Demonstration of human T-cell lymphotropic virus type I (HTLV-I) from an HTLV-I seronegative south Indian patient with chronic, progressive spastic paraparesis.

Here we describe a human T-cell lymphotropic virus type I (HTLV-I) seronegative patient from South India with a chronic, progressive spastic paraparesis from which HTLV-I has been isolated from peripheral blood lymphocytes. HTLV-I pol and tax viral sequences were detected in DNA from fresh peripheral blood lymphocytes (PBL) by polymerase chain reaction (PCR) and liquid hybridization techniques. Southern blot analysis of the PCR products demonstrated a low copy number of HTLV-I at the level of one viral copy per 10,000 fresh PBL. A long-term CD4+ T-cell line was established from PBL of this patient using recombinant interleukin-2, OKT3, and feeder cells. DNA from these cultured lines was amplified and portions of the HTLV-I long terminal repeat (U3), pol, env, and tax regions were sequenced (a total of 1,115 bp). The sequence data showed that the HTLV-I associated with this patient was 98.8% homologous to prototype HTLV-I. Southern blot analysis also confirmed the presence of full-length HTLV-I. These results indicate that HTLV-I can be demonstrated in an HTLV-I seronegative patient from South India with a chronic progressive neurological disorder.

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.

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.

Immunological studies in tropical spastic paraparesis.

Tropical spastic paraparesis (TSP) and other chronic-progressive myelopathies have been clearly associated with increased serum and cerebrospinal fluid antibody titers to human T-lymphotropic virus type I (HTLV-I). However, little is known about the cellular immune function in TSP. In the present study, activated T lymphocytes were found in the peripheral blood of patients with TSP. Specifically, there were increased numbers of large CD3+ cells that also expressed HLA-DR and interleukin-2-receptor molecules. A significantly elevated spontaneous lymphoproliferative response was demonstrated in all patients tested. Generation of measles virus-specific cytotoxic T-cell response was reduced in 4 of 4 patients. This was similar to previous findings in patients with multiple sclerosis. However, unlike multiple sclerosis, reduced generation of cytotoxic T-cell response to influenza and mumps viruses was observed in 2 of 4 patients. These observations confirm further the strong association between TSP and an HTLV-I-like virus and suggest that the observed abnormalities of the cellular immune response in TSP are related to infection of lymphocytes by the retrovirus.

Humoral and cellular immune responses to matrix protein of measles virus in subacute sclerosing panencephalitis.

The immune response to matrix (M) protein of measles virus was examined in patients with subacute sclerosing panencephalitis (SSPE) and controls. Antibodies specific for M and nucleocapsid (NC) proteins in 11 serum and 8 cerebrospinal fluid (CSF) samples from patients with SSPE were quantitated by enzyme-linked immunosorbent assay by using affinity-purified measles virus proteins. Geometric mean anti-NC antibody titers were higher in the serum (6.58 +/- 0.98 [mean +/- standard deviation]) and CSF (4.38 +/- 0.74) of SSPE patients compared with controls. Anti-M antibodies were present in the serum and CSF of all SSPE samples tested but in titers lower than those of anti-NC antibodies. Geometric mean anti-M antibody titer was 3.35 +/- 0.53 in sera from patients with SSPE compared with 3.05 +/- 0.66 in sera from patients with other neurological diseases and 3.12 +/- 0.74 in sera from healthy individuals. Geometric mean anti-M antibody titer was 2.59 +/- 0.86 in the CSF of eight patients with SSPE compared with a mean less than 1.00 for patients with other neurological disease (controls). Intrathecal synthesis of anti-M or anti-NC antibodies was established in four patients with SSPE. The cellular immune responses to M, F, HA, and NC proteins were examined in four of the patients with SSPE by lymphoproliferation and were not significantly different from those in five healthy controls. The results demonstrate humoral and cellular immune responses to M protein in patients with SSPE and indicate that it is unlikely that a defect in the immune response to this virus component accounts for the disease process in the patients studied.

Isolation of an HTLV-1-like retrovirus from patients with tropical spastic paraparesis.

Tropical spastic paraparesis (TSP) is a slowly progressive myelopathy associated with increased serum and cerebrospinal fluid antibodies to the human T-lymphotropic retrovirus type I (HTLV-I) (ref. 1), and has been observed in many regions of the world. A similar condition known as HTLV-I-associated myelopathy occurs in the Kagoshima prefecture of Japan. Recent but controversial reports suggest involvement of virus related to HTLV-I in multiple sclerosis. Magnetic resonance imaging and electrophysiological studies indicate that TSP lesions are like multiple sclerosis in that they are disseminated throughout the nervous system. Complete virus from patients with TSP has proved difficult to isolate using techniques successful in adult T-cell leukaemia cases associated with HTLV-I. Here we report the isolation of an HTLV-I-like virus from T-cell lines derived from the peripheral blood and cerebrospinal fluid of TSP patients. The monoclonal antibody OKT3 was used to generate non-transformed T-cell lines that express HTLV-I antigens. Infectious virus was demonstrated by co-cultivation and complete, replicating virions were visualized ultrastructurally.

Polypeptide specificities of measles virus-reactive T cell lines and clones derived from a patient with multiple sclerosis.

Eleven cloned and uncloned measles virus-specific T cell lines were generated from peripheral blood lymphocytes obtained from a patient with multiple sclerosis and were assayed for measles polypeptide specificity. Three clones reacted specifically with the fusion (F) protein and one recognized the hemagglutinin (HA). Two reacted with whole virus but not with any of the purified proteins. Five cell lines proliferated in response to multiple measles polypeptides. The addition of anti-HA or anti-F monoclonal antibodies to two of the multispecific cell lines each resulted in partial suppression of the proliferative response to whole virus by the cell lines. Two of the three F-reactive clones recognized antigen in association with a subgroup of HLA-DR4; the third responded to F only in the presence of autologous antigen-presenting cells. Of the two clones that reacted only with whole virus, one was restricted to DP3 and one to autologous cells. The HA-specific clone was DP3 restricted. Several cell lines recognized multiple measles polypeptides in association with a single HLA antigen. Recognition of individual measles polypeptides does not segregate with specific genetic restriction elements.

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 Freund's 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.

The lymphoproliferative response to measles virus in twins with multiple sclerosis.

The cellular immune response to measles virus, as measured by lymphocyte proliferation in normal individuals, is considerably lower than that to mumps or vaccinia viruses, and stable multiple sclerosis patients do not differ significantly from the norm. The response to these viruses was studied in 28 twin sets both concordant and discordant for multiple sclerosis. Normal responses to mumps and vaccinia viruses occurred throughout. Seven affected twins manifested a persistently elevated response to measles virus, whereas the unaffected twins had a (normal) low response. The differences were unrelated to differences in T cell subsets, unusual kinetics of the response, or differential susceptibility of lymphocytes to the effects of measles virus infection in vitro. The specificity of the response resides in an E+ subpopulation, and the addition of low-responder E+ cells to high-responder E+ cells failed to identify an active low-responder suppressor population. These findings suggest the presence of clonally expanded measles-specific T cell populations in the high responders with multiple sclerosis.

Immune reactivity of the purified hemagglutinin of measles virus.

The role of the immune response to measles virus in acute infection or in disease states associated with this virus is of major interest. The viral genome-specified surface antigens of measles, the hemagglutinin and fusion proteins, are likely to be of paramount importance with respect to the host immune response to the virus. This report describes initial studies aimed at assessing the immune response to the major surface glycoprotein, the hemagglutinin. This antigen was purified by affinity chromatography, using a monoclonal anti-hemagglutinin immobilized on Sepharose. The purified protein retained biological activity in hemagglutination assays. This activity could be specifically inhibited with a human antimeasles serum and with monoclonal antibody to the hemagglutinin. Lymphocytes from individuals known to proliferate to measles-infected monolayers also proliferated to the purified hemagglutinin. Thus, the immune-response to measles virus is, in part, directed to this surface antigen.

The response of human lymphocyte subpopulations to measles, mumps, and vaccinia viral antigens.

A proliferative assay employing virus-infected, fixed monolayers was used to examine the response of PBL and lymphocyte subpopulations to measles, mumps, and vaccinia viruses. The response obtained in this assay was shown to be virus specific. In individuals previously exposed to mumps or vaccinia viruses, a substantial proliferative response was elicited. In contrast, only a small number of individuals responded to measles virus even though they were seropositive for this virus. In the responders, the proliferation to each of the viruses was essentially limited to the T cell fraction and more specifically to the IgG Fc receptor-bearing T cell, the T gamma cell. The response was not dependent on the presence of antiviral antibody in the assay and was therefore apparently not related to the ADCC activity of the T gamma population. The failure of the T non-gamma cell to proliferate was not due to HLA restriction since a response in this cell population could not be elicited on infected autologous skin fibroblasts. The functional significance of the T gamma cell response is not established although a possible immunoregulatory role is considered.

Monospecific antibody to the haemagglutinin of measles virus.

A hybrid between a murine myeloma cell line and spleen cells from a mouse immunized with measles has been produced. Two stable clones produce antibody with identical immunochemical and biological properties. This antibody reacts with the 76,000 mol. wt. protein present in the lysates and on the surface of cells persistently infected with measles. It exhibits HAI and neutralizing activity.

Antibodies to the structural polypeptides of measles virus following acute infection and in SSPE.

A solid-phase radioimmunoassay was used to determine the specificity of IgG antibodies from normal sera, sera and CSF from patients with SSPE for the structural polypeptides of measles virus. The polypeptide specificity of antibodies from these sources were qualitatively similar; these results indicate antigenic cross-reactivity between SSPE-derived (Mantooth) and non-SSPE-derived strains of measles virus and stimulation of antibody formation by comparable antigens.

Detection of B cell antigens in multiple sclerosis. Use of serum from multiparous women.

Sera from two multiparous wives of patients with multiple sclerosis (MS) were used to detect B cell antigens in other patients. With serum X, 11 of 16 patients were positive as compared with ten of 16 controls (.05 less than P less than .1). With serum Y, a positive response was found in 11 of 16 patients and two of 23 controls (P less than .0005). Ten of the 11 patients who were positive with serum Y were also HLA-Dw2, which suggests that the B cell antigen detected by this serum is linked to Dw2. Three of four Dw2-positive controls were negative with serum Y, which raises two alternative hypothetical possibilities concerning the B cell antigen. These findings indicate that serum from multiparous wives may be an important tool in the investigation of the genetic components associated with MS.

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.

CSF 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.

The disposition and pharmacokinetics in humans of 5-azacytidine administered intravenously as a bolus or by continuous infusion.

The disposition of 5-[4-14C]azacytidine, administered i.v. as a bolus or continuous infusion, was studied in cancer patients. After bolus, plasma 14C levels exhibited as multiphasic disappearance pattern; half-life (t1/2, beta phase) = 3.4 to 6.2 hr. Of 14C in plasma, less than 2% was associated with 5-[4-14C]azacytidine 30 min after dose. The ratios of 14C levels were: red cells/plasma, approximately 0.8; leukocytes/plasma, 1.1 to 2.3; nucleic acids/leukocytes, 0.2 to 0.43; sputum/plasma, 0.05 to 0.17. Urinary excretion (3 days) accounted for 73 to 98% of 14C, LEss than 1% in feces. The relative concentration of 5-azacytidine in plasma with continuous infusion stayed higher than with bolus; urinary excretion was similar. Fewer side effects were observed with continuous infusion than with bolus. The stability of 5-azacytidine was determined in various media at several temperatures by thin layer chromatography and nuclear magnetic resonance. At 20 degrees in Ringer's lactate (pH 6.2), the t1/2 was 94 to 100 hr. Stability increased with lowering of temperature and pH. From our data we conclude that 5-azacytidine should be given by continuous infusion rather than as a bolus.