An amphipathic trans-acting phosphorothioate RNA element delivers an uncharged phosphorodiamidate morpholino sequence in mdx mouse myotubes.

An efficient method for the delivery of uncharged polyA-tailed phosphorodiamidate morpholino sequences (PMO) in mammalian cells consists of employing a synthetic 8-mer amphipathic trans-acting poly-2'-O-methyluridylic thiophosphate triester element (2'-OMeUtaPS) as a transfection reagent. Unlike the dTtaPS DNA-based element, this RNA element is potent at delivering polyA-tailed PMO sequences to HeLa pLuc 705 cells or to myotube muscle cells. However, much like dTtaPS, the 2'-OMeUtaPS-mediated internalization of PMO sequences occurs through an energy-dependent mechanism; macropinocytosis appears to be the predominant endocytic pathway used for cellular uptake. The transfected PMO sequences induce alternate splicing of either the pre-mRNA encoding luciferase in HeLa pLuc 705 cells or the excision of exon 23 from the pre-mRNA encoding dystrophin in myotube muscle cells of the mdx mouse model of muscular dystrophy with an efficiency comparable to that of commercial cationic lipid reagents but without detrimental cytotoxicity.

17 beta-estradiol, but not 5 alpha-dihydrotestosterone, augments antibodies to double-stranded deoxyribonucleic acid in nonautoimmune C57BL/6J mice.

The influence of sex hormones on the immune response to foreign antigens as well as self-antigens is now recognized. In this study, we investigated the influence of gender and sex hormones on the development of antibodies to double-stranded DNA in nonautoimmune C57BL/6J mice. Immunoglobulin G (IgG) anti-dsDNA antibodies are commonly present in lupus patients and several autoimmune disease-prone murine strains. We found that C57BL/6J mice have detectable antibodies (IgM and IgG, but not IgA) to dsDNA. Interestingly, the incidence and level of IgG anti-dsDNA antibodies were lower in male than in female mice. Orchidectomy or administration of 5 alpha-dihydrotestosterone to orchidectomized male mice had minimal effects on these antibodies. In contrast, administration of 17 beta-estradiol to orchidectomized or intact males significantly increased both the incidence and levels of anti-dsDNA antibodies. In female mice, ovariectomy decreased whereas administration of estrogen augmented the incidence and levels of anti-dsDNA antibodies in ovariectomized as well as intact female mice. Kinetic studies revealed that estrogen treatment of male and female mice induced earlier and sustained expression of IgG anti-dsDNA antibodies compared to controls. IgG subisotype analysis showed IgG2b to be predominant. In summary, our findings suggest that estrogen, but not dihydrotestosterone, promotes anti-dsDNA antibodies in normal mice.

Gender and risk of autoimmune diseases: possible role of estrogenic compounds.

A striking common feature of many autoimmune diseases in humans and experimental animals, despite differences in pathology, is that females are highly susceptible to autoimmune conditions compared to males. In several animal models, estrogens promote, whereas androgens abrogate, B-cell-mediated autoimmune diseases. To understand mechanisms by which estrogens regulate autoimmunity, it is first necessary to decipher estrogen effects on the normal immune system. Estrogen treatment of nonautoimmune mice diminished lymphocyte numbers in both developmental and mature lymphoid organs. Estrogen dysregulated T- and B-cell balance by inducing selective T-cell hypoactivity and B-cell hyperactivity. Even though estrogen did not alter the relative percentages of splenic T-cell subsets, splenic lymphocytes had a reduced proliferative response to T-cell stimulants and were refractory to rescue from activation-induced apoptosis compared to cells from placebo-treated mice. In contrast, estrogen induced B-cell hyperactivity (promoted autoantibodies to double-stranded DNA and phospholipids, increased numbers of plasma cells, and increased autoantibody yield per B cell). Note that treatment of normal mice with estrogen can alter T- and B-cell regulation and overcome B-cell tolerance to result in autoimmunity in normal individuals. Could environmental estrogens promote some human autoimmune disorders? Is there a link between environmental estrogens and autoimmune disorders, especially since these disorders are reported possibly more frequently? These provocative questions warrant investigation. Our findings on immunomodulatory effects may serve as a benchmark to examine whether endocrine-disrupting chemicals will have similar immunologic effects.

Sex hormones as immunomodulators in health and disease.

In addition to their effects on sexual differentiation and reproduction, sex hormones influence the immune system. This results in a gender dimorphism in the immune function with females having higher immunoglobulin levels and mounting stronger immune responses following immunization or infection than males. The greater immune responsiveness in females is also evident in their increased susceptibility to autoimmune diseases. However, a clear understanding of the myriad of effects that sex hormones have on the immune system is lacking. Studies in normal mice show that estrogen treatment induces polyclonal B cell activation with increased expression of autoantibodies characteristic of autoimmune diseases. Several mechanisms appear to contribute to the break in tolerance and the increase in plasma cell activity including a reduction of the mass of the bone marrow and the thymus, the emergence of sites of extramedullary hematopoiesis and altered susceptibility of B cells to cell death. In addition, sex hormone levels in both humans and experimental models correlated with the activity of their cytokine-secreting cells indicating that sex hormones influence the cytokine milieu and suggesting that altered sex hormonal levels in autoimmune patients contribute to the skewed cytokine milieu characteristic of systemic lupus erythematosus (SLE). While sex hormones alone do not cause autoimmune disease, abnormal hormone levels may provide the stage for other factors (genetic, infectious) to trigger disease. Understanding the physiology of the interaction between sex hormones and immune function and its potential pathological consequences may provide insight into the autoimmune diseases and new directions for their treatment.

Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotides.

Exposure to bacterial DNA generates a "danger signal" that stimulates cellular elements of the mammalian immune system to proliferate and/or secrete cytokines. Stimulation is critically dependent on hexameric motifs that contain an unmethylated CpG dinucleotide: these are commonly found in bacterial but not vertebrate DNA. Different motifs are optimally stimulatory in different species. This work examines whether oligodeoxynucleotides (ODNs) containing CpG motifs stimulate peripheral blood mononuclear cells from pigs. Results show that pigs respond to CpG ODN by proliferating and secreting IL-6, IL-12 and TNF-alpha. By screening a large panel (>100) of ODNs, the palindromic hexamer 'ATCGAT' was identified as being optimally active in all animals examined (N=10). These findings are the first to establish the immunostimulatory activity of CpG ODN in pigs, and suggest that the therapeutic uses envisioned for these ODNs (as vaccine adjuvants and immunoprotective agents) may be applicable to husbandry animals.

Assessment and reporting of the clinical immunogenicity of therapeutic proteins and peptides-harmonized terminology and tactical recommendations.

Immunogenicity is a significant concern for biologic drugs as it can affect both safety and efficacy. To date, the descriptions of product immunogenicity have varied not only due to different degrees of understanding of product immunogenicity at the time of licensing but also due to an evolving lexicon that has generated some confusion in the field. In recent years, there has been growing consensus regarding the data needed to assess product immunogenicity. Harmonization of the strategy for the elucidation of product immunogenicity by drug developers, as well as the use of defined common terminology, can benefit medical practitioners, health regulatory agencies, and ultimately the patients. Clearly, understanding the incidence, kinetics and magnitude of anti-drug antibody (ADA), its neutralizing ability, cross-reactivity with endogenous molecules or other marketed biologic drugs, and related clinical impact may enhance clinical management of patients treated with biologic drugs. To that end, the authors present terms and definitions for describing and analyzing clinical immunogenicity data and suggest approaches to data presentation, emphasizing associations of ADA development with pharmacokinetics, efficacy, and safety that are necessary to assess the clinical relevance of immunogenicity.

Regulatory role of suppressive motifs from commensal DNA.

The microbiota contributes to the induction of both effector and regulatory responses in the gastrointestinal (GI) tract. However, the mechanisms controlling these distinct properties remain poorly understood. We previously showed that commensal DNA promotes intestinal immunity. Here, we find that the capacity of bacterial DNA to stimulate immune responses is species specific and correlated with the frequency of motifs known to exert immunosuppressive function. In particular, we show that the DNA of Lactobacillus species, including various probiotics, is enriched in suppressive motifs able to inhibit lamina propria dendritic cell activation. In addition, immunosuppressive oligonucleotides sustain T(reg) cell conversion during inflammation and limit pathogen-induced immunopathology and colitis. Altogether, our findings identify DNA-suppressive motifs as a molecular ligand expressed by commensals and support the idea that a balance between stimulatory and regulatory DNA motifs contributes to the induction of controlled immune responses in the GI tract and gut immune homeostasis. Further, our findings suggest that the endogenous regulatory capacity of DNA motifs enriched in some commensal bacteria could be exploited for therapeutic purposes.

Sex hormone levels correlate with the activity of cytokine-secreting cells in vivo.

This work examines the correlation between serum levels of oestrogen, progesterone and dehydroepiandrosterone sulphate (DHEA-S) and the number of human peripheral blood cells actively secreting interleukin (IL)-2, IL-4, IL-6, IL-10, tumour necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma) in vivo. Simultaneous assessment of serum hormone levels and cytokine-secreting cell activity throughout the menstrual cycle showed that the number of peripheral blood mononuclear cells (PBMC) able to secrete IL-4 in response to stimulation correlated significantly (P < 0.0001) with oestrogen levels and fluctuated with the menstrual cycle in pre-menopausal women. The activity of IFN-gamma-secreting cells, on the other hand, varied as a function of serum DHEA-S levels in pre-menopausal women (P < 0.0001). Similarly, the number of cells secreting IFN-gamma in men correlated with serum DHEA-S levels (P < 0.001). In contrast, post-menopausal women had fewer cells actively secreting cytokines and the activity of these cells did not correlate with sex hormone levels. These results suggest that sex hormones may modulate cytokine production in vivo and contribute to gender-related differences in normal and pathological immune responses.

Antibodies to cardiolipin in normal C57BL/6J mice: induction by estrogen but not dihydrotestosterone.

Autoantibodies against cardiolipin, a phospholipid, have been demonstrated in a variety of pathological states including several autoimmune conditions in humans and in certain lupus-prone mice. In this study we detected antibodies reactive to cardiolipin in normal C57BL/6J mice by ELISA. The autoantibodies are detected less frequently in the serum of male than in female mice, suggesting the influence of sex hormones. The relative refractoriness of normal male mice to the induction of anticardiolipin antibodies is not due to the suppressive effects of male hormones, since prepubertal orchiectomy has little influence on this autoantibody. Further, dihydrotestosterone treatment of orchiectomized mice has minimal effect on anticardiolipin antibodies. However, orchiectomized mice when given estrogen develop a marked increase in the incidence as well as the levels of these autoantibodies. Similarly, estrogen treatment of female mice further augments the incidence and the levels of these autoantibodies. Estrogen-treated mice also have antibodies reactive against other membrane phospholipids including phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The intensity of binding of autoantibodies to the above phospholipids varies among individual mice. To our knowledge, this is the first report on the demonstration of antiphospholipid autoantibodies in normal mice and induction of these antibodies by estrogen.

Characterization of estrogen-induced autoantibodies to cardiolipin in non-autoimmune mice.

Antibodies to cardiolipin, in humans, have been associated with a variety of autoimmune disorders including anti-phospholipid syndrome, systemic lupus erythematosus and Sjögren's syndrome. These antibodies have also been demonstrated in autoimmune-prone MRL-Mp-lpr/lpr (MRL/lpr), BXSB-Mp-(+yaa) (BXSB) and (NZW x BXSB)F1 mice. In previous work, we had shown that gonadectomized or intact male and female non-autoimmune C57BL/6 mice, upon treatment with estrogen, express autoantibodies to cardiolipin. In this study, we extend these findings and show that the expression of these antibodies persists for months even after the exposure to exogenous estrogen has been terminated. These antibodies are of IgM and IgG, but not IgA, isotypes, and the predominant IgG subisotype is IgG2b. Estrogen-induced antibodies to cardiolipin only minimally cross-reacted with DNA, actin or ovalbumin. The binding of antibodies to cardiolipin from autoimmune human patients in general has been shown to depend upon the presence of a cofactor, beta2-glycoprotein I. We found that in estrogen-treated C57BL/6 mice, as well as in SLE-prone MRL/lpr and BXSB mice, the binding of anti-cardiolipin antibodies to cardiolipin was not enhanced, but rather reduced, in the presence of human beta2-glycoprotein I. Further, addition of exogenous human beta2-glycoprotein I to purified immunoglobulin fractions containing anti-cardiolipin antibodies reduces, rather than enhances, the binding to cardiolipin. Together, these data show that persistent detectable levels of IgG and IgM autoantibodies specific for cardiolipin can be induced in normal mice by estrogen treatment alone (i.e. without administration of autoantigens). Further, we characterize these antibodies regarding their kinetics, cross-reactivity, isotype distribution and cofactor (beta2-glycoprotein I) requirements.

Estrogen increases the number of plasma cells and enhances their autoantibody production in nonautoimmune C57BL/6 mice.

The immunological consequences of chronic estrogen exposure in normal individuals are not known, particularly in relation to B cells. In this study, by employing ELIspot, image cytometry, flow cytometry, cytology, and ELISA, we show that long-term exposure of normal mice to estrogen activates B cells to produce higher numbers of not only immunoglobulin-producing cells, but also autoantibody-producing cells. Estrogen promoted a decrease in B220(+) splenic lymphocytes, but resulted in a 10-fold increase in plasma cells. Further, the output of immunoglobulins including autoantibodies from individual plasma cells from estrogen-exposed mice was markedly increased, suggesting B cell hyperactivity. Importantly, our findings show that treatment of normal mice, solely with estrogen, can override B cell tolerance and promote autoreactive B cells in normal individuals.

Disassociation of sex hormone levels and cytokine production in SLE patients.

This study examines whether changes in the cytokine milieu of patients with systemic lupus erythematosus (SLE) are associated with abnormal levels of sex hormone levels in serum. The concentration of 17beta-estradiol (E2), progesterone (Pg) and dehydroepiandrosterone-sulphate (DHEAS) was monitored in sera from 128 lupus patients and 96 controls, and correlated with the activity of their cytokine secreting cells. Results indicate that SLE patients have (i) significantly fewer cells secreting IFNgamma, (ii) increased serum E2 and Pg levels, and (iii) reduced serum DHEAS levels compared to normal controls. However, the observed abnormalities in the cytokine milieu of SLE patients did not correlate with abnormalities in serum sex hormone levels. Instead, the association between IFNgamma production and DHEAS levels evident in healthy controls is absent in SLE patients, suggesting that cells from lupus patients are defective in their ability to produce IFNgamma in response to physiologic stimuli. Similarly, the normal correlation between IL-4 production and E2 levels was lost in patients with severe disease. Thus, while it remains possible that increased E2 and reduced DHEAS levels in lupus patients may help induce cytokine abnormalities early in disease, the subsequent cytokine imbalance does not correlate with sex hormone levels.

Activity and safety of DNA plasmids encoding IL-4 and IFN gamma.

Cytokine-encoding DNA plasmids can act as 'genetic adjuvants', improving the immune response stimulated by co-administered DNA vaccines. We examined whether plasmids encoding the Th1 cytokine IFN gamma (pIFN gamma) or the Th2 cytokine IL-4 (pIL-4) have long-term effects on immune homeostasis when administered to adult mice, or alter immune maturation in neonates. Both plasmids boosted immunity against a co-administered vaccine, with pIFN gamma promoting the development of a Th1 response (characterized by the production of IgG2a antibodies), and pIL-4 preferentially stimulating a Th2 response (characterized by increased IgG1 antibody production). Both pIFN gamma and pIL-4 influenced the ratio of cells actively secreting Th1 versus Th2 cytokines, consistent with an effect on Th cell maturation. Interestingly, this effect persisted for only a few weeks and was not magnified by repeated plasmid administration. Cytokine-encoding plasmids had no long-term effect on the immune response of newborn or adult mice to subsequent antigenic stimulation, nor did they selectively induce the production of pathogenic anti-DNA autoantibodies. These results suggest cytokine-encoding plasmids may be safe as immune adjuvants.

DNAse treatment does not improve the survival of lupus prone (NZB x NZW)F1 mice.

OBJECTIVE: To examine the efficacy of deoxyribonuclease I (DNAse) therapy in the (NZB x NZW)F1 murine model of lupus. METHODS: Lupus-prone female (NZB x NZW)F1 mice were treated daily with 0-15 microg/g of recombinant DNAse for 1-6 months. Parameters including anti-DNA autoantibody production, activation of cytokine secreting cells, kidney function and longevity were monitored. RESULTS: DNAse treatment selectively reduced the number of B cells secreting anti-dsDNA antibodies for approximately one month. However, neither short-term nor long-term treatment altered cytokine production, delayed the onset or reduced the severity of glomerulonephritis, or prolonged survival. CONCLUSION: DNAse treatment initiated before, during, or after the onset of murine lupus did not improve clinical outcome.

Human peripheral blood cells differentially recognize and respond to two distinct CPG motifs.

Oligodeoxynucleotides (ODN) that contain unmethylated CpG dinucleotides trigger a strong innate immune response in vertebrates. CpG ODN show promise as vaccine adjuvants, anti-allergens, and immunoprotective agents in animal models. Their transition to clinical use requires the identification of motifs that are optimally stimulatory in humans. Analysis of hundreds of novel ODN resulted in the identification and characterization of two structurally distinct "clusters" of immunostimulatory CpG ODN. One cluster ("D") preferentially stimulates IFN-gamma production by NK cells, whereas the other ("K") stimulates cell proliferation and the production of IL-6 and IgM by monocytes and B cells. The distinct immunostimulatory properties of K and D ODN can improve the design of CpG-based products to achieve specific therapeutic goals.

Response of peripheral blood mononuclear cells from lupus patients to stimulation by CpG oligodeoxynucleotides.

OBJECTIVES: Increased levels of hypomethylated CpG-containing DNA in sera from patients with systemic lupus erythematosus (SLE) may contribute to the initiation and/or perpetuation of the disease. This study characterizes the in vitro response of peripheral blood mononuclear cells (PBMC) from SLE patients to CpG DNA. METHODS: Secretion of cytokines and IgM, cell proliferation and up-regulation of co-stimulatory molecules were evaluated in PBMC from SLE patients (n=24) and normal controls (n=24) after stimulation with synthetic oligodeoxynucleotides (ODN) containing CpG motifs. RESULTS: Up-regulation of co-stimulatory molecules and the secretion of interferon-alpha and interleukin-6 (IL-6) in response to CpG ODN was significantly reduced in monocytes and dendritic cells from SLE patients. Secretion of interferon-gamma by natural killer (NK) cells was also reduced. In contrast, the IgM and IL-10 response of B cells to CpG ODN was normal. CONCLUSION: Monocytes, dendritic cells and NK cells from SLE patients respond abnormally to CpG ODN stimulation, which may contribute to the cytokine imbalance observed in SLE.