Control of immune-mediated disease of the central nervous system with monoclonal (CD4-specific) antibodies.

Chronic relapsing experimental allergic encephalomyelitis (CREAE) was induced in Biozzi AB/H (H-2dq1) mice by active sensitization with spinal cord antigens. A single i.p. injection of CD8-depleting (YTS169.4) monoclonal antibody (mAb) failed to affect the clinical course of CREAE when administered prior to and during the onset of both the initial clinical and subsequent relapse phase of the disease. By contrast similar treatment with both CD4-depleting (YTS191.1) or CD4-blocking/non-depleting (YTS177.9) mAb significantly inhibited disease progression. Treatment shortly before the anticipated onset of clinical EAE prevented the subsequent development of disease, although disease could be provoked following antigen-rechallenge. In contrast, treatment with these antibodies during post-acute remission phase mainly served to delay the incidence of relapse. This suggests that, unless tolerance can be re-induced, treatment of ongoing neuroimmunological disease will require 'pulse' therapy and thus potentiate the problems of long-term immunosuppresion. Despite the findings that CD4-specific antibodies can rapidly reverse overt clinical disease shortly after the onset of disease exacerbation, once neurological dysfunction becomes established anti-CD4 treatment fails to improve the animals clinically, possibly due to the inability to rapidly reverse established demyelination. Although this study does not exclude the potential central action of the injected mAb, the failure to significantly dissociate therapeutic benefit between mAb administered directly into the CNS and that given systemically suggests that a major action of these agents is probably by selectively removing T cells in the peripheral T cell pool.

Isolation and characterization of cells infiltrating the spinal cord during the course of chronic relapsing experimental allergic encephalomyelitis in the Biozzi AB/H mouse.

A novel method for the isolation of leukocytes from the spinal cords of mice during chronic relapsing experimental allergic encephalomyelitis (CREAE) was developed using discontinuous density gradients. Immunostaining of these cells, together with spinal cord sections and peripheral blood leukocytes, with a panel of monoclonal antibodies enabled a detailed profile of the kinetics and cell phenotype during CREAE to be developed. Overall, the kinetics of cell accumulation within the spinal cord correlated with disease severity. The number of cells increased from an average of 5 x 10(4) in unimmunized animals to 40 x 10(4) in paralysed animals during the initial disease episode. The cell numbers rapidly declined with clinical remission (6 x 10(4) cells/cord) and again dramatically increased during clinical relapse. Low numbers of CD3+ lymphocytes (50-150 cells) were consistently isolated from normal mice. However, the number of T cells infiltrating the spinal cord increased following immunization. The numbers of T cells, macrophages, B cells, neutrophils, and Ig-bearing cells all paralleled the clinical disease course, with T cells and macrophages (showing evidence of myelin breakdown) predominating. T cells infiltrating the spinal cord generally failed to express gamma delta T cell receptors and expressed low levels of IL-2 receptors (5% of infiltrating T cells). These cells were phenotypically dissimilar to peripheral blood leukocytes isolated in parallel, with the spinal cord having a consistently higher ratio (9:1) of CD4+ to CD8+ than the peripheral blood (7:3). The low expression of MEL-14 (L-selectin) and 16a antigen (CD45RBhigh) and the higher levels of Pgp-1 (CD44) expressed by the infiltrating T cells, compared with splenocytes, suggest a preferential recruitment/retention of distinct T cell subsets, possibly memory/primed cells, into the central nervous system from the periphery during neuroimmunological disease.

Control of immune-mediated disease of the central nervous system requires the use of a neuroactive agent: elucidation by the action of mitoxantrone.

Mitoxantrone was used as an immunosuppressive probe to elucidate a means for the control of experimental allergic encephalomyelitis (EAE) induced in Biozzi AB/H mice following injection of spinal cord homogenate emulsified in Freund's adjuvant. A single i.p. injection of 2.5 mg/kg of mitoxantrone, 1-2 days before the anticipated onset of EAE, failed to prevent the majority of animals from developing clinical disease, whereas when the compound was injected directly into the central nervous system (CNS), at this time point, significantly increased therapeutic benefit was evident, with most animals failing to develop clinical EAE. Although the clinical use of intrathecal mitoxantrone is strongly contraindicated, these data suggest that increased therapeutic benefit may be achieved in immune-mediated disease of the CNS by targeting immunosuppressive doses of suitable agents, on lymphocyte activation within the CNS. In addition, direct administration of immunosuppressive doses into the CNS may reduce potentially unwanted (side) effects in the periphery.

Therapy of chronic relapsing experimental allergic encephalomyelitis and the role of the blood-brain barrier: elucidation by the action of Brequinar sodium.

The immunosuppressive effect of the novel 4-quinoline carboxylic acid derivative Brequinar sodium on the chronic relapsing experimental allergic encephalomyelitis CREAE model in the Biozzi AB/H mouse was investigated. Although Brequinar sodium actively inhibited peripheral immune responses, it showed a limited potential to control an ongoing disease of the central nervous system (CNS). Doses of 25 mg/kg inhibited in vivo induced proliferative response and prevented EAE when treated from day 9 post-inoculation (p.i.). However, when administered from day 12 p.i. or during the post-acute remission phase-limited effects on the course of disease were observed. By comparison, treatment with a single high dose of cyclophosphamide (200 mg/kg) at these time points was significantly effective in controlling disease. As a possible explanation of the observed results it is suggested that for a compound to be effective in treating an ongoing immune response in the CNS, it must be capable of crossing the blood-brain barrier and act on the disease-inducing cells activated within the CNS. This hypothesis is supported by the finding that intracerebral injections of Brequinar sodium on day 12 p.i. significantly inhibited disease progression. This suggests that strategies aimed at controlling immune-mediated disease of the CNS require therapeutic doses of the compounds to be delivered into the CNS.

Suppression of demyelination by mitoxantrone.

The mode of action of the immunosuppressant mitoxantrone was examined in murine models of demyelinating disease. The drug has been shown to block antigen induced proliferative activity and to inhibit myelin degradation by leucocytes from paralysed mice. Mitoxantrone blocked myelin breakdown by macrophages although phagocytosis was not affected. Further evidence was obtained to indicate that mitoxantrone acts therapeutically in reducing, or at high dose, preventing signs of EAE developing in mice immunized with spinal cord homogenate and Freund's complete adjuvant. Mitoxantrone also significantly inhibited the incidence of relapse when treatment was initiated during the post-acute remission period.

Degradation of human myelin in vitro by leucocytes from patients with multiple sclerosis.

In order to study the possible autoimmune basis of multiple sclerosis (MS) a quantitative method has been used to investigate breakdown of human myelin in vitro. We found that serum from MS patients and controls was generally devoid of any myelin degradative activity. However, isolated peripheral blood mononuclear cells from 43% of MS patients showed significant myelin degradative activity as did those from 61.5% of patients with rheumatoid arthritis (RA). Myelin degradation by cells was found in only 13% of patients with other neurological diseases and in no healthy controls. It is proposed that this non-specific peripheral cellular immune degradative activity originates from cells activated within the central nervous system of MS patients or the joints of individuals with RA. As a result, activity in the blood only indirectly reflects the ongoing inflammatory response at the primary site, accounting for the lack of correlation between changes in the blood and the clinical status of the MS patient. We further propose that the lack of in vitro myelin degradative activity in cells recovered from the cerebrospinal fluid is due to autoaggressive cells being sequestered to the brain.

Nervous and immune system disorders in multiple sclerosis.

Multiple sclerosis is probably an acquired infectious disease with an autoimmune response relating to damage to the white matter of the central nervous system. There is evidence of continued intrathecal synthesis of oligoclonal antibody and there are perivascular inflammatory cell infiltrates close to areas of demyelination in the central nervous system. Following adoptive transfer, cells sensitized to the myelin basic protein can cause demyelinating disease in rodent recipients. Unlike the peripherally-mediated immune changes in the experimental model it is argued that autoaggressive cells are generated within the CNS in multiple sclerosis. The possible mechanism of cellular demyelination is discussed and the implication for therapy is reviewed.

Gamma-aminobutyric acid concentration in brain tissue at two stages of Alzheimer's disease.

The concentration of the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), was measured in the cerebral cortex obtained at diagnostic craniotomy from 10 patients with Alzheimer's disease of 3 yrs mean duration and 6 patients with other causes of dementia, and from 31 subjects undergoing other neurosurgical procedures (for which removal of apparently normal tissue was necessary). GABA content of 5 areas of the cerebral cortex and the cerebellar cortex was measured postmortem in the brains of 23 Alzheimer and 19 control subjects and 5 patients with other causes of dementia. Fourteen of these specimens, including 7 from patients with Alzheimer's disease of 8 yrs mean duration, were obtained within 3 h of death. These were processed in a similar manner to the neurosurgical specimens and are regarded also as fresh tissue samples. The remaining 33 specimens are regarded as conventional postmortem samples as the mean interval of death to autopsy was 21 h. GABA concentration in conventional autopsy specimens from Alzheimer subjects was not reduced as compared with controls in either cingulate or cerebellar cortex. In the inferior parietal cortex, agonal status confounded this comparison. The concentration was reduced in superior parietal, frontal and temporal cortex but there is a possibility that agonal state also confounded these comparisons. There was no deficit in GABA concentration in fresh cortical tissue from Alzheimer patients except for the temporal lobe from autopsy specimens. The content of somatostatin-like immunoreactivity was, like GABA, found to be comparable to control in some groups of Alzheimer specimens. It is argued that the deficits in autopsy samples and lack of change in surgical specimens is likely to be due to the duration of illness at the time of sampling. Losses of choline acetyltransferase activity were observed in all groups of Alzheimer specimens in all areas of brain studied. The data are consistent with other results which suggest that cholinergic under-activity is most closely related to the clinical course of Alzheimer's disease.

An in vitro micromethod for the quantitative assessment of central demyelination.

We report the development of a simple and reliable method for the study of demyelination in vitro based on the measurement of 2':3'-cyclic nucleotide 3'-phosphodiesterase in isolated myelin. Using only small quantities of myelin (equivalent to 100 micrograms of myelin protein) the system was tested under conditions that are believed to approximate those found at the site of an inflammatory demyelinating lesion. Treatment with a combination of trypsin, phospholipase A2, and lysophosphatidylcholine was used to evaluate the method. This microsystem has the potential not only for testing the myelinotoxicity of soluble factors but also for investigating the involvement of inflammatory cells in the demyelinating process. Myelin degradation by elicited peritoneal macrophages could be demonstrated at relatively high densities of these cells. Nylon wool purified lymph node T cells from myelin basic protein-primed SJL/J mice, after selective expansion with antigen and interleukin 2, failed to induce any significant myelin breakdown unless a limited number of syngeneic activated macrophages were also present. T cells from mice that had been inoculated with keyhole limpet haemocyanin failed to show any effect. The advantages of this technique over other in vitro systems are that it enables the study of demyelination using syngeneic sources of myelin and defined cell populations.

Effects of cyclosporin A on expression of IL-2 and IL-2 receptors in normal and multiple sclerosis patients.

The effects of the immunosuppressant Cyclosporin A (CsA) on T cell activation in vivo and in vitro were examined using the monoclonal antibody, anti-Tac, for interleukin 2 (IL-2) receptors and 3.9C2, for a peptide fragment of human IL-2. Peripheral blood lymphocytes (PBL) were stimulated with PHA in the presence of CsA. The expression of IL-2 receptors and production of IL-2 were reduced. PBL from CsA-treated MS patients had significantly lower proportions of Tac+ cells compared with untreated patients. This inhibition was not reflected in the CSF lymphocyte populations from CsA-treated patients and indicates the urgent need for an immunosuppressant drug which can enter the CNS in sufficient concentrations to inhibit local T cell activation.

Pathophysiology of ageing brain.

Even in nonfamilial cases of dementia there is some evidence of a genetic factor. This may be linked to defective expression of neurofilament protein and also abnormal phosphorylation of cytoskeletal proteins. In this respect there may be a link with accumulation of tangles and amyloid which have some degree of homology. It may be speculated that neurons containing tangles or undergoing granulovacuolar degeneration would not be able to release trophic factors and that transneuronal degeneration would result. However, the environmental or aetiological factors associated with Alzheimer's disease are not known. Although there has been a failure to transmit Alzheimer's disease to primates, it is possible that as in postencephalitic Parkinson's disease virus may be implicated at some stage in the pathogenesis. Finally, free radical formation has been considered as an alternative mechanism for death of large neurons within the CNS. Although tangles are found in several other dementing conditions (e.g. dementia puglistica, Parkinson-dementia complex of Guam), Alzheimer-type plaques and tangles are not invariably found in cases of cognitive deficit. For example, in dementia of Parkinson's disease there is a low neuritic plaque count and normal population of tangles. In addition, memory loss is not necessarily associated with defects in the cholinergic system and/or loss of nucleus basalis nerve cells. We have proposed that damage to or loss of cortical cells may be a more general finding in dementing illness.

Derivation of ganglioside-specific T cell lines of suppressor or helper phenotype from cerebrospinal fluid of multiple sclerosis patients.

In order to investigate the specificity of activated T cells in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS), we have cultured cells in the presence of mitogen-free IL-2 but without any antigen. Two T cell lines have been derived and showed specific reactivity to certain purified gangliosides (GM1, GD1a, GD1b and GQ1b). However, responses to other brain and viral antigens were not seen, and neither were T cell lines from peripheral blood lymphocytes (PBL) of normal, MS or other neurological disease patients stimulated by these gangliosides. Release of IL-2 could be detected after incubation of these CSF lines with specific gangliosides. One line exhibited predominantly helper/inducer (T4+) phenotype whilst the other was suppressor/cytotoxic (T8+), and further analysis indicated it could be of the suppressor phenotype. These data may have implications for T cell-induced demyelination in MS.

Immunocytochemical characterisation of the immune reaction in the central nervous system in multiple sclerosis. Possible role for microglia in lesion growth.

As there is evidence that in multiple sclerosis T-cell activation occurs in the central nervous system rather than outside, the inflammatory lesion may be extended through antigen presentation by cells at the edge of the plaque. In this study we present an immunocytochemical report on CNS tissue from an active case of MS, with an analysis of the distribution of CD4 and CD8 binding T cells and the expression of class I and II MHC determinants in plaques and white matter. Perivascular cuffs of early lesions, as judged by hypercellularity and minimal demyelination, contained activated T (Tac+) cells, which reacted with an anti-IL-2 monoclonal antibody. Thus sufficient T-cell growth factor would appear to be present to fuel the immune reaction in a growing lesion. The preponderance of T cells of the cytotoxic/suppressor (CD8) phenotype in the CNS parenchyma was found in conjunction with widespread staining of class I MHC antigen, a prerequisite for activity of cytotoxic T cells. Potential antigen presenting cells were demonstrated in MS plaques with a monoclonal antibody against the cytoplasmic, invariant chain of class II MHC. Macrophages and astrocytes, contributed to the staining in the hypercellular plaque border while the distribution of class II+ microglia in white matter suggest they may also be of importance in local antigen presentation.

Alzheimer's disease: a correlative study.

In a study of 17 patients with histologically proven Alzheimer's disease the relationship between psychological, pathological and chemical measures of disorder was examined. Severity of dementia, determined by mental test performance, correlated highly with pathological change in large cortical neurons (cell loss and reduction in nuclear and nucleolar volume and cytoplasmic RNA content), to a lesser extent with cortical senile plaque and neurofibrillary tangle frequency and reduction in acetylcholine (ACh) synthesis, and not with reduction in choline acetyltransferase (CAT) activity. A strongly significant relationship was demonstrated between cell loss and reductions in nuclear and nucleolar volume and cytoplasmic RNA content. Reduction in CAT activity and senile plaque frequency were significantly correlated, thereby linking changes in the sub-cortical projection system of the nucleus basalis with the cortical pathology. The pattern of correlations suggests that the dementia of Alzheimer's disease is largely a reflection of the state of large cortical neurons, and it is argued that abnormalities in the latter may not be directly related to primary loss of cholinergic neurons in the subcortex.

Neuropsychological syndromes in presenile dementia due to cerebral atrophy.

In a prospective study of 24 patients with presenile dementia associated with cerebral atrophy, clinical and psychological characteristics of patients' disorder were examined in relation to pathological and chemical findings obtained from tissue analysis following cerebral biopsy. The histological features of Alzheimer's disease were found in 75% of cases, but not in 25%. Distinctive patterns of neuropsychological breakdown emerged allowing clinical grouping of patients. While clinical patterns were helpful in differentiating Alzheimer's disease from non-Alzheimer's disease, there was not an absolute concordance between clinical and patho-chemical groupings. The findings, which support the notion that the "cerebral atrophies" represent a heterogeneous group of conditions, have relevance for the clinical diagnosis of presenile dementia.