anti-ssDNA Antibodies Research Articles

Decreased HLA-C1 alleles in couples of KIR2DL2 positive women with recurrent pregnancy loss

Specific killer cell immunoglobulin-like receptor (KIR) in women with recurrent pregnancy loss (RPL) and HLA ligands in couples invoke a susceptibility to RPL. However, the relationship between KIR2DL2 and its cognate ligand HLA-C1 has not been explored. In this prospective cohort study, 160 Caucasian women with RPL and 99 partners were included. KIR/HLA-C typing, NK assay, Th1/Th2 intracellular cytokine ratios, 25-(OH)-vitamin D level, and the presence of autoantibodies were analyzed. KIR2DL2 positive women (P = 0.023) and their partners (P = 0.017) had lower allele frequencies of HLA-C1 than those of KIR2DL2 negative women. KIR2DL2 positive women had significantly lower genotype frequency of HLA-C1C1 as compared to the North American Caucasian population controls (P < 0.05). In the partners of KIR2DL2 positive women, there was a substantially higher frequency of HLA-C2C2 than controls (P = 0.016). Besides, KIR2DL2 negative women had a higher prevalence of anti-ssDNA antibody as compared with that of KIR2DL2 positive women (P = 0.043). There were no differences in the distribution of HLA-C genotypes based on KIR2DL2, regardless of pregnancy outcome in women with RPL and their partners while on immunomodulation treatment. In conclusion, decreased ligands for inhibitory KIRs (inhKIR) could lead to insufficient inhibition of maternal uterine NK cells toward the trophoblast, thereby contributing to the pathogenesis of RPL. Specific KIR and HLA-C genotyping may predict the reproductive outcome of women with RPL.

Food Yellow4 reprotoxicity in relation to localization of DMC1 and apoptosis in rat testes: Roles of royal jelly and cod liver oil

Food Yellow 4 (FY4) is a lemon-yellow-colored synthetic organic azo dye, which is used widely for imparting pleasant and attractive appearance to foods and cosmetics. The present study aimed at evaluating the possible mechanism underlying the FY4-induced reprotoxicity in rats, and the potential supportive role of royal jelly (RJ) or cod liver oil (CLO), which is a natural remedy with several pharmacological benefits, against induced toxicity. Forty-eight male rats were divided into different groups-the control group, the CLO group (0.4 mL/kg), the RJ group (300 mg/kg), the FY4 group (500 mg/kg b.w.), and the co-treated groups (FY4 + CLO or FY4 + RJ). Semen analysis, serum hormones, and enzyme activities were estimated. Immunohistochemical staining was performed using anti-PCNA, anti-Sox 9, anti-STRA8, anti-DMC1, and anti-ssDNA antibody. The FY4 group exhibited a significant decrease in sperm concentration and motility percentage (%) and a substantial reduction in the TES and LH levels. Testicular LDH, ACP, and SDH were observed to be inhibited. Furthermore, co-localization of DMC1 and ssDNA, which reflected apoptotic induction in the leptotene and zygotene spermatocytes, respectively, was observed to have markedly elevated in the FY4 treated rats, with fewer PCNA-positive and SOX9-positive cells and higher ssDNA-positive cells in the seminiferous epithelium in comparison to the control groups. Interestingly, co-treatment with CLO or RJ exhibited healthy sperms and restored their features, activated the enzyme production, and raised the levels of sexual hormones. In addition, both RJ and CLO restored the features of the testicular tissue as observed under a light microscope, and limited the apoptosis as observed through antibody staining. Collectively, the results of the present study revealed that the co-administration of RJ or CLO with FY4 improved the biochemical, hormonal, and structural aspects of the testicular tissue in rats. Therefore, CLO and RJ may be considered promising agents that would be able to improve the testicular structure and function in the FY4-exposed individuals.

Long non-coding RNA expression analysis during mouse testis development using the FANTOM5 data

Specific killer cell immunoglobulin-like receptor (KIR) in women with recurrent pregnancy loss (RPL) and HLA ligands in couples invoke a susceptibility to RPL. However, the relationship between KIR2DL2 and its cognate ligand HLA-C1 has not been explored. In this prospective cohort study, 160 Caucasian women with RPL and 99 partners were included. KIR/HLA-C typing, NK assay, Th1/Th2 intracellular cytokine ratios, 25-(OH)-vitamin D level, and the presence of autoantibodies were analyzed. KIR2DL2 positive women (P = 0.023) and their partners (P = 0.017) had lower allele frequencies of HLA-C1 than those of KIR2DL2 negative women. KIR2DL2 positive women had significantly lower genotype frequency of HLA-C1C1 as compared to the North American Caucasian population controls (P < 0.05). In the partners of KIR2DL2 positive women, there was a substantially higher frequency of HLA-C2C2 than controls (P = 0.016). Besides, KIR2DL2 negative women had a higher prevalence of anti-ssDNA antibody as compared with that of KIR2DL2 positive women (P = 0.043). There were no differences in the distribution of HLA-C genotypes based on KIR2DL2, regardless of pregnancy outcome in women with RPL and their partners while on immunomodulation treatment. In conclusion, decreased ligands for inhibitory KIRs (inhKIR) could lead to insufficient inhibition of maternal uterine NK cells toward the trophoblast, thereby contributing to the pathogenesis of RPL. Specific KIR and HLA-C genotyping may predict the reproductive outcome of women with RPL.

Contribution of poly(ADP-ribose)polymerase-1 activation and apoptosis in trichloroethene-mediated autoimmunity

Trichloroethene (TCE), a common environmental toxicant and widely used industrial solvent, has been implicated in the development of various autoimmune diseases (ADs). Although oxidative stress has been involved in TCE-mediated autoimmunity, the molecular mechanisms remain to be fully elucidated. These studies were, therefore, aimed to further explore the contribution of oxidative stress to TCE-mediated autoimmune response by specifically assessing the role of oxidative DNA damage, its repair enzyme poly(ADP-ribose)polymerase-1 (PARP-1) and apoptosis. To achieve this, groups of female MRL +/+ mice were treated with TCE, TCE plus N-acetylcysteine (NAC) or NAC alone (TCE, 10 mmol/kg, i.p., every 4th day; NAC, 250 mg/kg/day in drinking water) for 6 weeks. TCE treatment led to significantly higher levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG) in the livers compared to controls, suggesting increased oxidative DNA damage. TCE-induced DNA damage was associated with significant activation of PARP-1 and increases in caspase-3, cleaved caspase-8 and -9, and alterations in Bcl-2 and Bax in the livers. Moreover, the TCE-mediated alterations corresponded with remarkable increases in the serum anti-ssDNA antibodies. Interestingly, NAC supplementation not only attenuated elevated 8-OHdG, PARP-1, caspase-3, cleaved caspase-9, and Bax, but also the TCE-mediated autoimmune response supported by significantly reduced serum anti-ssDNA antibodies. These results suggest that TCE-induced activation of PARP-1 followed by increased apoptosis presents a novel mechanism in TCE-associated autoimmune response and could potentially lead to development of targeted preventive and/or therapeutic strategies.

抗2本鎖DNA(IgG)抗体陰性,抗1本鎖DNA(IgG)抗体高値を呈した急速進行性ループス腎炎の1例

抗2本鎖DNA(IgG)抗体陰性,抗1本鎖DNA(IgG)抗体高値を呈した急速進行性ループス腎炎の1例

Cementocyte cell death occurs in rat cellular cementum during orthodontic tooth movement.

To clarify the mechanism of root resorption during orthodontic treatment, we examined cementocyte cell death and root resorption in the cellular cementum on the pressure side during experimental tooth movement. Using 8-week-old male Wistar rats, the right first molar was pushed mesiobuccally with a force of 40 g by a Ni-Ti alloy wire while the contralateral first molar was used as a control. Localization and number of cleaved caspase-3-positive and single-stranded DNA (ssDNA) - positive cells were evaluated using dual-label immunohistochemistry with anticleaved caspase-3 and anti-ssDNA antibodies. In addition, tartrate-resistant acid phosphatase (TRAP)-positive cells in the cellular cementum were evaluated using TRAP histochemical staining. Caspase-3- and ssDNA-positive cells appeared at 12 hours, but were restricted to the compressed periodontal ligament (PDL) and not the cellular cementum. Cleaved caspase-3-positive cementocytes were observed in the cellular cementum adjacent to the compressed PDL on day 1. From days 2 to 4, the number of caspase-3- and ssDNA-positive cementocytes increased. TRAP-positive cells appeared on the cellular cementum at the periphery of the hyalinized tissue on day 7, and resorption progressed into the broad surface of the cementum by day 14. Cementocytes adjacent to the hyalinized tissue underwent apoptotic cell death during orthodontic tooth movement, which might have been associated with subsequent root resorption.

Comparative protective effects of royal jelly and cod liver oil against neurotoxic impact of tartrazine on male rat pups brain

This study is aimed to evaluate the possible neurotoxic effect of tartrazine (T), an extensively used synthetic azo dye, as well as to determine the potential modulatory role of cod liver oil (CLO) or royal jelly (RJ) against such effects. For this purpose, thirty-six male rat pups were allocated into six groups. The 1st group received distilled water (control group), the 2nd group was given 300mg RJ/kg bw (RJ group), the 3rd group was given 0.4ml CLO/kg bw (CLO group), the 4th was given 500mg T/kg bw (T group). The 5th group was given T concurrently with RJ (TRJ group) and the 6th group was given T concurrently with CLO (TCLO group), at the same doses as the former groups. All treatments were given orally for 30 consecutive days. The concentrations of different brain neurotransmitters, gamma amino butyric acid (GABA), dopamine (DA) and serotonin (5HT) as well as the antioxidant and oxidative stress biomarkers were measured in the brain homogenates. An immunohistochemical staining of the cerebral cortex was applied with the anti-ssDNA antibody (an apoptotic cell marker) to reveal the changes in brain structure. The T group revealed a significant decrease in the concentration of the brain neurotransmitters, a sharp shortage in the level of antioxidant biomarkers (super oxide dismutase, catalase and the reduced glutathione), a marked increase in malondialdehyde levels, and numerous apoptotic cells in the brain cortex compared with the other groups. Interestingly, all the previously mentioned parameters were almost retrieved in both the TRJ and TCLO groups compared to the T group. These results conclusively demonstrate that RJ and CLO administration provides sufficient protection against the ruinous effects of T on rat pups brain tissue function and structure.

Anti‐non‐Gal‐specific combination treatment with an anti‐idiotypic Ab and an inhibitory small molecule mitigates the xenoantibody response

B-cell depletion significantly extends survival of α-1,3-galactosyltranferase knockout (GTKO) porcine organs in pig-to-primate models. Our previous work demonstrated that the anti-non-Gal xenoantibody response is structurally restricted. Selective inhibition of xenoantigen/xenoantibody interactions could prolong xenograft survival while preserving B-cell-mediated immune surveillance. The anti-idiotypic antibody, B4N190, was selected from a synthetic human phage display library after enrichment against a recombinant anti-non-Gal xenoantibody followed by functional testing in vitro. The inhibitory small molecule, JMS022, was selected from the NCI diversity set III using virtual screening based on predicted xenoantibody structure. Three rhesus monkeys were pre-treated with anti-non-Gal-specific single-chain anti-idiotypic antibody, B4N190. A total of five monkeys, including two untreated controls, were then immunized with GTKO porcine endothelial cells to initiate an anti-non-α-1,3-Gal (non-Gal) xenoantibody response. The efficacy of the inhibitory small molecule specific for anti-non-Gal xenoantibody, JMS022, was tested in vitro. After the combination of in vivo anti-id and in vitro small molecule treatments, IgM xenoantibody binding to GTKO cells was reduced to pre-immunization levels in two-thirds of animals; however, some xenoantibodies remained in the third animal. Furthermore, when treated with anti-id alone, all three experimental animals displayed a lower anti-non-Gal IgG xenoantibody response compared with controls. Treatment with anti-idiotypic antibody alone reduced IgM xenoantibody response intensity in only one of three monkeys injected with GTKO pig endothelial cells. In the one experimental animal, which displayed reduced IgM and IgG responses, select B-cell subsets were also reduced by anti-id therapy alone. Furthermore, natural antibody responses, including anti-laminin, anti-ssDNA, and anti-thyroglobulin antibodies were intact despite targeted depletion of anti-non-Gal xenoantibodies in vivo indicating that selective reduction of xenoantibodies can be accomplished without total B-cell depletion. This preliminary study demonstrates the strength of approaches designed to selectively inhibit anti-non-Gal xenoantibody. Both anti-non-Gal-specific anti-idiotypic antibody and small molecules can be used to selectively limit xenoantibody responses.

Vitamin D deficiency may be a risk factor for recurrent pregnancy losses by increasing cellular immunity and autoimmunity

Do women with recurrent pregnancy losses (RPL) and low vitamin D have increased prevalence of auto- and cellular immune abnormalities when compared with women with RPL who have normal vitamin D, and does vitamin D have any effect on cellular immunity in vitro? A high proportion of women with RPL have vitamin D deficiency and the risk of auto- and cellular immune abnormalities is increased in women with RPL and vitamin D deficiency. Vitamin D deficiency in pregnant women is associated with increased risk of obstetrical complications such as pre-eclampsia, bacterial vaginosis associated preterm delivery, gestational diabetes mellitus and small-for-gestational age births. A retrospective cross-sectional study of 133 women with RPL who were enrolled in a 2-year period, together with laboratory experiments. Women with three or more consecutive spontaneous abortions prior to 20 weeks of gestation who were enrolled at the University clinic. Serum vitamin D level, cellular activity and autoimmune parameters in vivo and in vitro were measured. Sixty-three out of 133 women (47.4%) had low vitamin D (<30 ng/ml). The prevalence of antiphospholipid antibody (APA) was significantly higher in low vitamin D group (VDlow) (39.7%) than in the normal vitamin D group (VDnl) (22.9%) (P< 0.05) and the adjusted odds ratio (OR) for APA in VDlow was 2.22 with the 95% confidence interval (CI) of 1.0-4.7. The prevalence of antinuclear antigen antibody (VDlow versus VDnl; 23.8% versus 10.0%, OR 2.81, 95% CI 1.1-7.4), anti-ssDNA (19.0% versus 5.7%, OR 3.76, 95% CI 1.1-12.4) and thyroperoxidase antibody (33.3% versus 15.7%, OR 2.68, 95% CI 1.2-6.1) was significantly higher in VDlow than those of VDnl (P < 0.05 each). Peripheral blood CD19(+) B and CD56(+) NK cell levels and NK cytotoxicity at effector to target cell (E:T) ratio of 25:1 were significantly higher in VDlow when compared with those of VDnl (P < 0.05 each). Reduction (%) of NK cytotoxicity (at E:T ratio of 50:1 and 25:1) by IgG (12.5 mg/dl) was significantly lower in VDlow than those of VDnl (P < 0.05, P < 0.01, respectively). There were no differences in Th1/Th2 ratios between VDlow and VDnl. When vitamin D3 was added in NK cytotoxicity assay in vitro, NK cytotoxicity at E:T ratio of 50:1 was significantly suppressed with 10 nMol/L (nM) (11.9 ± 3.3%) and 100 nM (10.9 ± 3.7%) of vitamin D3 when compared with controls (15.3 ± 4.7%) (P < 0.01 each). TNF-α/IL-10 expressing CD3(+)/4(+) cell ratios were significantly decreased with 100 nM of vitamin D3 (31.3 ± 9.4, P < 0.05) when compared with controls (40.4 ± 11.3) in vitro. Additionally, INF-γ/IL-10 expressing CD3(+)/4(+) cell ratio was significantly decreased with 100 nM of vitamin D3 (12.1 ± 4.0, P < 0.05) when compared with controls (14.8 ± 4.6). IFN-γ and TNF-α secretion from NK cells were significantly decreased (P < 0.01 each), and IL-10, IL-1β, vascular endothelial growth factor and granulocyte colony stimulating factor levels were significantly increased (P < 0.01 each) with vitamin D3 100 nM when compared with those of controls. The prevalence of vitamin D deficiency in women with RPL in this study is open to a possible type I error since women with vitamin D supplementation were excluded from this study. Assessment of vitamin D level is recommended in women with RPL. Vitamin D supplementation should be explored further as a possible therapeutic option for RPL. This work was supported by the intramural funding from Department of Microbiology and Immunology, Chicago Medical School at Rosalind Franklin University of Medicine and Science. None of the authors has any conflict of interest to declare. N/A.

Toll-Like Receptor 2 or Toll-Like Receptor 4 Deficiency Does Not Modify Lupus in MRLlpr Mice

Systemic lupus erythematosus is an autoimmune disease with a high morbidity and nephritis is a common manifestation. Previous studies in murine lupus models have suggest a role for Toll-like receptor 2 and 4. We examined the role of these molecules in MRL lpr mice which is one of the most established and robust murine models. We compared disease parameters in Toll-like receptor 2 or Toll-like receptor 4 deficient mice with their littermate controls. We found no difference in the severity of glomerulonephritis as assessed by histology, serum creatinine and albuminuria when Toll-like receptor 2 or Toll-like receptor 4 deficient MRLlpr mice were compared with Toll-like receptor sufficient controls. We also found similar levels of anti-dsDNA and anti-ssDNA antibodies. These results show that Toll-like receptor 2 and Toll-like receptor 4 do not play a significant role in MRLlpr mice, and therefore they may not be important in human lupus.

FRI0491 Circulating dna changes in rheumatoid arthritis patients blood

BackgroundEarly diagnostics of rheumatoid arthritis (RA) is of importance for the effective recovery, which is hampered due to the unspecific clinical manifestations. One of the clinically used RA markers is...

Structure–activity relationship of a guanine-free oligodeoxynucleotide as immunopotent inhibitor

Excessive innate immune response could contribute to the pathogenesis of inflammatory and autoimmune diseases. It is required to develop agents to inhibit the overwhelming innate immune response. SAT05f, an inhibitory ODN with CCT repeat sequence found in human microsatellite DNA, has been demonstrated to down-regulate TLR7/9-mediated innate immune response, protect mice from D-GalN/CpG ODN induced lethal shock, and reduce anti-ssDNA antibody level in the lupus-prone mice induced by chronic graft versus host disease (cGVHD). In this article, to explore the structure–activity relationship of SAT05f, we designed and synthesized a series of ODNs based on the sequence of SAT05f by changing repeat number of the CCT unit, substituting CCT unit with AAG at 3′ end or 5′ end or in the middle and by forming hairpin at 5′ or 3′ end, and tested their inhibitory effect on the CpG ODN induced proliferation and TNF-α production in murine immune cells. The results indicated that 1) at least 8 CCT units were required for a CCT repeat ODN to display its inhibitory activity; 2) CCT unit at 3′ end of SAT05f was necessary for its full inhibitory activity; and 3) 5′ end of SAT05f could be modified to design a more potent SAT05f derived inhibitory ODN. The data provided here would be helpful for finding a potent inhibitory ODN as a candidate medicament for the treatment of diseases associated with over-activated innate immune response.

Lack of IL-21R on donor cells attenuates chronic GVHD but promotes humoral autoimmunity in acute GVHD (56.11)

Abstract IL-21 enhances CD8 maturation into cytotoxic T lymphocytes (CTL), promotes the differentiation of T follicular helper cells (TFH) cells and Th17 cells, downregulates Tregulatory (Treg) cells and exerts stimulatory activities on B cells, suggesting a complex role for IL-21 in promoting autoimmunity. To assess the role of IL-21/IL-21R interaction on CD4 and CD8 T cells in promoting lupus-like disease we used the parent-into-F1 model of acute and chronic graft versus host disease (GVHD) using IL-21R KO and wild type B6 mice as donors. cGVHD induced with CD8 depleted splenocytes from IL-21R KO mice exhibited an attenuated disease phenotype characterized by significant decrease in the number of donor (d)CD4 and dTFH cells, impaired help for B cells as determined by anti-ssDNA antibody levels and less severe lupus-like glomerulonephritis. However the balance between IL-17 producing dTh17 cells and FoxP3 dTreg cells was not altered. Acute GVHD induced with IL-21R KO splenocytes resulted in an attenuated disease phenotype as indicated by a significant decrease in dCD4 and CD8 engraftment, decreased host B cell elimination (90%±0.3) compared to wt injected mice (57%±6; p=0.03), decreased anti-host CTL activity and increased anti-ssDNA Ab levels. These results suggest that IL-21/IL-21R interaction on T cells promotes lupus-like cGVHD by sustaining TFH cell number yet it downregulates the humoral component in aGVHD by enhancing host B cells elimination by CTL.

Effects of oligodeoxynucleotide with CCT repeats on chronic graft versus host disease induced experimental lupus nephritis in mice

A synthesized single-stranded oligodeoxynucleotide (ODN), designed as SAT05f with the sequence of human microsatellite DNA, has been studied for its capacity of alleviating the lupus nephritis in the chronic graft versus host disease (cGVHD) induced lupus-prone mice. In cGVHD model mice, both of continuous and discontinuous treatment with SAT05f was effective on reducing anti-ssDNA antibody production, decreasing renal IgG deposition and delaying the onset of lupus nephritis. In addition, SAT05f could down-regulate TLR9 mRNA expression in splenocytes of cGVHD model mice. These results indicated that SAT05f could be developed as a new therapeutic agent for the treatment of lupus nephritis by inhibiting TLR9 signaling pathways.

Defective B cell memory and autoantibodies in subjects of cardiac transplantation in infancy (169.27)

Abstract Aberrant functions of T cells and B cells may impair B cell memory and are thought to allow development of autoantibodies. Rarely, B cell memory defects and/or autoimmunity can be traced to specific mutations; however, in most cases which properties of T cells and B cells cause deficient B cell responses and/or autoimmunity are unclear. To answer these questions we studied child recipients of cardiac transplantation during infancy who, owing to removal of the thymus and T cell depletion at the time of surgery, have a greatly reduced T cell receptor repertoire diversity. We found these children to have impaired memory IgG antibody responses to polypeptide vaccines compared to controls, even though they appeared to mount IgM responses. These results suggest that the defective T cell compartment in recipients of cardiac transplantation in infancy impairs B cell memory and/or isotype class- switching. Remarkably, despite these defects and despite ongoing treatment with immunosuppressive drugs, 6 of the 9 subjects studied produced anti-ssDNA and rheumatoid factor antibodies as early as of 2 years of age. These findings suggest that the diversity of T cells and/or the availability of recent thymic emigrants are needed to generate B cell memory and prevent autoimmunity.

IL-21/IL-21R interaction on both CD4 cells and B cells is required to promote autoimmune parameters in chronic graft versus host disease. (44.10)

Abstract IL-21 stimulates B cell proliferation, plasma cell (PC) differentiation and germinal center (GC) expansion and it exerts autocrine effects on T follicular B helper cells (TFH) and Th17 cells while downregulating Treg cells. We assessed the effect of IL-21/IL-21R signaling on B cells independent from the effect on CD4 T helper cells using IL-21R-/- or IL-21R+/+ mice as donor or hosts in the P-into-F1 and Bm12-into-B6 models of chronic Graft Versus Host Disease (cGVHD). cGVHD induced by injection of IL-21R-/- CD4 cells from B6 mice into B6D2F1 hosts was characterized by a significant decrease in the expansion of donor CD4, TFH and Th17 cells and an increase in Treg cells compared to IL-21R +/+ donor cells. Furthermore, the number and size of GC, GC B cells and levels of anti-ssDNA antibody (Ab) were significantly decreased. When cGVHD was induced by injecting Bm12 spleen cells into IL-21R+/+ or IL-21R-/- B6 mice, parameters of cGVHD including B cell activation, number and size of GC, percentage of GC B cells and plasmablasts, IgG anti-dsDNA Ab production were significantly decreased compared to IL-21+/+ hosts. Long-term studies showed decreased severity of lupus-like renal disease in the IL-21R-/- hosts. These results suggest that IL-21/IL-21R interaction enhances autoimmune features in lupus by promoting the expansion of TFH and Th17 cells and the downregulation of Treg cells and by promoting the differentiation of autoreactive B cells into autoAb producing cells.

Analysing cell‐free plasma DNA and SLE disease activity

Over the years, the demonstration and confirmation of cell-free DNA in the circulation has increasingly been recognized as a valuable diagnostic tool. Likewise, it has been known for some time that DNA structures that are targeted by auto-antibodies play a central role in systemic lupus erythematosis (SLE) and that DNA-antibody complexes in the circulation are one of the hallmarks of SLE. Investigating whether and to what degree fluctuations in free plasma DNA levels in patients with SLE might correspond to disease severity was therefore the goal of this investigation. Blood from 13 patients with SLE and from 13 healthy controls was taken and analysed for the presence of anti-dsDNA, anti-ssDNA, anti-nucleosome, anti-histone antibodies as well as for cell-free DNA concentrations. For each patient, the SLE disease activity index (SLEDAI) was calculated. As demonstrated herein, compared to healthy subjects, cell-free DNA plasma levels in patients with SLE were significantly increased and so were anti-dsDNA, anti-ssDNA, anti-histone and anti-nucleosome antibodies. Furthermore, a statistically significant correlation was noted between cell-free DNA and anti-histone antibodies in patients with SLE. However, no correlation was noted between disease activity and anti-dsDNA, anti-ssDNA and anti-nucleosome antibody concentrations. Surprisingly, and more important in the context of this study, there was no correlation between cell-free DNA levels and SLEDAI scores. The presented data seem to exclude measuring free plasma DNA as an inexpensive, simple and quick tool to assess disease activity in patients with SLE. Further studies on a larger patient population would be needed to confirm our results.

老齢カニクイザルで検出される自己抗体（抗単鎖DNA抗体）の概要と産生機序に関する考察

老齢カニクイザルで検出される自己抗体（抗単鎖DNA抗体）の概要と産生機序に関する考察

IL-21/IL-21R interactions on host B cells but not on donor CD4 cells are critical for the development of autoimmune features in chronic graft versus host disease. (93.9)

Abstract Studies in murine models of lupus have indicated increased production of IL-21 and attenuation of autoimmune features following IL-21 blockade. IL-21 exerts an autocrine effect on T follicular B helper cells (TFH) cells and also stimulates B cell proliferation, plasma cell (PC) differentiation and germinal center (GC) expansion. To determine whether IL-21 promotes systemic lupus through effects on TFH cells or B cells, we assessed the effect of IL-21/IL-21R signaling on B cells independent from the effect on CD4 T helper cells using IL-21R-/- or IL-21R+/+ mice as donor or hosts in the P-into-F1 and Bm12-into-B6 models of cGVHD. CGVHD induced by injection of IL-21R-/- CD4 cells from B6 mice into B6D2F1 hosts was characterized by a decrease in the expansion of donor CD4, TFH and GC cells. However, other parameters of cGVHD such as host B cell expansion, MHC class II upregulation, PC differentiation and anti-ssDNA autoantibody production did not differ between groups. In contrast, when cGVHD was induced by injecting Bm12 spleen cells into IL-21R-/- B6 mice, parameters of cGVHD were markedly attenuated compared to IL-21R+/+ hosts. Specifically, MHC class II upregulation of B cells decreased by 32%, PC differentiation by 66%, GC cells by 60% and anti-ssDNA antibody level by 81%. These results suggest that IL-21R signaling on host B cells and possibly myeloid cells but not on Ag-specific CD4 T cells is critical for the initiation and progression of systemic autoimmunity in cGVHD.

Structure–function analysis and molecular modeling of DNase catalytic antibodies

There is great interest in the antibodies-to-DNA transformation, since this change is characteristic of autoimmune diseases and contributes to its pathology. After immunization and fusions, 14 hybridomas bearing DNA-hydrolysis activity against pUC19 plasmid DNA were obtained. Genes coding for V H and V L regions of the 14 monoclonal antibodies (mAbs) were cloned and sequenced. The sequences were compared with sequences of the Ig-Blast database to determine their germline and to identify potential mutations responsible for DNA binding and DNase activity. V genes of the H chains’ genes expressed four genes of the V H1/J558 family, three of V H5/V H7183, and three of V H8/V H3609. The genetic repertoire of these mAbs was examined by determining the nucleotide sequences of their H chain V regions. This V H and V L domain was most similar to an anti-ssDNA (DNA-1) antibody as well as to catalytic autoimmune mAb (m3D8). Computer-generated models of the three-dimensional structures of V H and V L (VHL4) of the IgG4 combinations were used to define the positions occupied by the important sequence motifs at the binding sites. The modeling structure showed that VHL4 binds to oligo (dT3) primarily by sandwiching thymine bases between Tyr L32, Tyr L49 and Tyr H97 side-chains. Superposing VHL4 with anti-nucleic acid m3D8 catAbs revealed a common ssDNA recognition module consisting of His L93, His H35 residues which are critical for DNA-hydrolyzing antibodies. This study demonstrates the potential usefulness of the protein DNA surrogate in the investigation of the origin of anti-DNA antibodies’ hydrolyzing activities.