Stephen L. Hauser

Intensive immunosuppression in progressive multiple sclerosis. A randomized, three-arm study of high-dose intravenous cyclophosphamide, plasma exchange, and ACTH.

Fifty-eight patients with severe, progressive multiple sclerosis were prospectively randomized to one of three treatments: 20 received intravenous ACTH, 20 received high-dose intravenous cyclophosphamide plus ACTH, and 18 were placed on a regimen consisting of plasma exchange, low-dose oral cyclophosphamide, and ACTH. The three groups were similar in age, sex, duration and type of disease, and degree of disability. Before treatment and six months and one year after treatment, a disability-status score, ambulation index, and functional-status score were determined, and a quantitative neurologic examination was performed. In the ACTH group, the number of patients stabilized or improved was 8 of 20 at six months and 4 of 20 at one year; in the cyclophosphamide-ACTH group, 18 of 20 at six months and 16 of 20 at one year; and in the plasma exchange group, 11 of 18 at six months and 9 of 18 at one year. High-dose cyclophosphamide plus ACTH was most effective in halting progression of the disease at both 6 and 12 months (at 12 months, cyclophosphamide-ACTH vs. ACTH, P = 0.0004; cyclophosphamide-ACTH vs. plasma exchange, P = 0.087). Thus, progressive multiple sclerosis may be stabilized by short-term, intensive immunosuppression with cyclophosphamide plus ACTH.

Cytokine accumulations in CSF of multiple sclerosis patients Frequent detection of interleukin‐1 and tumor necrosis factor but not interleukin‐6

We identified the cytokines interleukin-1 beta (IL-1β), tumor necrosis factor (TNF), and interleukin-6 (IL-6) by specific radioimmunoassays in the CSF of patients with multiple sclerosis (MS) and other neurologic diseases (OND). There was a high incidence of detectable IL-1β in patients with active MS compared with inactive MS or OND patients. TNF was also more frequently present in active MS than in OND CSF. By contrast, most MS CSF did not contain detectable IL-6. There was no correlation between the degree of CSF pleocytosis and the level of individual cytokines, suggesting that cytokine accumulations may be derived from CNS, and not CSF, cells. As IL-1β: and TNF experimentally induce astrogliosis, demyelination, temperature elevation, lassitude, and sleep, and results raise the possibility that these cytokines may contribute to a variety of manifestations in MS and in other disease states.

A simple method for DNA purification from peripheral blood

A new, simple, and inexpensive method for the rapid isolation of DNA from whole blood is described. Cell nuclei are prepared by lysis of cytoplasmic membranes and DNA within the nuclear pellet is dispersed with guanidine isothiocyanate and precipitated with isopropanol. DNA prepared in this way restricts completely and results in low backgrounds of nonspecific hybridization after Southern analysis. The yields of DNA are similar to those obtained by more tedious traditional procedures. Numerous genomic DNA samples can be prepared from whole blood in 2 h, thus facilitating gene linkage or other molecular studies in which large numbers of individuals are required.

Intermittent cyclophosphamide pulse therapy in progressive multiple sclerosis: Final report of the Northeast Cooperative Multiple Sclerosis Treatment Group

Previous studies reported that a 2- to 3-week course of IV cyclophosphamide plus adrenocorticotropic hormone (ACTH) induction can temporarily halt progressive MS for a period of 12 months in the majority of patients treated, after which reprogression occurs. The Northeast Cooperative Multiple Sclerosis Treatment Group was formed to determine whether outpatient pulse cyclophosphamide therapy could affect reprogression and whether there were differences between a modified induction regimen and the previously published regimen. Two hundred fifty-six progressive MS patients were randomized into four groups to receive IV cyclophosphamide/ACTH via the previously published versus a modified induction regimen, with or without outpatient IV cyclophosphamide boosters (700 mg/m2 every other month for 2 years). There were blinded evaluations performed every 6 months. Results demonstrate that (1) there were no differences between the modified and the published induction regimens either in terms of initial stabilization or subsequent progression; (2) without boosters, the majority of patients continued to progress; and (3) in patients receiving boosters, there was a statistically significant benefit at 24 months and 30 months (p = 0.04). Time to treatment failure after 1 year was also significantly prolonged in the booster versus the nonbooster group (p = 0.03). Age was the most important variable that correlated with response to therapy in that amelioration of disease progression occurred primarily in patients 40 years of age or younger. Boosters had a significant benefit on time to treatment failure in patients ages 18 to 40, p = 0.003, but not in patients ages 41 to 55, p = 0.97. In addition, patients with primary progressive MS had a poorer prognosis at 12 months than patients with secondarily progressive MS (p = 0.04). Our findings (1) support a role for immunosuppression in the treatment of MS, (2) begin to identify variables that may explain differences between studies of immunosuppression with cyclophosphamide in progressive MS, and (3) suggest that intermittent pulse therapy is an important method for the treatment of progressive MS and perhaps for earlier stages of MS as well.

A susceptibility locus for multiple sclerosis is linked to the T cell receptor β chain complex

Inheritance of T cell receptor beta chain (TCR beta) genes was analyzed in families of 40 sibling pairs concordant for the relapsing-remitting form of multiple sclerosis (MS). TCR beta haplotypes were determined by segregation analysis of polymorphic markers within the TCR beta complex. The mean proportion of TCR beta haplotypes identical by descent (IBD) inherited by MS sibling pairs was significantly increased compared with expected values (means test, p less than 0.004), whereas the distribution of haplotype sharing was random when MS patients were compared with their unaffected siblings. Furthermore, one allelic form of a TCR beta variable region gene segment was overrepresented on MS chromosomes compared with those parental chromosomes not transmitted to MS offspring both in the MS sibling pair families and in a second group of families containing only one individual affected with MS. These results demonstrate that a gene within the TCR beta complex or a closely linked locus influences susceptibility to MS.

Immunohistochemical staining of human brain with monoclonal antibodies that identify lymphocytes, monocytes, and the Ia antigen.

Using immunoperoxidase histochemistry, human brain sections obtained at biopsy were labeled with monoclonal antibodies which identify human lymphocyte subsets, monocytes, and the Ia antigen. Staining of a population of cells in white matter was present with the anti-Ia and the anti-M1 (monocyte-associated) antibodies but not with any of the 8 monoclonal antibodies which react with human T-cell subsets (anti-T1, 3, 4, 5, 6, 8, 10 and 12). The Ia antigen was present on 1-2% of cells in white matter, and approximately 5% of cells in white matter were M1-positive. Ia-positive cells demonstrated a pattern of diffuse surface membrane staining, whereas the M1 antigen appeared to cluster at proximal cell processes. Definitive identification of these cells as microglial cells, astrocytes or oligodendrocytes was not possible. These findings demonstrate that: (1) cells which bear the Ia and M1 determinants can be found in histologically normal human white matter, and (2) human oligodendrocytes do not react with monoclonal antibodies (anti-T5 and anti-T8) that identify human suppressor/cytotoxic cells.

Altered blood T-cell subsets in patients with multiple sclerosis☆

We have found an alteration in T-cell subsets in patients with active multiple sclerosis, specifically an increase in the T4:T8 ratio. These findings have been reproducibly obtained over the past four years, occurring in the majority of acute patients tested early in the course of an attack and in between 25 and 40% of chronic progressive patients, depending on their stage of illness. These changes correlate with pleocytosis in spinal fluid and with other abnormalities of immune function, such as spontaneous immunoglobulin production. They have been helpful in assessing disease activity in patients being treated on a variety of protocols and as part of research studies of immunoregulatory abnormality in multiple sclerosis, but have not been helpful as a diagnostic test for multiple sclerosis. The decrease of these cells in the peripheral blood of patients with active disease may be secondary to migration of these cells to the central nervous system, where they are sequestered.

Experimental allergic encephalomyelitis in cynomolgus monkeys. Quantitation of T cell responses in peripheral blood.

Chronic relapsing-remitting experimental allergic encephalomyelitis (EAE) was induced in cynomolgus monkeys by a single immunization with a homogenate of human brain white matter (BH) in adjuvant. Proliferative T lymphocyte responses to BH, to myelin basic protein (MBP), but not to proteolipid protein, were detected in peripheral blood mononuclear cells (PBMC) of all animals and persisted until their death or, in surviving animals, for greater than 10 mo postimmunization. Responses of higher magnitude tended to be associated with fatal, compared with nonfatal, episodes of clinical EAE. The frequency of MBP-reactive T cells in PBMC of animals with acute EAE was quantitated with a soft agar colony system; the ratio of T cells that proliferated specifically to MBP was estimated at between 5 and 20 per 10(6) PBMC. A similar frequency of peptide-specific T cells was estimated from PBMC of monkeys immunized with a synthetic 14-mer peptide corresponding to a region near the carboxy terminus of MBP. Thus, autoantigen-reactive T cells can be detected in the circulation throughout the course of chronic EAE, are predictive of disease severity, and occur at a frequency similar to that estimated to be present in humans with multiple sclerosis.

Segregation of immunoglobulin heavy chain constant region genes in multiple sclerosis sibling pairs

Multiple sclerosis (MS) is a disease of the central nervous system white matter in which demyelination and scarring occurs in association with chronic or recurrent inflammation (Matthews et al., 1985). Clear evidence implicates participation of the humoral immune system in MS. Levels of immunoglobulin (Ig) are increased in the nervous system of patients (Tourtellotte, 1970), and oligocional lg is present in the cerebrospinal fluid (CSF) indicating expression of a limited number of idiotypes (Link and Laurenzi, 19791. Oligoclonal Ig is produced locally in the CSF (SandbergWollheim, 1974). Despite extensive studies, antigenic targets of nervous system lg have not been identified. Another characteristic of MS is that genetic predisposition is a central determinant of disease susceptibility. Population studies suggest that multiple unlinked genes influence disease. A gene linked to the major histocompatibility complex (MHC) has been implicated in MS (Jersild et al., 1972; Tiwari and Terasaki, 1985), and other genes, including the T cell receptor beta chain complex (Seboun et al., 1989), have also been implicated in MS susceptibility. Involvement of both genetic factors and the humoral immune system has suggested that genes related to antibody complexes may be relevant to MS pathogenesis. This hypothesis is strengthened by the observation that, in MS subjects heterozygous for Ig gamma 1 (lgG1) allotypes, biased production of one allotype occurs in the nervous system (Salier et al., 19811.

The human T-cell receptor β-chain repertoire: longitudinal fluctuations and assessment in MHC matched populations

The influence of the environment and of the major histocompatibility complex (MHC) in shaping the human T-cell receptor β-chain variable region (TCRBV) repertoire has not been systematically studied. Here, expression of TCRBV gene families was estimated by a sensitive polymerase chain reaction (PCR)-based method. Serial studies of peripheral blood, performed at 2-week intervals over a 3-month period, revealed that fluctuation in the expression of many TCRBV genes occurred in healthy individuals and in the absence of clinically evident infections. Fluctuation of TCRBV4, TCRBV5.2, TCRBV9, and TCRBV13.1 genes were present in all subjects. Additional TCRBV genes fluctuated in some but not in other individuals. Comparison of the TCRBV repertoire between these unrelated individuals indicated differences in the mean expression of TCRBV5.1, TCRBV9, TCRBV11, TCRBV15, TCRBV17, and TCRBV20 genes. For any TCRBV gene, intersubject differences were generally of a magnitude of twofold or less. Larger differences characterized the TCRBV repertoire of CD4 compared to CD8 cells. Some differences, for example over-representation of TCRBV2 and TCRBV5.1 on CD4, and TCRBV10, TCRBV14, and TCRBV16 on CD8 cells, were present in most subjects. Individuals homozygous for DR2- or DR3-bearing extended MHC haplotypes displayed similar individual variability of TCRBV expression. These data indicate that the circulating TCRBV repertoire in humans is both dynamic and diverse. Both environment and MHC effects contribute to the diversity of TCRBV expression.