Beltrami-net: domain-independent deep D-bar learning for absolute imaging with electrical impedance tomography (a-EIT).

OBJECTIVE: To develop, and demonstrate the feasibility of, a novel image reconstruction method for absolute electrical impedance tomography (a-EIT) that pairs deep learning techniques with real-time robust D-bar methods and examine the influence of prior information on the reconstruction. APPROACH: A D-bar method is paired with a trained convolutional neural network (CNN) as a post-processing step. Training data is simulated for the network using no knowledge of the boundary shape by using an associated nonphysical Beltrami equation rather than simulating the traditional current and voltage data specific to a given domain. This allows the training data to be boundary shape independent. The method is tested on experimental data from two EIT systems (ACT4 and KIT4) with separate training sets of varying prior information. MAIN RESULTS: Post-processing the D-bar images with a CNN produces significant improvements in image quality measured by structural SIMilarity indices (SSIMs) as well as relative [Formula: see text] and [Formula: see text] image errors. SIGNIFICANCE: This work demonstrates that more general networks can be trained without being specific about boundary shape, a key challenge in EIT image reconstruction. The work is promising for future studies involving databases of anatomical atlases.

Activation of plasmacytoid dendritic cells by apoptotic particles - mechanism for the loss of immunological tolerance in Sjögren's syndrome.

Sjögren's syndrome (SS) is a common autoimmune disease targeting salivary and lacrimal glands. It is strongly female-dominant, characterized by low oestrogen levels combined with a local intracrine dihydrotestosterone defect. We hypothesized that these hormonal deficits lead to increased apoptosis of the epithelial cells and plasmacytoid dendritic cell (pDC)-mediated proinflammatory host responses. Expression of Toll-like receptors (TLRs)-7 and -9 and cytokine profiles was studied in pDCs treated with apoptotic particles collected in consecutive centrifugation steps of media from apoptotic cells. Expression and localization of SS autoantigens in these particles was also analysed. Furthermore, the effects of sex steroids were studied in pDCs cultured with several concentrations of dihydrotestosterone and 17-ß-oestradiol, and in saliva of patient treated with dehydroepiandrosterone. Apoptosis of the epithelial cells led to cleavage and translocation of SS-autoantigens, α-fodrin and SS-A, into apoptotic particles. The apoptosis-induced apoptotic particles also contained another SS-autoantigen, hy1-RNA. These particles were internalized by pDCs in a size-dependent manner and affected TLR-7 and -9 expression and the production of proinflammatory cytokines. The analysed androgens protected cells from apoptosis, influenced redistribution of autoantigens and diminished the apoptotic particle-stimulated increase of the TLRs in pDCs. Our findings suggest that the formation of apoptotic particles may play a role in loss of immune tolerance, manifested by production of autoantibodies and the onset of autoinflammation in SS.

Infections in MS: An innate immunity perspective.

Multiple sclerosis is a multifaceted inflammatory-autoimmune disease, which shows remarkable heterogeneity in its clinical presentation, disease progression and in tissue lesions in the CNS. Focal lesions in white matter consist of immune effector cells, antibodies, and complement deposits in varying combinations, suggesting that immune mechanisms related to CNS pathology are multiple. Although adaptive immunity to myelin antigens is essential in MS pathogenesis, innate immune mechanisms are likely involved in its initiation and perpetuation. One key question is if recognition of infectious agents and microbial products by innate immune mechanisms impacts on MS and if so, how and where? This short review aims at conceptualizing how interactions between microbes and innate immune mechanisms could contribute to MS pathogenesis. Consideration is given to initiation of local inflammation and to myelin-specific immune responses, and how innate immunity and microbes may contribute to these. Recent advances in our understanding of lymphatic drainage of CNS, its immune surveillance and effects of gut microbiota and obesity on systemic endotoxin levels and T-cell priming may open new perspectives to understanding the roles that infectious agents and microbes may have in MS.

The relationship between handling time and cortisol release rates changes as a function of brain parasite densities in California killifish Fundulus parvipinnis.

This study validated a technique for non-invasive hormone measurements in California killifish Fundulus parvipinnis, and looked for associations between cortisol (a stress hormone) and 11-ketotestosterone (KT, an androgen) release rates and the density or intensity of the trematode parasites Euhaplorchis californiensis (EUHA) and Renicola buchanani (RENB) in wild-caught, naturally infected F. parvipinnis. In experiment 1, F. parvipinnis were exposed to an acute stressor by lowering water levels to dorsal-fin height and repeatedly handling the fish over the course of an hour. Neither parasite was found to influence cortisol release rates in response to this acute stressor. In experiment 2, different F. parvipinnis were exposed on four consecutive days to the procedure for collecting water-borne hormone levels and release rates of 11-KT and cortisol were quantified. This design examined whether F. parvipinnis perceived the water-borne collection procedure to be a stressor, while also exploring how parasites influenced hormone release rates under conditions less stressful than those in experiment 1. No association was found between RENB and hormone release rates, or between EUHA and 11-KT release rates. The interaction between EUHA density and handling time, however, was an important predictor of cortisol release rates. The relationship between handling time and cortisol release rates was negative for F. parvipinnis harbouring low or intermediate density infections, and became positive for fish harbouring high densities of EUHA.

Peritoneal cavity is a route for gut-derived microbial signals to promote autoimmunity in non-obese diabetic mice.

Macrophages play a crucial role in innate immune reactions, and peritoneal macrophages (PMs) guard the sterility of this compartment mainly against microbial threat from the gut. Type 1 diabetes (T1D) is an autoimmune disease in which gut microbiota and gut immune system appear to contribute to disease pathogenesis. We have recently reported elevated free radical production and increased permeability of gut epithelium in non-obese diabetic (NOD) mice. Impaired barrier function could lead to bacterial leakage to the peritoneal cavity. To explore the consequences of impaired gut barrier function on extra-intestinal immune regulation, we characterized peritoneal lavage cells from young newly weaned NOD mice. We detected a rapid increase in the number of macrophages 1-2 weeks after weaning in NOD mice compared to C57BL/6 and BALB/c mice. Interestingly, this increase in macrophages was abrogated in NOD mice that were fed an antidiabetogenic diet (ProSobee), which improves gut barrier function. Macrophages in young (5-week-old) NOD mice displayed a poor TNF-α cytokine response to LPS stimulation and high expression of interleukin-1receptor-associated kinase-M (IRAK-M), indicating prior in vivo exposure to TLR-4 ligand(s). Furthermore, injection of LPS intraperitoneally increased T cell CD69 expression in pancreatic lymph node (PaLN), suggestive of T cell activation. Leakage of bacterial components such as endotoxins into the peritoneal cavity may contribute to auto-reactive T cell activation in the PaLN.

Islet-associated T-cell receptor-ß CDR sequence repertoire in prediabetic NOD mice reveals antigen-driven T-cell expansion and shared usage of VßJß TCR chains.

Autoimmune destruction of pancreatic islets in the nonobese diabetic (NOD) mouse is driven by T cells recognizing various autoantigens mostly in insulin-producing beta-cells. To investigate if T-cell accumulation in islets during early insulitis is clonally predetermined, we compared the complementarity determining regions (CDR3) of T-cell receptor (TCR)ß-chains present in islet-infiltrating T cells in young prediabetic NOD mice. High-throughput sequencing of TCRß-chain DNA extracted from islets of 7-wk old NOD mice revealed a biased TCRß-chain repertoire in all mice, as a restricted number of clones (17-36 clones) was highly overrepresented and made over 20% of total islet repertoire in each mouse. Among these clones, various Vß and Jß families were present but certain VßJß combinations such as TRBV19-0-TRBJ2-7 and TRBV13-3-TRBJ2-5 were highly shared between individual mice. On TCRß-chain CDR sequence level, many islet clones (72-146) were shared between at least two individual mice. None of them was among expanded clones in both, suggesting considerable stochasticity in the interactions between TCR and peptide-MHC, even with a limited range of autoantigens. A comparison of islet-CDR3-sequences with CRD-sequences from other tissues revealed clonal overlap with pancreatic lymph node and gut, but these repertoires did not overlap together. Our results suggest that antigen-specific T cells are expanded in pancreatic lymph node and islets, but different specificities expand in individual mice. Some islet-infiltrating T-cell specificities may have a distinct origin shared with gut-infiltrating T cells.

Casein hydrolysate diet controls intestinal T cell activation, free radical production and microbial colonisation in NOD mice.

AIMS/HYPOTHESIS: Dietary and microbial factors and the gut immune system are important in autoimmune diabetes. We evaluated inflammatory activity in the whole gut in prediabetic NOD mice using ex vivo imaging of reactive oxygen and nitrogen species (RONS), and correlated this with the above-mentioned factors. METHODS: NOD mice were fed a normal diet or an anti-diabetogenic casein hydrolysate (CH) diet. RONS activity was detected by chemiluminescence imaging of the whole gut. Proinflammatory and T cell cytokines were studied in the gut and islets, and dietary effects on gut microbiota and short-chain fatty acids were determined. RESULTS: Prediabetic NOD mice displayed high RONS activity in the epithelial cells of the distal small intestine, in conjunction with a proinflammatory cytokine profile. RONS production was effectively reduced by the CH diet, which also controlled (1) the expression of proinflammatory cytokines and colonisation-dependent RegIIIγ (also known as Reg3g) in ileum; (2) intestinal T cell activation; and (3) islet cytokines. The CH diet diminished microbial colonisation, increased the Bacteroidetes:Firmicutes ratio, and reduced lactic acid and butyric acid production in the gut. CONCLUSIONS/INTERPRETATION: Epithelial RONS production and proinflammatory T cell activation appears in the ileum of NOD mice after weaning to normal laboratory chow, but not after weaning to an anti-diabetogenic CH diet. Our data suggest a link between dietary factors, microbial colonisation and mucosal immune activation in NOD mice.

Brief report: first identification of H4 histamine receptor in healthy salivary glands and in focal sialadenitis in Sjögren's syndrome.

OBJECTIVE: The conventional H(1) and H(2) histamine receptors have >10,000-fold lower avidity for histamine than H(4) histamine receptor, which has been implicated in autoimmune diseases. This study was undertaken to compare H(4) histamine receptor levels in the salivary glands (SGs) of healthy controls with those in the SGs of patients with primary Sjögren's syndrome (SS). METHODS: H(4) histamine receptor messenger RNA (mRNA) was analyzed using real-time quantitative polymerase chain reaction, and the receptor protein was examined using immunostaining. Effects of the H(4) histamine receptor agonist ST-1006 on cytokine synthesis by human SG (HSG) cells were analyzed using xMAP technology and enzyme-linked immunosorbent assay. RESULTS: Healthy SGs contained H(4) histamine receptor mRNA. The receptor protein was localized to the acinar and ductal epithelial cells. H(4) histamine receptor agonist stimulated HSG cells to produce the cytokines interleukin-8 and vascular endothelial growth factor. SS patients had low H(4) histamine receptor levels. CONCLUSION: H(1) and H(2) histamine receptor antagonists are not effective in the treatment of autoimmune diseases. However, such antagonists do not affect the newly discovered H(4) histamine receptor. Dendritic cells and lymphocytes are nonprofessional histamine-producing cells, which produce histamine at 100-1,000-fold lower rates than mast cells do. Saliva contains only 0.31-12.4 ng/ml histamine, which is too low to stimulate H(1) or H(2) histamine receptor, but stimulates H(4) histamine receptor half maximally. Our findings show that H(4) histamine receptor is strongly expressed in tubuloacinar SG cells, which emphasizes the role of these cells in the pathogenesis of SS.

Vascular endothelial growth factor C acts as a neurotrophic factor for dopamine neurons in vitro and in vivo.

Neurotrophic factors regulate the development and maintenance of the nervous system and protect and repair dopaminergic neurons in animal models of Parkinson's disease (PD). Vascular endothelial growth factors A (VEGF-A) and B have also neurotrophic effects on various types of neurons, including dopaminergic neurons. We examined the ability of the key lymphangiogenic factor VEGF-C to protect dopaminergic cells in vitro and in vivo. The study was initiated by a finding from microarray profiling of Neuro2A-20 cells which revealed up-regulation of VEGF-C by glial cell-line-derived neurotrophic factor (GDNF). Next, we observed that VEGF-C can rescue embryonic dopaminergic neurons and activate the mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) pathway in vivo. VEGF receptors 1-2 and co-receptors, neuropilins 1-2, were expressed both in mouse embryonic cultures and adult midbrains. In vivo, VEGF-C had a robust functional effect in the rat unilateral 6-hydroxydopamine (6-OHDA) model of PD and there was a small additive effect on the survival of tyrosine hydroxylase (TH)-positive cells with GDNF. The neuroprotective effect of VEGF-C is most likely due to a combination of direct and indirect neurotrophic effects because, VEGF-C, unlike GDNF, induced also angiogenesis in the striatum following 6-OHDA insult as it did in human umbilical vein endothelial cells (HUVEC). However, we detected activation of astroglia and microglia as well as blood-brain barrier disruption after intracerebral delivery of VEGF-C, raising a concern of its safe usage as a therapeutic molecule. Our results provide evidence of VEGF-C as a neurotrophic factor that influences the dopaminergic system through multiple mechanisms.

Effects of a germ-free environment on gut immune regulation and diabetes progression in non-obese diabetic (NOD) mice.

AIMS/HYPOTHESIS: Microbial factors influence the development of diabetes in NOD mice. Studies in germ-free animals have revealed important roles of microbiota in the regulation of Th17 and forkhead box P3 (FOXP3)(+) T regulatory (Treg) activation in the intestine. However, the effects of intestinal microbiota in immune regulation and diabetes development in NOD mice are still poorly understood. METHODS: A colony of germ-free NOD mice was established to evaluate the effects of intestinal microbiota on regulatory immunity in the gut, and on the development of insulitis and diabetes in NOD mice. RESULTS: Diabetes developed in roughly equal numbers in germ-free and specific pathogen-free NOD mice. Insulitis was accentuated in germ-free NOD mice; yet insulin preservation was unaltered. Germ-free NOD mice showed increased levels of Il17 (also known as Il17a) mRNA in the colon, and of Th17 and Th1 cells in the mesenteric and pancreatic lymph nodes, while Foxp3 mRNA and FOXP3(+) Tregs were reduced. In the islet infiltrates, FOXP3(+)CD4(+) T cells were slightly increased in germ-free mice. B cells appeared less activated in the peritoneum and were less abundant in islet infiltrates. CONCLUSIONS/INTERPRETATION: These results indicate that lack of intestinal microbiota promotes an imbalance between Th1, Th17 and Treg differentiation in the intestine. This imbalance is associated with accelerated insulitis, but intact recruitment of FOXP3(+) Tregs into islets, suggesting: (1) a microbial dependence of local induction of Treg in the gut and draining lymph nodes; but (2) a potentially compensatory function of naturally occurring Tregs in the islets, which may help control diabetogenic T cells.

Medium-throughput computer aided micro-island method to assay embryonic dopaminergic neuron cultures in vitro.

In Parkinson's disease (PD) midbrain dopaminergic (DA) neurons degenerate and die, causing loss of motor function. Currently no therapies exist to ameliorate neurodegeneration or to restore DA neurons, although neurotrophic factors (NTFs) are promising leads. Prior in vivo studies the NTFs are routinely assessed in vitro by quantifying the survival of DA neurons from embryonic rodent midbrain cultures. Current in vitro methods are limited in terms of assay reliability, arduous workflow, low throughput, low statistical power and may obscure detection of molecules with minor yet critically important therapeutic effects. We have developed a medium-throughput, micro-island culture method. It permits analysis of 10-12 data points from a single embryo - several fold more than any previously published method - and enables comparisons of DA neurons from a single gene knockout (KO) embryo. It is computer-aided, improves statistical power and decreases the number of animals and workload per experiment. This method enhances testing capabilities of NTFs and other factors, and enables small scale screening of chemical drug libraries. We have validated the method by confirming the known effects of glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN), and demonstrated additive effects via simultaneous addition of GDNF and heparin binding growth associated molecule (HB-GAM). We also show for the first time that DA neurons isolated from GDNF receptor RET-deficient mice are still GDNF responsive, suggesting the presence of an alternative non-RET receptor for GDNF in the DA system. Finally, the method can be adapted for analyses of other low abundance neuronal systems.

Salivary glands - "an unisex organ'?

Usually no distinction is made between female and male salivary glands although cyclic changes of and / or differences in serum and salivary sex steroid concentrations characterize women and men. Moreover, sexual dimorphism is well recognized in salivary glands of rodents.Salivary glands contain estrogen and androgen receptors and are, according to modern high throughput technologies,subjected to gender differences not explainable by gene dose effects by the X chromosome alone. Because sex steroids are lipophilic, it is often thought that approximately 10% of them passively diffuse from plasma to saliva. Indeed, saliva can find use as sample material in sports medicine, pediatrics, veterinary medicine and behavioral sciences. Last but not least, humans and other primates are unique in that they have a reticular zone in their adrenal cortex, which produces dehydroepiandrosterone and androstendione pro-hormones. These are processed in peripheral tissues, not only in female breast and uterus and male prostate, but also in salivary glands by an intracrine enzymatic machinery to active 17b-estradiol,dihydrotestosterone and others, to satisfy and buffer against a constantly changing needs caused by circadian,menstrual, pregnancy and chronobiological hormonal changes in the systemic circulation. Female dominance of Sjögren's syndrome and certain forms of salivary gland cancer probably reflect these gender-based differences.

Enhanced trafficking to the pancreatic lymph nodes and auto-antigen presentation capacity distinguishes peritoneal B lymphocytes in non-obese diabetic mice.

AIMS/HYPOTHESIS: NOD.Igmicro ( null ) mice lacking mature B cells are highly resistant to diabetes and display poor CD4 T cell responses to autoantigens. Nevertheless, the degree to which different B cell subsets contribute to diabetes in NOD mice remains unresolved. Due to their role in the recognition of microbial and autoantigens, peritoneal B cell characteristics were examined in NOD mice to see if they differ developmentally, phenotypically or functionally in aspects relevant to diabetogenesis. METHODS: The population dynamics, activation state, migratory behaviour and antigen presentation function were investigated in NOD peritoneal B cells. RESULTS: NOD peritoneal B cells were found to express abnormally high levels of co-stimulatory molecules (CD40, CD86 and CD69). In contrast, the expression of L-selectin and integrin alpha4beta1 was markedly reduced in NOD mice compared with BALB/c and C57BL/6 mice. The number of B cells in the peritoneum was lower in NOD than in control mice throughout development; migration of B cells from the peritoneum to the pancreatic lymph nodes in NOD mice was enhanced tenfold. NOD B cells showed no chemotactic response to sphingosine-1-phosphate, which normally acts to retain B cells in the peritoneum. Peritoneal B cells of NOD mice also presented insulin autoantigen to CD4 T cells, inducing T cell proliferation. CONCLUSIONS/INTERPRETATION: NOD peritoneal B cells are hyperactivated, migrate to the pancreatic lymph nodes and are capable of driving insulin-specific CD4 T cell activation. These characteristics could make them important for inducing or amplifying T cell responses against islet-antigens.

High sensitivity measurement of CRP and disease progression in multiple sclerosis.

The authors measured serum C-reactive protein (CRP) serially in patients with multiple sclerosis (MS) who participated the PRISMS study using a high-sensitivity technique. CRP values were similar in patients with MS and in healthy controls but higher during MS relapses than in remission (p = 0.010). CRP levels were lower during treatment with high-dose interferon beta 1a than placebo (p = 0.035) and higher during first 12 months of study in patients who progressed by year 4 compared with stable patients (p = 0.007).

25-Hydroxyvitamin D levels in serum at the onset of multiple sclerosis.

Past sun exposure and vitamin D supplementation have been associated with a reduction in the risk of MS. We measured the serum concentration of 25-hydroxyvitamin D (25[OH]D) at the time of MS diagnosis in 40 MS patients and 40 controls. We found no difference in the serum levels of 25(OH)D between MS patients and controls when all samples or samples obtained during winter months were compared, but MS patients had significantly lower serum 25(OH)D concentrations in June to September than the controls. The vitamin D stores were adequate for bone metabolism (> 37 nmol/L) in 70% of MS patients throughout the year and within the hypovitaminosis level (< 37 nmol/L) in 30% of MS patients at some time of the year. During MS-relapses, 25(OH)D levels were lower than in remission, but mostly within the reference range observed in relation with normal bone metabolism. We conclude that the vitamin D stores in most MS patients are adequate for their normal bone metabolism. However, lower vitamin D levels during MS relapses than in remission suggest that vitamin D could be involved in the regulation of the clinical disease activity of MS. The optimal serum levels of vitamin D for the regulation of immune responses remain to be determined.

Hip fractures in Finland--a comparison of patient characteristics and outcomes in six hospitals.

BACKGROUND AND AIMS: To compare six Finnish hospitals for the quality of treatment of hip fractures and to obtain information for the development of care. MATERIAL AND METHODS: Data of 1179 consecutive hip fracture patients (about 200 patients per hospital) was collected prospectively, using similar standardized forms and focusing on background factors and the four-month functional outcome. RESULTS: There were significant differences between the hospitals in patient characteristics (age, place of residence, walking ability, use of walking aids, morbidity and type of fracture) and in the unadjusted outcome variables at four months' follow-up (place of residence, mobility, use of walking aids and pain in injured hip). After adjustment for baseline characteristics, there was a significant difference in the post-fracture walking ability between the centres but no significant differences in post-fracture place of residence. Unadjusted mortality did not vary between the centres, but adjustment resulted in significant differences. The most marked difference in surgical methods between the hospitals was seen in the use of either sliding hip screw or Gamma Nail for trochanteric fractures, but this difference was not reflected in the results of multivariate analysis. CONCLUSIONS: We found minor differences in mobility and mortality between the participating hospitals, and these might serve them as a stimulus for improving their standard of good practice. Continuous quality improvement by repeating the audit cycle is recommended in order to reach and then improve the prevalent standards in the care of hip fracture patients. Confounding factors should be adjusted when comparing the medical centres treating hip fractures, and the evaluation of the results should be multidimensional.

IL-10 is highly expressed in the cryptorchid cryptepididymal epithelium: a probable mechanism preventing immune responses against autoantigenic spermatozoa in the epididymal tubule.

The expression of several immunoregulatory adhesion proteins and cytokines was studied in the normal epididymis, cryptorchid cryptepididymis, the epididymis of oestrogen-treated mice and the epididymis of non-obese diabetic (NOD) mice at the protein level to see which of these immunoregulatory proteins may be involved in lymphocyte regulation in the normal or pathological epididymis and if cytokine balance in this organ is on the cellular or humoral side. The aim of the study was to characterize the immunological microenvironment of the epididymis to explain the survival of the autoantigenic spermatozoa in this site. In the 6-week-old BALB/c or NOD mouse epididymis there were some CD18 and CD44 expressing cells in the interstitial tissue. There were no differences between these strains in the expression of the studied antigens, except that some CD4 positive cells were present in the interstitial tissue of BALB/c mice. In the cryptorchid cryptepididymis CD4, CD8, CD18, CD44, CD54 and CD106 expressing cells were occasionally present in the connective tissue surrounding the epididymal tubule. In the epididymis of the oestrogen-treated mice these antigens were not expressed. In the cryptorchid cryptepididymis the epithelial cells expressed IL-10 highly and the myoid peritubular cells IL-6. The present results suggest that the epididymal epithelial IL-10 suppressing TH0, TH1 and TH2 immune responses may be involved in the protection of autoantigenic spermatozoa from immune destruction.

Surgical treatment of resistant tennis elbow. A prospective, randomised study comparing decompression of the posterior interosseous nerve and lengthening of the tendon of the extensor carpi radialis brevis muscle.

We compared decompression of the posterior interosseous nerve (PIN) and lengthening of the distal tendon of the extensor carpi radialis brevis (ECRB) for treatment of tennis elbow in a randomised trial of 28 patients. Fourteen underwent decompression of PIN and 14, lengthening of ERCB. The groups did not differ significantly with regard to age, sex and work activities. The average duration of preoperative symptoms was 23 months. The PIN was exposed in the groove between the brachioradialis and brachialis muscles and decompressed at the arcade of Frohse by means of a 1-2 cm incision through the supinator muscle. The ECRB tendon was lengthened by Z-plasty at the dorsilateral aspect of the forearm. No postoperative complications occurred. The outcome after the primary operation was successful in 50% of the PIN group and in 43% of the ECRB group. Four of the 5 patients with a poor outcome were reoperated in the former group and 3 in the latter. The overall outcome after a mean follow-up of 31 months after the primary operation was successful in 60% of the cases.

Mucosal antigen primes diabetogenic cytotoxic T-lymphocytes regardless of dose or delivery route.

Administration of antigens via mucosal routes, such as orally or intranasally, can induce specific immunological tolerance and has been used as a rational basis for the treatment of autoimmune diseases, including type 1 diabetes. Recently, however, orally delivered antigens were shown to induce CD8 cytotoxic T-lymphocytes (CTLs) capable of causing autoimmune diabetes. In this report, we have examined several mucosal routes for their ability to induce CTLs and autoimmune diabetes, with the aim of identifying approaches that would maximize tolerance and minimize CTL generation. In normal C57BL/6 mice, ovalbumin (OVA) delivered by either the oral or nasal routes or by aerosol inhalation was able to prime CTL immunity in both high- and low-dose regimens. To address the relevance of these CTLs to autoimmune disease, OVA was given to mice that transgenically expressed this antigen in their pancreatic beta-cells. Irrespective of antigen dose or the route of delivery, mucosal OVA triggered diabetes, particularly after intranasal administration. These findings suggest that CTL immunity is likely to be a consequence of mucosal antigen delivery, regardless of the regimen, and should be considered in the clinical application of mucosal tolerance to autoimmune disease prevention.