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Laboratoire de Virologie-Immunologie and Service de Neurologie, Institut National de la Santé et de la Recherche Médicale UMR, Centre Hospitalier Universitaire de Fort-de-France, Martinique
Human T lymphotropic virus type I (HTLV-I)associated myelopathy/tropical spastic paraparesis (HAM/TSP) is associated with accumulation of HTLV-Iinfected T cells in the central nervous system (CNS). However, data on HTLV-I proviral load in the CNS at the asymptomatic stage are still lacking. We measured HTLV-I proviral load in cerebrospinal fluid (CSF) cells from 17 patients with HAM/TSP and 25 asymptomatic carriers. The percentage of HTLV-Iinfected cells in CSF cells and the CSF cell : peripheral blood mononuclear cell HTLV-I proviral load ratio were always >10% and >1, respectively, in the patients with HAM/TSP but were always <10% and <1, respectively, in the asymptomatic carriers. We propose that determination of HTLV-I proviral load in CSF cells should be included as a new parameter for the diagnosis of HAM/TSP.
Human T lymphotropic virus type I (HTLV-I) is the etiologic agent of adult T cell leukemia/lymphoma and HTLV-Iassociated myelopathy/tropical spastic paraparesis (HAM/TSP) [1]. The vast majority of HTLV-Iinfected individuals remain asymptomatic throughout life, and the factors that determine the outcome of HTLV-Iassociated diseases are still unclear. CD4+ and, to a lesser extent, CD8+ T lymphocytes represent the main reservoirs of HTLV-I in vivo [2]. The level of virus-infected cell burden is suspected to play a major role in the pathogenesis of HTLV-Iassociated inflammatory conditions. Indeed, HTLV-I proviral load in peripheral blood mononuclear cells (PBMCs) has been shown to be higher in patients with HAM/TSP than in asymptomatic carriers [3].
HTLV-I sequences have been detected in cerebrospinal fluid (CSF) cells from patients with HAM/TSP, and infiltrating CD4+ T cells appear to be the main reservoir of HTLV-I in spinal cord lesions [4, 5]. HTLV-Ispecific CD8+ cytotoxic T lymphocytes have been detected in CSF cells (reviewed in [4]). Histopathological studies have shown that HAM/TSP lesions are associated with perivascular and parenchymal infiltration of T cells (reviewed in [5]). The relative levels of infiltrating CD4+ T cells, CD8+ T cells, macrophages, and microglial cells in and around central nervous system (CNS) lesions fluctuate with disease duration [6]. The progressive decline in CD4+ T cell infiltration has been shown to correlate with levels of HTLV-I proviral DNA in spinal cord lesions [7]. Such variations are probably associated with changes in the percentage of HTLV-Iinfected cells in CSF cells, and the HTLV-I proviral load in this compartment might accurately reflect events in the CNS during the course of HAM/TSP.
Previous studies of the relative levels of HTLV-I proviral load in paired PBMC and CSF cell samples from patients with HAM/TSP have led to conflicting conclusions [8, 9]. Moreover, data on HTLV-I proviral load in CSF cells from asymptomatic carriers are still lacking. In the present study, using an accurate quantitative real-time polymerase chain reaction (PCR) assay [10], we demonstrated that HTLV-I proviral load was higher in CSF cell samples than in PBMC samples, from the patients with HAM/TSP but not from the asymptomatic carriers, and showed that patients with HAM/TSP harbored significantly higher HTLV-I proviral loads in CSF cells than did asymptomatic carriers. Both the HTLV-I DNA proviral load in CSF cells and the CSF cell : PBMC HTLV-I proviral load ratio should be taken into consideration as additional criteria for the diagnosis of HAM/TSP, and they could provide critical information on disease pathogenesis and progression.
PATIENTS AND METHODS
Patients.
The present study was performed at the University Hospital of Fort-de-France, Martinique (French West Indies). Diagnosis of definite HAM/TSP was based on World Health Organization diagnostic guidelines [11], which comprise (1) slowly progressive spastic paraparesis with symmetrical pyramidal signs, (2) disturbance of bladder function, (3) no radiologic evidence of significant spinal cord compression, and (4) intrathecal synthesis of antiHTLV-I antibodies. The asymptomatic HTLV-I carriers, who were selected from among HTLV-Iseropositive blood donors, did not display any neurological symptoms. CSF cell samples, collected after informed consent had been obtained, were coded during a previous study [12]. The residual samples were used in the present laboratory methodological study, in accordance with the French ethical guidelines relating to biological collections. CSF cell samples from 25 asymptomatic carriers and 17 patients with HAM/TSP were centrifuged at 1200 g for 10 min, and then the supernatants and pellets were immediately frozen at -80°C until use. Paired peripheral blood samples were available from 11 of the asymptomatic carriers and from all 17 patients with HAM/TSP. PBMCs were isolated from EDTA blood by density gradient centrifugation and were immediately cryopreserved at -80°C until use.
Measurement of HTLV-I proviral load.
DNA was extracted by use of a spin column DNA-extraction system (GFX PCR DNA; Amersham Pharmacia Biotech). HTLV-I proviral load was quantified by use of a real-time TaqMan PCR method, as described elsewhere [10]. Briefly, SK110/SK111 primers were used to amplify a 186-bp fragment of the pol gene, and the dual-labeled TaqMan probe (5-FAM and 3-TAMRA) was located at 48294858 bp of the HTLV-I reference sequence (HTLVATK). Albumin DNA was quantified in parallel, to determine the input cell number, and was used as an endogenous reference, to avoid variations due to differences in either the PBMC count or the DNA extraction method used. Standard curves were generated by use of 10-fold serial dilutions of a double standard plasmid (pcHTLV-ALB) containing 1 copy of the target regions of both the HTLV-I pol gene and the cellular albumin gene. The HTLV-I proviral load was reported as (HTLV-I average copy number/albumin average copy number) × 100 and was expressed as the number of HTLV-I copies per 104 cells.
Measurement of antiHTLV-I antibodies in CSF cells.
CSF cell supernatants were tested for HTLV-Ispecific antibodies by use of an ELISA (Ortho Diagnostics). The optical density (OD) : threshold OD ratio was calculated for each sample.
Quantification of neopterin.
The CSF cell level of neopterin was determined by fluorometric detection, after high-performance liquid chromatography separation with isocratic elution, as described elsewhere [12].
Statistical analysis.
The Mann-Whitney U test, Wilcoxon rank sum test, and Spearman's rank correlation were used, as appropriate. P < .05 was considered to be statistically significant.
RESULTS
The mean ± SD and median HTLV-I proviral loads in PBMCs were 383 ± 274 and 397 copies/104 cells in the asymptomatic carriers and 1109 ± 633 and 1011 copies/104 cells in the patients with HAM/TSP; the difference was statistically significant (P < .0001, Mann-Whitney U test). The mean ± SD and median CSF cell HTLV-I proviral loads were 202 ± 292 and 0 copies/104 cells in the asymptomatic carriers and 2991 ± 1621 and 2271 copies/104 cells in the patients with HAM/TSP; again the difference was statistically significant (P < .0001, Mann-Whitney U test). In the patients with HAM/TSP, HTLV-I proviral loads were significantly higher in CSF cells than in paired PBMCs (P = .0003, Wilcoxon rank sum test). In contrast, in the asymptomatic carriers, no significant difference in HTLV-I proviral loads was found between PBMCs and CSF cells.
The CSF cell : PBMC HTLV-I proviral load ratio was determined for the 28 paired samples, and the median ratio was found to be 0.59 for samples from the asymptomatic carriers and 2.46 for samples from the patients with HAM/TSP (figure 1B) (P = .0014, Mann-Whitney U test). The ratio was <1 for samples from all asymptomatic carriers and >1 for samples from all patients with HAM/TSP.
Correlation between HTLV-I proviral load, levels of HTLV-Ispecific antibody, and levels of neopterin in CSF cells.
HTLV-Ispecific antibodies were detected in CSF cells from 17 (100%) of the patients with HAM/TSP and 4 (16%) of the 25 asymptomatic carriers. The mean ± SD and median levels of neopterin were 5.9 ± 9.1 and 3.1 ng/mL (range, 145 ng/mL) in the asymptomatic carriers and 15.4 ± 9.5 and 14.4 ng/mL (range, 3.237.5 ng/mL) in the patients with HAM/TSP (P < .0001, Mann-Whitney U test). HTLV-I proviral load in CSF cells was positively correlated with CSF cell levels of antiHTLV-I antibody (P < .0001, Spearman's rank correlation) and neopterin (P < .0001) (figure 2), and the CSF cell : PBMC HTLV-I proviral load ratio was positively correlated with the CSF cell level of neopterin (P < .0001; data not shown). These correlations were found when analyzing the samples as a whole but not when analyzing the samples from the asymptomatic or HAM/TSP subgroup alone.
Correlation between HTLV-I proviral load and the evolution rate of HAM/TSP.
The clinical and biological features of the patients with HAM/TSP are summarized in table 1. There was no correlation between HTLV-I proviral load in either PBMCs or CSF cells and disease duration or stage according to Osame's disability score. However, a trend toward significance (P = .05, Spearman's rank correlation) was seen in the correlation between CSF cell : PBMC HTLV-I proviral load ratio and evolution rate (Osame's score/duration of the disease) for patients with HAM/TSP.
DISCUSSION
In the present study, we confirmed that, in the patients with HAM/TSP, HTLV-I proviral load was increased in the CSF cell compartment, compared with that in PBMCs, as previously reported by Nagai et al. [9]. That study and the present study underline the reliability of real-time PCR-based approaches for quantification of HTLV-I load in the CSF cell compartment. In the patients with HAM/TSP, the proportion of p40tax proteinexpressing T cells in CSF cells was also higher than that in PBMCs [13]. If we assume that most HTLV-Iinfected cells carry 1 copy of the provirus, our results showed that up to 60% of CSF cells from patients with HAM/TSP were infected. A similar percentage of provirus-carrying cells in CSF cells from patients with HAM/TSP was reported by Nagai et al. [9] and, more recently, in a case report of acute HTLV-Iassociated myelopathy [14]. T cell migration across the blood-brain barrier is a pivotal event in the neuropathogenesis of HTLV-I disease [5]. The higher CSF cell : PBMC HTLV-I proviral load ratio in the patients with HAM/TSP suggests a selective infiltration of HTLV-Iinfected T cells. HTLV-I has been shown to cross the blood-brain barrier via HTLV-Iinfected T cells, and oligoclonal expansion of these cells might occur within the CSF compartment [15]. Enhanced activation and expression of adhesion molecules contribute to the increased adherence of HTLV-Iinfected T lymphocytes to spinal cord blood vessels in patients with HAM/TSP [5]. Moreover, HTLV-Iinfected T lymphocytes have been shown to increase the paracellular permeability of the endothelial monolayer. Cell-free traffic, by transcytosis of viral particles or endothelial cell infection, may also occur and may increase the entry of HTLV-I into the CNS [16].
HTLV-Iinfected T cells produce proinflammatory cytokinessuch as interferon-, tumor necrosis factor, and interleukin-1which are potentially involved in the deterioration of the blood-brain barrier seen in patients with HAM/TSP [16]. Cytokine-induced production of matrix metalloproteinases may help disrupt the endothelial extracellular matrix in patients with HAM/STP [12]. Neopterin level is a commonly used parameter for assessing cell-mediated immune activation in neuroinflammatory diseases, including HAM/TSP [12]. Nagai et al. reported a relationship between CSF cell levels of neopterin and HTLV-I proviral load in PBMCs [3]. The correlation that we observed between HTLV-I proviral load in CSF cells and levels of neopterin in the same compartment is consistent with the hypothesis of an inflammatory cascade driven by a massive infiltration of HTLV-Iinfected cells into the CNS.
In the present study, HTLV-I proviral load in PBMCs was found to be higher in the patients with HAM/TSP than in the asymptomatic carriers. However, the values between the 2 groups overlapped, as described elsewhere [3], making this factor unsuitable as a diagnostic criterion. We also observed an increased HTLV-I proviral load in CSF cells from patients with HAM/TSP, compared with that in CSF cells from asymptomatic carriers. Interestingly, the HTLV-I proviral load in CSF cells (i.e., the percentage of HTLV-Iinfected cells in CSF cells) was always >10% in the patients with HAM/TSP and was always <10% in the asymptomatic carriers. Thus, it is possible to define a threshold for HTLV-I proviral load in CSF cells that allows patients with HAM/TSP and non-HAM/TSP HTLV-I carriers to be classified into the appropriate groups. Moreover, the CSF cell : PBMC HTLV-I proviral load ratio was always <1 in the asymptomatic carriers and was always >1 in the patients with HAM/TSP and may also be applied to the diagnosis of HAM/TSP in patients with suggestive symptoms. The detection of antiHTLV-I antibodies in CSF cells is currently one of the criteria for diagnosis of HAM/TSP [11], but this parameter has poor specificity and limited sensitivity [17]. In the present study, antiHTLV-I antibodies were detected in CSF cells from some asymptomatic carriers, probably as a result of a break in the cerebrospinal barrier during puncture and subsequent blood contamination. Thus, with regard to HTLV-I carriers, serologic analysis of CSF cells alone cannot be used to distinguish between HAM/TSP and a progressive form of multiple sclerosis, which is an emerging disease in the French West Indies [18]. To achieve better predictive values, Puccioni-Sohler et al. [17] proposed an algorithm including determination of the intrathecal synthesis of antiHTLV-I antibodies, detection of oligoclonal IgG bands in CSF cells, and PCR-based detection of HTLV-I DNA in CSF cells. Our data suggest that quantification of HTLV-I proviral load in CSF cells by real-time PCR provides a consistent additional criterion for confirmation of the diagnosis of HAM/TSP.
HTLV-I proviral load in PBMCs reaches an equilibrium set point, which does not fluctuate by >24-fold over the course of a decade and correlates with progression of HAM/TSP [19]. In the present study, there was a tendency toward a correlation between CSF cell : PBMC HTLV-I proviral load ratio and the evolution rate of HAM/TSP. Large-scale and longitudinal studies are required to accurately evaluate the relative prognosis values of the HTLV-I proviral load (either circulating or in CSF cells) in patients with HAM/TSP. In addition, the subgroup of asymptomatic carriers with a detectable HTLV-I proviral load in CSF cells identified in the present study will be followed to see whether they develop HAM/TSP.
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