Elizabeth S. Mingioli

Nucleotide sequence analysis of a provirus derived from an individual with tropical spastic paraparesis

Human T-cell lymphotropic virus type 1 (HTLV-1), the cause of inapparent infections and T-cell leukemias and lymphomas, has also been implicated in two chronic neurological diseases, tropical spastic paraparesis (TSP) and HTLV-1 associated myelopathy (HAM). We initiated a search for a neurotropic variant of HTLV-1 that might be responsible for these chronic progressive myelopathies by cloning and sequencing a provirus from a T-cell line from an individual with TSP. The LTRs and genes of the TSP provirus differ from HTLV-1 by 20-30 nucleotides in each region, but none of the substitutions ostensibly affect functional sites with the exception of the env gene. We document one substitution in the region encoding gp46 common to TSP and HAM proviruses and a mutation that introduces two stop codons in the region encoding gp21. The latter should delete about 100 amino acids from the transmembrane anchor, and, for this reason, the progeny of the sequenced provirus are likely to be defective viruses, maintained in the culture through coinfection of cells with wild-type non-defective HTLV-1. While defective viruses could be responsible for persistent infection of the nervous system in TSP, this cannot be generally the case as we show that HTLV-1 DNA amplified from cell lines from two other individuals with TSP lacked the stop codons. Similarly, comparisons of DNA amplified from HTLV-1 DNA in cases of ATL, HAM, and TSP did not establish a correlation between the mutation in gp46 and neurological disease. The issue of neurotropic variants in HTLV-1 associated neurological disease thus remains an open one which may be resolved in the future by examining proviruses in cells in the lesions in the nervous system; or proviruses in ATL and HAM/TSP which differ in their ability to replicate in glial or neuronal cells.

Quantitation of IgG, IgA and IgM in the CSF by radioimmunoassay

A radioimmunoassay (RIA) for quantitating IgG, IgA and IgM in unconcentrated CSF has been developed. The amounts and percentages of these immunoglobulins in CSF from 31 normal individuals were determined. Using these values as normal, CSF from patients with syphilis, encephalitis, subacute sclerosing panencephalitis (SSPE), and multiple sclerosis (MS) was studied. Abnormalities were detected, indicating the potential relevance of more extensive study of the CSF immunoglobulins. CSF from patients with myotonic dystrophy and myasthenia gravis was normal. RIA was compared with rocket electroimmunodiffusion (EID) for the quantitation of IgG. Although RIA consistently gave lower absolute values, both assays reliably detect elevated IgG in CSF. However, an advantage of RIA is its capacity to quantitate IgA and IgM.

Increased CSF IgM in multiple sclerosis

C S F IgM levels have been measured by radioimmunoassay in 56 patients with MS, 62 patients with other neurologic diseases, and 31 normal controls. Forty-eight percent of the patients with MS had a raised CSF IgM level compared with 5 percent of the patients with other diseases. The IgM level did not correlate with the IgG level. Forty percent of the MS patients with normal IgG levels had high IgM levels. No relationship was found between the CSF IgM level and length or severity of the MS, relapses, or steroid therapy. Attempts to identify the IgM as being anti-measles were unsuccessful.

Myelin basic protein-specific T cell lines and clones derived from SJL/J mice with experimental allergic encephalomyelitis.

Myelin basic protein (BP)-specific T-cell lines and clones have been derived from SJL/J mice which had been sensitized with BP in complete Freunds adjuvant. Cell lines which were initiated and maintained in the presence of BP were specific for this antigen. Cell lines specific for tuberculin-purified protein derivative (PPD) were also established. BP-reactive cell lines maintained for 1 month in culture produced experimental allergic encephalomyelitis (EAE) when transferred to recipient mice. The number of cells required was only slightly less than that necessary for transfer of disease after 3-day culture of sensitized lymph node cells. In contrast, proliferative responses to BP were significantly enhanced after 1 month in culture. Cell lines lost the capacity to transfer EAE after 4 months in culture, but retained a vigorous proliferative response to BP. Similarly, cloned BP-reactive T cells failed to transfer disease, even when recipient mice were treated with IL-2, pertussis vaccine, or low-dose irradiation. Serial FACS analyses demonstrated alterations in cell surface antigen expression, particularly loss of reactivity with anti-Ia antibody, which correlated temporally with loss of ability to transfer disease. Persistence of antigen-induced proliferation by both cloned and uncloned T-cell lines should render these populations suitable for detailed study of the T-cell BP receptor.

Leukocyte surface antigens in patients with multiple sclerosis

Lymphocyte phenotypes have been measured in 20 normal females, 19 normal males, 3 females with acute exacerbations of MS and 21 females and 17 males with chronic progressive MS. Using a FACS IV, lymphocytes were gated by light scattering properties, and fluorescence was analyzed using a log amplifier. No abnormalities were found in the 3 females with acute exacerbations. In male patients the percentage of OKT8 was significantly reduced, the percentage of OKT4 was significantly increased, and the ratio of OKT4/T8 was increased. In females with chronic progressive disease the percentage of OKT8 was not statistically altered, but the percentage of OKT4 and the OKT4/T8 ratio were elevated. Interpretation of these findings requires more extensive study in control populations.

Changes in leukocyte recirculation, NK cell activity, and HLA-DR expression in peripheral blood mononuclear cells of MS patients treated with poly ICLC

To investigate the cellular immune effects of the interferon inducer, Poly ICLC, in humans, peripheral blood mononuclear cells from patients with multiple sclerosis receiving Poly ICLC as part of a preliminary clinical trial were studied. Peripheral blood mononuclear cell phenotype analysis using fluoresceinated monoclonal antibodies and flow microfluorometry showed decreases in the percentages and absolute numbers of all lymphocyte subsets 24 h after infusion. These changes returned toward baseline at 48 h except the percentage of CD-4 positive cell which increased above baseline levels. The percentage of HLA-DR antigen positive cells and CD-16 (Leu 11a) positive cells were increased 24 h after infusion but returned to baseline at 48 h. NK activity as determined by chromium release from K562 target cells was decreased at 24 h but increased 48 h after drug infusion. The increases in percentages of HLA-DR antigen and CD-16 positive cells at 24 h and NK activity at 48 h are consistent with the in vitro effects of IFN while the decreases in peripheral blood mononuclear cells are suggestive of changes in cell recirculation.