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Activation of macrophage procoagulant activity (MPCA) is involved in the manifestation of EAE and EAN in susceptible guinea pigs and provides a mechanism for the deposition of fibrin, which is a feature of histologic lesions of EAE. Peritoneal exudate cells (PEC) from susceptible (strain 13) guinea pigs immunized with either central or peripheral nervous tissue antigens produce procoagulant activity when incubated with the immunogen in vitro. The production of the procoagulant is quantitative and antigen-specific and is maximal at the time of clinical signs of the disease. After recovery, the production of procoagulant activity decreased. The MPCA test was able to discriminate the biochemical differences existing between chicken and mammalian peripheral nerve proteins, thus providing a quantitative and sensitive indicator of cell-mediated immunity in EAE and EAN. The autoimmune response to brain and nerve antigens in nonsusceptible (strain 2) guinea pigs was coincident with the antigen-specific production of a cell-bound anticoagulant activity by stimulated mononuclear cells. The production of anticoagulant activity followed the same sequence of time changes after immunization as that of the MPCA in susceptible guinea pigs, and high immunizing doses of nerve antigens induced high levels of anticoagulant activity. The same cells produced high levels of procoagulant when incubated with tuberculin or lipopolysaccharide. The recalcification time of normal plasma was prolonged by the anticoagulant, and the decreased clotting time of plasma induced by the procoagulant activity obtained by incubating sensitized strain 13 PEC with myelin basic protein was suppressed by the anticoagulant produced by culturing sensitized strain 2 PEC with myelin basic protein. Preliminary evidence indicates that the anticoagulant has properties similar to antithrombin III. The anticoagulant could play a role in the control of effector cell function, and therefore in recovery from clinical features of EAE and EAN in susceptible guinea pigs.

Juvenile strain-13 guinea pigs challenged with whole central nervous system (CNS) tissue in complete Freund's adjuvant (CFA) developed chronic-relapsing (CR) experimental allergic encephalomyelitis (EAE). The animals that recovered from the first clinical episode were divided into three groups. One group was left untreated, one group was treated with three intracardiac injections of 100 micrograms glutaraldehyde-fixed myelin basic protein (MBP)-liposomes (MBP-L-GA) given once a week, and one group was treated with cytochrome c-liposomes (CYC-L-GA). The animals treated with MBP-liposomes were very well protected against further relapses. In vitro proliferative responses of peripheral blood lymphocytes (PBL) were performed repeatedly on most animals. The lymphocytes exhibited excellent proliferative responses to MBP, proteolipid apoprotein (PLP) and whole myelin, as well as to purified protein derivative (PPD) and concanavalin-A (ConA). High proliferative responses were recorded over the entire period of observation which lasted 12-22 months, each time the animals were tested in remission or in full relapse. However, a sharp decrease in proliferative responses was observed in most animals when the assay was performed 24-48 h before to 24 h after entering a relapse. The results demonstrate the presence of long-term and sustained cell-mediated responses to two distinct neuroantigens, and show fluctuations of both neuroantigen-specific and nonspecific responses concordant with a well-defined phase of the disease. Isoelectric focusing and immunofixation was performed on sera and cerebrospinal fluids obtained at the time of sacrifice. The pattern showed clear oligoclonal IgG bands (OB) in the samples obtained from untreated, CYC-L-GA-treated as well as in the MBP-L-GA-treated animals.

Lewis rats with experimental allergic encephalomyelitis (EAE), induced either by the subcutaneous injection of guinea pig myelin basic protein (MBP) or by the adoptive transfer of MBP-primed spleen cells, suffer from a single episode of paralysis from which they recover spontaneously. Animals developing EAE were found to have greatly elevated levels of corticosterone in the blood. This endogenous increase in steroid production was accompanied by lymphopenia and depressed delayed-type hypersensitivity responses to OVA, indicating that rats with EAE are immunosuppressed in an antigen-nonspecific fashion. Adrenalectomized rats given subcutaneous implants of corticosterone to maintain basal steroid levels invariably died when EAE was induced. However, if the steroid replacement therapy was adjusted to mimic the hormone levels that were observed in intact rats developing EAE, then the disease followed a nonfatal course closely resembling that seen in the nonadrenalectomized controls. Replacement therapy that achieved serum corticosterone levels slightly higher than those found in intact rats with EAE virtually suppressed the disease completely. It is concluded that endogenous corticosterone release in rats with EAE plays an essential role in the spontaneous recovery that is observed in this condition. However, the subsequent refractory phase that is characteristic of rats that have recovered from EAE induced by active immunization with MBP is not associated with chronically elevated corticosterone levels. This finding is discussed in the light of other data that suggest that unlike the spontaneous recovery, the refractory state has an immunological basis rather than an endocrinological basis.

Myelinated cultures of mouse spinal cord were exposed to sera obtained from rabbits affected by experimental allergic encephalomyelitis following challenge with whole white matter in complete Freund's adjuvant. In the presence of complement, the tissue response begins with an increased birefringence of its myelin sheaths. This is rapidly followed by a gamut of changes leading to demyelination. This study reports that, in the absence of complement, the response is arrested at the stage of increased birefringence. In this way, this early stage of the demyelinating process was available for detailed examination by light and electron microscopy. The brightened myelin sheaths appeared with a few hours of exposure and were seen around all axons and sometimes around cell bodies. This was often accompanied by abrupt breaks in the sheaths and angularly shaped myelin figures. Examination by electron microscope revealed a uniform increase in the myelin period from 11 nm. to 22 nm. The normally double intraperiod line was increased to four electron-dense leaflets, the additional two appearing to be derived from the close apposition of an additional electron-dense layer on the outer surface of the myelin sheath or oligodendrocytic membrane. Oligondendrocytes responsed with a prolific growth of processes whose membranes compacted to form swollen myelin. Neurons, astrocytes, and neuropil showed no changes. In its early stages, at least, the swelling was reversible. It would appear, therefore, that we have isolated the first stage of antiserum-induced demyelination in vitro, a stage which is now available for further study.

To study immune responses in inflammatory diseases of the central nervous system (CNS) we have devised a method of removing lymphocytes from brain and spinal cord. Experimental allergic encephalomyelitis was induced in adult Hartley guinea pigs to produce CNS inflammation. Brain and cervical spinal cord were removed and minced. The single-cell suspension was centrifuged on a modified Ficoll-Hypaque gradient. Lymphocytes passed through to the pellet, while brain cells stayed at the top of the gradient. An average of 1.7 x 10(6) lymphocytes was obtained from a single guinea pig brain and cervical spinal cord. When the CNS lymphocytes were cultured in the presence of phytohemagglutinin (PHA) or purified protein derivative (PPD) proliferative responses were obtained. No proliferative responses occurred when CNS lymphocytes were cultured with myelin basic protein. Using this technique, viable lymphocytes can be separated from inflamed CNS and can be cultured in vitro to measure cell-mediated immune responses.

The importance of humoral antibodies formed in the course of neuroallergic diseases characterized by a cytergic type of reaction and especially in the course of experimental allergic encephalomyelitis (EAEM) is discussed, special attention being given to the importance of pathogenetic, immunodynamic, diagnostic, and therapeutic factors. The circulating antibodies of EAEM induced by cellular and humoral immunoprocesses are assumed to produce either intensifying or mitigating effects. Among the most important functions are the influence of immune globulins on the blood/brain boundary lesion, the gliotoxic and myelinotoxic effects, and the detection of a blockade of the intraneural transmission. The therapeutical effect to be discussed in the light of the protective effects of humoral antibodies is a mere conception in view of incompletely understood mechanisms including those governing the chronology and dynamics of immunoprocesses associated with multiple sclerosis, especially since the antigen-specific induction of tolerance appears to be far more essential.

Age-related concentrations of myelin basic protein serum factor (MBP-SF), an endogenous neuroantigen detected and quantitated by inhibition of binding of rat myelin basic protein (RMBP) antibody with 125I-RMBP reagent antigen and immunochemically indistinguishable from native RMBP in this respect, reach peak levels as high as 21 ng/microliter among 2-3-wk-old normal suckling Lewis rats. Levels then progressively decline to low, usually undetectable levels of less than or equal to 0.6 ng/microliter MBP-equivalents in adult animals by 7 wk of age. MBP-SF levels are inversely related to the age-related increasing capacity of maturing Lewis rats to develop experimental allergic encephalomyelitis (EAE) after sensitization to MBP of syngeneic, but not xenogeneic, origin. MBP-SF appears to be an endogenous neuroimmunoregulatory product of potential importance for immunologic tolerance to autologous RMBP in Lewis rats.

Cholesterol esters and free cholesterol were determined in the serum of guinea-pigs with experimental allergic encephalomyelitis (EAE). EAE was induced by immunization with myelin basic protein (MBP). The results showed a marked decrease in cholesterol esters (CHE) 4 days after immunization. Seven days after immunization the values of esters increased and reached a maximum during the period of manifest clinical signs of EAE. After the cessation of clinical signs, after 2 weeks, the values of esters returned to the initial or somewhat lower values. The level of cholesterol was inverse to the values of cholesterol esters. Histological analyses showed characteristic changes in the brain and spinal cord. A possible correlation between the changes in cholesterol esters and cholesterol of the brain and serum in EAE is suggested.

The presence of tumor necrosis factor alpha (TNF alpha)/cachectin was investigated in 180 paired cerebrospinal fluid (CSF) and serum samples from patients with neurological diseases, and in five paired CSF and serum samples of Macaca cynomolgus monkeys with acute monophasic experimental autoimmune encephalomyelitis (AMEAE). TNF alpha was never detected in human CSF, even when an extensive demyelination was documented (active multiple sclerosis, acquired immunodeficiency syndrome (AIDS) dementia complex). Only one Macaca with AMEAE had detectable levels of TNF alpha in CSF but not in serum, suggesting an intrathecal synthesis of this cytokine in AMEAE.

It is postulated that multiple sclerosis might be an autoimmune demyelinating disease of the central nervous system (CNS). The mechanisms involved are unknown but, since the blood-brain barrier (BBB) is damaged, injury to endothelial cells is likely to have occurred. Our previous studies have led us to investigate the autoimmune effect of injuring the blood-brain barrier by immunizing rhesus monkeys with an endothelial cell membrane from the same kind of animals. The immunized animals developed a chronic or a relapsing neurological illness. Histological and ultrastructural examinations of the brain in the acute stage showed infiltrates of mononuclear cells around the blood vessels of the white matter of cerebrum, cerebellum, pons and midbrain, while in the chronic phase, large areas of demyelination and remyelination, especially in the white matter regions, were present. The animals immunized with extraneural antigen, an endothelial cell membrane obtained from human umbilical cord, developed no neurological illness. This results indicate that the brain endothelial cell membrane has an inflammatory encephalitogenic activity which could produce widespread demyelination in animals. The animal model described here may prove to be useful in the pathogenetic investigation of human autoimmune demyelinating diseases.

Stable leukocytosis and lymphocytosis were observed in the peripheral blood of guinea pigs from the 14th day after their sensitization with B. pertussis oil suspension. A shift to the left occurred in the leukocytic count. After the contact of leukocytes with the specific cerebral antigen both in vivo and in vitro leukolysis and allergic alteration of leukocytes which occurred at the last stages of sensitization i.e. at the period of clinical manifestation of the experimental allergic encephalomyelitis (EAE) involved mainly the granulocytes. These phenomena are supposed to be caused by the anticerebral antibodies which appear in the blood at this stage of EAE, i.e. in the mechanism of development of EAE allergic reactions of the immediate type in combination with allergic reactions of the delayed type.

The myelin/oligodendrocyte glycoprotein (MOG) is a target antigen for autoantibody-mediated demyelination in chronic relapsing experimental allergic encephalomyelitis (CREAE) in the Strain 13 guinea pig. Anti-idiotypic antibodies directed against a demyelinating MOG-specific monoclonal antibody (8-18C5) have identified a cross-reactive idiotype in 10/10 CREAE sera. In nine of these sera inhibition studies demonstrated that this idiotype was at or in close proximity to the combination site of guinea pig anti-MOG autoantibodies. This cross-reactive anti-MOG idiotype may represent an important target for the anti-idiotypic regulation of demyelinating antibody responses in CREAE.

A monoclonal antibody against the human interleukin-2 receptor (anti-Tac) has been found to cross-react with an antigen on the surface of guinea pig leucocytes. Cells marking with anti-Tac and with an anti-pan T cell monoclonal antibody have been quantitated in the peripheral blood and cerebrospinal fluid (CSF) of guinea pigs with chronic relapsing experimental allergic encephalomyelitis (CR-EAE). T cells account for about 90% of peripheral blood leucocytes in all animals whilst in the CSF, T cells are the major contributor only when there is a pleocytosis. The proportion of T cells marking with anti-Tac, a measure of T cell activation, in blood and CSF of control animals is 12%, rising to 23% in blood in the post-acute phase of the disease. However, a fall in the blood Tac/T ratio to 13% occurs during the first 10 days of relapse with a subsequent rise to 30-35%. This change is related to the time after onset of relapse irrespective of the subsequent course of the disease. From first relapse onwards CSF lymphocytes show a greater level of activation than lymphocytes from paired peripheral blood samples but the proportion of Tac+ cells in CSF does not increase with increasing CSF pleocytosis. The data is consistent with migration of activated T cells from blood to CSF at the onset of relapse.

The spontaneous chemiluminescence activity (CL-A) of peripheral mononuclear cells (MNC) was examined in Lewis rats with acute experimental allergic encephalomyelitis (EAE), compared to rats immunized with complete adjuvant (n = 11) and healthy animals (n = 16). In rats with EAE, CL-A increased sharply 8-9 days after immunization (3420 +/- 3124 counts/10 s, n = 16) at the time of flattening of the weight curve. This CL-A peak was compared to that of animals immunized with complete adjuvant: 765 +/- 441 counts/10 s (P = 0.01) and healthy rats: 450 +/- 172 counts/10 s (P = 0.0001). After this initial peak in EAE rats, CL-A decreased almost to normal values when animals lost weight (746 +/- 251 counts, n = 19) and tail paralysis developed (557 +/- 251 counts/10 s, n = 15). CL-A increased again with the onset of paralysis of the extremities (1527 +/- 990 counts/10 s, n = 11), followed by a decrease as the clinical course deteriorated. Finally, CL-A approached normal values as the animals improved. A significant increase in the number of meningeal (P less than 0.01) and perivascular (P less than 0.01) cells in the CNS coincided with the initial CL-A peak. Gel filtration of the serum of rats with increased CL-A revealed at least one substance, with a molecular weight between 13,700 and 43,000 Da, which stimulated the CL-A of normal mononuclear cells.