ESR, leukopenia and significant proteinuria. Analysis of the Th1=Th2 cytokine response in the FWGE -treated mice compared with the untreated mice with experimental lupus showed an interdependence between the balance in Th1=Th2 cytokine response and the clinical picture. FWGE caused a decrease in the Th2 cell response (IL-4, IL-10 production) in the mice which were exposed to the treatment, as compared to the non-treated group of lupus mice. Our data are in line with other groups of researchers who have tried to improve the clinical manifestations of SLE by diet in mouse models as well as in human, by immunomodulating the Th1=Th2 cell dynamic balance.21 Th1 T cells produce IL-2 and IFNg cytokines while Th2 cells generally produce IL-4 and IL-10 cytokines which immunomodulate an immune reaction.28

 Previous studies demonstrated that the expression of IL-4 and IFNg play a prominent role in the pathogenesis of murine lupus.29,30 Administration of anti-IL-4 antibody was effective in preventing the onset of lupus nephritis in these mice. Furthermore, mice with constitutive transgenic expression of IL-4 developed experimental lupus nephritis which could be abrogated by IL-4 neutralizing antibody.31 Kinetics of cytokine production in experimental SLE model, induced by idiotypic manipulation— immunization with a human anti-ssDNA mAb (16=6 Id)— revealed dysregulation of Th1=Th2 cytokine network at different stages of disease development.9 At the early stage of the disease, a preferential increase in Th1 cytokine IL-2, IFNg production was documented, while IL-4 and IL-10 (Th2 cytokines) production predominated later in the disease course.9 Our results are in line with the above data. FWGE was found to affect preferentially the very early stage of the disease induction by prolonging the Th1 response and reversing the early stage of the Th2 response towards Th1 (eg enhancing IL-2 and IFNg production and decreasing IL-4 and IL-10 production). Diet manipulation in NZBNZWF1 mice, which develop lupus, showed that fish oil administration delayed the onset of autoimmune kidney disease by suppressing both Th1 (IL2, IFNg) and Th2 (IL-5, IL-10) cytokine production.32 High IL-4 and IL-10 gene expression was observed in lupus patients in the active stage of disease.33 More interesting are the observations that SLE patients produced abnormally large amounts of IL-10,34 – 36 and serum levels of IL-10 correlated with disease activity.

37 – 39 Treatment of lupus patients with IL-10 antagonist (IL-10 mAb) resulted in a decrease in SLE disease activity index, and emphasized the importance of IL-10 involvement in the pathogenesis of the disease.40 Furthermore, employing gene therapy, IL4 and IL-10 genes were found to be most frequently protective.41,42 Referring to our results, administration of AVEMAR starting prior to disease induction, delayed the Th2 response. Taken together, the results of administration of AVEMAR are encouraging enough to propose a diet based on AVEMAR for a high-risk population to develop lupus, as a prevention measure.