Ellsworth C. Alvord

Brain malformations related to prenatal exposure to ethanol.

Microcephaly and mental retardation have been principal features of the fetal alcohol syndrome. This article describes the neuropathologic findings in four human neonates who were exposed to large quantities of ethanol at frequent intervals during gestation. The findings suggest that intrauterine exposure to ethanol can result in structural abnormalities of the brain. All four brains displayed similar malformations stemming from errors in migration of neuronal and glial elements. Hydrocephalus was one consequence of the malformations in two of the infants. Futhermore, the brain alterations may be the only distinct abnormality produced by in utero ethanol exposure. Only two of the four subjects were diagnosed as having the fetal alcohol syndrome from external criteria.

The in vitro demyelinating activity of sera from guinea pigs sensitized with whole CNS and with purified encephalitogen

Experimental allergic encephalomyelitis (EAE) was induced in guinea pigs with either homologous whole spinal cord in Freunds complete adjuvant, or purified homologous myelin basic protein (BP) in complete adjuvant. In order to induce high levels of anti-BP-antibody, another group of guinea pigs was hyperimmunized by multiple injections of BP in incomplete adjuvant, followed by challenge with BP in complete adjuvant. The presence of antibodies to BP was determined by a specific binding assay capable of detecting antibodies in all three γ-globulin classes. Sera from animals with EAE resulting from sensitization with whole CNS tissue contained no detectable levels of anti-BP-antibody, but caused demyelination of cultures of mouse cerebellum. Sera from guinea pigs with EAE after sensitization with BP and sera from hyperimmunized animals with no EAE contained detectable levels of anti-BP-antibodies, but failed to cause demyelination in vitro. This complete dissociation of EAE, antibodies to BP, and demyelinating antibodies indicates that antibodies induced by the encephalitogenic BP are not the serum factors responsible for in vitro demyelinating activity, and that the demyelinating antibodies must be evoked by some antigen in whole spinal cord other than encephalitogenic myelin BP.

Experimental allergic encephalomyelitis: Activation of myelin basic protein-sensitized spleen cells by specific antigen in culture

Abstract In vitro culture with myelin basic protein (BP) permits transfer of experimental allergic encephalomyelitis (EAE) with as few as 5 × 10 6 sensitized Lewis rat spleen cells. Activation of the sensitized cells is effective with as little as 0.1 μg/ml BP. No activation is obtained after 4 hr in culture with BP but is significant after 12 hr; optimal transfer occurs after 72 hr of culture. Homogenized cells or concentrated culture supernatants do not transfer disease. Spleen cells capable of activation by BP are found at the height of disease as well as after recovery from EAE. The use of NH 4 Cl or distilled water to lyse erythrocytes (RBC) prior to culture abolishes BP-induced [ 3 H]TdR uptake but does not adversely affect transfer of EAE. Further studies on the role of cellular proliferation suggest that a stage of cell division is necessary for enhanced transfer of disease, but that this may occur in the recipient animals after cell transfer.

Experimental allergic encephalomyelitis in rabbits. A major encephalitogenic determinant within residues 1-44 of myelin basic protein

Summary Experimental allergic encephalomyelitis could be induced in rabbits by injection in Freunds complete adjuvant of either peptide 1–44 or peptide 45–87 of rabbit myelin basic protein. In order to localize the encephalitogenic determinant present in peptide 1–44, several smaller derivative peptides were prepared and examined. Peptic peptide 15–44 and thrombic peptide 1–31 were as active as peptide 1–44, whereas peptic peptides 1–14 and 18–38 and BrCN peptide 22–44 were virtually inactive. Weak activity was shown by BrCN peptide 1–21. These results provide evidence that a major encephalitogenic determinant present in peptide 1–44 lies within sequence 15–31. The encephalitogenic activity of peptide 15–44 was essentially destroyed by oxidation of methionine-21 to methionine sulfoxide; methylation of Met-21, on the other hand, appeared to be relatively ineffective in eliminating the encephalitogenicity of peptide 1–44.

In vivo administration of anti-CD4 monoclonal antibody prolongs survival in longtailed macaques with experimental allergic encephalomyelitis

The in vivo administration of monoclonal antibody (mAb) to the CD4 antigen associated with helper T cells has been successful in prolonging the survival of nonhuman primates with experimental allergic encephalomyelitis (EAE). EAE was induced in 17 outbred longtailed macaques (Macaca fascicularis) by inoculation of homologous myelin basic protein (BP) in complete Freunds adjuvant (CFA). Treatment was begun at the onset of clinical signs. Eleven animals were treated with anti-CD4 mAb Leu3a (eight) or OKT4a (three). Of the six control animals, two received anti-CD8 mAb (Leu2a), and four were treated with saline. Specific T- and B-cell subsets which have been implicated in the development of EAE were monitored throughout the course of the disease by one- and two-color immunofluorescence (IF). The monkey anti-BP antibody and anti-mouse immunoglobulin (IgG) responses were measured by enzyme-linked immunoassay (ELISA) techniques, as were the levels of free-circulating murine IgG. The nature of the infiltrating lymphocytes in the brain was evaluated histologically post mortem. Our results indicate that anti-CD4 mAb can prolong survival and in some cases completely reverse the clinical appearance of the disease; however, relapses did occur. Treatments with Leu3a or OKT4a anti-CD4 mAbs reversed the ongoing depletion of CD4+ and CD8+ cells caused by the development of EAE and appeared to reduce the size and degree of inflammation in brain lesions. These treatments did not induce immunologic tolerance to mouse IgG since all of the anti-CD4-treated animals produced high titers of anti-mouse IgG antibodies. Treatment with Leu2a (anti-CD8) had no effect on the development of EAE. These results suggest that CD4+ cells are important to the pathogenesis of EAE in macaques and that manipulation of this subset with monoclonal antibodies may provide effective treatment of human demyelinating disease.

Enhanced transfer of experimental allergic encephalomyelitis with Lewis rat lymph node cells.

Lewis rat lymph node cells (LNC) are greatly enhanced in their ability to transfer experimental allergic encephalomyelitis (EAE) after culture with myelin basic protein (BP). As few as 10(6) LNC transfer disease after culture with antigen. In contrast to spleen cells, enhanced transfer with LNC is not seen after culture with concanavalin A. Neither cell homogenates nor culture supernatants were capable of transferring EAE. Removal of B-cells had no adverse effect on disease transfer. LNC capable of enhanced transfer were found in rats after recovery from, as well as at the height of, clinical disease.

Fluctuations of T- and B-cell subsets in basic protein-induced experimental allergic encephalomyelitis (EAE) in long-tailed macaques☆

Experimental allergic encephalomyelitis (EAE) was induced in long-tailed macaques (Macaca fascicularis) by inoculation of autologous myelin basic protein (BP) in complete Freunds adjuvant. Natural killer (NK) cell activity and lymphocyte subsets detected by one- and two-color immunofluorescence were monitored longitudinally in these animals. A decrease in NK cell activity was detected at the onset of clinically defined disease. During the preclinical phase of EAE (5-7 days before the onset of clinical signs) the absolute number of T helper (CD4+) and T suppressor (CD8+) cells in the peripheral blood decreased significantly. Analysis of peripheral blood B cells revealed a selective depletion of IgD+ B cells and a corresponding increase in the number of IgD- B cells prior to and during the onset of clinical signs. Total B-cell numbers were not significantly different between EAE and normal groups. The increased proportion of IgD- B cells in BP-sensitized animals corresponded with the appearance of high titers of circulating anti-BP antibodies. Thus two-color analysis of B-cell subsets may be a sensitive indicator of B-cell activation and of abnormal immune status in EAE. Changes in lymphocyte subsets in macaques with EAE are compared with those in humans with multiple sclerosis.

Region-specific immunoassays for human myelin basic protein

Three monoclonal antibodies reactive with human myelin basic protein have been used to develop immunoradiometric assays for this protein. Clone 1, a mouse IgG2a, is reactive with an epitope in the region 129-138. Clone 2, a mouse IgG1, is reactive with the region 119-131. Clone 12, a rat IgG, is reactive with the region 86-96. Competition experiments show that the reactions of Clone 1 and Clone 2 are mutually exclusive, probably because of steric effects. In contrast, when either Clone 1 or Clone 2 react they cause minimal interference with the subsequent binding of Clone 12. Less than 1 ng/ml of myelin basic protein can be detected in each of the two immunoradiometric assays developed. Clone 12 can also be used on its own in a competitive immunoassay to detect around 2 ng/ml. Using an extraction technique before the assay, serum or plasma can also be investigated. Assays for defined regions of myelin basic protein should prove valuable in identifying the products of myelin catabolism in patients with demyelinating disease.

Experimental Allergic Encephalomyelitis as a Model of Multiple Sclerosis

Opinions have changed many times during the past several decades as to whether experimental allergic encephalomyelitis (EAE) is a good model of multiple sclerosis (MS) or not. It is not likely today that everyone’s thoughts are synchronized, indeed probably quite likely that each of us has retained our first or last impression based on thoughts of several or many years ago. Since the situation has changed rapidly in the last few years, I welcome this opportunity to try to bring everyone up to speed. I hope that I can convince most of you that EAE is a good, perhaps even a perfect, model of MS (Fig. 1) — but we must be careful in defining just which form of EAE we are talking about.

What Experimental Allergic Encephalomyelitis Teaches Us About Multiple Sclerosis

After 37 years of study of experimental allergic encephalomyelitis (EAE) and multiple sclerosis (MS) I obviously know more about these conditions than I did when I began, but two questions arise: 1) Could I have predicted where I am now sufficiently clearly to have convinced any granting agency to support my research for these 37 years, and 2) from my vantage point can I predict how and when the puzzle will be solved? Of course, the answer to both questions is no — the first, emphatically no; the second, more questioningly so. I have the impression I am looking over a photographer’s shoulder, watching a picture being developed, with the more heavily exposed portions appearing first, apparently randomly scattered and only slowly coalescing to provide some details while the whole picture is still not recognizable.