Consensus-based reporting standards for diagnostic test accuracy studies for paratuberculosis in ruminants.

The Standards for Reporting of Diagnostic Accuracy (STARD) statement (www.stard-statement.org) was developed to encourage complete and transparent reporting of key elements of test accuracy studies in human medicine. The statement was motivated by widespread evidence of bias in test accuracy studies and the finding that incomplete or absent reporting of items in the STARD checklist was associated with overly optimistic estimates of test performance characteristics. Although STARD principles apply broadly, specific guidelines do not exist to account for unique considerations in livestock studies such as herd tests, potential use of experimental challenge studies, a more diverse group of testing purposes and sampling designs, and the widespread lack of an ante-mortem reference standard with high sensitivity and specificity. The objective of the present study was to develop a modified version of STARD relevant to paratuberculosis (Johne's disease) in ruminants. Examples and elaborations for each of the 25 items were developed by a panel of experts using a consensus-based approach to explain the items and underlying concepts. The new guidelines, termed STRADAS-paraTB (Standards for Reporting of Animal Diagnostic Accuracy Studies for paratuberculosis), should facilitate improved quality of reporting of the design, conduct and results of paratuberculosis test accuracy studies which were identified as "poor" in a review published in 2008 in Veterinary Microbiology.

Risk of autoimmune disease: challenges for immunotoxicity testing.

Autoimmunity represents a potentially diverse and complex category among the range of adverse outcomes for detection with immunotoxicity testing. For this reason, the risk of autoimmune disease is discussed in this overview chapter with additional mention among the later specific protocol chapters. Improvements in clinical diagnostic capabilities and disease recognition have led to a more accurate picture of the extent of autoimmune diseases across different human populations. While the risk of any single autoimmune disease remains modest when compared with that of lung or heart disease, the cumulative prevalence of autoimmune diseases is both significant and increasing. Autoimmune diseases are usually viewed in the context of the damaged tissue or organ (e.g., as a thyroid, gastrointestinal, cardiovascular or neurological disease). But improved recognition that underlying immune dysfunction can connect the risks for these as well as other diseases is critical for optimizing risk assessment. Since autoimmune diseases are chronic in nature with many first appearing in children or in young adults, these diseases exert a serious impact on both health care costs and quality of life. This chapter provides a discussion of the issues that should be considered with immunotoxicity testing for risk of autoimmunity.

The induction of the lupus phenotype by estrogen is via an estrogen receptor-alpha-dependent pathway.

In order to investigate the roles of ER subtypes in the estrogen-induced lupus phenotype, ERalpha-deficient (ERalpha(-/-)) and wild-type mice (WT) were injected monthly with estradiol (E-2) starting at 8 weeks. In WT mice, E-2 treatment induced a lupus phenotype, with accelerated death and increased kidney damage, as well as Th2-type serum cytokine and autoantibody production. In contrast, only minimal changes were observed in ERalpha(-/-) mice after E-2 treatment. In a separate study, we found that in immune cells of autoimmune-prone SNF(1) and non-autoimmune DBF(1) mice, both ERalpha and ERbeta were differentially expressed and modulated by E-2. In SNF(1) mice, there were more CD4(+) and CD8(+) T cells constitutively expressing ERalpha, and the percentages of ERalpha+ dendritic cells and macrophages were increased after E-2 exposure compared to DBF(1) mice. Taken together, these observations strongly suggest a role for ERalpha in E-2-induced development of the lupus phenotype.

Gestational exposure to mercury leads to persistent changes in T-cell phenotype and function in adult DBF1 mice.

Previously, we showed that in utero exposure to mercury induced phenotypic changes in fetal immune cells. Here, we sought to determine whether the effects of in utero exposure on immune cells persisted in the adult. After overnight breeding to DBA/1 males, pregnant BALB/c dams were given either mercuric chloride in drinking water at 10 mg/L ad libitum for the duration of gestation or plain water. At the time of parturition, all dams were placed on regular drinking water. The pups (DBF(1)) were weaned and thymic and splenic tissues were harvested at 10 wk-of-age to assess T-cell phenotypes and function. Significant changes in the CD4/CD8 subsets in the thymus and spleen among mercury-exposed male and female mice were not observed. However, there was a significant reduction in splenic CD4(+)CD25(+) cells in mercury-exposed female, but not in male, mice. ConA-stimulated splenocytes from mercury-exposed mice showed significant increases in proliferative responses relative to cells from control mice, regardless of sex. Cytokine secretion was also modulated in the mercury-exposed mice. In particular, the production of IL-4 and IFN by ConA-stimulated splenocytes from mercury-exposed male and female mice was significantly increased, while IL-2 and IL-10 levels were unaffected. The results of our study revealed that exposure of the developing immune system to relatively low levels of inorganic mercury could lead to persistent alterations in adult immune cell phenotypes and functions. These changes could pose a relevant health risk, if they contribute to impaired responses to pathogens and/or an increased risk for the development of atopy, asthma or possibly autoimmune diseases.

Estrogen signaling is not required for prostatic bud patterning or for its disruption by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Estrogens play an important role in prostatic development, health, and disease. While estrogen signaling is essential for normal postnatal prostate development, little is known about its prenatal role in control animals. We tested the hypothesis that estrogen signaling is needed for normal male prostatic bud patterning. Budding patterns were examined by scanning electron microscopy of urogenital sinus epithelium from wild-type mice, mice lacking estrogen receptor (ER)alpha, ERbeta, or both, and wild-type mice exposed to the antiestrogen ICI 182,780. Budding phenotypes did not detectably differ among any of these groups, strongly suggesting that estrogen signaling is not needed to establish the prototypical prostatic budding pattern seen in control males. This finding contributes to our understanding of the effects of low-level estrogen exposure on early prostate development. In utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can greatly alter the pattern in which prostatic buds form and reduce their number. For several reasons, including a prior observation that inhibitory effects of TCDD on prostatic budding in rats depend heavily on the sex of adjacent fetuses, we tested the hypothesis that estrogen signaling is needed for TCDD to disrupt prostatic budding. However, budding did not detectably differ among wild-type mice, or mice lacking ERalpha, ERbeta, or both, that were exposed prenatally to TCDD (5 microg/kg on embryonic day 13.5). Nor did ICI 182,780 detectably affect the response to TCDD. These results strongly suggest that estrogen signaling is not needed for TCDD to inhibit prostatic epithelial budding.

Immunization with peptides derived from the idiotypic region of lupus-associated autoantibodies delays the development of lupus nephritis in the (SWR x NZB)F1 murine model.

Systemic lupus erythematosus (SLE) is a multiorgan autoimmune disease affecting 40-50/100,000 Americans. Although most of the research on pathogenic antibodies focuses on antigenic specificity, there is increasing evidence that specific immunoglobulin idiotypes may mediate lupus nephritis independent of autoantigen specificity. In previous work, our laboratory characterized a set of nephritogenic monoclonal antibodies with substantial idiotypic cross-reactivity, produced by the spontaneous SLE model (SWR x NZB)F(1) (SNF(1)), termed Id(LN)F(1). Peptides derived from one of these antibodies, Id540, was previously shown to stimulate pathogenic T-cells from prenephritic SNF(1) mice, similar to what has been seen for pathogenic A6.1 antibody produced by the (NZB x NZW)F(1) model. In this study, we immunized pre-nephritic SNF(1) mice with p62-73, a peptide derived from the variable region of Id540 and, in separate experiments, with p58-69, a peptide derived from the variable region of A6.1. In both cases, immunization resulted in increased survival and delayed nephritis; however, while both peptides affected levels of anti-DNA antibodies, immunization with p62-73 only affected levels of Id(LN)F(1) antibodies. These findings confirm the roles of pathogenic idiotypes in the pathogenesis of lupus nephritis and suggest that therapies that target specific idiotypes might be a potential tool in the management of SLE.

Prenatal HgCl(2) Exposure Alters Fetal Cell Phenotypes.

Here we sought to determine whether prenatal exposure to subtoxic levels of inorganic mercury induced modulations in the fetal immune repertoire. After overnight breeding of female BALB/c and male DBA/1 mice, pregnant females were exposed to 10 mg/l mercuric chloride in drinking water. DBF(1) pups were examined at day 16 of gestation for immunophenotypic changes in the fetal thymus and liver. While total thymocyte counts remained comparable to unexposed controls, intrauterine mercury exposure was found to modulate several immune phenotypes in the fetal thymus. Specifically, we saw an increase in the percentages of double negative (DN, CD4(-)CD8(-)) cells as well as a reduction in the numbers of cells representing activation phenotypes (CD4(+)CD25(+)). In the liver, we observed modulations that suggested skewing of the development of B220(+)-expressing cells with mercury exposure. Further, we saw increased populations of thymic and splenic lymphocytes reactive with a pathogenic idiotypic determinant Id(LN)F(1,) which is associated with spontaneous autoimmune nephritis in (NZB x SWR)F(1) (SNF(1)) mice. Previous studies from our laboratory have shown that dysregulation of the Id(LN)F(1) idiotypic network, particularly the expansion of idiotype-reactive T- and B-lymphocytes, is key in the development of lupus nephritis in the autoimmune SNF(1) mouse. These studies show that acute exposure to relatively low levels of inorganic mercury in utero may alter the fetal immune repertoire which could potentially modulate post-natal immune responses.

Accelerated macrophage apoptosis induces autoantibody formation and organ damage in systemic lupus erythematosus.

Increased monocyte/macrophage (Mphi) apoptosis occurs in patients with systemic lupus erythematosus (SLE) and is mediated, at least in part, by an autoreactive CD4(+) T cell subset. Furthermore, autoreactive murine CD4(+) T cells that kill syngeneic Mphi in vitro induce a lupus-like disease in vivo. However, it is unclear whether increased Mphi apoptosis in SLE per se is sufficient to accelerate/promote autoimmunity. We have investigated whether increased Mphi apoptosis in vivo, induced by the administration of clodronate liposomes, can exacerbate the autoimmune phenotype in NZB x SWR (SNF(1)) lupus-prone mice, and induce autoantibody production in haplotype-matched BALB/c x DBA1 (DBF(1)) non-lupus-prone mice. Lupus-prone mice SNF(1) mice that were treated with clodronate liposomes, but not mice treated with vehicle, developed significant increases in autoantibodies to dsDNA, nucleosomes, and the idiotypically related family of nephritic Abs Id(LN)F(1), when compared with untreated SNF(1) mice. Furthermore, clodronate treatment hastened the onset of proteinuria and worsened SNF(1) lupus nephritis. When compared with vehicle-treated controls, clodronate-treated non-lupus-prone DBF(1) mice developed significantly higher levels of anti-nucleosome and Id(LN)F(1) Abs but did not develop lupus nephritis. We propose that Mphi apoptosis contributes to the pathogenesis of autoantibody formation and organ damage through both an increase in the apoptotic load and impairment in the clearance of apoptotic material. This study suggests that mechanisms that induce scavenger cell apoptosis, such as death induced by autoreactive cytotoxic T cells observed in SLE, could play a pathogenic role and contribute to the severity of the disease.

Molecular identification of pathogenetic IdLNF+1 autoantibody idiotypes derived from the NZBxSWR F1 model for systemic lupus erythematosus.

The acceleration of nephritis in SNF(1) mice by CD4(+) T-cell clones reactive with a nephritogenic idiotype, Id(LN)F(1) [1], as well as the ability of anti-Id(LN)F(1) antisera to down-regulate the production of Id(LN)F(+)(1) immunoglobulin (Ig) in vivo and delay nephritis [2], suggests that dysregulation of this idiotype may contribute to the development of SNF(1) nephritis. Herein, we show that a monoclonal Id(LN)F(1)-expressing antibody, 540, significantly (P< or = 0.01) stimulated Id(LN)F(1)-reactive T-cell clones B6 and D2 to proliferate, while other Id(LN)F+1 antibodies did not. Further, injection of 540-producing hybridoma cells into nonautoimmune (SWRxBalb/c)F(1) mice resulted in the deposition of Id(LN)F(+)(1) Ig in the kidneys, in a pattern indicative of early nephritis. To identify the pathogenetic Id(LN)F(1) epitope(s) at the molecular level, we compared the deduced amino acid sequences of the heavy and light chain variable regions of pathogenetic and non-pathogenetic Id(LN)F(1)-expressing Igs 540, 317, and 533. Two overlapping peptides derived from the V(H) sequence of 540 (aa 54-66 and 62-73), which both contain the triple basic amino acid motif K(X)K(X)K, stimulated SNF(1) T cells and T-cell clones B6 and D2. These results further support the involvement of a subset of Id(LN)F(1)-expressing Ig in SNF(1) nephritis.