Increased HTLV-I proviral DNA in HTLV-I-associated myelopathy: a quantitative polymerase chain reaction study

Using the polymerase chain reaction, we quantitated the amount of human T-lymphotropic virus type I (HTLV-I) proviral DNA in peripheral blood mononuclear cells from 18 patients with HTLV-I--associated myelopathy/tropical spastic paraparesis; 17 HTLV-I carriers without HTLV-I--associated myelopathy/tropical spastic paraparesis, with or without other autoimmune or inflammatory diseases; and 19 seronegative control subjects. The HTLV-I proviral DNA was 10- to 100-fold higher in the patients and in the HTLV-I carriers without HAM/TSP who had autoimmune or inflammatory diseases than in the carriers without autoimmune or inflammatory diseases. The patients who had had onset of myelopathy at a younger age (15 to 39 years) had an extremely high level of HTLV-I proviral DNA in the early phase, as compared with findings in those with a late onset of myelopathy (at 44 to 61 years). The large increase in HTLV-I proviral DNA in peripheral blood mononuclear cells is presumably closely related to the development of autoimmune or inflammatory processes in HTLV-I carriers, including HTLV-I--associated myelopathy/tropical spastic paraparesis.     

Mimicry between HTLV-I and myelin basic protein: no response in HTLV-I-associated myelopathy patients.

The reactivity to a peptide from the HTLV-I polyprotein (FKLPGLNSR) and a similar sequence from myelin basic protein (MBP) (FKLGGRDSR) was examined in relation to the proposal that mimicry of MBP by HTLV-I could be involved in autoimmune responses in HTLV-I-associated myelopathy (HAM). It was found that rabbit antibodies raised against the HTLV-I peptide recognised both peptides, with a titre of 1/10240 to the HTLV-I peptide and 1/5220 to the MBP peptide. Human sera from HAM patients and a HTLV-I carrier without HAM showed slightly higher responses to the HTLV-I peptide compared to the responses from uninfected human sera. HAM patients had greater responses to the HTLV-I peptide than to the similar MBP peptide and an unrelated bovine MBP peptide. There was no recognition of the peptides by peripheral blood lymphocytes from HAM patients or a HTLV-I carrier without HAM. It was concluded that although cross-reactivity was demonstrated in rabbits and the HTLV-I peptide was recognised by sera from HAM patients, the epitope does not appear to evoke a mimicking response to the similar region in MBP. Hence it is not likely to be involved in the pathogenesis of HAM through molecular mimicry.     

HTLV-I sequences are not detected in peripheral blood genomic DNA or in brain cDNA of multiple sclerosis patients

Human T-cell lymphotropic virus (HTLV-I) was recently reported to be etiologically associated with multiple sclerosis (MS). Genomic DNA from peripheral blood lymphocytes and brain plaques of patients with MS was analyzed for the presence of sequences homologous to the HTLV-I pol gene using the polymerase chain reaction and dot blot techniques. Comparison of DNA amplification patterns between patients with MS, and with control subjects who have other autoimmune conditions, with those in healthy control subjects and with an HTLV-I-infected cell line indicates that HTLV-I pol sequence is not present in the peripheral blood of patients with MS, and that the virus is not active in MS brain plaques.     

Active production of anti-human T-lymphotropic virus type I (HTLV-I) IgM antibody in HTLV-I-associated myelopathy

We investigated the presence of anti-human T-lymphotropic virus type I (HTLV-I) IgM in sera and cerebrospinal fluid from patients with HTLV-I-associated myelopathy (HAM) by Western blot analysis. Analyses of 36 serum samples revealed that most patients (31/36; 86.1%) had anti-HTLV-I IgM, whereas only four of 23 (17.4%) HTLV-I carriers had it. In studies of cerebrospinal fluid, anti-HTLV-I IgM was detected in 24 of 36 (66.7%) HAM patients, whereas none was detected in nine HTLV-I carriers. The differences were statistically significant (p less than 0.01). These results suggest that persistent active replication of HTLV-I occurs in the central nervous system as well as in the peripheral blood of HAM patients, and may contribute to the development of HAM.     

An inverse correlation of HTLV-I viral load in CSF and intrathecal synthesis of HTLV-I antibodies in TSP/HAM.

The authors measured human T-cell lymphotrophic virus type I (HTLV-I) proviral load and intrathecal synthesis of antibodies to HTLV-I in CSF of 13 Brazilian patients with tropical spastic paraparesis/ HTLV-I-associated myelopathy (HAM). The authors also measured HTLV-I proviral load in peripheral blood mononuclear cells of five of these patients and found that it was 10- to 100-fold higher than that in CSF cells. The combination of HTLV-I proviral load and intrathecal synthesis of antibodies to HTLV-I appears to be a useful marker of disease progression. Patients with high viral load and no intrathecal synthesis of antibodies to HTLV-I had more rapidly progressing, serious clinical disease.     

Peripheral neuropathy in HTLV-I infected individuals without tropical spastic paraparesis/HTLV-I-associated myelopathy

Abstract. Tropical spastic paraparesis/ HTLV-I-associated myelopathy (TSP/HAM) is the classical neurological manifestation of HTLV-I. Only a few studies have described isolated peripheral neuropathy (PN) among HTLV-I infected individuals. 335 infected individuals without TSP/HAM were evaluated for the presence of PN and 45 of them showed evidences of peripheral nervous system involvement. Of these 21 patients had isolated PN, defined by clinical and/or electrophysiological criteria. Sural nerve biopsies revealed inflammatory infiltrates in 2, axonal degeneration in 2 and segmental demyelination in 1. Therefore, peripheral neuropathy can be found as an isolated manifestation of HTLV-I infection. We conclude that HTLV-I infection should be investigated in patients with PN of unknown origin.     

HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) with amyotrophic lateral sclerosis-like manifestations

To clarify the existence of HAM/TSP presenting amyotrophic lateral sclerosis (ALS)-like manifestations, we assayed HTLV-I proviral load in peripheral blood mononuclear cells (PBMC) in 15 patients with anti-HTLV-I antibody in serum and ALS-like manifestations (upper motor neuron involvement in at least one region and lower motor neuron involvement in at least two limbs) by quantitative PCR, and compared the proviral load with that of 233 HAM/TSP patients and of 213 HTLV-I carriers. Five of 15 patients with ALS-like manifestations had proviral loads as high as those in the 233 patients with HAM/TSP. Anti-HTLV-I antibody in cerebrospinal fluid (CSF) was present in all of five patients. The proviral load in the remaining 10 patients was similar to that in HTLV-I carriers. Four of five patients with a high proviral load met the diagnostic criterion of HAM/TSP except for lower motor neuron involvement. These four patients showed high neopterin levels in CSF. On the basis of HTLV-I proviral load in PBMC and the clinical symptoms, our tentative conclusion is that these four patients are HAM/TSP presenting ALS-like manifestations.     

Steroid-responsive myeloneuropathy in a man dually infected with HIV-1 and HTLV-I

Two human retroviruses, HIV-1 and HTLV-I, have been associated with myelopathies in addition to other neurologic disorders. We report an American dually infected with HIV-1 and HTLV-I who developed steroid-responsive myeloneuropathy. This 28-year-old bisexual man developed interstitial pneumonitis and a transient midthoracic sensory level followed by the evolution of a slowly progressive spastic paraparesis and sensorimotor neuropathy. Serologic studies demonstrated coinfection with both HIV-1 and HTLV-I. Peripheral blood absolute CD4 count was persistently within the normal range. Cranial MRI was normal and spinal MRI showed T3-T10 atrophy. Serial CSF analyses demonstrated marked intrathecal synthesis of anti-HTLV-I IgG, lymphocytic pleocytosis, elevated protein and immunoglobulin G, and oligoclonal bands. HIV-1 was isolated from CSF but not from peripheral nerve. Lymphoproliferative studies confirmed spontaneous proliferation in both blood and CSF. Soluble interleukin 2 receptor and soluble CD8 were greatly elevated in blood and CSF when compared with patients with HIV-related vacuolar myelopathy and seronegative patients with other causes of myelopathy. Nerve biopsy showed epi- and endoneurial CD8+ lymphocytic infiltration without vasculitis; muscle biopsy showed features of acute and chronic denervation. A 6-week course of prednisone produced sustained improvement in leg strength and walking times. We speculate that the myeloneuropathy was caused by HTLV-I in the setting of coinfection with HIV-1.     

Reduced Foxp3 expression with increased cytomegalovirus-specific CTL in HTLV-I-associated myelopathy

Human T-lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients show high immune responses to HTLV-I. However, it is unclear whether the cytotoxic T lymphocyte (CTL) responses to other chronic viruses also increase. We investigated the responses in the peripheral blood by using HLA-A*0201/peptide pentamers. The frequency of cytomegalovirus (CMV)-specific CTL tended to be higher in HAM/TSP patients than in healthy controls (HCs). The frequency of CMV-specific CTL positively correlated with that of HTLV-I Tax-specific CTL. The frequency of Foxp3+ cells in CD4+ lymphocytes tended to be higher in HAM/TSP patients than in ACs and HCs. The expression level of Foxp3 was lower in HAM/TSP patients than in HCs and was inversely correlated with the CMV-specific CTL frequency. A percentage of Foxp3+ cells showed a positive correlation with the HTLV-I proviral load. These results suggest that a decrease in the Foxp3 expression may contribute to the high immune response to CMV and that the Foxp3+ regulatory T cells may play a role in the immune surveillance of HTLV-I.     

Altered subsets of peripheral blood lymphocytes in patients with HTLV-I associated myelopathy (HAM)

We studied subsets of peripheral blood lymphocytes (PBL) from 12 patients with human T-lymphotropic virus type I (HTLV-I) associated myelopathy (HAM). The percent of OKT8+ cells was significantly decreased, and the percentages of OKDR+ cells and IL-2R+ cells and the ratio of OKT4+ cells to OKT8+ cells were significantly increased compared with either HTLV-I seronegative healthy adults (controls) or HTLV-I seropositive non-HAM subjects (carriers). The subsets of PBL from eight carriers showed no differences from those in controls. The patients with HAM showed significantly higher antibody titers to HTLV-I compared with carriers. These altered PBL subsets and high HTLV-I antibody titers in the patients suggest that immune functions are in an activated state in HAM.     

Possible association of HTLV-I infection and dementia

We report a Swedish patient with progressive dementia possibly associated with human T cell-lymphotropic virus type I (HTLV-I) infection. The clinical investigation revealed no typical sings of other neurological disorders. The patient was probably infected in East-Asia 35 years before onset of the disease. High titers of specific HTLV-I antibodies were detectable with solid-phase peptide ELISA in serum (1:1.600) and cerebrospinal fluid (CSF) (1:20), and the CSF/serum anti-HTLV-I antibody ratio indicated intrathecal HTLV-I antibody synthesis. Western blot for HTLV-I and polymerase chain reaction with primers selected for the HTLV-I pol gene were positive in both peripheral blood and cerebrospinal fluid. HTLV-I antigen was also demonstrated after in vitro co-cultivation of mononuclear cells from peripheral blood. Thus, our findings indicate that HTLV-I infection also may be associated with dementia. In addition, this case report calls attention upon HTLV-I as a possible etiologic agent to neurological diseases in countries previously spared from the infection.     

Expression of HTLV-I antigen in cultured peripheral blood mononuclear cells from patients with HTLV-I associated myelopathy

The HTLV-I antigen (Ag) was detected in short-term cultured peripheral blood mononuclear cells (PBMNC) from 44% of patients with HTLV-I-associated myelopathy (HAM), using the indirect immunofluorescence method. The HTLV-I Ag-positive cells accounted for less than 4% of cultured PBMNC in all but one case. The kinetics and mechanism of HTLV-I Ag expression in cultured cells were these studied in this individual with about 20% positive cells. HTLV-I Ag was 0.3% at 6 h after the culture and the number of positive cells increased to 9.3% at 48 h. The sera from HAM patients had a suppressive effect on the expression of HTLV-I Ag in the cultured cells. This suppression was more potent in sera from patients with a high than with low antibody titer. There were no correlations between the HTLV-I Ag expression in cultured cells and the various clinical and laboratory findings, in each case.     

HTLV-I-associated myelopathy endemic in Texas-born residents and isolation of virus from CSF cells

We report three Texas-born patients with spastic paraparesis and well-documented infection with HTLV-I. CSF examination showed moderate pleocytosis, protein elevation, and elevated IgG index. Oligoclonal bands were present in two patients. On MRI, one patient had frontal lobe lesions that were low intensity on T1- and high intensity on T2-weighted images. HTLV-I immunoblot studies of serum and CSF revealed reactivity to p19, p24, p53, gp46, or gp68 from all three patients. Titration studies of serum and CSF antibodies on ELISA and immunoblot assays indicated an intrathecal virus-specific response. HTLV-I-specific p19 antigen capture assay and polymerase chain reaction (PCR) demonstrated HTLV-I in lymphocyte cultures derived from each patient's peripheral blood mononuclear cells (PBMC) or CSF cells. Using HTLV-I- and HTLV-II-specific pol and gag primers, PCR studies of PBMC cells obtained directly from the patients demonstrated that the patients were infected with HTLV-I and not HTLV-II. These three cases are to our knowledge the only US cases in whom virus isolation from the CSF has been accomplished. Importantly, two patients may be the first US cases of myelopathy arising from endemic infection.     

Antibody titers to HTLV-I-p40tax protein and gag-env hybrid protein in HTLV-I-associated myelopathy/tropical spastic paraparesis: correlation with increased HTLV-I proviral DNA load.

We studied the relationship between antibody titers to recombinant HTLV-I p40tax protein and gag-env hybrid protein in serum (by an enzyme-linked immunosorbent assay) and HTLV-I proviral DNA load in peripheral blood mononuclear cells (by a quantitative polymerase chain reaction method) in 18 patients with HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP), 17 HTLV-I carriers without HAM/TSP and 16 HTLV-I uninfected controls. The IgG and IgA antibody titers to either of the proteins correlated significantly with the HTLV-I pX (coding p40tax protein) and pol DNA amounts in HTLV-I infected subjects. HAM/TSP patients had significantly higher titers of IgG and IgA antibodies to the HTLV-I proteins than did the HTLV-I carriers without HAM/TSP. While the IgM antibodies to the HTLV-I proteins were found in only 6% of HTLV-I carriers without HAM/TSP, they were found in 40% of HAM/TSP patients, especially those having both a high HTLV-I proviral DNA load and high titers of the IgG and IgA antibodies. HAM/TSP patients with the IgM antibodies had a tendency to deteriorate more frequently on the Kurtzke's disability status scale and magnetic resonance imaging of the brain (leukoencephalopathy) than did those without in the two-year follow-up. Thus, the presence of IgM antibody and high titers of IgG and IgA antibodies to the HTLV-I proteins, together with the increased HTLV-I proviral DNA load, appears to distinguish HAM/TSP patients from HTLV-I carriers without HAM/TSP.     

HTLV-I-specific cytotoxic T lymphocytes in the cerebrospinal fluid of patients with HTLV-I-associated neurological disease.

Recently, it has been shown that in patients with human T-cell lymphotropic virus type I (HTLV-I)-associated neurological disease, high levels of HTLV-I-specific cytotoxic T lymphocytes (CTLs) could be detected in the peripheral blood. These CTLs predominantly recognized products of the pX region of HTLV-I, had a CD8+ phenotype, and were human leukocyte class I restricted. Moreover, these responses were not detected in asymptomatic, HTLV-I-seropositive individuals. This implied a role for these CTLs in the pathogenesis of the neurological disorder associated with HTLV-I. We have extended these observations by demonstrating HTLV-I-specific CTLs directly from lymphocytes obtained from the cerebrospinal fluid of patients with HTLV-I-associated myelopathy/tropical spastic paraparesis. Uncultured cerebrospinal fluid lymphocytes were used directly as effectors on a variety of targets expressing HTLV-I. These cells were lysed in a virus-specific and HLA class I-restricted manner. Moreover, the cerebrospinal fluid lymphocytes were sorted into purified CD8+ populations, cloned by limiting dilution, and assayed for CTL activity. An exceedingly high proportion of these resultant lines were shown to be cytolytic and precursor frequency analysis indicated that as many as 1 in 500 cells were HTLV-I-specific CTLs. The majority of these CTL lines recognized HTLV-I gene products encoded within the pX region of HTLV-I. The significance of these HTLV-I-specific CTLs in the central nervous system of patients with HTLV-I-associated neurological disease is discussed with regard to the potential role of CTLs in the pathogenesis of this disease.     

HTLV-I specific IFN-gamma+ CD8+ lymphocytes correlate with the proviral load in peripheral blood of infected individuals

Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurological disease caused by HTLV-I infection. It has been shown that HAM/TSP patients have high proviral loads and an extraordinarily high frequency of circulating CD8 + cytotoxic T lymphocytes specific for HTLV-I in their peripheral blood when compared to asymptomatic HTLV-I carriers (AC). We have previously described an intracellular cytokine detection assay, in which interferon-gamma (IFN-gamma) + CD8 + lymphocytes are specific for HTLV-I in infected individuals. Here, we have established a competitive polymerase chain reaction assay to measure the proviral load of patients and investigate a potential relationship between proviral load and virus-specific CD8 + lymphocytes. Genomic DNA was extracted from peripheral blood lymphocytes (PBL) from eight HAM/TSP patients and seven AC for the measurement of HTLV-I measuring proviral loads. The same PBL were analyzed for intracellular IFN-gamma expression by flow cytometry. In the HAM/TSP patients and AC, the average proviral loads were 34,482 and 9784 copy/microg DNA (P = 0.021), and the average of IFN-gamma + CD8 + lymphocytes in total PBL were 1.47 and 0.08% (P = 0.001), respectively. It was confirmed that HAM/TSP patients have both high proviral loads and increased HTLV-I-specific CD8 + lymphocytes. Furthermore, we found a positive correlation between both factors in the patients with HAM/TSP (P = 0.044) but not in the AC (P = 0.508). These findings suggest that the high number of HTLV-I-specific lymphocytes may result from the increased proviral load in HAM/TSP patients.     

Polymyositis inpatients infected with human T-cell leukemia virus type I: The role of the virus in the cause of the disease

To investigate the mechanism of polymyositis in human T-cell leukemia virus type 1 (HTLV-I) infection, we studied 6 HTLV-I–positive patients, 3 with polymyositis and 3 with adult T-cell leukemia but without clinical signs of muscle disease, by (a) quantitative single or double immunocytochemistry on serial 4-μm-thick muscle biopsy sections using antibodies to lymphocyte subsets, major histocompatibility complex (MHC) antigens, and HTLV-I proteins; (b) polymerase chain reaction using HTLV-I primers in the RNA and DNA extrcted from 50 μg of muscle tissue or from serial 5-μm-thick fresh-frozen tissue sections; and (c) cocultures of the patients' HTLV-I–positive peripher blood lymphocytes with their homologous muscles searching for replication of HTLV-I within the myotubes. In the muscle of patients with HTLV-I–associated myopathy, the predominant endomysial cells surrounding healthy muscle fibers were CD8⁺ cells followed by CD4⁺ cells and macrophages. MHC-I antigens were ubiquitous in the muscles of all 6 patients, even in those without endomysial inflammation. HTLV-I sequences were amplified from the whole muscle biopsy specimens but the cells harboring viral antigens were rare endomysial macrophages and not muscle fibers. Although HTLV-I sequences were amplified from all the patients' peripheral blood lymphocytes, these cells did not exert myotoxicity or resulted in viral replication in cocultures with their homologous myotubes. We conclude that in HTLV-I polymyositis (a) the HTLV-I sequences amplified from the whole muscle biopsy specimens are related to scattered HTLV-I–positive endomysial macrophages; (b) HTLV-I and HTLV-I–positive lymphocytes do not infect the muscle in vivo or in vitro; and (c) the virus, which perists in tissues other than muscle, triggers the activationof autoaggressive T cells that cause a T-cell–mediated and MHC-I–restricted myocytotoxicity.     

Demonstration of human T-cell lymphotrophic virus type I (HTLV-I) from an HTLV-I seronegative South Indian patient with chronic,progessive spastic paraparesis

Here we describe a human T-cell lymphotopic 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 peripheal 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.     

Activated T-lymphocytes with polyclonal gammopathy in patients with human T-lymphotropic virus type I--associated myelopathy

Human T-lymphotropic virus type I (HTLV-I)--associated myelopathy (HAM) has been shown to be closely related to HTLV-I infection. However, the mechanism by which this disease occurs after infection with HTLV-I is still obscure. We found that HAM patients have unusually high proportions of CD4+ HLA-DR+ cells, CD8+HLA-DR+ cells, OKT9+ cells, and CD38(OKT10)+ cells in their peripheral blood, all of which suggest the presence of activated T-lymphocytes. Antibody titer against HTLV-I was much higher in HAM patients than in HTLV-I carriers without evident neurological disease (p less than 0.01). Polyclonal gammopathy was also observed in most HAM patients, and 6 of 10 patients were positive for rheumatoid factor. These observations, coupled with the previous observation that corticosteroid therapy improves clinical symptoms in some patients, make it likely that continuous activation of the immune system, initiated by HTLV-I infection, plays a key role in the pathogenesis of HAM.