Irradiation potentiation of cellular transfer of EAE: time course and locus of effect in irradiated recipient Lewis rats.

Further studies of the potentiating effect of 500 rads total body irradiation on cellular transfer of experimental allergic encephalomyeliatis (EAE) in Lewis rats have revealed two findings bearing on underlying mechanisms. First, the effect is transitory, potentiation of disease being observed in recipients irradiated 1 or 4 days before transfer of syngeneic sensitized donor lymphoid cells but not among animals irradiated 7 or 14 days before cell transfer. Second, lead shielding selectively excluding the central neuraxis from irradiation results in relatively little augmentation of EAE compared to that observed in non-shielded irradiated animals. We believe irradiation potentiation of EAE results from transitory alterations in central nervous system target tissue rendering it more vulnerable to host immunologic attack.     

Adoptive transfer of experimental allergic encephalomyelitis in mice with the aid of pertussigen from Bordetella pertussis

Adoptive transfer of experimental allergic encephalomyelitis (EAE) in (SJL X BALB/c)F1 mice was accomplished by an iv injection of 2.4 to 4.7 X 10(7) lymph node cells (LNC) from mice immunized with mouse spinal cord emulsified in complete Freund's adjuvant when both donors and recipients had been treated iv with 400 ng of pertussigen at the time of immunization for the donors and on transfer of cells for the recipients. Pertussigen was essential in both donors and recipients for development of frank EAE. Signs of EAE in recipients were delayed, appearing 21-23 days after cell transfer; the maximum response at about Day 27 is considerably delayed in comparison with other reported studies on passive transfer of EAE. Histologically, recipient mice with paralysis due to EAE had typical perivascular infiltrates of mononuclear cells in the brain and spinal cord. The mechanisms by which pertussigen promotes the development of EAE after adoptive transfer of sensitized LNC are uncertain.     

Regulation of experimental allergic encephalomyelitis. II. Appearance of suppressor cells during the remission phase of the disease

Lewis rats are susceptible to experimental autoimmune encephalomyelitis (EAE). Most rats recover from paralysis and are subsequently resistant to the disease. In an adoptive transfer system, we found that lymph node cells (LNC) from rats that had recovered from EAE protect syngeneic recipients from the disease when the latter are challenged with encephalitogenic myelin basic protein and adjuvant after receiving donor cells. Suppression is antigen-specific and requires viable LNC. In contrast to the suppressor cells we previously studied in tolerized rats, which were nonadherent T lymphocytes, the suppressor cells found in rats that have recovered from EAE adhere to glass wool. However, they are not retained on Sephadex G-10 columns to which macrophages adhere. Suppressor activity is enriched in the nylon wool-adherent LNC population (which consists of approximately 80% Ig+ cells). Our findings suggest that activation of adherent suppressor cells may be implicated in recovery from EAE. These may be adherent T cells, or B cells that produce anti-BP blocking antibodies.     

Recipient contributions to serial passive transfer of experimental allergic encephalomyelitis

EAE can be adoptively transferred into normal recipients by the transfer of BP-specific EAE effector cells. After cell transfer, a series of ill-defined events occur in the recipient that culminate in the development of paralysis associated with neural tissue damage. We investigated the subsequent recipient response to the adoptively transferred disease and examined the role that recipient lymphocytes play in the development of adoptively transferred EAE. Recipient involvement was assessed by the transfer of EAE through a series of normal F1 animals as recipients and at the endpoint of the experiment, determining the MHC restriction pattern of the BP-sensitive cells present. The serial transfer of EAE from BP-CFA-sensitized LEW----(LEW X F-344)F1----(LEW X P2)F1, and from BP-CFA sensitized LEW----(LEW X F344)F1----(LEW X F-344)F1, resulted in the development of BP-sensitive cells in the spleens of the secondary recipients that were able to transfer disease into normal LEW recipients. To test directly for the development of host-derived BP-sensitive cells that might arise in the F1 animal, the serial transfer of EAE from LEW----(LEW X ACI)F1----(LEW X ACI)F1 was performed. When BP-sensitive cells obtained from the secondary (LEW X ACI)F1 recipient animal were transferred into either normal LEW and ACI, or irradiated LEW and ACI animals as final recipients, transfer of disease was successful only into the LEW parental. These results suggest that the development of passive EAE is due solely to the transferred BP-sensitive cells originating from the actively immunized donor, and that no host-derived lymphocytes are recruited into the pool of EAE effector precursor cells found in the spleen of animals after the development of adoptively transferred EAE.     

Alterative and repair processes in lymph nodes under the direct and mediated effects of radiation]

Under study were the popliteal lymph nodes of rats at different times after total irradiation of animals (the 1st series), total irradiation with screening the left node (the 2nd series) and local screening of other parts of the body (the 3rd series). X-ray irradiation in all experiments was performed under standard conditions in dosage of 800 r. The amount of mitoses (MC 0/00) in light centers and cortical substance was counted in addition to histological alterations. In shielded lymph nodes (2nd series) mediate effects of irradiation were observed characterized by a decrease of the MK amount and massive death of lymphocytes in later terms than after direct effects (1st series). In irradiated nodes (3rd series) the reparative process was more rapid than in the first series due to migration of lymphocytes from non-irradiated parts of the body. The mediate effect of radiation results also in increased amount of plasma cells in lymphatic nodes of animals subjected to total irradiation (1st and 2nd series). It is suggested by the absence of such increase of amount of plasma cells in locally irradiated lymphatic nodes when screening other parts of the body (3rd series). availability of individual distinctions in the character of the lymphoid tissue response to effects of ionizing radiation puts a question of division of experimental animals at least into 2 subgroups which have different indices of proliferative processes.     

Interleukin 1 and myelin basic protein synergistically augment adoptive transfer activity of lymphocytes mediating experimental autoimmune encephalomyelitis in Lewis rats

This investigation focused on the role of adherent accessory cells and their cellular product, interleukin 1 (IL 1), in cellular immune responses associated with experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Guinea pig myelin basic protein (GPMBP)-sensitized lymph node cells (LNC) responded in culture with GPMBP by undergoing activation as measured by augmented transfer of EAE to syngeneic recipients, and proliferation as measured by [3H]thymidine incorporation. GPMBP-sensitized LNC, after depletion of adherent accessory cells, no longer responded to GPMBP in the EAE transfer activation assay. In contrast, aliquots of the same LNC preparation exhibited proliferative responses to GPMBP that were only partially reduced. Addition of irradiated thymocytes to adherent cell-depleted cultures fully reconstituted responsiveness to GPMBP in the activation assay and restored full reactivity to GPMBP in the proliferation assay. Furthermore, addition of either purified human IL 1 or recombinant human IL 1 to adherent cell-depleted cultures reconstituted reactivity to GPMBP in the EAE transfer activation assay and augmented GPMBP-specific proliferative responses. Anti-Ia monoclonal antibodies blocked GPMBP + IL 1-induced cellular activation of nonadherent LNC. These results demonstrate that both IL 1 and Ia molecules are important in the pathway leading to GPMBP-induced activation of EAE-inducing T lymphocytes. Furthermore, these results suggest that different accessory signals may be required for optimal induction of GPMBP-induced lymphocyte activation vs GPMBP-specific proliferative responses.     

Neurotoxin Quinolinic Acid Is Selectively Elevated in Spinal Cords of Rats with Experimental Allergic Encephalomyelitis

Abstract: Experimental allergic encephalomyelitis (EAE) is an autoimmune, animal model of multiple sclerosis (MS) in which demyelination and paralysis are evident. Quinolinic acid (QUIN) is a neurotoxin and endogenous N -methyl- d -aspartate receptor agonist formed from tryptophan. The role of neurotoxins in general and QUIN in particular in EAE or MS is unknown. Lewis rats inoculated with myelin basic protein developed signs of EAE by day 12, were killed, and their tissues assayed for QUIN by gas chromatography with mass spectrometry. QUIN levels were significantly elevated in the more caudal regions of the spinal cords of animals with EAE. Brain, serum, and liver levels of QUIN were not altered. In a similar manner, QUIN in mylin basic protein-injected, asymptomatic animals was not different from control animals. The time course for QUIN was similar to the neurological signs of the disorder; however, the initial elevation in QUIN occurred before the appearance of behavioral signs. Last, treatment with the glucocorticoid dexamethasone prevented both the signs of EAE and the elevation in spinal cord QUIN. It is not known whether QUIN contributes to the paralysis in EAE. However, if QUIN is pathogenic in EAE, this finding could have therapeutic implications for MS.     

Structural and functional changes in the intestine of irradiated and hypothermic irradiated rats: A scanning and transmission electron microscopic study

Severe destructive changes in the intestine of rats following whole body exposure to gamma rays (832 rads) were observed by light microscope, scanning and transmission electron microscope studies. Hypothermia (15‡C rectal temperature) induced prior to irradiation protected the intestinal mucosa from destruction. A simultaneous study showed that glucose absorption decreased significantly in irradiated rats, whereas it was increased in hypothermic irradiated animals.     

Induction and clinical scoring of chronic-relapsing experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that commonly affects young adults. It is characterized by demyelination and glial scaring in areas disseminated in the brain and spinal cord. These lesions alter nerve conduction and induce the disabling neurological deficits that vary with the location of the demyelinated plaques in the CNS (e.g. paraparesis, paralysis, blindness, incontinence). Experimental autoimmune encephalomyelitis (EAE) is a model for MS. EAE was first induced accidentally in humans during vaccination against rabies, using viruses grown on rabbit spinal cords. Residues of spinal injected with the inactivated virus induced the CNS disease. Following these observations, a first model of EAE was described in non-human primates immunized with a CNS homogenate by Rivers and Schwenther in 1935. EAE has since been generated in a variety of species and can follow different courses depending on the species/strain and immunizing antigen used. For example, immunizing Lewis rats with myelin basic protein in emulsion with adjuvant induces an acute model of EAE, while the same antigen induces a chronic disease in guinea pigs. The EAE model described here is induced by immunizing DA rats against DA rat spinal cord in emulsion in complete Freund's adjuvant. Rats develop an ascending flaccid paralysis within 7-14 days post-immunization. Clinical signs follow a relapsing-remitting course over several weeks. Pathology shows large immune infiltrates in the CNS and demyelination plaques. Special considerations for taking care for animals with EAE are described at the end of the video.     

Suppressor cell control of unresponsiveness to experimental allergic encephalomyelitis.

Lymph node cells (LNC) from Lewis rats rendered unresponsive to experimental allergic encephalomyelitis (EAE) by pretreatment with myelin basic protein markedly suppressed clinical (but not histologic) EAE in normal recipients later challenged with an encephalitogenic emulsion. Unresponsiveness was immunologically specific, and required viable LNC; serum transfer was ineffective. These findings suggest that suppressor cells exert control over this autoimmune disease.     

Passive induction of experimental allergic encephalomyelitis

Experimental allergic encephalomyelitis (EAE) is a widely used animal model of the human demyelinating disease multiple sclerosis. EAE is initiated by immunization with myelin antigens in adjuvant or by adoptive transfer of myelin-specific T cells, resulting in inflammatory infiltrates and demyelination in the central nervous system. Induction of EAE in rodents typically results in ascending flaccid paralysis with inflammation primarily targeting the spinal cord. This protocol describes passive induction of EAE by adoptive transfer of T cells isolated from mice primed with myelin antigens into na?ve mice. The advantages of using this method versus active induction of EAE are discussed.     

Immunosuppression of experimental allergic encephalomyelitis. III. In vitro evidence for induction of suppressor T lymphocytes in draining lymph node cells of animals immunized with myelin basic protein complexed to lipopolysaccharides

We recently demonstrated that Lewis rats immunized with bacterial lipopolysaccharides (LPS) precomplexed to guinea pig myelin basic protein (BP) in complete Freund's adjuvant were less effective in inducing experimental allergic encephalomyelitis (EAE) than BP-immunized controls. When tested in vitro both lymph node cells (LNC) and spleen cells (SpC) of animals immunized with BP-LPS were less effective in proliferative responses to various mitogens, which included phytohemagglutinin, concanavalin A, purified protein derivative of tuberculin, LPS, and BP. Of importance immunization of rats with BP complexed to LPS results in the generation of cells in lymph nodes of these animals that suppress the mitogenic response of BP-immunized LNC and also SpC in mixed lymphocyte cultures. The suppressive effect of these cells in mixed lymphocyte culture reaction was found specifically in response to BP and to a lesser extent to LPS in LNC. SpC of BP-LPS immunized animals did not suppress the proliferative response to SpC of BP-immunized animals. Treatment of these LNC with antithymocyte serum and complement abolished this suppressive effect of LNC, suggesting that the immunoregulatory cells in LNC of BP-LPS immunized animals are suppressor T lymphocytes. The parallel between the in vitro induction of suppressor T lymphocytes in the draining LNC and the function of LPS in the development of EAE in Lewis rats suggests a possible immunologic significance of the effect.     

Lymphocytes from SJL/J mice immunized with spinal cord respond selectively to a peptide of proteolipid protein and transfer relapsing demyelinating experimental autoimmune encephalomyelitis.

Relapsing experimental autoimmune encephalomyelitis (R-EAE) can be induced in SJL/J mice by immunization with spinal cord homogenate and adjuvant. The specific Ag(s) responsible for acute disease and subsequent relapses in this model is unknown. Myelin basic protein (BP), an encephalitogenic peptide of BP (BP 87-99), and proteolipid protein (PLP) can each induce R-EAE in SJL/J mice, and a peptide of PLP (PLP 139-151) has been reported to induce acute EAE. To determine the encephalitogens in cord-immunized mice with R-EAE, the in vitro proliferative responses of lymph node cells (LNC) and central nervous system mononuclear cells to BP, BP peptides, and PLP peptides were examined during acute EAE and during relapses. LNC responded only to PLP peptides 139-151 and 141-151 and did not respond to BP or its peptides during acute or chronic disease. Central nervous system mononuclear cells also preferentially responded to PLP 139-151 and 141-151 during acute and relapsing disease. A PLP 139-151 peptide-specific Th cell line was selected from LNC of cord-immunized donors. Five million peptide-specific line cells transferred severe relapsing demyelinating EAE to naive recipients. We conclude that PLP peptide 139-151 is the major encephalitogen for R-EAE in cord-immunized SJL/J mice. We demonstrate for the first time that Th cells specific for this peptide are sufficient to transfer relapsing demyelinating EAE. The predominance of a PLP immune response rather than a BP response in SJL/J mice suggests that genetic background may determine the predominant myelin Ag response in human demyelinating diseases such as multiple sclerosis.     

Modification of radiation myelopathy by the transplantation of neural stem cells in the rat

In a novel approach, neural stem cells were transplanted to ameliorate radiation-induced myelopathy in the spinal cords of rats. A 12-mm section of the cervical spinal cord (T2-C2) of 5-week-old female Sprague-Dawley rats was locally irradiated with a single dose of 22 Gy of (60)Co gamma rays. This dose is known to produce myelopathy in all animals within 6 months of irradiation. After irradiation, the animals were subdivided into three groups, and at 90 days after irradiation, neural stem cells or saline (for controls) were injected into the spinal cord, intramedullary, at two sites positioned 6 mm apart on either side of the center of the irradiated length of spinal cord. The injection volume was 2 microl. Group I received a suspension of MHP36 cells, Group II MHP15 cells, and Group III (controls) two injections of 2 microl saline. All rats received 10 mg/kg cyclosporin (10 mg/ml) daily i.p. to produce immunosuppression. All animals that received saline (Group III) developed paralysis within 167 days of irradiation. The paralysis-free survival rates of rats that received transplanted MHP36 and MHP15 cells (Groups I and II) were 36.4% and 32% at 183 days, respectively. It was concluded that transplantation of neural stem cells 90 days after irradiation significantly (P = 0.03) ameliorated the expression of radiation-induced myelopathy in the spinal cords of rats.     

Autoimmune effector cells. III. Role of adjuvant and accessory cells in the in vitro induction of autoimmune encephalomyelitis

The present investigation shows that autoreactive effector cells that transfer experimental allergic encephalomyelitis (EAE) can be activated from spleens and lymph nodes of Lewis rats given a single injection of 25 micrograms myelin basic protein (BP) in incomplete Freund's adjuvant (IFA), despite the fact that the cell donors do not develop EAE. Rather, these donor rats are unresponsive to EAE when given an encephalitogenic emulsion of BP in complete Freund's adjuvant (CFA). Lymphoid cells from rats given a single injection of BP-IFA were almost as effective as cells from BP-CFA-treated rats with respect to transferring EAE after in vitro activation with BP or concanavalin A (Con A). Irrespective of whether donors received BP in IFA or CFA, BP-cultured spleen and lymph node cells (SpC and LNC, respectively) transferred EAE, whereas Con A-cultured SpC but not LNC exhibited effector cell activity. Con A-cultured LNC were able to transfer EAE if the cultures were reconstituted with irradiated adherent phagocytic cells (which could be obtained from normal Lewis rat spleens) or with conditioned medium from these adherent SpC. These findings indicate that accessory cells are required for in vitro induction of this T cell-mediated autoimmune response.     

Haematocytometrical changes in chicken blood to acute 60Co gamma radiation

Newly hatched white leghorn chicks (Gallus domesticus) subjected to single whole body 2.25 Gy (225 rads) gamma radiation exposure at the dose rate of 0.50 Gy/sec (50 rads/sec), were studied for changes in a number of haematological parameters at days 1,3,5,7,14 and 28 post irradiation during development. The sudden decline and gradual recovery in total RBC and WBC counts and the level of Hb and Hct along with MCV, MCH and MCHC values evaluation indicates a high regenerative capability of leghorn chicks.     

It is still not for the old iron: adjuvant effects of carbonyl iron in experimental autoimmune encephalomyelitis induction

Experimental autoimmune encephalomyelitis (EAE) is a model of multiple sclerosis. Dark Agouti rats immunized with spinal cord homogenate (SCH) and carbonyl iron (CI), as an adjuvant, develop severe hyperacute form of EAE. They succumb to EAE earlier and have higher clinical scores and lethality rate in comparison to counterparts immunized with SCH + complete Freund's adjuvant. There is no difference in the number of cells or in histological presentation of the CNS infiltrates of rats immunized with the two adjuvants. However, there are more granulocytes, NK and NKT cells, and less CD4(+) T cells in the spinal cord infiltrates of SCH + CI-immunized animals. Nitric oxide (NO)-generating enzyme inducible NO synthase have higher expression in spinal cord of SCH + CI-immunized rats, and this corresponds to more intensive nitrotyrosine formation in the CNS tissue of these rats. Abundant infiltration of granulocytes and NK cells into the CNS and excessive generation of peroxynitrite within the CNS of SCH + CI-immunized rats might account for the severe neurological deficits induced by immunization with CI. These factors should be closely examined in the fulminant forms of multiple sclerosis and acute disseminated encephalomyelitis, as they could represent a promising targets for therapy.     

Prevention of experimental autoimmune encephalomyelitis and induction of tolerance with acute immunosuppression followed by syngeneic bone marrow transplantation.

Experimental autoimmune encephalomyelitis (EAE) is an inducible autoimmune disease widely used as a model of the acute/relapsing stage of multiple sclerosis. In the present study we examined the effect of acute immunosuppression induced by total body irradiation (TBI) (900 to 1100 centigray (cGy)) or by a single high dose of cyclophosphamide (CY) (300 mg/kg), followed by syngeneic bone marrow transplantation (SBMT), on the development of EAE in SJL/J mice. EAE was induced in SJL/J mice by immunization with spinal cord homogenate in adjuvant. Treatment with TBI (900 cGy) and SBMT on day 6 postimmunization caused a delayed onset and a marked reduction in the incidence and severity of EAE. A higher dose of irradiation (1100 cGy) or the administration of CY followed by SBMT completely abrogated the development of paralysis. None of the 21 mice treated with CY and SBMT, and only 1 of 7 mice treated with TBI (1100 cGy) and SBMT developed clinical signs of EAE during a period of 3 months. Furthermore, mice treated with CY and SBMT became resistant to rechallenge with the same encephalitogenic inoculum. In addition, the lymphocytes obtained from these mice did not proliferate in vitro in response to myelin basic protein or tuberculin-purified protein derivative, unlike lymphocytes from immunized but untreated animals. This absence of reactivity was not associated with alterations in the proportion of the L3T4 and Lyt-2 T-cell subsets nor with a loss in T cell competence as evidenced by the full response of lymphocytes to the T cell mitogen Con A and to a nonrelevant Ag (OVA). Our results indicate that the elimination of effector lymphocytes either by myeloablative doses of CY or ionizing irradiation followed by rescue with SBMT inhibits the development of the autoimmune process in EAE and leads to induction of tolerance to the immunizing Ag by newly developing lymphocytes. This approach of combining immunoablation and reconstitution with autologous bone marrow transplantation may be applicable in the treatment of life-threatening neurologic autoimmune diseases.     

Effectiveness of hypoglycemic agents in guinea pigs exposed to mixed gamma-neutron radiations

The effect of 200, 1000, and 5000 rads of mixed gamma-neutron radiations on total blood reducing sugar and blood glucose levels in guinea pigs was investigated 2 and 24 hours, and 9, 22, and 60 days postirradiation. In addition, the effectiveness of insulin and tolbutamide in these animals was evaluated before and after irradiation. Glucose increased to a lesser degree and later than did the nonglucose fraction of the blood sugar. Insulin and tolbutamide were at least as effective in irradiated animals as in unirradiated ones except after 5000 rads, when tolbutamide was significantly less effective. These results suggest that: (1) insufficient insulin is released by the pancreas in response to elevated blood sugar levels following irradiation; (2) the pancreas does produce insulin at these times and is able to release it in response to tolbutamide; and (3) a decrease in insulin production occurs following supralethal doses of radiation.     

Chronic permeability of the central nervous system to mononuclear cells in experimental allergic encephalomyelitis in the Lewis rat.

In order to assess whether experimental allergic encephalomyelitis (EAE), a putative animal model for multiple sclerosis (MS), is an ongoing chronic disorder, we have studied the permeability of spinal cords of Lewis rats with EAE to 3H-uridine- or 3H-thymidine-labeled lymphoid cells obtained from thymuses of naive donors or from draining lymph nodes of donors injected with guinea pig spinal cord + complete Fruend's adjuvant (CFA), guinea pig myelin basic protein + CFA, or with CFA alone. During the acute clinical phase of EAE there is a high-level infiltration of 3H-thymidine- or 3H-uridine-labeled cells into the spinal cords. After clinical recovery from EAE up to 58 days post-inoculation, there is a low-level infiltration of 3H-thymidine-labeled cells into the spinal cords. A similar infiltration into the spinal cords by 3H-uridine-labeled cells was not detected. Donor cells from animals immunized with CFA alone showed similar levels of infiltration into the spinal cords of animals with EAE as donor cells from animals immunized with the encephalitogenic emulsion. Spinal cords from recipients immunized with CFA alone showed no increased permeability to labeled cells. Heat-killed labeled cells did not migrate into the spinal cords of animals with EAE. We conclude that a) EAE is a chronic disease and in this regard is a valid model for MS; and B) in the chronic phase of EAE, recently divided cells (3H-thymidine-labeled cells) show higher levels of migration into the target tissue than 3H-uridine-labeled cells.