The postnatal maternal environment influences diabetes development in nonobese diabetic mice.

When nonobese-diabetic (NOD) mouse embryos were implanted into pseudopregnant mothers of a nonautoimmune mouse strain, the progeny had a reduced type 1 diabetes (T1D) incidence, suggesting that transmission of maternal autoantibodies is important for T1D development. Whether eliminating islet autoantibody transmission in utero, or postnatally (through milk), prevented T1D is unknown. Herein, we show that fostering newborn NOD mice on B-cell deficient NOD.Igmu-/- dams does not prevent T1D, demonstrating that postnatally transmitted islet autoantibodies are not required for disease pathogenesis. Additionally, NOD.Igmu-/- mice reared on NOD dams did not develop T1D, indicating that autoantibody transmission to B-cell deficient NOD neonates is insufficient to trigger T1D. Interestingly, newborn NOD mice that were reared by ICR (but not NOD or C57BL/6) dams had reduced T1D incidence, although not as reduced as that reported after embryo transfer to ICR mice, suggesting that both prenatal and postnatal factors contribute to the observed reduction in T1D incidence. Thus, NOD mice have different risks for developing T1D depending on the strain of their foster mother, and both prenatal and postnatal maternal factors, other than islet autoantibodies, influence their T1D incidence. The results may be relevant for understanding the increasing incidence of T1D and designing interventions.

Report from the 1st International NOD Mouse T-Cell Workshop and the follow-up mini-workshop.

A workshop on autoreactive T-cell responses in NOD mice was held to optimize autoreactive T-cell detection methodologies. Using different proliferation assay protocols, 1 of the 11 participating laboratories detected spontaneous T-cell responses to GAD(524-543) and insulin(9-23) in their NOD mice. Two other laboratories were able to detect autoreactive responses when using enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA) analysis of cytokines in culture supernatants, suggesting that these assays provided greater sensitivity. To address the divergent findings, a follow-up mini-workshop tested NOD mice from four different colonies side-by-side for T-cell proliferative responses to an expanded panel of autoantigens, using the protocol that had enabled detection of responses in the 1st International NOD Mouse T-Cell Workshop. Under these assay conditions, 16 of 16 NOD mice displayed proliferative responses to whole GAD65, 13 of 16 to GAD(524-543), 9 of 16 to GAD(217-236), 7 of 16 to insulin(9-23), and 5 of 16 to HSP277. Thus, spontaneous proliferative T-cell responses can be consistently detected to some beta-cell autoantigens and peptides thereof. Overall, the results suggest that more sensitive assays (e.g., ELISPOT, ELISA analysis of cytokines in supernatants, or tetramer staining) may be preferred for the detection of autoreactive T-cells.

Lipopolysaccharide-activated B cells down-regulate Th1 immunity and prevent autoimmune diabetes in nonobese diabetic mice.

B cells can serve dual roles in modulating T cell immunity through their potent capacity to present Ag and induce regulatory tolerance. Although B cells are necessary components for the initiation of spontaneous T cell autoimmunity to beta cell Ags in nonobese diabetic (NOD) mice, the role of activated B cells in the autoimmune process is poorly understood. In this study, we show that LPS-activated B cells, but not control B cells, express Fas ligand and secrete TGF-beta. Coincubation of diabetogenic T cells with activated B cells in vitro leads to the apoptosis of both T and B lymphocytes. Transfusion of activated B cells, but not control B cells, into prediabetic NOD mice inhibited spontaneous Th1 autoimmunity, but did not promote Th2 responses to beta cell autoantigens. Furthermore, this treatment induced mononuclear cell apoptosis predominantly in the spleen and temporarily impaired the activity of APCs. Cotransfer of activated B cells with diabetogenic splenic T cells prevented the adoptive transfer of type I diabetes mellitus (T1DM) to NOD/scid mice. Importantly, whereas 90% of NOD mice treated with control B cells developed T1DM within 27 wk, <20% of the NOD mice treated with activated B cells became hyperglycemic up to 1 year of age. Our data suggest that activated B cells can down-regulate pathogenic Th1 immunity through triggering the apoptosis of Th1 cells and/or inhibition of APC activity by the secretion of TGF-beta. These findings provide new insights into T-B cell interactions and may aid in the design of new therapies for human T1DM.

The frequency of high avidity T cells determines the hierarchy of determinant spreading.

Autoimmunity often spreads in a predefined pattern during the progression of T cell-mediated autoimmune diseases. This progression has been well described in animal models and in man, but the basis for this phenomenon is little understood. To gain insight into the factors that determine this spreading hierarchy, we characterized the binding affinity of a panel of beta cell-autoantigenic peptides to I-Ag7, as well as the precursor frequency, functional avidity, and phenotype of the T cells that recognize these peptides in type 1 diabetes-prone nonobese diabetic mice. We observed that autoimmunity gradually spreads from a beta cell determinant, which had the largest precursor pool of high avidity T cells, to beta cell determinants with progressively smaller and lower avidity T cell precursor pools. This correlation between the sequential development of spontaneous T cell autoimmunity and the frequency and avidity of autoantigen-reactive T cells suggests that the extent to which T cells were negatively selected by the self-determinants is the key factor determining the spreading hierarchy.

Antisense oligonucleotides to C-fos reduce postictal seizure susceptibility following fully kindled seizures in rats.

In a previous study we demonstrated that increased FOS expression in the amygdala induced by partial kindling seizures could be attenuated by administering c-Fos specific antisense oligonucleotides. In addition, we found that the administration of c-Fos antisense oligonucleotides at the beginning of the amygdala kindling process facilitated the appearance of stage V kindled seizures. In the present study, we evaluated the effect of the suppression of FOS on the expression and severity of the generalized fully kindled seizures as well as on the susceptibility to additional ictal events during the postictal and interictal periods. We observed that the administration of c-Fos antisense oligonucleotides did not modify the behavioral and electrographic manifestations of generalized stage V kindled seizures. However, c-Fos antisense oligonucleotides significantly reduced the susceptibility to additional ictal events during the postictal refractory period. Hence, the increased FOS protein induced by generalized tonic clonic seizures may participate in postictal mechanisms.

GABA(A) receptors mediate inhibition of T cell responses.

We describe the presence of functional GABA(A) receptors on T cells. GABA inhibited anti-CD3 and antigen-specific T cell proliferation in vitro in a dose-dependent manner that was 1) mimicked by the GABA(A) receptor agonist muscimol (but not the GABA(B) receptor agonist baclofen), 2) blocked by GABA(A) receptor antagonists and a GABA(A) receptor Cl- channel blocker (picrotoxin) and 3) enhanced by pentobarbital. These data suggest that GABA(A) receptors mediate this immune inhibition and that these receptors can be modulated in a similar fashion to their neuronal counterparts. Finally, GABA inhibited DTH responses in vivo. Thus, pharmacological modulation of GABA(A) receptors may provide new approaches to modulate T cell responses in inflammation and autoimmune disease.

Antigen-based immunotherapy for autoimmune disease: from animal models to humans?

Insights into tolerance and autoimmune processes have led to novel immunotherapeutics for inhibiting autoimmune disease in animal models. However, recent studies question the immune basis of some of these therapeutic strategies and raise concerns about their efficacy and safety. Here, we discuss the feasibility of extending the success of antigen-based immunotherapeutics for T-cell-mediated autoimmune diseases from animal models to humans.

Attenuation of inducible Th2 immunity with autoimmune disease progression.

Autoantigen-based immunotherapeutics have been shown to activate regulatory responses capable of inhibiting T cell-mediated autoimmune disease in animal models. However, their efficacy generally declines, as treatment occurs later in the disease process, and their mechanism of action is a matter of intense debate. Here, we report that the early administration of beta cell autoantigens (betaCAAs) to nonobese diabetic (NOD) mice broadly diverts the natural development of potentially pathogenic Thl-biased autoimmune responses toward the Th2 phenotype through Th2 spreading. With disease progression, there was a steady decline in the ability of betaCAA treatment to promote Th2-type cellular and humoral autoimmunity. Late in the disease process, some betaCAAs were still able to induce Th2 responses and Th2 spreading (although to a much lesser extent), while other autoantigens were not. This attenuation of inducible Th2 immunity with disease progression is likely to reflect a reduction in the availability of uncommitted autoantigen-reactive precursor T cells. These findings suggest that there are inherent differences in the frequency of betaCAA-reactive T cells and that, in advanced stages of autoimmune disease, regulatory responses may be best elicited with target tissue Ags against which large uncommitted T cell pools are still available. Since individuals presenting the first signs of autoimmune disease are likely to already have an advanced disease process, these findings may be useful for the rational design of Ag-based immunotherapeutics.

Infectious Th1 and Th2 autoimmunity in diabetes-prone mice.

In the non-obese diabetic (NOD) mouse, a Th1-biased autoimmune response arises spontaneously against glutamic acid decarboxylase, concurrent with the onset of insulitis. Subsequently, Th1-type autoreactivity spreads intra- and intermolecularly to other beta-cell autoantigens (beta CAAs), suggesting that a spontaneous Th1 cascade underlies disease progression. Induction of Th2 immunity to a single beta CAA results in the spreading of Th2-type T-cell and humoral responses to other beta CAAs in an infectious manner. Thus, both Th1 and Th2 autoimmunity can evolve in amplificatory cascades defined by site-specific, but not antigen-specific, positive feedback circuits. Despite the continued presence of Th1 autoimmunity, the induction of Th2 spreading is associated with active tolerance to beta CAAs and reduced disease incidence. With disease progression there is an attenuation of beta CAA-inducible Th2 spreading, presumably because of a reduced availability of uncommitted beta CAA-reactive precursor T cells. We discuss the implications of these findings for the rational design of antigen-based immunotherapeutics.

In vivo administration of c-Fos antisense oligonucleotides accelerates amygdala kindling.

Repeated subconvulsive electrical stimulation of the amygdala leads to generalized seizures and provides an experimental model of epileptogenesis. Following electrical kindling stimulation the expression of c-Fos is rapidly induced. To evaluate the role of FOS protein in epileptogenesis, we used an antisense oligonucleotide strategy designed to inhibit its expression in the brain. Experimental and control oligonucleotides were delivered directly into the amygdala just prior to electrical stimulation. Immunocytochemical analysis showed that the administration of c-Fos antisense (but not sense) oligonucleotides inhibited expression of FOS in the amygdala following electrical stimulation. Behaviorally, treatment with c-Fos antisense oligonucleotides significantly accelerated the development of fully kindled (stage V) seizures. These data suggest that the increased FOS expression following electrical stimulation may be part of a protective mechanism which acts to inhibit epileptogenesis in the amygdala.

Insulin selectively primes Th2 responses and induces regulatory tolerance to insulin in pre-diabetic mice.

Little is known about the immunological impact of insulin administration other than it can boost insulin autoantibody levels. In particular, while the subcutaneous administration of a soluble foreign antigen (without adjuvant) is generally only weakly immunogenic in a naive animal, it is unknown what effect the subcutaneous administration of a soluble self-antigen has in animals with established autoimmune responses to the antigen. Addressing these questions in pre-diabetic nonobese diabetic (NOD) mice, we examined the effects of administering insulin, as well as the metabolically inactive B-chain of insulin, on insulin-specific cellular and humoral immune responses. We show that pre-diabetic NOD mice have a spontaneous Th1-biased response against insulin. Administering insulin, or the insulin B-chain, rather than boosting the established Th1 response, primed Th2 cellular and humoral immunity to insulin, shifting the predominant insulin response toward a Th2 phenotype. Despite the presence of a Th1 response against insulin, insulin treated mice failed to mount proliferative T-cell responses following immunization and challenge with insulin, demonstrating that the treatment induced an active form of tolerance to this autoantigen. Thus, the subcutaneous administration of a soluble antigen can engage Th2 responses and induce self-tolerance, even after the establishment of autoreactive Th-1 responses. Such immune deviation and induced regulatory tolerance may contribute to the protective effects of prophylactic insulin therapy, as well as the establishment of a "honeymoon" phase in new-onset insulin-dependent diabetic patients.

Determinant spreading of T helper cell 2 (Th2) responses to pancreatic islet autoantigens.

The nature (Th1 versus Th2) and dynamics of the autoimmune response during the development of insulin-dependent diabetes mellitus (IDDM) and after immunotherapy are unclear. Here, we show in nonobese diabetic (NOD) mice that the autoreactive T cell response starts and spreads as a pure Th1 type autoimmunity, suggesting that a spontaneous Th1 cascade underlies disease progression. Surprisingly, induction of antiinflammatory Th2 responses to a single beta cell antigen (betaCA) resulted in the spreading of Th2 cellular and humoral immunity to unrelated betaCAs in an infectious manner and protection from IDDM. The data suggest that both Th1 and Th2 autoimmunity evolve in amplificatory cascades by generating site-specific, but not antigen-specific, positive feedback circuits. Determinant spreading of Th2 responses may be a fundamental mechanism underlying antigen-based immunotherapeutics, explaining observations of infectious tolerance and providing a new theoretical framework for therapeutic intervention.

Modulating autoimmune responses to GAD inhibits disease progression and prolongs islet graft survival in diabetes-prone mice.

In nonobese diabetic (NOD) mice, beta-cell reactive T-helper type 1 (Th1) responses develop spontaneously and gradually spread, creating a cascade of responses that ultimately destroys the beta-cells. The diversity of the autoreactive T-cell repertoire creates a major obstacle to the development of therapeutics. We show that even in the presence of established Th1 responses, it is possible to induce autoantigen-specific anti-inflammatory Th2 responses. Immune deviation of T-cell responses to the beta-cell autoantigen glutamate decarboxylase (GAD65), induced an active form of self-tolerance that was associated with an inhibition of disease progression in prediabetic mice and prolonged survival of syngeneic islet grafts in diabetic NOD mice. Thus, modulation of autoantigen-specific Th1/Th2 balances may provide a minimally invasive means of downregulating established pathogenic autoimmune responses.

Nasal administration of glutamate decarboxylase (GAD65) peptides induces Th2 responses and prevents murine insulin-dependent diabetes.

We previously demonstrated that a spontaneous Th1 response against glutamate decarboxylase (GAD65) arises in NOD mice at four weeks in age and subsequently T cell autoimmunity spreads both intramolecularly and intermolecularly. Induction of passive tolerance to GAD65, through inactivation of reactive T cells before the onset of autoimmunity, prevented determinant spreading and the development of insulin-dependent diabetes mellitus (IDDM). Here, we examined whether an alternative strategy, designed to induce active tolerance via the engagement of Th2 immune responses to GAD65, before the spontaneous onset of autoimmunity, could inhibit the cascade of Th1 responses that lead to IDDM. We observed that a single intranasal administration of GAD65 peptides to 2-3-wk-old NOD mice induced high levels of IgG1 antibodies to GAD65. GAD65 peptide treated mice displayed greatly reduced IFN gamma responses and increased IL-5 responses to GAD65, confirming the diversion of the spontaneous GAD65 Th1 response toward a Th2 phenotype. Consistent with the induction of an active tolerance mechanism, splenic CD4+ (but not CD8+) T cells from GAD65 peptide-treated mice, inhibited the adoptive transfer of IDDM to NOD-scid/scid mice. This active mechanism not only inhibited the development of proliferative T cell responses to GAD65, it also limited the expansion of autoreactive T cell responses to other beta cell antigens (i.e., determinant spreading). Finally, GAD65 peptide treatment reduced insulitis and long-term IDDM incidence. Collectively, these data suggest that the nasal administration of GAD65 peptides induces a Th2 cell response that inhibits the spontaneous development of autoreactive Th1 responses and the progression of beta cell autoimmunity in NOD mice.

Characterization of the murine mu opioid receptor gene.

The analgesic and addictive properties of morphine and other opioid drugs are thought to result from their interaction with mu opioid receptors. Using a delta opioid receptor cDNA as a probe, we have isolated a murine mu opioid receptor cDNA clone (mMOR). Stable expression of mMOR in Chinese hamster ovary cells conferred high binding affinity for mu receptor ligands including morphine and [D-Ala2,N-methyl-Phe4,Gly5-ol]-enkephalin and low affinity for delta and kappa preferring ligands. Treatment of these cell lines with morphine and other mu agonists inhibited forskolin-induced cAMP accumulation, demonstrating a functional coupling of mMOR to the inhibition of adenylate cyclase. The predicted amino acid sequence of mMOR shares approximately 55% overall amino acid identity with the delta receptor and approximately 97% identity with the recently reported rat mu opioid receptor. Expression of the mu receptor in mouse brain as revealed by in situ hybridization parallels the reported pattern of distribution of mu-selective ligand binding sites. Chromosomal localization (to mouse chromosome 10) and Southern analysis are consistent with a single mu opioid receptor gene in the mouse genome, suggesting that the various pharmacologically distinct forms of the mu receptor arise from alternative splicing, post-translational events, or from a highly divergent gene(s).

Cellular immunity to a determinant common to glutamate decarboxylase and coxsackie virus in insulin-dependent diabetes.

Insulin-dependent diabetes (IDD) results from the autoimmune destruction of the insulin-producing pancreatic beta cells. Autoreactive T-lymphocytes are thought to play a pivotal role in the pathogenesis of IDD; however, the target antigens of these cells, as well as the inductive events in the disease, are unclear. PBMC in persons with or at increased risk for IDD show elevated reactivity to the beta cell enzyme glutamate decarboxylase (GAD). To identify the T-lymphocyte-reactive determinants of GAD, an overlapping set of synthetic peptides was used to stimulate the PBMC from these individuals, PBMC responsiveness to GAD peptides was not restricted to those with IDD, and a number of peptides elicited responses in PBMC. However, the major determinant of GAD recognized by persons at increased risk for IDD was amino acids 247-279, a region which has significant sequence similarity to the P2-C protein of Coxsackie B virus (47% of 15 increased risk [islet cell autoantibody-positive relatives]; 25% of 16 newly diagnosed IDD patients; and 0% of 13 healthy control subjects). Responses to tetanus and insulin antigens were not different between the study groups. In addition, PBMC from individuals responding to GAD peptides within 247-279 also responded to a Coxsackie viral peptide (i.e., P2-C amino acids 32-47), an observation supporting potential molecular mimicry in this immune response. Although the role of environmental agents in the pathogenesis of the disease remains unclear, these cellular immunological findings support the epidemiological evidence suggesting an inductive role for enteroviruses like Coxsackie B in the autoimmunity underlying IDD.

T cell cross-reactivity between coxsackievirus and glutamate decarboxylase is associated with a murine diabetes susceptibility allele.

Limited regions of amino acid sequence similarity frequently occur between microbial antigens and host proteins. It has been widely anticipated that during infection such sequence similarities could induce cross-reactive T cell responses, thereby initiating T cell-mediated autoimmune disease. However, the nature of major histocompatibility complex (MHC)-restricted antigen presentation confers a number of constraints that should make this type of T cell cross-reactivity a rare, MHC allele-dependent event. We tested this prediction using two insulin-dependent diabetes mellitus (IDDM)-associated antigens, coxsackievirus P2-C (Cox P2-C) protein and glutamate decarboxylase (GAD65), which share a prototypic sequence similarity of six consecutive amino acids within otherwise unrelated proteins. We surveyed a panel of 10 murine MHC class II alleles that encompass the spectrum of standard alleles for the ability to cross-reactively present Cox P2-C and GAD65. Out of the 10 restriction elements tested, the sequence similarity regions were both dominant determinants and were cross-reactively displayed after the natural processing of whole antigens, only in the context of I-Anod. These data show that cross-reactive T cell recognition of sequence similarity regions in unrelated proteins is confined to certain MHC alleles, which may explain MHC association with autoimmune disease. It is striking that these two diabetes-associated antigens were cross-reactively recognized only in the context of a diabetes susceptibility allele. Since the human and the murine class II alleles associated with IDDM share conserved features, cross-reactive T cell recognition of GAD65 and Cox P2-C may contribute to the pathogenesis of human IDDM and account for the epidemiological association of coxsackievirus with IDDM.

Intrauterine oxygen tension during the oestrous cycle in the hamster: patterns of change.

Oxygen tension (pO2) within the uterine lumen of the hamster during the oestrous cycle was measured in vivo with a polarographic oxygen sensor. Mean oxygen tension underwent cyclic variation; maximum pO2 occurred during dioestrus (35.2 +/- 1.9 mmHg). Minute-to-minute alterations in pO2 also occurred; the frequency of change in pO2 was maximal during early dioestrus (94.4 +/- 8.9 peaks/hr) and minimal during pro-oestrus (31.2 +/- 5.6 peaks/hr). The amplitude of change in pO2 was maximal during late dioestrus (40.7 +/- 3.6 mmHg) and minimal during pro-oestrus (5.1 +/- 1.2 mmHg). Thus, intrauterine pO2 increases during periods of progesterone dominance and decreases under oestrogenic stimulation. Arterial and venous pO2 were 89.9 +/- 4.8 and 38.9 +/- 1.8 mmHg, respectively. Thus, mean intrauterine pO2 was consistently less than venous blood levels and varied as much as 30 mmHg during the oestrous cycle. The level of pO2 prevailing during the interval of the cycle in which insemination is permitted is intermediate to the maximum and minimum levels occurring during the oestrous cycle and is clearly sufficient to meet the metabolic requirements of spermatozoa during their passage through the uterus.

Comparative localization of two forms of glutamic acid decarboxylase and their mRNAs in rat brain supports the concept of functional differences between the forms.

Two isoforms of glutamic acid decarboxylase (GAD67 and GAD65) and their mRNAs were localized in the rat brain by immunohistochemistry and nonradioactive in situ hybridization methods with digoxigenin-labeled cRNA probes. In most brain regions, both GAD isoforms were present in neuronal cell bodies as well as axon terminals. A few populations of neurons, such as those in the reticular nucleus of the thalamus, exhibited similar cell body labeling for both GADs. However, in many brain regions, the cell bodies that were immunoreactive for GAD67 were often more numerous than those that were immunoreactive for GAD65. In contrast, the density (quantity) of GAD65-immunoreactive axon terminals was higher than that of GAD67-immunoreactive terminals. Strong parallels were observed between the intensity of immunohistochemical labeling of cell bodies and the levels of mRNA labeling for both GAD isoforms. Many groups of GAD-containing cell bodies were distinctly labeled for GAD67, and these same groups of neurons were heavily labeled for GAD67 mRNA. Such neurons included Purkinje cells of the cerebellar cortex, nonpyramidal cells in the cerebral cortex, and neurons of the reticular nucleus of the thalamus. Similar parallels in labeling were observed for GAD65 and its mRNA. Distinct cell body labeling for the protein and associated high levels of GAD65 mRNA were found in neurons of the reticular nucleus of the thalamus and periglomerular cells in the olfactory bulb. However, many cell bodies were not readily labeled for GAD65 with immunohistochemical methods. Such absence or weakness of cell body labeling for the protein was associated with low or moderate levels of GAD65 mRNA. Even though light cell body staining was frequently observed for GAD65 and its mRNA, strong axon terminal labeling for GAD65 was present. Thus, in the deep cerebellar nuclei to which the Purkinje cells of the cerebellar cortex project, strong terminal labeling was observed for both GAD isoforms even though only light cell body labeling of the Purkinje cells was obtained for GAD65 and its mRNA. The findings suggest that the two isoforms of GAD are present in most classes of GABA neurons but that they are not similarly distributed within the neurons. GAD67 is present in readily detectable amounts in many GAD-containing cell bodies whereas GAD65 is particularly prominent in many axon terminals. In addition, neurons that express either form of GAD mRNA also express the corresponding protein. Levels of labeling for the GAD mRNAs suggest that, under normal conditions, the synthesis of GAD65 is frequently lower than that of GAD67.(ABSTRACT TRUNCATED AT 400 WORDS)

Horizontal cells in cat and monkey retina express different isoforms of glutamic acid decarboxylase.

The neurotransmitter used by horizontal cells in mammals has not been identified. GABA has been the leading candidate, but doubt has remained because of failure to clearly demonstrate the GABA synthetic enzyme, glutamic acid decarboxylase (GAD) in these cells. Because GAD was recently shown to exist as two isoforms, 65 kDa and 67 kDa, we considered whether there might be a mismatch between the forms of GAD expressed in horizontal cells and the probes used to detect it. Accordingly, we stained sections of mammalian retina with antibodies specific for each isoform. Cat horizontal cells of both types (A and B) were immunoreactive for GAD67 but negative for GAD65; monkey horizontal cells of both types (H(I) and HII) were positive for GAD65 and negative for GAD67. The findings reconcile previous, apparently conflicting, observations and strengthen considerably the hypothesis that mammalian horizontal cells are GABAergic.