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Dysregulation of retinal pigment epithelium (RPE) cells is the main cause of a variety of ocular diseases. Potentially heat shock proteins, by preventing molecular and cellular damage and modulating inflammatory disease, may exert a protective role in eye disease. In particular, the inducible form of heat shock protein 70 (Hsp70) is widely upregulated in inflamed tissues, and in vivo upregulation of Hsp70 expression by HSP co-inducing compounds has been shown to be a potential therapeutic strategy for inflammatory diseases. In order to gain further understanding of the potential protective effects of Hsp70 in RPE cells, we developed a method for isolation and culture of canine RPE cells. Identity of RPE cells was confirmed by detection of its specific marker, RPE65, in qPCR, flow cytometry, and immunocytochemistry analysis. The ability of RPE cells to express Hsp70 upon experimental induction of cell stress, by arsenite, was analyzed by flow cytometry. Finally, in search of a potential Hsp70 co-inducer, we investigated whether the compound leucinostatin could enhance Hsp70 expression in stressed RPE cells. Canine RPE cells were isolated and cultured successfully. Purity of cells that strongly expressed RPE65 was over 90%. Arsenite-induced stress led to a time- and dose-dependent increase in Hsp70 expression in canine RPE cells in vitro. In addition, leucinostatin, which enhanced heat shock factor-1-induced transcription from the heat shock promoter in DNAJB1-luc-O23 reporter cell line, also enhanced Hsp70 expression in arsenite-stressed RPE cells, in a dose-dependent fashion. These findings demonstrate that leucinostatin can boost Hsp70 expression in canine RPE cells, most likely by activating heat shock factor-1, suggesting that leucinostatin might be applied as a new co-inducer for Hsp70 expression.

Background: Elephant endotheliotropic herpesviruses (EEHV) can cause an acute highly fatal hemorrhagic disease in young Asian elephants (Elephas maximus), both ex situ and in situ. Amongst eight EEHV types described so far, type 1 (subtype 1A and 1B) is the predominant disease-associated type. Little is known about routes of infection and pathogenesis of EEHV, and knowledge of disease prevalence, especially in range countries, is limited., Methods: A large cross-sectional serological survey was conducted in captive elephants (n = 994) throughout Thailand using an EEHV-1A glycoprotein B protein antigen specific antibody ELISA., Results: Antibody seroprevalence was 42.3%, with 420 of 994 elephants testing positive. Associations between seropositivity and potential risk factors for EEHV infection were assessed and included: elephant age, sex, camp cluster size, management type (extensive versus intensive), sampling period (wet vs. dry season) and location of camp (region). Univariable regression analysis identified management system and region as risk factors for the presence of EEHV antibodies in elephants, with region being significant in the final multivariable regression model. Prevalence was highest in the North region of the country (49.4%)., Conclusions: This study produced baseline serological data for captive elephants throughout Thailand, and showed a significant EEHV burden likely to be maintained in the captive population.

The skin is immunologically very potent because of the high number of antigen-presenting cells in the dermis and epidermis, and is therefore considered to be very suitable for vaccination. However, the skin's physical barrier, the stratum corneum, prevents foreign substances, including vaccines, from entering the skin. Microneedles, which are needle-like structures with dimensions in the micrometer range, form a relatively new approach to circumvent the stratum corneum, allowing for minimally invasive and pain-free vaccination. In this study, we tested ceramic nanoporous microneedle arrays (npMNAs), representing a novel microneedle-based drug delivery technology, for their ability to deliver the subunit vaccines diphtheria toxoid (DT) and tetanus toxoid (TT) intradermally. First, the piercing ability of the ceramic (alumina) npMNAs, which contained over 100 microneedles per array, a length of 475 µm, and an average pore size of 80 nm, was evaluated in mouse skin. Then, the hydrodynamic diameters of DT and TT and the loading of DT, TT, and imiquimod into, and subsequent release from the npMNAs were assessed in vitro . It was shown that DT and TT were successfully loaded into the tips of the ceramic nanoporous microneedles, and by using near-infrared fluorescently labeled antigens, we found that DT and TT were released following piercing of the antigen-loaded npMNAs into ex vivo murine skin. Finally, the application of DT- and TT-loaded npMNAs onto mouse skin in vivo led to the induction of antigen-specific antibodies, with titers similar to those obtained upon subcutaneous immunization with a similar dose. In conclusion, we show for the first time, the potential of npMNAs for intradermal (ID) immunization with subunit vaccines, which opens possibilities for future ID vaccination designs.

The fundamental problem of autoimmune diseases is the failure of the immune system to downregulate its own potentially dangerous cells, which leads to destruction of tissue expressing the relevant autoantigens. Current immunosuppressive therapies offer relief but fail to restore the basic condition of self-tolerance. They do not induce long-term physiological regulation resulting in medication-free disease remissions. Heat shock proteins (HSPs) have shown to possess the capacity of inducing lasting protective immune responses in models of experimental autoimmune diseases. Especially mycobacterial HSP60 and HSP70 were shown to induce disease inhibitory IL-10-producing regulatory T cells in many different models. This in itself may seem enigmatic, since based on earlier studies, HSPs were also coined sometimes as pro-inflammatory damage-associated molecular patterns. First clinical trials with HSPs in rheumatoid arthritis and type I diabetes have also indicated their potential to restore tolerance in autoimmune diseases. Data obtained from the models have suggested three aspects of HSP as being critical for this tolerance promoting potential: 1. evolutionary conservation, 2. most frequent cytosolic/nuclear MHC class II natural ligand source, and 3. upregulation under (inflammatory) stress. The combination of these three aspects, which are each relatively unique for HSP, may provide an explanation for the enigmatic immune tolerance promoting potential of HSP.

Vaccines often contain adjuvants to strengthen the response to the vaccine antigen. However, their modes of action at the site of injection (SOI) are poorly understood. Therefore, we assessed the local effects of adjuvant on the innate immune system in mice. We investigated the safe, widely used adjuvants MF59 and aluminum hydroxide (alum), as well as trehalose-6,6'-dibehenate (TDB), Complete Freund's Adjuvant (CFA) and the Toll-Like-Receptor-ligands lipopolysaccharide (LPS) and Pam3CysSerLys4 (Pam 3 CSK 4 ). We assessed muscle immune cell infiltration after adjuvant injection and observed 16h post immunization (hpi) an increased influx with CFA, MF59 and TDB, but not with alum, LPS or Pam 3 CSK 4 . An elevated influx with the latter three became visible only 72hpi. Contribution of granulocytes, macrophages and dendritic cells to the influx differed per adjuvant and in time. Adjuvants generally induced a local pro-inflammatory micro-milieu that was transient except for CFA and TDB. The gene expression of CXCL-1, CCL-2 and CCL-5, involved in recruitment of immune cells, varied per adjuvant and corresponded grossly with the observed influx of granulocytes and monocytes/macrophages. Muscles injected with CFA or MF59 (when co-injected with peptide) resulted in APC ex vivo capable to induce proliferation of peptide-specific T-cells. By adding in vitro an excess of peptide to the APC/T cell co-cultures, we observed an adjuvant-enhanced co-stimulation or antigen presentation by APC after CFA- but not MF59-injection. After TDB-injection this effect was observed only at 72hpi, but not 24hpi. Thus the cellular influx profile and the local cytokine and chemokine micro-milieu in the muscle were strongly influenced by the type of adjuvant. Additionally, the capacity of muscle APC to load and present antigen was affected by the adjuvant. These findings may assist the development of novel adjuvanted vaccines in a more rational manner., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Chicken Ig-like receptors (CHIRs) represent a multigene family encoded by the leukocyte receptor complex that encodes a variety of receptors that are subdivided into activating CHIR-A, inhibitory CHIR-B, and bifunctional CHIR-AB. Apart from CHIR-AB, which functions as an Fc receptor, CHIR ligands are unknown. In the current study, we used a panel of different BWZ.36 CHIR reporter cells to identify an interaction between specific CHIRs and avian influenza virus (AIV). The specificity of the CHIR-AIV interaction was further demonstrated using CHIR fusion proteins that bound to AIV-coated plates and were able to reduce the interaction of reporter cells with AIV. There was no difference in binding of CHIR to different AIV strains. Furthermore, CHIR fusion proteins reduced AIV-induced in vitro activation of NK cells obtained from lungs of AIV-infected animals, as judged by the lower frequency of CD107 + cells. Because the original CHIR reporter lines were generated based on sequence information about extracellular CHIR domains, we next identified a full-length CHIR that displayed similar binding to AIV. The sequence analysis identified this CHIR as a CHIR-A. Neuraminidase treatment of coated CHIR-human Ig proteins reduced binding of trimeric H5 proteins to CHIR. This suggests that the interaction is dependent on sialic acid moieties on the receptor. In conclusion, this article identifies AIV as a ligand of CHIR-A and describes the functional consequences of this interaction., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by a chronic relapsing-remitting joint inflammation. Perturbations in the balance between CD4 + T cells producing IL-17 and CD4 + CD25(high)FoxP3 + Tregs correlate with irreversible bone and cartilage destruction in RA. APL1 is an altered peptide ligand derived from a CD4+ T-cell epitope of human HSP60, an autoantigen expressed in the inflamed synovium, which increases the frequency of CD4 + CD25(high)FoxP3+ Tregs in peripheral blood mononuclear cells from RA patients. The aim of this study was to evaluate the suppressive capacity of Tregs induced by APL1 on proliferation of effector CD4+ T cells using co-culture experiments. Enhanced Treg-mediated suppression was observed in APL1-treated cultures compared with cells cultured only with media. Subsequent analyses using autologous cross-over experiments showed that the enhanced Treg suppression in APL1-treated cultures could reflect increased suppressive function of Tregs against APL1-responsive T cells. On the other hand, APL1-treatment had a significant effect reducing IL-17 levels produced by effector CD4+ T cells. Hence, this peptide has the ability to increase the frequency of Tregs and their suppressive properties whereas effector T cells produce less IL-17. Thus, we propose that APL1 therapy could help to ameliorate the pathogenic Th17/Treg balance in RA patients.

Antigen-specific regulatory T cells (Tregs) directed at self-antigens are difficult to study since suitable specific tools to isolate and characterize these cells are lacking. A T cell receptor (TCR)-transgenic mouse would generate possibilities to study such -antigen-specific T cells. As was shown previously, immunization with the mycobacterial heat shock protein (Hsp) 70-derived peptide B29 and its mouse homologs mB29a and mB29b induced anti-inflammatory responses. Furthermore, B29 induced antigen--specific Tregs in vivo. To study mB29b-specific Tregs, we isolated the TCR from T cell hybridomas generated against mB29b and produced a TCR transgenic mouse that expresses a MHC-class II restricted mB29b-specific TCR. These TCR transgenic CD4(+) T cells were found to cross-react with the B29 epitope as identified with peptide-induced proliferation and IL-2 production. Thus, we have successfully generated a novel mouse model with antigen-specific CD4(+) T cells that recognize self and bacterial Hsp 70-derived peptides. With this novel mouse model, it will be possible to study primary antigen-specific T cells with specificity for a regulatory Hsp70 T cell epitope. This will enable the isolation and characterization CD4(+)CD25(+) Tregs with a proven specificity. This will provide useful knowledge of the induction, activation, and mode of action of Hsp70-specific Tregs, for instance, during experimental arthritis.

Bovine Neonatal Pancytopenia (BNP), a fatal bleeding syndrome of neonatal calves, is caused by maternal alloantibodies absorbed from colostrum and is characterized by lymphocytopenia, thrombocytopenia and bone marrow hypoplasia. An inactivated viral vaccine is the likely source of alloantigens inducing BNP-associated alloantibodies in the dam. In this study the specificity of BNP alloantibodies was assessed and was linked to the pathology of BNP. We demonstrated that Major Histocompatibility Complex class I (MHC I) and Very Late Antigen-3, an integrin α3/β1 heterodimer, were the major targets of BNP alloantibodies. However, alloantibody binding to various bovine cell types correlated with MHC I expression, rather than integrin β1 or α3 expression. Likewise, alloantibody-dependent complement-mediated cell lysis correlated strongly with MHC I expression. Examination of several tissues of third trimester bovine foetuses revealed that cells, shown to be affected in calves with BNP, were characterized by high MHC class I expression and high levels of alloantibody binding. We conclude that in spite of the heterogeneous specificity of BNP associated maternal alloantibodies, MHC I-specific antibodies mediate the pathogenicity of BNP in the calf and that cells with high MHC I expression were preferentially affected in BNP.

Background: Feather pecking and cannibalism are major concerns in poultry farming, both in terms of animal welfare and farm economics. Genetic selection and introduction of (aspects of) maternal care have been suggested as potential interventions to reduce feather pecking in laying hens. Altered brain development has been proposed to reflect welfare states in animals, and can provide more insight into the underlying processes involved in feather pecking. Both vasotocin (the avian homologue of vasopressin) and dopaminergic neural circuitry have roles in control of social behaviors as well as in the stress response, and may be linked to feather pecking. Thus, the hypothalamus of adult laying hens selected for low early mortality (LML), which show low feather pecking, was examined and compared with a control line of adult laying hens selected for production characteristics only (CL). The effect of foster hen rearing on the two genetic lines and their hypothalamic morphology was also investigated., Results: We demonstrated an increase in the number of neurons positive for the rate-limiting enzyme in dopamine production, tyrosine hydroxylase, in the periventricular area of the hypothalamus in the LML hens compared to CL hens. Hen-reared chicks showed more vasotocin -positive neurons in the medial pre-optic area compared to the hens raised without a hen. No correlations were found between behavior in an open field at 5-6 weeks of age, and the histology of the same hens at adulthood., Conclusion: The hypothalamic dopaminergic and vasotinergic systems are altered in hens following genetic selection or maternal care, indicating a potential role for these systems in feather pecking.

Intravenous immunoglobulin (IVIg), a therapeutic preparation containing pooled human immunoglobulin (Ig) G, has been suggested to inhibit differentiation and maturation of dendritic cells (DCs); however, controversies exist on this issue. We aimed to reinvestigate the effects of IVIg on human DC maturation and cytokine production, and to determine whether an artifactual determinant is involved in the observed effects. Human monocyte-derived DCs or freshly isolated blood myeloid DCs were cultured in the presence of IVIg in vitro, and the expression of maturation markers CD80, CD86, CD83, and Human Leukocyte Antigen-DR were determined by flow cytometry, whereas production of interleukin (IL)-12 and IL-10 was measured by enzyme-linked immunosorbent assay, and T-cell stimulatory capacity was determined in cocultures with allogeneic CD4(+) T cells. Interestingly, we observed that IVIg did not inhibit, but instead stimulated, spontaneous maturation and T-cell stimulatory ability of human DCs, while leaving lipopolysaccharide-induced DC maturation and cytokine production unaffected. Strikingly, prevention of IVIg binding to culture plate surface, or blocking of the activating Fcγ receptor IIa on DC, abrogated the stimulatory effect of IVIg on costimulatory molecule expression and on T-cell stimulatory capacity of DCs, suggesting that IVIg activates DCs on IgG adsorption to the plastic surface. This study warrants for careful study design when performing cell culture studies with IVIg to prevent artifactual effects, and shows that IVIg does not modulate directly costimulatory molecule expression, cytokine production, or allogeneic T-cell stimulatory capacity of human DCs., (Copyright © 2014 Mosby, Inc. All rights reserved.)

Synthetic oligodeoxynucleotides containing cytosine phosphatidyl guanine-rich DNA sequences (CpG ODN) can promote T-helper type 1 (Th1) responses, reduce T-helper type 2 (Th2) responses and/or favour regulatory T cell (Treg) responses in vitro and in vivo in humans and animals, by acting via Toll-like receptor 9 (TLR9). Since CpG ODN can be used as immune-modulators for canine atopic dermatitis (AD), the aim of the current study was to investigate their immunostimulatory potential on peripheral blood mononuclear cells (PBMC) and their subsets, from AD and healthy dogs. Expression of TLR9 and cytokine mRNA in CpG ODN-stimulated and unstimulated cells was assessed by real-time quantitative PCR. Stimulation of PBMC with CpG class C ODN upregulated mRNA expression of interleukin (IL)-6, interferon (IFN)-γ and IL-12p40 in AD dogs (P<0.05). It also stimulated IFN-γ protein secretion by PBMC of atopic and healthy dogs as measured by ELISA. In healthy dogs only, CpG class C ODN stimulated IFN-α mRNA production by CD21(+) cells, and IL-10, IL-13 and IFN-γ mRNA production by CD3(+) cells. Increased expression of TLR9 mRNA was only observed in CD3(+) cells from AD dogs. No significantly increased gene expression was found in the CD11c(+) subset upon stimulation, for those genes evaluated. The results indicate that PBMC of healthy and atopic dogs are sensitive to stimulation with CpG ODN class C, with a resulting Th1 cytokine response in AD dogs and a mixed Th1/Th2/Treg cytokine response in healthy dogs. From this study, little evidence was found to support the use of CpG ODN class C for therapeutic purposes in dogs affected with AD., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Exposure to environmental toxicants can alter a range of cellular functions involved in the immune response. Increased expression of the stress protein metallothionein 1 (MT1) is one example hereof. Previously, it has been reported that MT1 has several immunosuppressive properties. Furthermore, we earlier showed that functionally tolerogenic dendritic cells (DCs) expressed increased mRNA levels of MT1. Here, we demonstrate that dexamethasone-treated murine DCs are functionally tolerogenic and produce MT1. However, these DCs do not actively transport MT1 to the cell membrane and their regulatory function does not depend on MT1. Alternatively, ZnCl2-treated murine DCs transport MT1 to the cell surface are tolerogenic and promote the expansion of T cells with a regulatory phenotype. Moreover, the membrane-bound MT1 was shown to be essential for ZnCl2-treated DCs to exert their regulatory function. On the basis of this, MT1 can be used as a new marker for functionally tolerogenic DCs. Additionally, we have found a new mechanism for tolerogenic DCs to exert their immune regulatory function.

Kisspeptin (KISS1) and its receptor (KISS1r) are essential for normal reproductive function in many species, but the role of kiss1/kiss1r signalling in the dog has not yet been elucidated. The aims of this study were to identify the canine kiss1 and kiss1r genes and to determine gonadotrophin and oestradiol stimulatory activity of KP-10, the shortest biologically active form of KISS1. Canine kiss1 and kiss1r genes were localized by comparing the reference dog genome with relevant human cDNA sequences, using BLASTn software. The amino acid sequence of canine KP-10 (YNWN V FGLR Y ) differs at two positions from human KP-10 (YNWN S FGLR F ). A single bolus of canine KP-10 was administered intravenously to anoestrous Beagle bitches in dosages of 0, 0.1, 0.2, 0.3, 0.5, 1, 5, 10, and 30 μg/kg. Blood samples were collected before and after canine KP-10 administration for the measurement of plasma luteinizing hormone (LH, all doses), follicle-stimulating hormone (FSH) and oestradiol (1-30 μg/kg). From 0.2 μg/kg onwards, canine KP-10 resulted in a rapid and robust rise in plasma LH concentration (max. at 10 min). KP-10 also resulted in a rapid and robust rise in plasma FSH concentration (max. at 10-20 min). Plasma oestradiol concentration increased significantly after dosages of 1, 5, and 10 μg/kg and reached a maximum at 60-90 min. In conclusion, canine KP-10 is a potent kisspeptin which elicits robust gonadotrophin and oestradiol responses in anoestrous bitches, suggesting that canine kiss1/kiss1r are cogent targets for modulating reproduction in dogs., (© 2014 S. Karger AG, Basel.)

Innate-like murine B-1a cells are well known for their ability to secrete natural IgM. Their non-Ab mediated functions, including Ag presentation to CD4(+) T cells, are less well explored. Using combined adoptive transfer experiments with peptide-pulsed peritoneal cavity (PerC)-derived B-1a cells and CFSE-labeled T cells, we show that B-1a cells present Ag to CD4(+) T cells from the periphery in vivo. In vitro characterization, using co-cultures in which B-1a or splenic B cells presented whole OVA protein to OVA-specific Tg T cells, shows that B-1a cells differentially promote intracellular cytokine-expressing T cells. PerC-derived B-1a cells increase the percentage of IL-10-producing T cells along with IL-4- and IFN-γ-producing CD4(+) T cells. These data suggest that B cells in the PerC have the potential to influence peripheral immune responses without the necessity to migrate out of this location. This, to our knowledge previously undescribed, immuno-logical pathway potentially plays a role in the presentation of gut microbiota-derived Ags to peripheral T cells., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Since the initial discovery of the protective role of heat shock protein (HSP) 60 in arthritis, T cell recognition of endogenous HSP was found to be one of the possible underlying mechanisms. Recently we have uncovered potent disease-suppressive Tregs (anti-inflammatory immunosuppressive T cells) recognizing HSP70 self-antigens, and enabling selective targeting of such Tregs to inflamed tissues. HSP70 is a major contributor to the major histocompatibility complex (MHC) Class II ligandome and we have shown that a conserved HSP70-epitope (B29) is abundantly present in murine MHC Class II. Upon transfer, B29-induced CD4+CD25+Foxp3+T cells suppressed established proteoglycan-induced arthritis (PGIA) in mice. These self-antigen specific Tregs were activated in vivo and as little as 4.000 cells sufficed to fully inhibit arthritis. Furthermore, in vivo depletion of transferred Tregs abrogated disease suppression. Given that B29 can be presented by most human MHC class II molecules and that B29 inhibited arthritis in HLA-DQ8 (human MHC) transgenic mice, we feel that therapeutic vaccination with selected HSP peptides can be an effective route for induction of anti-inflammatory Tregs as a novel intervention in chronic inflammatory diseases., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Pneumoviruses such as pneumonia virus of mice (PVM), bovine respiratory syncytial virus (bRSV) or human (h)RSV are closely related pneumoviruses that cause severe respiratory disease in their respective hosts. It is well-known that T-cell responses are essential in pneumovirus clearance, but pneumovirus-specific T-cell responses also are important mediators of severe immunopathology. In this study we determined whether memory- or pre-existing, transferred virus-specific CD8(+) T-cells provide protection against PVM-induced disease. We show that during infection with a sublethal dose of PVM, both natural killer (NK) cells and CD8(+) T-cells expand relatively late. Induction of CD8(+) T-cell memory against a single CD8(+) T-cell epitope, by dendritic cell (DC)-peptide immunization, leads to partial protection against PVM challenge and prevents Th2 differentiation of PVM-induced CD4 T-cells. In addition, adoptively transferred PVM-specific CD8(+) T-cells, covering the entire PVM-specific CD8(+) T-cell repertoire, provide partial protection from PVM-induced disease. From these data we infer that antigen-specific memory CD8(+) T-cells offer significant protection to PVM-induced disease. Thus, CD8(+) T-cells, despite being a major cause of PVM-associated pathology during primary infection, may offer promising targets of a protective pneumovirus vaccine., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Reestablishing self-tolerance in autoimmunity is thought to depend on self-reactive regulatory T cells (Tregs). Exploiting these antigen-specific regulators is hampered by the obscure nature of disease-relevant autoantigens. We have uncovered potent disease-suppressive Tregs recognizing Heat Shock Protein (Hsp) 70 self-antigens, enabling selective activity in inflamed tissues. Hsp70 is a major contributor to the MHC class II ligandome. Here we show that a conserved Hsp70 epitope (B29) is present in murine MHC class II and that upon transfer, B29-induced CD4(+)CD25(+)Foxp3(+) T cells suppress established proteoglycan-induced arthritis in mice. These self-antigen-specific Tregs were activated in vivo, and when using Lymphocyte Activation Gene-3 as a selection marker, as few as 4,000 cells sufficed. Furthermore, depletion of transferred Tregs abrogated disease suppression. Transferred cells exhibited a stable phenotype and were found in joints and draining lymph nodes up to 2 mo after transfer. Given that (i) B29 administration by itself suppressed disease, (ii) our findings were made with wild-type (T-cell receptor nontransgenic) Tregs, and (iii) the B29 human homolog is presented by HLA class II, we are nearing translation of antigen-specific Treg activation as a promising intervention for chronic inflammatory diseases.

Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic intestinal infection of ruminants and has been associated with the etiology of human Crohn's disease. A MAP Hsp70/DDA subunit vaccine previously showed a significant reduction in fecal shedding of MAP in cattle, concomitant with pronounced antibody production against MAP Hsp70, rather than T cell reactivity. Our hypothesis is that if Hsp70-specific antibodies are able to confer protection, the first requisite would be that the Hsp70 molecule is accessible for antibodies in intact MAP bacteria. In the current study monoclonal antibodies identified MAP Hsp70 B cell epitopes. Two linear epitopes were also recognized by antibodies of vaccinated calves and goats. These epitopes showed to be accessible by antibodies in the bacterial cell wall and in intestinal lesional tissue during natural infection. These results indicate that vaccination-induced antibodies can bind intact bacteria and have the potential to contribute to the protective effect of Hsp70/DDA subunit vaccination against bovine paratuberculosis., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Natural killer (NK) cell activity is conserved throughout vertebrate development, but characterization of non-mammalian NK-cells has been hampered by the absence of specific mAbs for these cells. Monoclonal antibodies were generated against in vitro IL-2 expanded sorted CD3-CD8alpha+ peripheral blood lymphocytes, previously described to contain chicken NK-cells. Screening of embryonic and adult splenocytes with hybridoma supernatants resulted in five candidate NK markers. Activation of chicken NK-cells with PMA/Ionomycin or with the NK target cell-line LSCC-RP9 resulted in increased expression of CD107 (LAMP-1) and a newly developed flow cytometry based cytotoxicity assay showed that NK-cells were able to kill target cells. Combining NK markers with functional assays indicated that marker positive cells showed NK-cell function. In conclusion, we generated new monoclonal antibodies and developed two functional assays which will enhance our understanding of the role of NK-cells in healthy and diseased chickens., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Objective: Stress proteins, such as members of the heat-shock protein (HSP) family, are up-regulated by cells in inflamed tissue and can be viewed functionally as "biomarkers" for the immune system to monitor inflammation. Exogenous administration of stress proteins has induced immunoregulation in various models of inflammation and has also been shown to be effective in clinical trials in humans. This study was undertaken to test the hypothesis that boosting of endogenous HSP expression can restore effective immunoregulation through T cells specific for stress proteins., Methods: Stress protein expression was manipulated in vivo and in vitro with a food component (carvacrol), and immune recognition of stress proteins was studied., Results: Carvacrol, a major compound in the oil of many Origanum species, had a notable capacity to coinduce cellular Hsp70 expression in vitro and, upon intragastric administration, in Peyer's patches of mice in vivo. As a consequence, carvacrol specifically promoted T cell recognition of endogenous Hsp70, as demonstrated in vitro by the activation of an Hsp70-specific T cell hybridoma and in vivo by amplified T cell responses to Hsp70. Carvacrol administration also increased the number of CD4+CD25+FoxP3+ T cells, systemically in the spleen and locally in the joint, and almost completely suppressed proteoglycan-induced experimental arthritis. Furthermore, protection against arthritis could be transferred with T cells isolated from carvacrol-fed mice., Conclusion: These findings illustrate that a food component can boost protective T cell responses to a self stress protein and down-regulate inflammatory disease, i.e., that the immune system can respond to diet.

Interleukin-7 (IL-7) is a central regulator of T cell survival and homeostasis and its expression is indicative for naïve and memory T cells. We cloned chicken IL-7Ralpha (CHIL-7Ralpha) and determined its expression profile in chicken lymphocyte subpopulations. The predicted protein sequence contained 460 amino acids. The extracellular domain exhibited features typical of a type I cytokine receptor; a fibronectin type III domain and the GXWSXWS motif were conserved. ChIL-7Ralpha mRNA is highly expressed in lymphoid organs and in CD4+, CD8alpha+ and CD8beta+ cells. A monoclonal antibody was generated and expression of the protein investigated. ChIL-7Ralpha was expressed on CD4+ and CD8alpha+, but not CD8beta+, T cells, in contrast to the high mRNA expression levels in all of these cells. Upon polyclonal stimulation with ConA, IL-7Ralpha was rapidly down-regulated on T cells, suggesting that in the chicken expression of this receptor might also be correlated to the T cell activation status.

Background: The anti-inflammatory capacity of heat shock proteins (HSP) has been demonstrated in various animal models of inflammatory diseases and in patients. However, the mechanisms underlying this anti-inflammatory capacity are poorly understood. Therefore, the possible protective potential of HSP70 and its mechanisms were studied in proteoglycan (PG) induced arthritis (PGIA), a chronic and relapsing, T cell mediated murine model of arthritis., Methodology/principal Findings: HSP70 immunization, 10 days prior to disease induction with PG, inhibited arthritis both clinically and histologically. In addition, it significantly reduced PG-specific IgG2a but not IgG1 antibody production. Furthermore, IFN-gamma and IL-10 production upon in vitro restimulation with HSP70 was indicative of the induction of an HSP70-specific T cell response in HSP70 immunized mice. Remarkably, HSP70 treatment also modulated the PG-specific T cell response, as shown by the increased production of IL-10 and IFN-gamma upon in vitro PG restimulation. Moreover, it increased IL-10 mRNA expression in CD4+CD25+ cells. HSP70 vaccination did not suppress arthritis in IL-10(-/-) mice, indicating the crucial role of IL-10 in the protective effect., Conclusions/significance: In conclusion, a single mycobacterial HSP70 immunization can suppress inflammation and tissue damage in PGIA and results in an enhanced regulatory response as shown by the antigen-specific IL-10 production. Moreover, HSP70 induced protection is critically IL-10 dependent.

The purpose of this study was to investigate the immunoregulatory potential of Hsp60 in the skin of dogs with atopic dermatitis. Three dogs with chronic atopic dermatitis and four healthy dogs were injected intradermally with Hsp60 and phosphate-buffered saline. Biopsies were taken before testing from non-injected control skin, lesional and non-lesional atopic skin, and 48 and 72 h after injection. Analysis of cytokine messenger RNA was performed using quantitative real-time polymerase chain reaction. Forty-eight hours after Hsp60 injection, a rise in interleukin (IL)-10 was found (P = 0.034) with the highest expression levels in non-lesional atopic and control skin. A rise of transforming growth factor beta (P = 0.015) and IL-12p40 (P = 0.017) was noticed 72 h after Hsp60 injection in control skin. No significant differences were observed for the expression of IL-4, IL-12p35, and interferon gamma. The results indicate that Hsp60 is able to induce cytokines of a regulatory and Th1 phenotype in the skin. Furthermore, this study seems to provide a first indication of deficient Hsp60 response in atopic dermatitis affected skin.

Systemic administration of agents that neutralize or antagonize Th1-mediated pro-inflammatory responses has been demonstrated to ameliorate inflammation in chronic autoimmune disease. However, systemic administration of such immunosuppressive biologicals causes serious side effects and has only limited success. To minimize these side effects, autoantigen-specific lymphocytes have been proposed as a carrier to deliver immunosuppressive agents to sites of inflammation. Here we studied the effects of primary cartilage proteoglycan-specific CD4+ T cells that were transduced using an efficient method of viral transduction with active genes encoding IL-1beta receptor antagonist, soluble TNF-alpha receptor-Ig, IL-4 or IL-10 in chronic proteoglycan-induced arthritis in mice. This is the first study describing such gene therapy using primary CD4+ T cells in a chronic arthritis. Moreover, the impact of proteoglycan-specific Th1, Th2 or naïve T cells was studied. Although proteoglycan-TCR transgenic CD4+ T cells can transfer arthritis to lymphopenic recipients, none of the proteoglycan-TCR transgenic T cell phenotypes that were tested induced worsening of arthritis in wild type hosts. Proteoglycan-specific T cells ameliorated arthritis when expressing the transduced IL-10 gene, and not when expressing the other transgenes/phenotypes. Although all of the tested biologicals can suppress in a wide range of different inflammatory disorders, especially IL-10 would therefore serve as a promising candidate to be used in cellular gene therapy for chronic arthritis.

Deficient T cell regulation can be mechanistically associated with development of chronic autoimmune diseases. Therefore, combining the regulatory properties of IL-10 and the specificity of autoreactive CD4(+) T cells through adoptive cellular gene transfer of IL-10 via autoantigen-specific CD4(+) T cells seems an attractive approach to correct such deficient T cell regulation that avoids the risks of nonspecific immunosuppressive drugs. In this study, we studied how cartilage proteoglycan-specific CD4(+) T cells transduced with an active IL-10 gene (T(IL-10)) may contribute to the amelioration of chronic and progressive proteoglycan-induced arthritis in BALB/c mice. TCR-transgenic proteoglycan-specific T(IL-10) cells ameliorated arthritis, whereas T(IL-10) cells with specificity for OVA had no effect, showing the impact of Ag-specific targeting of inflammation. Furthermore, proteoglycan-specific T(IL-10) cells suppressed autoreactive proinflammatory T and B cells, as T(IL-10) cells caused a reduced expression of IL-2, TNF-alpha, and IL-17 and a diminished proteoglycan-specific IgG2a Ab response. Moreover, proteoglycan-specific T(IL-10) cells promoted IL-10 expression in recipients but did not ameliorate arthritis in IL-10-deficient mice, indicating that T(IL-10) cells suppress inflammation by propagating the endogenous regulatory IL-10 response in treated recipients. This is the first demonstration that such targeted suppression of proinflammatory lymphocyte responses in chronic autoimmunity by IL-10-transduced T cells specific for a natural Ag can occur via the endogenous regulatory IL-10 response.

Dendritic cells are believed to play an essential role in regulating the balance between immunogenic and tolerogenic responses to mucosal Ags by controlling T cell differentiation and activation via costimulatory and coinhibitory signals. The CD28/CTLA-4-CD80/CD86 signaling pathway appears to be one of the most important regulators of T cell responses but its exact role in responses to orally administered proteins remains to be elucidated. In the present study, the involvement of the CD28/CTLA-4-CD80/CD86 costimulatory pathway in the induction of allergic sensitization and oral tolerance to peanut proteins was investigated. In both an established C3H/HeOuJ mouse model of peanut hypersensitivity and an oral tolerance model to peanut, CD28/CTLA-4-CD80/CD86 interactions were blocked using the fusion protein CTLA-4Ig. To examine the relative contribution of CD80- and CD86-mediated costimulation in these models, anti-CD80 and anti-CD86 blocking Abs were used. In the hypersensitivity model, CTLA-4Ig treatment prevented the development of peanut extract-induced cytokine responses, peanut extract-specific IgG1, IgG2a, and IgE production and peanut extract-induced challenge responses. Blocking of CD80 reduced, whereas anti-CD86 treatment completely inhibited, the induction of peanut extract-specific IgE. Normal tolerance induction to peanut extract was found following CTLA-4Ig, anti-CD86, or anti-CD80 plus anti-CD86 treatment, whereas blockade of CD80 impaired the induction of oral tolerance. We show that CD28/CTLA-4-CD80/CD86 signaling is essential for the development of allergic responses to peanut and that CD86 interaction is most important in inducing peanut extract-specific IgE responses. Additionally, our data suggest that CD80 but not CD86 interaction with CTLA-4 is crucial for the induction of low dose tolerance to peanut.

Objective: To better understand the role of antigen (arthritogenic epitope)-specific T cells in the development of autoimmune arthritis., Methods: A transgenic (Tg) mouse expressing the T cell receptor (TCR) Valpha1.1 and V(beta)4 chains specific for a dominant arthritogenic epitope (designated 5/4E8) of human cartilage proteoglycan (HuPG) aggrecan was generated. This TCR-Tg mouse strain was backcrossed into the PG-induced arthritis (PGIA)-susceptible BALB/c strain and tested for arthritis incidence and severity., Results: CD4+ TCR-Tg T cells carried functionally active TCR specific for a dominant arthritogenic epitope of HuPG (5/4E8). T cells of naive TCR-Tg mice were in an activated stage, since the in vitro response to HuPG or to peptide stimulation induced interferon-gamma and interleukin-4 production. TCR-Tg mice uniformly, without exception, developed severe and progressive polyarthritis, even without adjuvant. Inflamed joints showed extensive cartilage degradation and bone erosions, similar to that seen in the arthritic joints of wild-type BALB/c mice with PGIA. Spleen cells from both naive and HuPG-immunized arthritic TCR-Tg mice could adoptively transfer arthritis when injected into syngeneic BALB/c.SCID recipient mice., Conclusion: TCR-Tg BALB/c mice display increased arthritis susceptibility and develop aggravated disease upon in vivo antigen stimulation. This model using TCR-Tg mice is a novel and valuable research tool for studying mechanisms of antigen (arthritogenic epitope)-driven regulation of arthritis and understanding how T cells recognize autoantigen in the joints. This type of mouse could also be used to develop new immunomodulatory strategies in T cell-mediated autoimmune diseases.

Proteoglycan (PG)-induced arthritis (PGIA), a murine model for rheumatoid arthritis (RA), is driven by antigen (PG)-specific T and B cell activation. In order to analyze the pathogenic role of antigen-specific T cells in the development of autoimmune arthritis, we have generated a transgenic (Tg) mouse. The CD4(+) T cells of this TCR-5/4E8-Tg line express a functional T cell receptor (TCR) composed of the Valpha1.1 and Vbeta4 chains with specificity for the dominant arthritogenic T cell epitope of human cartilage PG. Adoptive transfer of naive TCR-5/4E8-Tg cells induced arthritis with severe clinical symptoms in syngeneic immunodeficient BALB/c.RAG2(-/-) mice. In vivo activation of TCR-5/4E8-Tg CD4(+)Vbeta4(+) cells with cartilage PG seemed to be critical for arthritis induction. Arthritis never developed after transfer of naive wild-type cells. The arthritis was characterized as a chronic progressive disease with intermittent spontaneous exacerbations and remissions. Inflamed joints showed extensive cartilage damage and bone erosions leading to massive ankylosis in peripheral joints. These PG epitope-specific TCR-5/4E8-Tg mice can be valuable research tools for studying antigen-driven T cell regulation in arthritis, and migration of T cells to the joints. In addition the model may be used for the development of immune modulating strategies in T cell-mediated autoimmune diseases.

The use of mAbs to abrogate costimulatory interactions has attracted much attention with regard to prevention and modulation of adverse (auto)immune-like reactions. However, the role of costimulatory molecules and possible therapeutic use of Ab-treatment in drug-induced immunostimulation is poorly elucidated. In the present studies, we show that CD28/CTLA-4-CD80/CD86 costimulatory interactions differently regulate drug-induced type 1 and type 2 responses to an identical bystander Ag, TNP-OVA, in BALB/c mice using the reporter Ag popliteal lymph node assay. The antirheumatic drug D-Penicillamine, which may induce lupus-like side-effects, stimulated type 2 responses against TNP-OVA, characterized by the production of IL-4 and TNP-specific IgG1 and IgE. These responses were abrogated in CD80/CD86-deficient mice and in wild-type mice that were treated with anti-CD80 and anti-CD86, or CTLA-4-Ig. Anti-CTLA-4 intensively enhanced the D-Penicillamine-induced effects. In contrast, the type 1 response (IFN-gamma, TNF-alpha, IgG2a) to TNP-OVA induced by the diabetogen streptozotocin still developed in the absence of CD80/CD86 costimulatory signaling. In addition, it was demonstrated that coadministration of anti-CD80 and anti-CD86 mAbs slightly enhanced streptozotocin-induced type 1 responses, whereas the CTLA-4-Ig fusion protein completely abrogated this response. In conclusion, different drugs may stimulate distinct types of immune responses against an identical bystander Ag, which are completely dependent on (type 2) or independent of (type 1) the CD28/CTLA-4-CD80/CD86 pathway. Importantly, the effects of treatment with anti-CD80/CD86 mAbs and CTLA-4-Ig may be considerably different in responses induced by distinct drugs.

Although food allergy has emerged as a major health problem, the mechanisms that are decisive in the development of sensitization to dietary Ag remain largely unknown. CTLA-4 signaling negatively regulates immune activation, and may play a crucial role in preventing induction and/or progression of sensitization to food Ag. To elucidate the role of CTLA-4 signaling in responses to food allergens, a murine model of peanut allergy was used. During oral exposure to peanut protein extract (PPE) together with the mucosal adjuvant cholera toxin (CT), which induces peanut allergy, CTLA-4 ligation was prevented using a CTLA-4 mAb. Additionally, the effect of inhibition of the CTLA-4 pathway on oral exposure to PPE in the absence of CT, which leads to unresponsiveness to peanut Ag, was explored. During sensitization, anti-CTLA-4 treatment considerably enhanced IgE responses to PPE and the peanut allergens, Ara h 1, Ara h 3, and Ara h 6, resulting in elevated mast cell degranulation upon an oral challenge. Remarkably, antagonizing CTLA-4 during exposure to PPE in the absence of CT resulted in significant induction of Th2 cytokines and an elevation in total serum IgE levels, but failed to induce allergen-specific IgE responses and mast cell degranulation upon a PPE challenge. These results indicate that CTLA-4 signaling is not the crucial factor in preventing sensitization to food allergens, but plays a pivotal role in regulating the intensity of a food allergic sensitization response. Furthermore, these data indicate that a profoundly Th2-biased cytokine environment is insufficient to induce allergic responses against dietary Ag.

Heat shock proteins (hsps) are known to be immunodominant antigens of bacteria. Hsps are evolutionarily strongly conserved proteins present in all eukaryotic and prokaryotic cellular organisms and upregulated by several forms of stress. Despite (the paradigm of) self-tolerance, hsp-epitopes homologous to endogenous host hsp sequences have been implicated as T cell epitopes to endow crossreactive, hsp-specific T cells with the capacity to regulate inflammation, such as in experimentally induced autoimmune diseases. Such T cells were found to produce regulatory cytokines like IL10, in contrast to T cells induced with other conserved microbial proteins that are not upregulated by stress. Hsps have been implicated in immune regulation not only as upregulated targets of adaptive immunity during inflammatory stress, but recently also as triggering factors for innate immunity through activation via Toll-like receptors (TLRs).

Objective: To prevent and treat experimental arthritis via nasal administration of an altered peptide ligand (APL) from the major arthritogenic epitope in adjuvant-induced arthritis (AIA) and to explore the mechanisms involved., Methods: Peptides were administered nasally before and after induction of arthritis. Splenocytes and lymph node cells draining both the site of inflammation and the site of tolerance induction were used for cell transfer and were studied for antigen-specific T cell characteristics. In addition, attempts were made to stop T cell tolerance in vitro, using anticytokine antibodies., Results: Nasal administration of a modulatory APL of the heat-shock protein 60 (Hsp60) 180-188 T cell epitope, alanine 183, had a suppressive effect in AIA that far exceeded that of the wild-type epitope. In addition to its effectiveness in preventing AIA, alanine 183 may be effective in the treatment of ongoing AIA. The protective effect of alanine 183 can be passively transferred using activated splenocytes. Nasal administration of alanine 183 did not lead to detectable T cell proliferation or interleukin-2 (IL-2) production in mandibular lymph node cells, while transforming growth factor beta (TGF beta), IL-10, and IL-4 were readily produced. Likewise, after nasally induced tolerance, followed by induction of arthritis, inguinal lymph node cells produced IL-4, TGF beta, and IL-10. After neutralizing in vitro the individual cytokines with anticytokine antibodies, only blocking of IL-10 production led to reversal of tolerance, at the site of tolerance induction and the site of inflammation., Conclusion: Nasal administration of an APL of Hsp60 180-188 induces highly effective protection against AIA through generation of regulatory cells that produce IL-4, TGF beta, and IL-10, whereas the induced tolerance is driven mainly by production of IL-10.

CD154 is transiently expressed by activated T cells and interacts with CD40 on B cells, dendritic cells, macrophages, and monocytes. This costimulatory receptor-ligand couple seems decisive in Ag-driven immune responses but may be differentially involved in type 1 vs type 2 responses. We studied the importance of CD40-CD154 in both responses using the reporter Ag popliteal lymph node assay in which selectively acting drugs generate clearly polarized type 1 (streptozotocin) or type 2 (D-penicillamine, diphenylhydantoin) responses to a constant coinjected Ag in the same mouse strain. Treatment of mice with anti-CD154 reduced characteristic immunological parameters in type 2 responses (B and CD4(+) T cell proliferation, IgG1 and IgE Abs, and IL-4 secretion) and only slightly affected the type 1 response (small decrease in IFN-gamma production, influx of CD11c(+) and F4/80(+) cells, and prevention of architectural disruption of the lymph node, but no effect on IgG2a Ab and TNF-alpha secretion or B and CD4(+) T cell proliferation). The findings indicate that the CD40-CD154 costimulatory interaction is a prerequisite in drug-induced type 2 responses and is only marginally involved in type 1 responses. The observed expression patterns of CD80 and CD86 on different APC (B cells in type 2 and dendritic cells in type 1) may be responsible for this discrepancy.