Biomedical subjects

H Offner

Publications and source records attributed to H Offner.

At least 37 records · Page 2Linked to original sources

PubMed1998

Immunity to T cell receptor peptides in multiple sclerosis. III. Preferential immunogenicity of complementarity-determining region 2 peptides from disease-associated T cell receptor BV genes.

Vaccination with synthetic TCR peptides from the BV5S2 complementarity-determining region 2 (CDR2) can boost significantly the frequency of circulating CD4+ peptide-specific Th2 cells in multiple sclerosis (MS) patients, with an associated decrease in the frequency of myelin basic protein (MBP)-reactive Th1 cells and possible clinical benefit. To evaluate the immunogenicity of CDR2 vs other regions of the TCR, we vaccinated seven MS patients with overlapping BV5S2 peptides spanning amino acids 1-94. Six patients responded to at least one of three overlapping or substituted CDR2 peptides possessing a core epitope of residues 44-52, and one patient also responded to a CDR1 peptide. Of the CDR2 peptides, the substituted (Y49T)BV5S2-38-58 peptide was the most immunogenic but cross-reacted with the native sequence and had the strongest binding affinity for MS-associated HLA-DR2 alleles, suggesting that position 49 is an MHC rather than a TCR contact residue. Two MS patients who did not respond to BV5S2 peptides were immunized successfully with CDR2 peptides from different BV gene families overexpressed by their MBP-specific T cells. Taken together, these results suggest that a widely active vaccine for MS might well involve a limited set of slightly modified CDR2 peptides from BV genes involved in T cell recognition of MBP.

Adult↗

PubMed1998

Neonatal exposure of TCR BV8S2 transgenic mice to recombinant TCR BV8S2 results in reduced T cell proliferation and elevated antibody response to BV8S2, and increased severity of EAE.

Transgenic (Tg) mouse models are unique tools for investigating regulatory mechanisms of the immune system. Mice bearing a T cell receptor (TCR) BV8S2 transgene derived from an encephalitogenic T cell clone are highly susceptible to experimental autoimmune encephalomyelitis (EAE), a T cell-mediated neurological disorder. Although the pathogenesis of EAE is not yet fully understood, TCR-specific regulatory T cells seem to play a role in its remission and/or recovery process. In previous studies, we showed that immunization of BV8S2 Tg mice with recombinant BV8S2 protein induced TCR-specific T cells and protection against EAE, clearly indicating the persistence of a functional TCR regulatory network in spite of the highly skewed T cell repertoire. To further investigate the natural regulatory role of TCR-specific T cells, we evaluated the effect on EAE of inducing neonatal tolerance to heterologous (rat) and homologous BV8S2 proteins in Tg mice. Neonatal exposure to rat BV8S2 protein induced "split" tolerance, characterized by decreased T cell proliferation but increased antibody responses to both rat and mouse BV8S2 proteins that are known to be cross-reactive. When challenged as adults with an encephalitogenic emulsion, Tg mice tolerized with rat but not mouse BV8S2 protein developed more severe EAE compared to control mice. These results demonstrate that immunity to BV8S2 determinants in BV8S2 Tg mice is naturally induced and functions to limit the severity of EAE.

Animals↗

PubMed1998

Gender differences in experimental autoimmune encephalomyelitis develop during the induction of the immune response to encephalitogenic peptides.

Multiple sclerosis (MS) strikes women more often than men. Gender differences in experimental autoimmune encephalomyelitis (EAE) parallel those seen in MS. We utilized the adoptive transfer model of EAE to determine the role of gender on the induction and effector phases of disease. PLP 139-151-sensitized spleen cells from female SJL mice were more effective at transferring disease than male cells. However, there were no gender differences in the frequency of PLP 139-151-specific T cells. PLP 139-151-specific female T cell lines induced more severe disease than male T cell lines. Disease severity was more strongly linked to the sex of the donor T cells, indicating that gender influences the immune response primarily during the induction phase.

Adoptive Transfer↗

PubMed1998

Gonadal hormones influence the immune response to PLP 139-151 and the clinical course of relapsing experimental autoimmune encephalomyelitis.

Females have an increased incidence of multiple sclerosis (2:1). This gender dimorphism can be studied effectively using a murine model of relapsing experimental autoimmune encephalomyelitis (R-EAE). We demonstrated previously that male SJL mice immunized with proteolipid protein (PLP) peptide 139-151 developed an initial episode of paralysis, but failed to relapse. In the present study, clinical EAE relapses were induced by orchidectomy. Relapses in castrated mice were accompanied by an influx of activated CD4+, Th1 cells into the CNS which were absent in sham mice. Our data suggests an important regulatory role for androgens on the immune response to PLP 139-151 and the clinical course of R-EAE.

Animals↗

PubMed1998

Novel adjuvants for induction of T-cell and antibody responses to encephalitogenic and regulatory determinants in Lewis rats.

Treatment of human autoimmune diseases may be enhanced by using adjuvants that can selectively induce immunoregulatory responses. Two versions of a novel nonionic block copolymer adjuvant suitable for human use, Optivax Oil Formulation (OF) and Optivax Aqueous Formulation (AF), were evaluated for induction of immunity to encephalitogenic and regulatory T-cell receptor (TCR) V-gene determinants. In Lewis rats immunized with myelin basic protein (BP), Optivax OF was more efficient than Optivax AF for inducing delayed type hypersensitivity (DTH), T-cell proliferation, antibodies, and even mild clinical signs of experimental autoimmune encephalomyelitis (EAE). Similarly, Optivax OF was more efficient for inducing inflammatory T-cell and antibody responses to immunoregulatory V beta 8.2 proteins and peptides than Optivax AF, which induced a noninflammatory Th2 response. In general, DTH response to the various immunogens was reflected by increased cellularity and mRNA levels for IFN-gamma in draining lymph nodes, whereas LN cell proliferation without DTH was characterized by increased IL-2 mRNA levels but low or absent IFN-gamma message. These data suggest important differential adjuvant effects of Optivax OF versus Optivax AF on the respective induction of Th1 versus Th2 responses that may be useful in the selective treatment of human immune disorders.

Adjuvants, Immunologic↗

PubMed1998

Effects of vaccination with T cell receptor peptides: epitope switching to a possible disease-protective determinant of myelin basic protein that is cross-reactive with a TCR BV peptide.

Immunization of Lewis rats with guinea-pig myelin basic protein (Gp-MBP) induced T cell responses to primary and secondary encephalitogenic determinants, as well as to a third non-encephalitogenic epitope, residues 55-69. This sequence is of interest due to its protective activity against experimental autoimmune encephalomyelitis. Protection involved induction of MBP-55-69-specific T cells expressing cross-reactive TCR BV8S6 genes that activated regulatory T cells specific for TCR BV8S2 determinants expressed on encephalitogenic T cells. We here present and discuss new evidence suggesting a possible immunological cross-reactivity between the protective Gp-MBP-55-69 peptide and the regulatory BV8S2-39-59 peptide. This cross-reactivity, which may also occur between the human MBP-55-74 peptide and the BV12S2-38-58 sequence, has potentially important implications for human diseases such as multiple sclerosis.

Animals↗

PubMed1998

The OX-40 receptor provides a potent co-stimulatory signal capable of inducing encephalitogenicity in myelin-specific CD4+ T cells.

The OX-40 receptor, a member of the nerve growth factor/tumor necrosis factor receptor gene family, is expressed preferentially on autoreactive CD4+ T cells isolated from the site of inflammation in rats with clinical signs of experimental autoimmune encephalomyelitis (EAE). To examine whether the OX-40 receptor has biologic relevance to T cell function, we evaluated the ability of a rat OX-40 receptor-specific antibody to co-stimulate a myelin basic protein (MBP)-reactive CD4+ T cell line. The anti-OX-40 antibody provided a potent co-stimulatory signal to CD4+ T cells when added in conjunction with a submitogenic dose of anti-CD3, but the anti-OX-40 antibody alone did not produce a mitogenic response. The magnitude and dose-response of anti-OX-40 co-stimulation was virtually identical to the signal delivered to T cells when cultured with anti-CD28 in conjunction with anti-CD3. MBP-specific T cells stimulated with both anti-CD3 and anti-OX-40 antibodies expressed increased mRNA and protein for IL-2 when compared to anti-CD3 alone. MBP-specific T cells stimulated with both anti-CD3 and anti-OX-40 antibodies were also able to induce EAE when transferred into naive Lewis rats. In contrast, cells stimulated with anti-CD3 alone were not encephalitogenic. These data suggest that the function of the OX-40 receptor on activated T cells is to provide an alternative pathway for T cell co-stimulation that may be similar in potency to the CD28-mediated signal.

Animals↗

PubMed1997

Similar pattern of MCP-1 expression in spinal cords and eyes of Lewis rats with experimental autoimmune encephalomyelitis associated anterior uveitis.

Monocyte chemoattractant protein-1 (MCP-1) is a member of the CC chemokine family responsible for the recruitment of T cells that have been found during inflammation of the spinal cord in experimental autoimmune encephalomyelitis (EAE) in Lewis rats immunized with myelin basic protein (MBP). Lewis rats injected with MBP also developed anterior uveitis (AU), which coincided with the onset of EAE. In the present studies, we examined the expression and distribution of MCP-1 in the eye and spinal cord during disease and compared it to the expression of Th1 cell type cytokines. Initially, MCP-1 expression was detected at the preclinical phase in the iris/ciliary body and lumbar spinal cord and increased during the course of EAE/AU. Mononuclear infiltrating cells and endothelial cells and astrocytes of the CNS could be identified as a source of MCP-1 by in situ hybridization. Kinetics of expression of Th1 characteristic cytokines such as IL-2 and IFNgamma was in agreement with the expression of MCP-1 chemokine. Moreover, induction of the gene expression of MCP-1 seemed to occur earlier than that of MIP-2, and it correlated with increasing disease severity. MCP-1 seems to contribute to the initial recruitment of inflammatory cells into both the tissues of the eye and CNS over the course of disease.

Animals↗

PubMed1997

Multiple class I motifs revealed by sequencing naturally processed peptides eluted from rat T cell MHC molecules.

Class I major histocompatibility complex (MHC) molecules interact with a diverse array of self and foreign peptides. Displayed on the cell surface, the class I/peptide complex provides an extracellular indication of the intracellular milieu. We have characterized the Lewis rat Vbeta8.2+ T cell hybridoma C14/BW12-12A1 by FACS analysis and have used immunoaffinity chromatography to purify class I molecules from these cells. Peptides eluted from the class I molecules have been fractionated by HPLC and sequenced. Self-peptide mixtures indicate two distinct peptide motifs, suggesting the possibility of multiple class I loci. The majority of the naturally processed peptide ligands were nonamers. Naturally processed peptide ligands fitting the first motif contained a hydrophobic leucine anchor residue at position three and a carboxyl-terminal serine anchor residue. A second motif was characterized by a tyrosine or phenylalanine residue at position three and a phenylalanine or isoleucine carboxyl-terminal residue. Four peptides derived from the Vbeta8.2 T cell receptor have sequences that fit these motifs, providing a mechanistic explanation for their immunoregulatory role. Identification of these class I peptide binding motifs will be useful for predicting potential CTL epitopes in studies on autoimmunity, immunoregulation and transplantation in the Lewis rat.

Amino Acid Sequence↗

PubMed1997

A TCR V alpha CDR3-specific motif associated with Lewis rat autoimmune encephalomyelitis and basic protein-specific T cell clones.

To investigate TCR V alpha gene expression in the Lewis rat model of experimental autoimmune encephalomyelitis, we obtained V alpha chain sequences from two V beta8.2+-encephalitogenic, BP72-89-specific T cell clones. Two different V alpha genes, a V alpha2 gene and a V alpha23 gene, are utilized, but both were found to contain an asparagine repeat (Asn3+) sequence present in the V alpha CDR3 region. This Asn3+ motif is also present in the previously reported sequence of a BP68-88-specific hybridoma, 510, which utilizes a different V alpha2 gene family member. In further experiments, spinal cord T cells were isolated at the onset of basic protein (BP)-induced disease and sorted for the OX-40 activation marker, which we have previously used to enrich for specifically activated T cells. Analysis of V alpha expression in the OX-40+ population revealed the biased use of three V alpha genes, V alpha1, V alpha2, and V alpha23. The Asn3+ motif was present in the V alpha CDR3 region of V alpha1, V alpha2, and V alpha23 cDNA derived from OX-40+ spinal cord T cells but found to be generally absent in the OX-40- spinal cord population. Since these Asn3+ motif-bearing V alpha chain sequences are nearly identical to those utilized by the BP-specific encephalitogenic clones described, it is likely that these V alpha sequences are derived from disease-associated T cells in the spinal cord. Thus, we demonstrate that the Asn3+ V alpha CDR3 motif is strongly associated with experimental autoimmune encephalomyelitis in the Lewis rat and propose that it plays a role in TCR recognition of a specific BP peptide/MHC complex.

Amino Acid Sequence↗

PubMed1997

Myelin basic protein-specific and TCR V beta 8.2-specific T-cell lines from TCR V beta 8.2 transgenic mice utilize the same V alpha and V beta genes: specificity associated with the V alpha CDR3-J alpha region.

Our analysis of TCR V gene usage in mice transgenic for the V beta 8.2 gene has demonstrated that T cells isolated from the spinal cord of these transgenic mice during active experimental autoimmune encephalomyelitis were significantly biased for V alpha 2 expression. This V alpha 2 bias was noted in T cells derived from the periphery as well but only after stimulation with specific antigen. Thus, spinal cord-derived pathogenic T cells had already undergone activation and expansion within the central nervous system environment of these mice. As part of an investigation of regulatory function in these V beta 8.2 transgenic mice, two T cell lines were selected. The first T cell line is encephalitogenic and specific for the dominant myelin basic protein peptide NAc1-11, while the second T cell line is specific for the V beta 8.2 protein. Surprisingly, polymerase chain reaction and sequence analysis of the TCR from both the T cell lines demonstrated that they utilize identical V beta, D beta, J beta, and V alpha gene segments. The only difference found was in their use of the J alpha gene segment, indicating that this region of the TCR molecule can play a key role in determining antigen specificity.

Animals↗

PubMed1996

Male SJL mice do not relapse after induction of EAE with PLP 139-151.

SJL mice immunized with proteolipid protein (PLP) develop relapsing experimental autoimmune encephalomyelitis (R-EAE). R-EAE is a CD4+, Th1 cell-mediated demyelinating disease of the central nervous system (CNS) that is used as a model for the human disease multiple sclerosis (MS). Previous studies showed that young (< 8 weeks) male SJL mice were resistant to active induction of EAE with CNS homogenate, while female mice were susceptible. We have recently observed that young male SJL mice immunized with a major encephalitogenic peptide of myelin, PLP 139-151, developed initial clinical and histological symptoms of EAE with a severity similar to age-matched females; however, unlike females, male mice did not relapse. Significant T cell proliferation to PLP 139-151, but not to other PLP and myelin basic protein (MBP) epitopes, was observed in both males and females during the initial episode, recovery, and first relapse of clinical disease. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of lymphokine mRNA revealed differences in IFN-gamma and IL-4 synthesis consistent with the hypothesis that Th2 T cells develop in young male SJL mice that regulate the relapsing phase of the disease. These data suggest that immunization of young male SJL mice with PLP 139-151 overrides a defect in antigen presentation responsible for the previously observed resistance to EAE, and that natural processing and presentation of neuroantigens during the course of acute EAE induces Th2 cells that prevent the relapse of disease.

Amino Acid Sequence↗

PubMed1996

Variation in H-2K(k) peptide motif revealed by sequencing naturally processed peptides from T-cell hybridoma class I molecules.

Class I major histocompatibility complex (MHC) molecules interact with a diverse array of self and foreign peptides, displaying them on the cell surface and providing an extracellular indication of intracellular invasion. The most clearly defined role for these class I/peptide complexes is to cause effector responses upon binding to antigen-specific receptors of cytotoxic T cells. We have characterized the mouse thymoma/rat V beta 8.2+ T-cell hybridoma C14/BW12-12A1 by fluorescence-activated cell sorting analysis and have used immunoaffinity chromatography to purify class I molecules from these cells. The peptides bound to the class I molecules were fractionated by high-performance liquid chromatography and sequenced. Self-peptide mixtures eluted from the mouse H-2Kk class I allele revealed a dominant primary sequence motif, with a carboxyl-terminal residue that appeared to be invariantly valine and a secondary or auxiliary anchor residue at position 2 that could be either glutamate or proline. The majority of naturally processed peptide ligands appeared to be octamers. Although peptides eluted off H-2Kk molecules from tissue derived from a number of different inbred mouse strains also appeared to be octamers, others have reported that isoleucine is the dominant carboxyl-terminal residue. Thus, different cell types displayed distinct differences in naturally processed peptides bound by the same class I alleles. The variation in naturally processed peptides loaded onto the same class I allele most likely reflects the nature of the pool of peptides within the cell available for loading class I molecules.

Alleles↗

PubMed1996

MHC-restriction, cytokine profile, and immunoregulatory effects of human T cells specific for TCR V beta CDR2 peptides: comparison with myelin basic protein-specific T cells.

HLA-DR2+ patients with multiple sclerosis (MS) that respond to vaccination with TCR V beta 5.2-38-58 peptides have increased frequencies of TCR peptide-specific T cells, reduced frequencies of myelin basic protein (MBP)-specific T cells, and a better clinical course than non-responders. To evaluate possible network regulation of MBP responses by TCR peptide-specific T cells, we compared properties of both cell types. Both MBP- and TCR peptide-specific T cell clones were CD4+ and predominantly HLA-DR restricted. HLA-DR2, which is in linkage disequilibrium in MS patients, preferentially restricted TCR peptide-specific clones as well as MBP-specific responses in HLA-DR2 and DR2,3+ donors. Within the DR2 haplotype, however, both DR beta 1*1501 and DR beta 5*0101 alleles could restrict T cell responses to V beta CDR2 peptides, whereas responses to MBP were restricted only by DR beta 5*0101. TCR peptide-specific clones expressed message for Th2 cytokines, including IL-4, IL-5, IL-6, IL-10, and TGF-beta, whereas MBP-specific T cell clones expressed the Th1 cytokines IFN-gamma and IL-2. Consistent with the Th2-like cytokine profile, TCR peptide-specific T cell clones expressed higher levels of CD30 than MBP-specific T cells. Culture supernatants from TCR peptide-specific T cell clones, but not from MBP- or Herpes simplex virus-specific T cells, inhibited both proliferation responses and cytokine message production of MBP-specific T cells. These results demonstrate distinct properties of MBP and TCR peptide-specific T cells, and indicate that both target and bystander Th1 cells can be inhibited by Th2 cytokines secreted by activated TCR peptide-specific T cells. These data support the rationale for TCR peptide vaccination to regulate pathogenic responses mediated by oligoclonal T cells in human autoimmune diseases.

Adult↗

PubMed1996

Prevention and treatment of relapsing autoimmune encephalomyelitis with myelin peptide-coupled splenocytes.

Injection of antigen cross-linked accessory cells has proven to be an efficient and highly selective approach for inducing epitope-specific peripheral tolerance. This approach has been used successfully to inhibit induction of experimental autoimmune encephalomyelitis (EAE) and to dissect the relative dominance of component encephalitogenic determinants that contribute to both acute and relapsing EAE. In this study, we evaluated the tolerogenic effect of the dominant encephalitogenic epitope for SJL/J mice, residues 139-151 of myelin proteolipid protein (PLP), on the induction and relapses of EAE induced actively with PLP139-151/CFA. Our results demonstrate the powerful protective effect of treating mice before induction of EASE with PLP139-151-conjugated splenocytes (SPL) on the incidence and severity of both the initial episode and the first relapse of EAE. Moreover, treatment of mice on the first day of onset of clinical signs of EAE reduced the severity of the first relapse, apparently by reducing T cell recognition of PLP139-151, although no significant therapeutic effect was observed during the initial treated clinical episode. These data demonstrate the utility of using neuroantigen-coupled accessory cells to prevent and treat relapsing EAE.

Amino Acid Sequence↗

PubMed1996

Neonatal injection of Lewis rats with recombinant V beta 8.2 induces T cell but not B cell tolerance and increased severity of experimental autoimmune encephalomyelitis.

In Lewis rats with experimental autoimmune encephalomyelitis (EAE) mediated by V beta 8.2 effector cells, anti-idiotypic T cells and antibodies could be boosted by injection of V beta 8.2 peptides, inducing both T cells and antibodies that reduced the severity and shortened the course of disease. However, EAE in Lewis rats is self-limiting, and we sought to determine if the anti-idiotypic response contributed to the natural recovery process. In a previous study, we found that adult tolerance induced to one of the regulatory idiotopes, V beta 8.2-44-54, caused worsening of EAE, implicating response to this epitope in recovery from EAE. However, neonatally-induced tolerance to V beta 8.2-44-54 did not alter the course of EAE, suggesting either compensation by additional V beta 8.2 determinants, or mechanistic differences in tolerization protocols. In this report, we reevaluate the role of V beta 8.2 determinants in recovery from EAE, using two recombinant V beta 8.2 constructs to induce neonatal tolerance to the comprehensive set of V beta 8.2 epitopes prior to adult induction of EAE. We found that neonatal exposure to either of the recombinant V beta 8.2 molecules induced "split" tolerance-specific T cell tolerance but enhanced antibody responses- and a more severe course of EAE. In contrast, neonatal exposure to a V beta 8.2 + T cell hybridoma or a control protein did not induce T cell tolerance to V beta 8.2 determinants and did not alter the EAE disease course. These results are consistent with those obtained by inducing adult tolerance, and suggest that our previous result (normal recovery from EAE in rats neonatally tolerized to V beta 8.2-44-54) was probably due to a compensatory response to other V beta 8.2 determinants. In both studies, the data clearly implicate T cell recognition of V beta 8.2 determinants in the natural EAE recovery process.

Amino Acid Sequence↗

PubMed1996

Treatment of relapsing autoimmune encephalomyelitis with T cell receptor V beta-specific antibodies when proteolipid protein is the autoantigen.

Monoclonal antibodies (mAbs) directed against the V beta chain of the T cell receptor (TCR) of pathogenic T cells have been used to treat acute murine experimental autoimmune encephalomyelitis (EAE) induced by myelin basic protein (BP). We evaluated anti-V beta mAb for the treatment of relapsing EAE (R-EAE) induced in SJL/J mice by the myelin proteolipid protein (PLP) peptide 139-151. Spinal cord mononuclear cells isolated from mice immunized for R-EAE with PLP 139-151 were shown to express a predominance of V beta 2 and V beta 17 during acute and relapsing disease. T cell lines specific for PLP 139-151 were magnetically sorted to express 80-90% V beta 2. These V beta 2-enriched lines induced typical relapsing demyelinating EAE in naive recipient mice. SJL/J mice with R-EAE induced by a PLP 139-151-specific T cell line expressing 88% V beta 2 were treated with anti-V beta 2 mAb. Anti-V beta 2 mAb markedly reduced clinical and histological disease severity when given at the time of cell transfer or when given at clinical disease onset. In contrast, anti-V beta mAbs showed only a mild clinical effect on R-EAE induced by immunization with PLP 139-151 or R-EAE transferred by a PLP 139-151-specific T cell line expressing multiple V beta s. A cocktail of mAbs directed against V beta 2, V beta 4, and V beta 17 significantly reduced the numbers of spinal cord T cells expressing these V beta s during acute EAE but had little effect on disease course, suggesting that pathogenic T cells expressing other V beta s were producing disease. These findings may have implications for the treatment of multiple sclerosis with V beta-selective therapy.

Analysis of Variance↗

PubMed1996

Myelin basic protein specific T-helper cells induce experimental anterior uveitis.

Immunopathological changes in the eyes were examined in Lewis rats after active and passive induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic proteins (MBP) at various stages of EAE. The onset of anterior uveitis (AU) coincided with hind limb paralysis, but uveitis persisted after clinical signs of EAE had subsided. A mild form of uveitis was characteristic for the majority of rats. The changes within the iris and ciliary body consisted of an accumulation of inflammatory cells lining the anterior surface of iris, the trabecular meshwork, and, in some cases, within the ciliary body and the aqueous humor. A similar histopathological picture was observed when rats were injected with the secondary encephalitogenic determinant for Lewis rats, MBP peptide 87-99. Flow cytometry analysis of T cells from the anterior segment of the inflamed eyes after immunization with MBP revealed the presence of CD4+ cells exclusively expressing V beta 8.2 and OX-40 markers. Our data suggest that MBP are encephalitogenic and uveitogenic in Lewis rats and that the V beta 8.2-positive T cells in the eye represent encephalitogenic T cells. Many of those T cells were distributed in the iris and the anterior chamber. These findings indicate that these MBP-specific T cells may play a critical role in EAE as well as in AU.

Animals↗