The influence of cyclosporin A on the development of actively induced and passively transferred experimental allergic encephalomyelitis

The immunosuppressive effects of cyclosporin A (CsA) were tested on actively induced and passively transferred experimental allergic encephalomyelitis (EAE). Actively induced EAE could be inhibited if CsA was administered per os at 25 mg/kg/day but not at 10 mg/kg/day. Passive transfer of clinical EAE occurred in all cell recipients including those fed CsA at either 25 or 50 mg/kg/day. Cyclosporin A could inhibit the development of transfer active cells in vitro and in vivo, however, inhibition of transfer active populations by CsA required the presence of CsA during the initial stage of cell response. If CsA was added to Con A-stimulated spleen cell cultures after a delay of 24 hr then these cells transferred clinical disease. Similarly, animals fed CsA concurrently with basic protein sensitization did not develop cell populations capable of transferring EAE. If CsA feeding commenced 2 or 4 days following sensitization all basic proteinsensitized animals still failed to develop EAE; however these latter groups of animals were a suitable source of cells capable of transferring some signs of clinical EAE.     

Prevention of passively transferred experimental autoimmune myasthenia gravis by an in vitro selected RNA aptamer

Myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are mainly caused by autoantibodies directed against acetylcholine receptors (AChR) located in the postsynaptic muscle membrane. Previously, we isolated an RNA aptamer with 2'-fluoropyrimidines using in vitro selection techniques that acted as an effective decoy against both a rat monoclonal antibody called mAb198, which recognizes the main immunogenic region on the AChR, and a significant fraction of patient autoantibodies with MG. To investigate the therapeutic potential of the RNA, we tested the ability of the RNA aptamer to protect the receptors in vivo from mAb198. Clinical symptoms of EAMG in rats engendered by passive transfer of mAb198 were efficiently inhibited by a truncated RNA aptamer that was modified with polyethylene glycol, but not by control scrambled RNA. Moreover, the loss of AChR in the animals induced by the antibody was also significantly blocked with the modified RNA aptamer. These results suggested that RNA aptamers could be applied for antigen-specific treatment for autoimmune diseases including MG.     

Differential susceptibility to actively induced experimental allergic encephalomyelitis and experimental allergic orchitis among BALB/c substrains

Experimental allergic orchitis (EAO) and experimental allergic encephalomyelitis (EAE) are animal models of organ-specific autoimmune disease. In this study, BALB/cByJ and BALB/cAnNCr mice were susceptible to both autoimmune diseases whereas BALB/cJ subline mice were resistant. Disease resistance in BALB/cJ mice did not appear to be a reflection of either (i) a nonspecific generalized impairment of cellular immunity or (ii) an alteration in the phenotypic expression of Bordetella pertussis-induced histamine sensitization, a phenotype which has been shown to be associated with susceptibility to both diseases. Susceptibility to both EAE and EAO was inherited as a dominant trait in F1 hybrid animals. Segregation analysis in a (BALB/cByJ X BALB/cJ) X BALB/cJ backcross population suggested that disease resistance may be associated with a single genotypic difference in a common regulatory gene affecting susceptibility to both diseases. Linkage analysis of the backcross population failed to demonstrate an association of disease resistance with the mutant raf-1b allele carried by BALB/cJ mice. The results of these studies support previous observations that multiple genotypic differences may in fact exist in mice of the BALB/cJ subline and that such differences play a significant role in the genetic control of susceptibility to EAE and EAO.     

Prazosin treatment suppresses increased vascular permeability in both acute and passively transferred experimental autoimmune encephalomyelitis in the Lewis rat

Prazosin, an antagonist of the alpha 1-adrenoceptor, has been found to suppress the clinical and histologic expression of experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. This effect appears to be specific for the alpha 1-receptor. To determine the effect of this drug on vascular permeability to serum proteins and inflammatory cells, leakage of serum proteins into the central nervous system (CNS) was measured with [125I]albumin, and quantitation of cellular inflammation was determined by an estimation of total DNA. The results show that in both actively induced and passively transferred models of the disease, treatment with prazosin significantly suppresses leakage of serum proteins into the CNS but does not significantly suppress the increase of DNA. The results of the [125I]albumin studies additionally support the conclusion that the extent of vascular permeability to serum proteins in the spinal cord is a significant correlate of clinical disease. The results of the DNA estimation were at variance with the histologic evidence of cellular infiltration. We conclude that treatment with prazosin has a significant effect on the development of vascular edema in EAE. These results additionally validate a role for the adrenergic receptor in the development of EAE, and support the hypothesis that the primary site of action of prazosin is on the vascular alpha 1-adrenoceptor.     

Proinflammatory effects of exogenously administered IL-10 in experimental autoimmune orchitis

We studied the effects of exogenously administered recombinant murine interleukin (IL)-10 on the development of experimental autoimmune orchitis (EAO) in C3H/He mice. IL-10 significantly augments histological signs of EAO when administered for 6 consecutive days from days 15 to 20 after primary immunisations with testicular germ cells. These data demonstrate that IL-10, in addition to its well-known antiinflammatory property, also has proinflammatory functions capable of up-regulating testicular immunoinflammatory processes in vivo.     

Immunoluminescent detection of antibodies to testicular antigens in experimental autoimmune orchitis]

Antibodies interacting with spermatid structures in the testis sections and with acrosome and caudal regions of spermatozoa in the smears of sperm were determined in the sera of guinea pigs and mice with experimental autoimmune orchitis by the indirect immunofluorescent method. The antibodies were found in mice from the 14th to the 30th day and in guinea pigs from the 14th to the 60th day after immunization. Circulation time and the level of the fluorescent antibodies were significantly decreased in immunized female guinea pigs and mice. Fluorescent antibodies did not show cytotoxic effect and belonged to the IgG fraction. The fluorescent antibodies of guinea pigs did not react with the mouse testicular antigens in cross reaction. The analogous results were seen in the cross reaction of the mouse antibodies with the guinea pig antigens.     

The cellular basis for the Ia restriction in murine experimental autoimmune thyroiditis

Susceptibility to experimental autoimmune thyroiditis (EAT) in the mouse is linked to the I-A subregion of the major histocompatibility complex. EAT can be induced in susceptible strains of mice by immunization with mouse thyroglobulin (MTg) and adjuvant. We have described a cell transfer system wherein spleen cells from EAT-susceptible CBA/J mice primed in vivo with MTg and lipopolysaccharide (LPS) can be activated in vitro with MTg to transfer EAT to naive syngeneic recipients. This cell transfer system was used to elucidate the cellular basis for the I-A restriction in EAT. While the cell active in transferring EAT was Thy 1+ I-A-, depletion of I-A+ cells from the in vitro culture prevented the activation of EAT effector T cells. MTg-pulsed mitomycin C-treated naive syngeneic spleen cells as antigen-presenting cells (APCs) could replace the I-A+ cells in vitro. Allogeneic (Balb/c) APCs were ineffective. Using APCs from several recombinant inbred strains of mice, it was shown that C3H/HEN and B10.A(4R) APCs were effective in activating MTg/LPS-primed CBA/J spleen cells to transfer EAT while B10.A(5R) APCs were ineffective. This maps the H-2 restriction to the K or I-A subregions. Addition of polyclonal anti-Iak or monoclonal anti-I-Ak or anti-L3T4 during in vitro activation inhibited both the generation of EAT effector cells and the proliferative response to MTg. Irrelevant anti-Ia reagents, monoclonal anti-I-Ek, and monoclonal anti-I-Jk were ineffective. Thus the I-A restriction in murine EAT appears to result from an I-A restricted interaction between Ia+ APCs and Ia- EAT effector T cells.     

Activation requirements of donor T cells and host T cell recruitment in adoptive transfer of murine experimental autoimmune orchitis (EAO)

The relative roles of donor and host T lymphocytes and the T cell activation requirements in adoptive transfer of experimental autoimmune orchitis (EAO) in (C57BL/6 x A/J)F1 mice were investigated in order to gain an understanding of the pathogenesis of this disease. Depletion of T cell subsets in recipients by adult thymectomy and treatment with monoclonal antibodies against CD4 or CD8 had no effect on the incidence of EAO following adoptive transfer of activated T cells from donors immunized with testis homogenate (TH) and adjuvants. In contrast, such depletion of CD4+ T cells inhibited development of EAO in actively immunized mice. Thus, CD4+ cells are required for induction of EAO, but donor CD4+ cells are sufficient by themselves without a comparable contribution from the recipient. Adoptive transfer of EAO required that donor splenic and lymph node T cells be activated in vitro before transfer. We found that exposure to antigen (TH) for as little as 4 hr allowed EAO to occur in 25% of recipients, and by 24 hr the cells were fully competent to induce disease. Proliferation of the cells could not be measured until 2 days later. In serial double-transfer experiments, it was found that the cells must be cultured with TH before each transfer in order for the secondary recipients to develop EAO. However, it was not necessary for the transferred T cells to "see" antigen in vivo in the primary recipients, since transfer to castrated primary recipients had no effect on EAO incidence in secondary recipients. Lymphocytes isolated from diseased testes of immunized donors were competent to transfer EAO without activation in vitro, suggesting that, unlike spleen and lymph node cells, these orchitic lymphocytes were already capable of trafficking to the testis.     

Role of beta 2-adrenoceptors in the biological effect of autoimmune orchitis spleen cells

Autoimmune orchitis induced an increment in beta 2-adrenoceptor populations in intact mouse spleen lymphocytes. Normal and autoimmune lymphocytes incubated with soterenol increased the mechanical response of isolated atria. Autoimmune cells were more effective than normal cells in inducing this response. Soterenol or spleen cells alone did not modify the contractility at the concentration used. Inhibitors of beta 2-adrenoceptors of spleen cells completely blunted the reaction between soterenol and lymphocytes, while when atria were exposed to butoxamine, mechanical activity induced by soterenol plus lymphocytes was not affected. Cell-free supernatants of lymphocytes exposed to soterenol elicited the reaction in the same way as soterenol-treated lymphocytes. Direct contact of cells with the assay organ was not necessary. Inhibitors of cyclooxygenase on lymphocytes blocked the reaction of soterenol-treated lymphocytes on atria, while inhibitors of lipoxygenase(s) completely blocked the reaction of atria exposed to soterenol-stimulated lymphocytes or supernatants. These results suggest that soterenol reacts with beta 2-adrenoceptors of normal and autoimmune cells. From this reaction, soluble factors are released that in turn trigger stimulation of the atrial contractility as a consequence of the release of oxidative products of the lipoxygenase(s) pathway of arachidonic acid from atria. The high activity of atria in the presence of autoimmune spleen cells is probably related to the increment in number of beta 2-adrenoceptors of these cells.     

Experimental autoimmune orchitis: in vitro induction of an autoimmune disease.

Experimental autoimmune orchitis (EAO) can be induced in vitro. Normal lymph node lymphocytes cultured with autologous dissociated testis cells form rosette-like aggregates and later undergo blast transformation and proliferation. These stimulated lymphocytes cause in vivo EAO lesions, when injectd into syngeneic recipients. Moreover, their autoimmune reactivity can be monitored by an in vitro cytostasis assay. Density gradient analysis of the early lymphocyte-testis cultures reveals that the autoimmune reactive lymphocytes are enriched in the rosette populations. It therefore appears that testicular self-antigens are recognized by clonally preformed autologous lymphocytes.     

Comparing seed removal rates in actively and passively restored tropical moist forests

High rates of seed removal can impede forest recovery, but tropical seed removal studies are few and mainly from the neotropics. Little is known about the comparative influences of active restoration (i.e. planting) and passive restoration (i.e. protection of natural regrowth) on seed removal. We conducted an evaluation of seed removal in grasslands, natural forests (tropical moist semideciduous forest), and actively (21‐, 17‐, 16‐, 11‐, 8‐, and 6‐year‐old) and passively (21‐year‐old) restored forests in Kibale National Park, Uganda. We wanted to compare the effect of vegetation type, time since restoration and restoration actions (i.e. active vs. passive) on removal of seeds of five animal‐dispersed tree species during wet and dry seasons. Seeds were either fully exposed or placed in closed mesh cages or under a mesh roof. We used differential removal rates between these treatments to attribute seed removal to different animal taxa. Seed removal rate (percentage of seed removed over a 4‐day period) was highest in passively restored forests, compared with actively restored forests, grasslands, and natural forests. We detected no significant relationship between time since restoration and seed removal rates within actively restored sites. Seed removal rate from roofed treatments was not significantly different from removal from open treatments but was significantly higher than removal from closed treatments, which we interpret as reflecting the greater effect of small mammals versus insects. Smaller seeds tended to be removed at a greater rate than larger seeds. We discuss the implications of these findings for forest regeneration.     

Suppression of generation of concomitant antitumor immunity by passively transferred suppressor T cells from tumor-bearing donors

Summary Infusion of normal recipient mice with suppressor T cells from donors bearing a progressive Meth A fibrosarcoma results in a diminished capacity of the recipients to generate concomitant and postexcision antitumor immunity. The passive transfer of suppressor cells which prevented the generation of immunity to the Meth A fibrosarcoma did not affect the capacity of the recipients to reject an allogeneic tumor. The data provides direct evidence in support of the hypothesis that suppressor T cells, generated at later stages of growth of Meth A fibrosarcoma, function to down-regulate an already acquired mechanism of concomitant immunity.This work was supported by Grants CA-16642 and CA-17794 from the National Cancer Institute, Grant RR-05705 from the Division of Research Resources, NIH, and a grant from R. J. Reynolds Industries, Inc     

Distribution analysis of transferred donor cells in avian blastodermal chimeras.

Blastodermal chimeras were constructed by transferring quail cells to chick blastoderm. Contribution of donor cells to host were histologically analyzed utilizing an in situ cell marker. Of the embryos produced by injection of stage XI-XIII quail cells into stage XI-2 chick blastoderm, more than 50 percent were definite chimeras. The restriction on the spatial arrangement of donor cells was induced by varying the stage of host. Ectodermal chimerism was limited to the head region and no mesodermal chimerism was shown when the quail cells were injected into stage XI-XIII blastoderm. Mesodermal and ectodermal chimerisms were limited to the trunk, not to the head region, when the quail cells were injected into the stage XIV-2 blastoderm. In these chimeras, however, some of the injected quail cells formed ectopic epidermal cysts. Consequently, the stage XIV-2 blastoderm may become intolerant of the injected cells. Our results suggest that it is possible to obtain chimeras that have chimerism limited to a particular germ layer and region by varying the stage of donor cell injection. Injected quail cells contributed to endodermal tissues and primordial germ cells regardless of the injection site. The quail-chick blastodermal chimeras could be useful in the production of a transgenic chicken and in the investigation of immunological tolerance.     

Experimental allergic orchitis in mice. V. Resistance to actively induced disease in BALB/cJ substrain mice is mediated by CD4 + T cells

Previous studies have shown that differential susceptibility to actively induced experimental allergic orchitis (EAO) exists among various BALB/c substrains. Of 13 substrains studied, BALB/cJ mice consistently exhibit greater resistance to disease induction. Such resistance is associated with a single recessive genotypic difference in an immunoregulatory locus which is unlinked to any of the known alleles distinguishing the BALB/cJ substrain. In this study, gene complementation protocols were used to study the genetics of susceptibility and resistance to EAO. The results indicate that resistance in BALB/cJ mice is not due to a mutation in theH-2D ^d linked gene which governs the phenotypic expression of autoimmune orchitis. The mechanistic basis for disease resistance was examined using reciprocal bone marrow radiation chimeras generated between the disease-susceptible BALB/ cByJ (ByJ) substrain and BALB/cJ (Jax) mice. All constructs, including Jax - Jax and Jax - ByJ, developed severe EAO following inoculation with mouse testicular homogenate (MTH) and adjuvants whereas control chimeras immunized with adjuvants alone did not. These results suggest that an active immunoregulatory mechanism rather than a passive one, such as the lack of T cells and/or B cells with receptors for the aspermatogenic autoantigens relevant in the induction of EAO, is responsible for disease resistance in BALB/cJ mice. The role of immunoregulatory cells was examined by pretreating BALB/cJ mice with either cyclophosphamide (20 mg/kg) or low-dose whole body or total lymphoid irradiation (350 rads) 2 days prior to inoculation. BALB/cJ mice immunized with MTH plus adjuvants generate immunoregulatory spleen cells (SpCs) that, when transferred to naive BALB/cByJ recipients, significantly reduce the severity of autoimmune orchitis observed during actively induced EAO. Treatment of such cells with either cytotoxic monoclonal anti-Thy-1.2 or anti-CD4 plus C' before transfer abrogates the ability of BALB/cJ spleen cells to inhibit disease. In contrast, neither SpCs from adjuvantimmunized BALB/cJ nor MTH plus adjuvant-primed BALB/cByJ donors significantly influenced the severity of disease observed in recipients. Taken together, these results suggest that genetically controlled resistance to EAO in BALB/cJ mice is associated with a mutation in an immunoregulatory locus whose effects appear to be mediated through a cyclophosphamide and low-dose radiation-sensitive CD4⁺ T-cell population.     

Unbalanced thermoregulation in experimental autoimmune encephalitis induced in Lewis rats

Thermoregulation in patients suffering from multiple sclerosis (MS) is impaired and may result in either increases or decreases in body temperature. We have found that rat experimental autoimmune encephalitis (EAE), being a model of MS, is associated with body temperature disturbances as well.The purpose of the current study was to examine whether the altered body temperature in EAE-induced rats is due to either a deficit in thermoregulation or a controlled change in its set point.Subcutaneous injection of encephalitogenic emulsion into both pads of hind feet of the Lewis rats provoked EAE symptoms. Body temperature (Tb) of 6 rats was measured using biotelemetry system, and ambient temperature (Ta) preferred by 6 rats of another group was analyzed using thermal gradient system.Symptoms of EAE started 11 days postinjection and progressed quickly, culminating in a complete paralysis in rats placed in the gradient, which was associated with behavioural fever (accordingly, selected Ta raised to as much as 32.8 ± 0.5 °C vs 27.2 ± 0.6 °C in control rats). On the other hand, EAE rats, placed at a constant Ta of 24 °C, were able to generate fever (Tb of 37.8 ± 0.1 °C) at the start of the illness and then paralysis compromised fever (most likely due to an impairment of thermogenesis), which, surprisingly, resulted in recovery.We conclude that EAE onset in rats is associated with fever and its behavioural supporting leads to aggravation of the autoimmune neurotoxicity.