Exogenous citric acid enhances drought tolerance in tobacco (Nicotiana tabacum).

Organic acids play a pivotal role in improving plant response to long-term drought stress. External application of organic acids has been reported to improve drought resistance in several species. However, whether organic acids have similar effects in tobacco remains unknown. A screening study of the protective function of organic acids in tobacco and understanding the underlying molecular mechanism would be useful in developing a strategy for drought tolerance. Several physiological and molecular adaptations to drought including abscisic acid, stomatal closure, reactive oxygen species homeostasis, amino acid accumulation, and drought-responsive gene expression were observed by exogenous citric acid in tobacco plants. Exogenous application of 50 mm citric acid to tobacco plants resulted in higher chlorophyll content, net photosynthesis, relative water content, abscisic acid content and lower stomatal conductance, transpiration and water loss under drought conditions. Moreover, reactive oxygen species homeostasis was better maintained through increasing activity of antioxidant enzymes and decreasing hydrogen peroxide content after citric acid pretreatment under drought. Amino acids involved in the TCA cycle accumulated after external application of citric acid under drought stress. Furthermore, several drought stress-responsive genes also dramatically changed after application of citric acid. These data support the idea that external application of citric acid enhances drought resistance by affecting physiological and molecular regulation in tobacco. This study provides clear insights into mechanistic details of regulation of amino acid and stress-responsive gene expression by citric acid in tobacco in response to drought, which is promising for minimizing growth inhibition in agricultural fields.

The investigation of genetic and clinical features in Chinese patients with juvenile amyotrophic lateral sclerosis.

Juvenile amyotrophic lateral sclerosis (JALS) occurs at an age of onset below 25 years with a heterogeneous disease onset location, variable progression and survival time. To investigate whether an ALS gene profile could resolve any aspects of clinical symptom heterogeneity, we have used targeted sequencing technology in a cohort of 12 JALS patients of Chinese descent. We detected 5 likely pathogenic mutations, 2 in familial probands and 3 in sporadic patients. One was a known TARDBP mutation (p.G348V) and 4 were FUS frameshift mutations including a known p.Gln519Ilefs*9 mutation and 3 novel mutations, p.Gly515Valfs*14, p.Gly486Profs*30, and p.Arg498Alafs*32. Of the 4 FUS mutations, 2 were able to be confirmed as de novo mutations. The TARDBP mutation carrier showed a classic ALS phenotype. All patients with FUS mutations experienced limb weakness at an early age and developed bulbar symptoms during the disease course. FUS mutations have previously been associated with increased JALS disease progression, however, we found a large range 12 to 84 months in disease survival (mean 58.2 months). Our results justify future screening for variants in FUS as it remains the most frequent genetic determinant of early onset, JALS (found in 30% of our patients).

Clinical efficacy and immunological impact of tacrolimus in Chinese patients with generalized myasthenia gravis.

In this multicenter, open-label pilot study, the efficacy, safety, and immunological impact of tacrolimus in Chinese patients with generalized myasthenia gravis are assessed. Forty-seven generalized myasthenia gravis (MG) patients were enrolled into this study and given 3mg/day tacrolimus for 24 weeks. The primary efficacy measurements used to monitor response to tacrolimus in MG patients were the Osserman grade, the quantitative MG score (QMGS) recommended by the MGFA, the MG-specific manual muscle testing (MMT) score, and the MG-related activities of daily living (MG-ADL) scale. Also, reduction in steroid doses was used to monitor the effect of tacrolimus. Clinical evaluations were conducted at weeks 4, 8, 12, 16, 20, and 24, while immunological parameters were measured at weeks 4, 12, and 24. Measurements of the Osserman grade, QMGS, MMT, and MG-ADL all suggested improvement in patient health by the fourth week of treatment. Steroid dosage was reduced during the course of the study in 74.2% of the forty-three patients who completed the study. There were thirty-one reported adverse events in the study. Only one was considered serious. We found that tacrolimus reduced levels of the IFN-γ, IL-2, IL-10, and IL-13 cytokines and induced the proliferation of tolerogenic plasmacytoid dendritic cells after treatment. Tacrolimus did not change the population of T cell subtypes but did steadily reduce the population of BAFF-R(+) CD19(+) B cells over the course of the study. Our results show that tacrolimus improves the clinical condition of MG patients and is well tolerated. The decrease in IL-13 and reduction of BAFF-R(+) CD19(+) B cells may be related to the therapeutic effect of tacrolimus.

A putative mechanism on remission of multiple sclerosis during pregnancy: estrogen-induced indoleamine 2,3-dioxygenase by dendritic cells.

The basis for the reduced relapse rate of multiple sclerosis (MS) during pregnancy remains unexplained but, if defined, could create novel treatment options. Estrogen constitutes one candidate molecule, but the mechanism by which estrogen may affect MS during pregnancy is unclear. In this study, we used monocyte-derived dendritic cells (DCs) from MS patients to explore the estrogen (17-beta-estradiol)-related pathway of immune modulation. Estrogen induced the expression of indoleamine 2,3-dioxygenase (IDO) on DCs, limiting T-cell proliferation and both Th1 and Th2 cytokine production. The suppression of T-cell proliferation mediated by estrogen-exposed DCs was partly abolished by the IDO-inhibitor, 1-methyl-dl-tryptophan, indicating that estrogen-exposed DCs induced IDO-dependent T-cell suppression. Our data support the hypothesis that the change in the clinical course of MS observed in pregnancy may be related to the estrogen-DC-IDO axis, which could represent a novel target for MS therapy.

G-CSF and neuroprotection: a therapeutic perspective in cerebral ischaemia.

In several experimental studies of cerebral ischaemia, G-CSF (granulocyte colony-stimulating factor) exerted neuroprotective effects through different mechanisms, including mobilization of haemopoietic stem cells, anti-apoptosis, neuronal differentiation, angiogenesis and anti-inflammation. Hence, G-CSF not only inhibits neuron death, but also generates 'new' neural tissue formation. A small pilot trial reports on the safety and feasibility of G-CSF therapy in stroke patients. According to this evidence, we can speculate that G-CSF, being used either alone or in combination with another agent, should have a dual activity beneficial both to acute neuronal protection and long-term plasticity after cerebral ischaemia, thus proposing that G-CSF is an ideal new drug for stroke and neurodegenerative diseases.

Interferon-gamma-modified dendritic cells suppress B cell function and ameliorate the development of experimental autoimmune myasthenia gravis.

This study was designed to investigate the therapeutic effects of interferon (IFN)-gamma-modulated dendritic cells (DC) in experimental autoimmune myasthenia gravis (EAMG). We induced EAMG in Lewis rats by immunization with Torpedo nicotinic acetylcholine receptor (nAChR) and adjuvant. On day 33 post-immunization (p.i.), splenic DC were prepared, exposed to IFN-gamma alone (IFN-gamma-DC) or to IFN-gamma in combination with 1-methyl-DL-tryptophan (1-MT), the specific inhibitor of indoleamine 2,3-dioxygenase (IDO) (IFN-gamma + 1-MT-DC), and injected subcutaneously into rats with incipient EAMG on day 5 p.i. A control group of EAMG rats received naive DC on day 5 p.i., while another group received 1-MT every other day, intraperitoneally (p.i.), from days 5 to 41 p.i. The severity of clinical signs of EAMG was reduced dramatically in IFN-gamma-DC-treated rats compared to rats receiving naive DC, IFN-gamma + 1-MT-DC or 1-MT alone. The number of plasma cells secreting nAChR antibodies was reduced and the expression of B cell activation factor (BAFF) on splenic and lymph node mononuclear cells (MNC) was down-regulated in rats treated with IFN-gamma-DC. In vitro co-culture of MNC derived from EAMG rats with IFN-gamma-DC produced relatively few cells secreting nAChR antibodies. Addition of 1-MT to the co-culture significantly increased the number of cells secreting nAChR antibodies. We conclude that IFN-gamma-DC reduced the number of plasma cells secreting nAChR antibodies in an IDO-dependent manner and ameliorated the development of EAMG in Lewis rats.

Bone marrow-derived dendritic cells from experimental allergic encephalomyelitis induce immune tolerance to EAE in Lewis rats.

We have previously shown that dendritic cells (DC), upon being pulsed in vitro with encephalitogenic myelin basic protein peptide 68-86 (MBP 68-86) and injected subcutaneously (s.c.) back to healthy Lewis rats, transfer immune tolerance to experimental allergic encephalomyelitis (EAE) induced by immunization with MBP 68-86 and Freund's complete adjuvant (FCA). We here assumed that DC become pulsed in EAE rats, and that expansion in vitro of such 'in vivo pulsed EAE-DC' might also have the capacity to induce immune tolerance to EAE, thereby eliminating the need for in vitro pulsing of DC with autoantigens which are still unknown in many autoimmune diseases in the human. In the present study, EAE-DC were generated from bone marrow of Lewis rats, with EAE induced with MBP 68-86 + FCA, and expanded in vitro by culture with GM-CSF and IL-4. In comparison with DC from normal rats, EAE-DC exhibited higher viability in the absence of growth factors, and presented specific antigen to naïve T cells in vitro. The DC derived from both EAE and healthy rats stimulated strong proliferation in an antigen-independent manner, lasting for 4 weeks after DC were s.c. injected into healthy rats. During this time, injection of EAE-DC did not induce clinical EAE. However, when these rats were immunized with MBP 68-86 + FCA, subsequent EAE was dramatically suppressed, and was associated with increased IFN-gamma expression, nitric oxide production, gradually reduced proliferation and cell apoptosis, compared with PBS-injected control EAE rats. LPS-treated DC did not induce tolerance, suggesting that the tolerance is mediated by an immature stage of DC. These observations support the hypothesis that EAE-DC can transfer immune tolerance to EAE, thereby omitting the step of characterizing specific autoantigen. Omitting the step of loading DC with antigen not only eliminates the extremely complex procedure of defining pathogenically-relevant autoantigens, but also avoids the risk of inducing immunogenicity of DC in the treatment of autoimmune diseases.

Vaccination with autologous dendritic cells: from experimental autoimmune encephalomyelitis to multiple sclerosis.

Autoimmune diseases such as multiple sclerosis (MS) are characterized by the loss of tolerance to self-determinants, activation of autoreactive lymphocytes and subsequent damage to single or multiple organs. The mechanisms by which autoimmune responses are triggered, and how activation of autoreactive lymphocytes is initiated and maintained, are not fully understood. Therapeutic approaches in autoimmune diseases have so far concentrated on antigens and T cells. Given the exceptional capacity of dendritic cells (DCs) to induce immunity in vivo, recent reports of the first successful clinical trials based on vaccination of tumor patients with autologous blood DCs pulsed in vitro with tumor antigen come as no surprise. The recent identification of tolerogenic subsets of DCs and their generation in culture may allow a novel approach to induce tolerance in autoimmune diseases. By selective in vitro manipulation of DCs and their subsequent reinfusion, DC-mediated tolerance has been achieved in animal models of human autoimmune diseases, including experimental autoimmune encephalomyelitis in Lewis rats and SJL/J mice and spontaneous diabetes in NOD mice. In vitro observations of human blood DCs are promising for DC-based treatment of MS and other diseases with an autoimmune component. Data from animal models and human materials suggest that DC-based immunotherapy could be beneficial at least as a complement to conventional therapy. Molecular-biological approaches to tolerogenic DCs could provide a rationale for designing immunotherapeutic strategies in autoimmune diseases.

Interferon-beta induces the development of type 2 dendritic cells.

Suppression of interleukin 12 (IL-12) production by dendritic cells (DCs) has been hypothesized to be a principal mechanism underlying the biological action of interferon (IFN)-beta used for treatment of multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system with possible autoimmune origin. How IFN-beta interacts with DCs to inhibit IL-12 production remains unclear. In this study, we found that DCs derived from human blood monocytes, upon culture in the presence of IFN-beta with granulocyte-macrophage colony- stimulating factor (GM-CSF) and IL-4, differentiated into a population expressing CD14- CD1a- HLA-DR+. This population expressed CD123 (IL-3Ralpha). IFN-beta dose-dependently increased IL-3Ralpha+ DCs and decreased CD1a+ DCs. After 7 days' culture with IFN-beta at a concentration of 10 000 U/ml, more than 40% of DCs expressed IL-3Ralpha. IFN-beta, together with GM-CSF and IL-4, also induced maturation of IL-3Ralpha-expressing cells, as reflected by upregulation of HLA-DR and of the costimulatory molecules CD40, CD80 and CD86. In contrast to control DCs, IFN-beta-treated DCs produced predominantly IL-10 but only low levels of IL-12p40. Correspondingly, IFN-beta-treated DCs strongly suppressed IFN-gamma production but enhanced IL-10 production by allogeneic blood mononuclear cells. Our data suggest that IFN-beta in vitro can induce the development of DC2, which provide a permissive environment for Th2 differentiation. This finding represents a novel mechanism for action of IFN-beta in MS.

Rat models as tool to develop new immunotherapies.

Rat models (experimental autoimmune encephalomyelitis, EAE; myasthenia gravis, EAMG) have been developed that mimic certain aspects of the pathophysiology of multiple sclerosis (MS) and myasthenia gravis (MG) in humans. Rat EAE and EAMG are, therefore, widely used to evaluate immunotherapeutic strategies. Mucosal tolerance induced by oral or nasal administration of autoantigen effectively suppresses rat EAE and EAMG. Nasal administration of the cytokines interleukin (IL)-4, IL-10 or transforming growth factor (TGF)-beta1 also ameliorates clinical signs of rat EAE. However, neither mucosal tolerance nor cytokines affect clinical disease if administered during ongoing rat EAE or EAMG. Dendritic cells (DC) pulsed in vitro with autoantigen can mediate peripheral tolerance against rat EAE and EAMG, but do not affect ongoing EAE or EAMG. DC from healthy rats, modified in vitro by exposure to IFN-gamma or TGF-beta1, effectively suppress ongoing EAE and EAMG when given by the subcutaneous route. Most importantly, DC from EAMG rats, after being modified in vitro with IL-10, also inhibit ongoing rat EAMG. These results demonstrate that different strategies influencing T- and/or B-cell responses in rat EAE or EAMG are available, but that DC-based immunotherapies are the most promising, in order to ameliorate ongoing organ-specific autoaggressive immunity Based on these observations, autologous DC, after being modified in vitro with cytokines, constitute a basis for new immunotherapeutic strategies in MS, MG and other diseases with autoimmune background.

Autoantigen-pulsed dendritic cells induce tolerance to experimental allergic encephalomyelitis (EAE) in Lewis rats.

Dendritic cells (DC) can modulate the nature of immune responses in a stimulatory or tolerogenic fashion. Great attention has been given to the induction of immunity to tumour and infection. In this study, bone marrow-derived DC from healthy Lewis rats were pulsed in vitro with encephalitogenic myelin basic protein peptide 68-86 (MBP 68-86), and injected subcutaneously (1 x 106/rat) into normal Lewis rats. Upon observation of the rats pretreated in this way for 4 weeks, when no clinical signs of EAE occurred, these rats were immunized with MBP 68-86 and Freund's complete adjuvant. The pretreated rats failed to develop clinical EAE. This tolerance was associated with augmented proliferative responses, interferon-gamma secretion, inducible nitric oxide synthase (iNOS) expression and NO production. The frequency of apoptotic cells was increased in the rats receiving MBP 68-86-pulsed DC compared with unpulsed control DC. Few infiltrating inflammatory cells were observed in spinal cord sections from rats that had received MBP 68-86-pulsed DC. The data are compatible with the interpretation that MBP 68-86-pulsed DC induce tolerance to EAE possibly through up-regulation of iNOS expression and NO production, which mediate cell apoptosis, thereby reducing infiltration of inflammatory cells within the central nervous system.

Oral administration of cholera toxin B subunit conjugated to myelin basic protein protects against experimental autoimmune encephalomyelitis by inducing transforming growth factor-beta-secreting cells and suppressing chemokine expression.

The efficacy and mechanism of immunosuppression against experimental autoimmune encephalomyelitis (EAE) by oral low-dose administration of myelin basic protein (MBP) conjugated to cholera toxin B subunit (CTB) were investigated in Lewis rats immunized with MBP together with complete Freund's adjuvant 4 days before the start of treatment. Oral treatment with CTB-MBP conjugate gave almost complete protection against disease, an effect that was totally abrogated by including a low dose of cholera holotoxin (CT). The protection by CTB-MBP was associated with a dramatic reduction in the number of leukocytes staining for CD4, CD8, IL-2R or MHC class II in the spinal cord as examined by immunohistochemistry. The mRNA expressions of T(h)1 cytokines IFN-gamma, IL-12 and tumor necrosis factor-alpha, as well as of chemokines monocyte chemotactic protein (MCP)-1 and RANTES in the spinal cord were also reduced by 76-94%, as assessed by in situ hybridization. In contrast, transforming growth factor (TGF)-beta mRNA-expressing cells were strongly increased in the spinal cord from animals treated orally with the CTB-MBP conjugate. In the draining peripheral lymph nodes, the number of MBP-specific TGF-beta mRNA-expressing cells was also increased, whereas there was a decrease in cells expressing T(h)1 or T(h)2 cytokine mRNA. Protection against EAE could be transferred by injection of cells from the mesenteric lymph nodes of animals fed with CTB-MBP into naive animals exposed to encephalitogenic T cells. The results indicate that the protective anti-inflammatory effect by oral treatment with CTB-MBP conjugate is, to a large extent, due to the induction of TGF-beta-secreting suppressive-regulatory T cells and to local down-regulation of MCP-1 and RANTES in the spinal cord.

Suppression of ongoing experimental allergic encephalomyelitis (EAE) in Lewis rats: synergistic effects of myelin basic protein (MBP) peptide 68-86 and IL-4.

Mucosal myelin autoantigen administration effectively prevented EAE, but mostly failed to treat ongoing EAE. Patients with multiple sclerosis (MS), for which EAE is considered an animal model, did not benefit from oral treatment with bovine myelin. We anticipated that autoantigen, administered together with a cytokine that counteracts Th1 cell responses, might ameliorate Th1-driven autoimmune disease, and that nasal administration might considerably reduce the amounts of antigen + cytokine needed for treatment purposes. Lewis rats with EAE actively induced with myelin basic protein peptide (MBP 68-86) and Freund's complete adjuvant (FCA), received from day 7 post-immunization, i.e. after T cell priming had occurred, 120 microg MBP 68-86 + 100 ng IL-4 per rat per day for 5 consecutive days. These rats showed later onset, lower clinical scores, less body weight loss and shorter EAE duration compared with rats receiving MBP 68-86 or IL-4 only, or PBS. EAE amelioration was associated with decreased infiltration of ED1+ macrophages and CD4+ T cells within the central nervous system, and with decreased interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) and enhanced IL-4, IL-10 and transforming growth factor-beta (TGF-beta) responses by lymph node cells. Simultaneous administration of encephalitogenic peptide + IL-4 by the nasal route thus suppressed ongoing EAE and induced IL-4, IL-10 and TGF-beta-related regulatory elements.

Multiple sclerosis is associated with high levels of circulating dendritic cells secreting pro-inflammatory cytokines.

Recent evidence emphasises a pivotal role for dendritic cells (DC) in the control of immunity by priming and tolerising T cells. DC capture and process antigens, express co-stimulatory molecules, migrate to lymphoid organs and secrete cytokines to initiate immune responses. In multiple sclerosis (MS), autoreactive T cells are proposed to play a pathogenic role by secreting pro-inflammatory cytokines, but studies on DC are lacking. To evaluate the involvement of DC in patients with MS, a modified procedure was used to prepare DC from blood of patients with MS and healthy subjects. DC were found to be potent stimulators of T cells in allogeneic and, to a lesser extent, in autologous mixed leukocyte reaction (MLR). Enzyme-linked immunospot (ELISPOT) assays were adopted to determine levels of IFN-gamma, TNF-alpha, IL-6 and IL-10 secreting DC vs. mononuclear cells (MNC). Proportionally more DC than MNC secreted IFN-gamma and IL-10 in both MS and healthy subjects. Patients with MS had higher levels of IFN-gamma, TNF-alpha and IL-6 secreting DC than healthy subjects. The differences for IFN-gamma and TNF-alpha secreting cells were confined to the subgroup of untreated MS patients and not observed in the subgroup examined during ongoing treatment with IFN-beta. Circulating DC secreting pro-inflammatory cytokines may represent another focus for the study of both immuno-pathogenesis and therapeutic interventions in MS.

Mechanisms of recovery from experimental allergic encephalomyelitis induced with myelin basic protein peptide 68-86 in Lewis rats: a role for dendritic cells in inducing apoptosis of CD4+ T cells.

Spontaneous remission of experimental allergic encephalomyelitis (EAE) is usually associated with prominent apoptosis. The mechanisms behind apoptosis are unknown. We examined the functions of dendritic cells (DC) from Lewis rats with EAE induced by immunization with myelin basic protein peptide 68-86 (MBP68 - - 86). Recovery from EAE was associated with three major functional changes of freshly prepared DC: (1) elevated proliferation, (2) increased nitric oxide (NO) production, and (3) augmented IFN-gamma secretion. In Freund's complete adjuvant (FCA)-immunized control rats, no increase of proliferation, NO production or IFN-gamma secretion was observed on day 21 post-immunization (p.i.), i.e., recovery from EAE. In vitro effects of IFN-gamma, TNF-alpha, TGF-beta1, IL-4 and IL-10 on DC were examined. IFN-gamma enhanced proliferation and NO production by DC, while TNF-alpha and IL-4 induced only slight DC proliferation. DC from recovering EAE rats (day 21 p.i.) suppressed MBP68 - - 86-induced T cell proliferation compared to DC obtained at other time points in EAE and FCA-immunized rats. DC-derived NO induced apoptosis of CD4+ T cells, thereby inhibiting autoreactive T cell responses. Besides IFN-gamma stimulation, NO production by DC was mainly induced in an antigen-dependent manner when DC were co-cultured with T cells. The results suggest that spontaneous recovery from EAE is associated with augmented DC functions. Overproduction of NO by DC results in apoptosis of autoreactive CD4+ T cells, thereby decreasing autoreactive T cell reactivities. The existence of such a NO negative feedback loop may contribute to remission of EAE.

Mice with IFN-gamma receptor deficiency are less susceptible to experimental autoimmune myasthenia gravis.

IFN-gamma can either adversely or beneficially affect certain experimental autoimmune diseases. To study the role of IFN-gamma in the autoantibody-mediated experimental autoimmune myasthenia gravis (EAMG), an animal model of myasthenia gravis in humans, IFN-gammaR-deficient (IFN-gammaR-/-) mutant C57BL/6 mice and congenic wild-type mice were immunized with Torpedo acetylcholine receptor (AChR) plus CFA. IFN-gammaR-/- mice exhibited significantly lower incidence and severity of muscle weakness, lower anti-AChR IgG Ab levels, and lower Ab affinity to AChR compared with wild-type mice. Passive transfer of serum from IFN-gammaR-/- mice induced less muscular weakness compared with serum from wild-type mice. In contrast, numbers of lymph node cells secreting IFN-gamma and of those expressing IFN-gamma mRNA were strongly augmented in the IFN-gammaR-/- mice, reflecting a failure of negative feedback circuits. Cytokine studies by in situ hybridization revealed lower levels of lymphoid cells expressing AChR-reactive IL-1beta and TNF-alpha mRNA in AChR + CFA-immunized IFN-gammaR-/- mice compared with wild-type mice. No differences were found for AChR-reactive cells expressing IL-4, IL-10, or TGF-beta mRNA. These results indicate that IFN-gamma promotes systemic humoral responses in EAMG by up-regulating the production and the affinity of anti-AChR autoantibodies, thereby contributing to susceptibility to EAMG in C57BL/6-type mice.

Phenotypic and functional properties of dendritic cells isolated from human peripheral blood in comparison with mononuclear cells and T cells.

Dendritic cells (DCs) are pivotal for antigen presentation, T-cell priming and B-cell functions. Few studies have been carried out on DCs in human diseases, partly because the current procedures used for DC preparation include elaborate negative selection with monoclonal antibodies (MoAb) and prolonged culture in cytokine-enriched milieu, which may influence DC functions. Using physical density and their adherent properties, DCs were prepared from the blood of healthy subjects. Approximately 2% of human blood mononuclear cells (MNC) were shown to consist of DCs, yielding DCs of 80-90% purity. They expressed markers related to DCs (CD1a, CD11c, CD32 and CD83), costimulatory molecules (CD40, CD80, CD86), human leucocyte antigen (HLA) class I and II molecules and inducible nitric oxide (NO) synthase (NOS2), and lacked lymphocyte and monocyte markers (CD3, CD19, CD20, CD56 and CD14). Compared with blood MNC and T cells, DCs showed a high level of spontaneous proliferation and nitric oxide production, as well as strong proliferative responses in mixed leucocyte reactions. Enzyme-linked immunospot (ELISPOT) assays revealed higher levels of interleukin (IL)-4-, IL-10- and interferon-gamma (IFN-gamma)-secreting cells among DCs than among MNC or T cells obtained from the same blood specimens, while levels of tumour necrosis factor-alpha (TNF-alpha)- and IL-6-secreting cells did not differ. The results demonstrate that the method used is fast, effective and competitively priced, and should be useful for studies of DCs in disease states.

Decrease of LFA-1 is associated with upregulation of TGF-beta in CD4(+) T cell clones derived from rats nasally tolerized against experimental autoimmune myasthenia gravis.

Tolerance to experimental autoimmune myasthenia gravis by nasal administration of microgram amounts of acetylcholine receptor (AChR) has been reported. To elucidate the mechanisms behind tolerance induction via the respiratory tract and the involvement of CD4(+) T cells, we established AChR-specific CD4(+)CD8(-) T cell clones from nasally tolerized rats. Nasal tolerance decreased leukocyte function-associated antigen-1 (LFA-1) expression in CD4(+) T cells from tolerized rats. There was no difference between nasally tolerized and control rats in expression of intercellular adhesion molecule-1. The levels of transforming growth factor-beta (TGF-beta) mRNA-expressing cells were upregulated in CD4(+) T cell clones after tolerance induction. These findings suggest that decreased LFA-1 expression in CD4(+) T cells contributes to reduction of the infiltration of inflammatory CD4(+) T cells, while upregulated TGF-beta may inhibit lymphocyte functions.

Regulation of beta-chemokine mRNA expression in adult rat astrocytes by lipopolysaccharide, proinflammatory and immunoregulatory cytokines.

Astrocytes constitute a part of the blood-brain barrier. Chemokine expression by astrocytes may contribute to leucocyte infiltration within the central nervous system (CNS) during inflammation. To investigate factor(s) regulating chemokine expression by astrocytes, we studied the induction of beta-chemokine mRNA expression in adult rat astrocytes. Astrocyte-derived monocyte chemoattractant protein- (MCP-1), RANTES, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta mRNA were induced by interferon-gamma (IFN-gamma). Tumour necrosis factor-alpha (TNF-alpha) induced MCP-1, RANTES and MIP-1beta mRNA expression, and lipopolysaccharide (LPS) induced MCP-1, MIP-1alpha and MIP-1beta mRNA expression in astrocytes. LPS-induced MCP-1, MIP-1alpha and MIP-1beta mRNA expression by astrocytes was antagonized by transforming growth factor (TGF)-beta1 and interleukin (IL)-10. TGF-beta1 and IL-10 also down-regulated MCP-1 and RANTES mRNA expression induced by TNF-alpha. IL-10, but not TGF-beta1, inhibited MIP-1beta mRNA expression induced by TNF-alpha. The results of this in vitro study suggest that beta-chemokine mRNA expression by adult rat astrocytes can be induced by LPS or proinflammatory cytokines, while regulatory cytokines, such as TGF-beta1 and IL-10, down-regulate astrocyte-derived beta-family chemokine mRNA expression induced by LPS, IFN-gamma and TNF-alpha. Further study of CNS chemokines will enhance our understanding of leucocyte recruitment to the CNS and suggest therapeutic strategies for neurological disorders.

Inhibition of experimental autoimmune encephalomyelitis in Lewis rats by nasal administration of encephalitogenic MBP peptides: synergistic effects of MBP 68-86 and 87-99.

Induction of mucosal tolerance by inhalation of soluble peptides with defined T cell epitopes is receiving much attention as a means of specifically down-regulating pathogenic T cell reactivities in autoimmune and allergic disorders. Experimental autoimmune encephalomyelitis (EAE) induced in the Lewis rat by immunization with myelin basic protein (MBP) and Freund's adjuvant (CFA) is mediated by CD4+ T cells specific for the MBP amino acid sequences 68-86 and 87-99. To further define the principles of nasal tolerance induction, we generated three different MBP peptides (MBP 68-86, 87-99 and the non-encephalitogenic peptide 110-128), and evaluated whether their nasal administration on day -11, -10, -9, -8 and -7 prior to immunization with guinea pig MBP (gp-MBP) + CFA confers protection to Lewis rat EAE. Protection was achieved with the encephalitogenic peptides MBP 68-86 and 87-99, MBP 68-86 being more potent, but not with MBP 110-128. Neither MBP 68-86 nor 87-99 at doses used conferred complete protection to gp-MBP-induced EAE. In contrast, nasal administration of a mixture of MBP 68-86 and 87-99 completely blocked gp-MBP-induced EAE even at lower dosage compared to that being used for individual peptides. Rats tolerized with MBP 68-86 + 87-99 nasally showed decreased T cell responses to MBP reflected by lymphocyte proliferation and IFN-gamma ELISPOT assays. Rats tolerized with MBP 68-86 + 87-99 also had abrogated MBP-reactive IFN-gamma and tumor necrosis factor-alpha mRNA expression in lymph node cells compared to rats receiving MBP 110-128 nasally, while similar low levels of MBP-reactive transforming growth factor-beta and IL-4 mRNA expressing cells were observed in the two groups. Nasal administration of MBP 68-86 + 87-99 only slightly inhibited guinea pig spinal cord homogenate-induced EAE, and passive transfer of spleen mononuclear cells from MBP 68-86 + 87-99-tolerized rats did not protect naïve rats from EAE. Finally, we show that nasal administration of MBP 68-86 + 87-99 can reverse ongoing EAE induced with gp-MBP, although higher doses are required compared to the dosage needed for prevention. In conclusion, nasal administration of encephalitogenic MBP peptides can induce antigen-specific T cell tolerance and confer incomplete protection to gp-MBP-induced EAE, and MBP 68-86 and 87-99 have synergistic effects. Non-regulatory mechanisms are proposed to be responsible for tolerance development after nasal peptide administration.