e ur o l og ical Dis o rd e rs Human T - L y mph ot r o pic Virus - Ass o cia te dN 17

Human T-Lymphotropic Virus-Associated Neurological Disorders

Pathogenesis, Immunology, and Clinical Management

Michael D. Lairmore, Bindhu Michael, Lee Silverman, and Amrithraj Nair

1. INTRODUCTION

Human T-cell lymphotropic virus type 1 (HTLV-1), the first human retrovirus discovered, has a tropism for T lymphocytes and has been established as the causative agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive CD4 T-cell malignancy (1–4). HTLV-2 was subse- quently isolated and found to share approximately 60% nucleotide sequence homology with HTLV-1 (5). In the mid-1980s, HTLV-1 began to be associated through epidemiologic studies as a contributing factor in chronic neurodegenerative disorders. These clinical syndromes were first reported in Japan and Martinique (French West Indies), both regions that were endemic for HTLV-1 infection (6,7). Through important international cooperative studies, both syndromes were revealed to be identical in clinical presentation and are now known collectively as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-2 has been sporadically associated with lymphoproliferative diseases and recently has been implicated in isolated case reports of neurological disease (8,9).

Currently, a total of 10 to 20 million people worldwide are estimated to be infected with HTLV-1, and HTLV-1 is considered endemic in southwestern Japan, the Caribbean basin, Central and West Africa, the Middle East, Melanesia, and South America (10). It is also a serious public health problem among the at-risk groups in Europe and North America and therefore is a target of intervention policies, including blood-donor screening (10–16). Although the majority of the infected persons (~95%) remain lifelong asymptomatic carriers of the virus, 2 to 3% of those infected develop ATLL, and another 2 to 3% develop chronic inflammatory diseases, such as HAM/TSP (11,17,18). Natural transmission of HTLV-1 and HTLV-2 occurs through cell-associ- ated transmission by three primary routes: orally via breast milk from infected mothers to chil- dren, via sexual contact (predominantly from male to female), and by exposure to infected blood or whole-cell blood products (e.g., transfusion, use of contaminated needles) (9,10,19).

Although the molecular events of virus replication are beginning to be unraveled and knowl- edge about HTLV-1- and HTLV-2-associated diseases has increased, many questions regarding the pathogenesis of neurologic diseases associated these retroviruses remain. For example, it is unclear why some HTLV-1-infected subjects develop ATLL or HAM/TSP, whereas the majority remains

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From: Current Clinical Neurology: Inflammatory Disorders of the Nervous System:

Pathogenesis, Immunology, and Clinical Management

Edited by: A. Minagar and J. S. Alexander © Humana Press Inc., Totowa, NJ

asymptomatic. What factors contribute to the pathogenesis of these two widely divergent clinical disorders? In HAM/TSP patients, why is there preferential damage to the thoracic spinal cord?

What is the mechanism of viral persistence in the face of a strong immune response? What is the nature of the cell-mediated immune response to HTLV-1 infection, and is this response protective or could it lead to lymphocyte-mediated lesion development if it is uncontrolled? In this chapter, we focus on the pathogenesis of HAM/TSP in terms of its clinical features, pathologic lesions, patient susceptibility factors, and immune responses and review the proposed mechanisms for the development of this chronic neurological disorder.

2. CLINICAL FEATURES OF HAM/TSP

In evaluation of the clinical presentation of HTLV-associated neurological disorders, it is important to understand that HTLV-1 has been associated not only with HAM/TSP but also with several inflammatory diseases, such as alveolitis, polymyositis, arthritis, uveitis, and peripheral myopathy (11,18). HAM/TSP is strongly associated with HTLV-1 infection and is characterized as a progressive chronic myelopathy, with preferential damage of the thoracic spinal cord (11,20–23). In affected patients, the myelopathy presents as paraparesis with spasticity in the lower extremities, hypereflexia, muscle weakness, and sphincter disorders, including dysfunction of the urinary bladder and intestines (Table 1). Less frequently, it may precede or give rise to a cerebellar syndrome with ataxia and intention tremor (24). Some HAM/TSP patients exhibit a predominance of sympathetic nervous system dysfunction (25). HAM/TSP patients may exhibit prolonged central motor conduction time (CMCT) in all the limbs and central sensory conduction time in lower limbs, with CMCT in upper limbs correlating with the clinical severity of HAM/TSP (26,27). Magnetic resonance imaging (MRI) of HAM/TSP patients reveals the pres- ence of multiple white-matter lesions in both the spinal cord and the brain, involving perivascular demyelination and axonal degeneration (28–31). In addition, recent studies show that the carriers and patients with HAM/TSP exhibit mild cognitive impairments in verbal and visual memory, attention, and visuomotor abilities (32).

Table 1

Summary of HTLV-1- and HTLV-2-Associated Inflammatory Neurological Diseases Associated

Viral neurological Typical presenting Host factors associated

etiology diseases symptoms Treatments with disease

HTLV-1 HAM/TSP Spasticity in the lower Corticosteroids, Certain patient HLA extremities, hypereflexia, plasmapheresis, haplotypes muscle weakness, interferon, and (HLA-A*02 and sphincter disorders antiretroviral Cw*08) lead to a (urinary bladder and drugs. lower risk of HAM/TSP, intestines), and less Experimentally, whereas HLA-B*5401 frequently, cerebellar BPHA inhibitors and DRB1*0101 syndrome with ataxia of MMP-2 and are associated and intention tremor MMP-9 with an elevated

risk of HAM/TSP HTLV-2 Isolated cases Spastic ataxia Corticosteroids Unknown

of spastic and

pararesis symptomatic

and ataxia support

HTLV, human T-lymphotropic virus; HAM/TSP, HTLV-1-associated myelopathy/tropical spastic paraparesis;

BPHA, N-biphenyl sulphonyl-phenylalanine hydroxamic acid; MMP, matrix metalloproteinase.

3. SPINAL CORD AND BRAIN PATHOLOGY ASSOCIATED WITH HAM/TSP

In patients with HAM/TSP, the primary site of lesion development is the spinal cord; lesions are particularly severe in the middle to lower thoracic regions (11,33,34). This lesion distribution is consistent with neurological symptoms, such as spastic paraparesis of the lower limbs. Brain lesions less commonly may occur simultaneously with spinal cord involvement and may be detected using MRI, which reveals abnormal-intensity lesions in the brain (white matter) of patients with HAM/TSP (28,29,31,35). Within the spinal cord, regions mainly affected are the so- called watershed zones, suggesting a nonrandom distribution (36). These include degenerative lesions in the lateral corticospinal tract and spinocerebellar or spinothalamic tract of the lateral col- umn. Damage to the anterior and posterior columns is less extensive and more variable than that of the lateral column, in correlation with the clinical findings. In addition, there is also perivascular and parenchymal cellular infiltration, consisting of mainly T cells (both CD4

and CD8

T cells), macrophages, astrocytes, and glial cells (33). Similar perivascular inflammatory infiltration is also present in the brain, particularly in the deep white matter and the marginal area of the cortex and white matter (33). Interestingly, the nature of perivascular-infiltrate and cytokine expression varies depending on the duration of the disease. Patients with short duration of illness, extending from 2.5 to 4.5 yr, exhibit distribution of CD4

cells, CD8

cells, and macrophages with the presence of proinflammatory cytokines, such as interleukin (IL)-1 β, tumor necrosis factor (TNF)-α, and inter- feron (IFN)- γ (37,38), whereas in those with more prolonged duration of illness, extending from 8 to 10 yr, there are greater numbers of CD8

cells compared to CD4

cells, with fewer proinflam- matory cytokines (33,39). The distribution of macrophages and microglia varies depending on the duration of illness (40). Intriguingly, patients with shorter duration of spinal cord lesions and with a predominance of CD4

cells appear to contain more HTLV-1 pX and pol DNA than do those with longer duration of illness who exhibit fewer CD4

cells (41). HTLV-1 tax RNA is also found in infiltrating CD4

T lymphocytes in active central nervous system (CNS) lesions in patients with HAM/TSP (42), particularly within astrocytes, but not in perivascular infiltrating cells (43). These reports collectively suggest that viral proteins may elicit the early inflammatory lesions associated with HAM/TSP, but subsequent lesions may develop as a result of secondary injury caused by cytokines and other inflammatory mediators from infiltrating leukocytes.

Within the myelopathy, widespread loss of myelin and axons has been observed (33,44).

β-Amyloid precursor protein (APP), an early axonal damage marker, is more intensively expressed in areas of active-inflammatory lesions than in those of inactive-chronic lesions, suggest- ing that axonal damage is closely associated with inflammation in active-chronic lesions (34). Addi- tionally, the presence of APP

axons without relation to inflammatory cells in inactive-chronic lesions suggests that soluble neurotoxic factors might induce the axonal changes (34). This same study reported that myelinated fibers in the anterior and posterior spinal roots in lower thoracic to lumbar levels had occasional APP

axons, suggesting that spinal nerve roots can be affected in HAM/TSP, especially in lower thoracic to lumbar levels.

4. ANIMAL MODELS OF HAM/TSP

A variety of animal models of HTLV-1 infection have been reported, although the virus consis-

tently infects only rabbits (45,46), some nonhuman primates (47,48) and, to a lesser extent, rats

(49,50). Viral transmission in mice using typical methods of infection produces inconsistent

infections and limited virus expression in tissues (51–55). Nonhuman primates have been infected

with HTLV-1, and certain species have a natural infection with simian T-lymphotropic virus infec-

tion type 1 (56–62). The squirrel monkey has been successfully infected with HTLV-1 and offers

an attractive nonhuman primate model of HTLV-1 for vaccine testing (63–66). Rats have been

infected with HTLV-1 cell lines and offer a model of the neurological disease associated with the

viral infection (67–70). In addition, rats have been successfully used to test the role of cell-mediated immunity to the infection (71–73), although controversy exists regarding the reproducibility of the viral infection in rats (50).

The WKA strain of rat appears to be uniquely susceptible to an inflammatory neurological disease resembling HAM/TSP when is infected with HTLV-1-infected cell lines via inoculation (74). This strain appears susceptible to the infection with approximately 80% infection efficiency when rats are exposed at 16 wk of age with HTLV-1 cell lines derived from rat, human, or rabbit cells (74–76). In this model, affected rats develop spastic paraparesis of the hind legs with lesions confined primarily to the lateral and anterior funiculi of the spinal cord. Lesions include extensive symmetrical white- matter degeneration characterized by loss of myelin, axon damage, vacuolar degeneration, infiltra- tion with foamy macrophages, and astrocytic gliosis, most severe within the thoracic cord and continuing from the cervical to the lumbar area with degeneration of nerve roots and peripheral nerves (70,75). Electron microscopic studies of these lesions indicated symmetrical alterations mainly confined to the marginal areas of white matter with distribution in the anterior and the lateral columns. Additional ultrastructural features included separation of myelin lamellae and vacuolation of myelin sheaths with debris and the presence of both demyelinated and remyelinated axons, occa- sionally with tubuloreticular inclusions (74,75). Ultrastructural alterations in the peripheral nerve were similar to those of the spinal cord with the addition of apoptosis of oligodendrocytes and Schwann cells. Similar to the human infection virus, particles were absent in either the spinal cord or peripheral nerve lesions (75). Antibodies against HTLV-1 antigens were demonstrated in the plasma and cerebrospinal fluid (CSF) of these rats, whereas HTLV-1 provirus was less frequently detected from the peripheral blood mononuclear cells (PBMCs) and spinal cords of these rats; however, many other strains of rats do not develop inflammatory neurological lesions under the same conditions, suggesting that the development of the lesions in rats is under strict genetic restriction (69).

Although the clinical and neuropathological features of the WKA rat model of HAM/TSP generally mimic those of HAM/TSP in humans, the inflammatory cell infiltration is generally reduced or absent in the affected rat spinal cord lesions, unlike the disease in humans (77).

Using a variety of promoters and enhancers to drive gene expression, transgenic mouse models of HTLV-1-associated diseases have provided new tools to understand the role of tax and enve- lope proteins in the pathogenesis of HTLV-1-associated lesions. Nerenberg et al. (78,79) produced neurofibromas associated with peripheral nerves in tax transgenic mice by 8 mo of age. These mice also exhibit skeletal lesions similar to that in patients with neurofibromatosis who have a mutation in the NF-1 gene. Dysregulation of cytokines, in particular granulocyte-macrophage colony-stimulating factor (GM-CSF), may have resulted in the infiltration of neutrophils associ- ated with these lesions (80). Kitajima and Nerenberg (81) decreased the size of these neurofi- broma tumors with antisense inhibition of NF- κB, directly demonstrating the importance of this transcription factor in the development of the tumors. A limitation of this line using mice as a model of HTLV-1 is the lack of reports associating the virus with lesions of peripheral nerves.

Seeking to understand how the virus targets nervous system tissue, Coscoy and Ozden et al. (82) demonstrated that the HTLV-1 promoter directs the expression of lacZ markers in choroid plexus cells, ependymal cells and, occasionally, in neurons. Some lines of tax transgenic mice have increased nerve growth factor-, GM-CSF-, and IL-2-receptor expression in their tissues (83,84).

The wide array of inflammatory lesions associated with HTLV-1 have long been hypothesized to be mediated by autoimmune reactions triggered by the virus (78,85–87).

5. VIRAL AND HOST FACTORS THAT CONTRIBUTE TO HAM/TSP

For carriers in Japan, the lifetime risk of HAM/TSP is estimated to be 0.23%, based on multiple

risk factors (88). Significant progress has been made in the understanding of the risk factors that con-

tribute to the development of HAM/TSP. These studies collectively indicated that the disease is influ-

enced by a complex interplay between host factors, in particular the immune response of infected

subjects and viral properties that allow HTLV-1 and HTLV-2 to persist in the presence of a vigorous immune response.

5.1. Human T-Cell Lymphotropic Virus Proviral Load and Patient Gender Among the factors that have been linked to a higher risk of HAM/TSP is a high HTLV-1 proviral load (51,89–92). PBMCs of HAM/TSP patients have ~16-fold more proviral DNA than do asymptomatic carriers (93,94). Patients with proviral loads representing more than 1% of the total PBMC may have a significantly higher risk of HAM/TSP, suggesting that increased viral loads contribute to the development of lesions (11). In patients with HAM/TSP, the anti-HTLV-1 anti- body titer, which parallels the number of viral-infected PBMC, is a useful correlate to predict HAM/TSP in HTLV-1 carriers (89,91).

5.2. Host Genetic Factors

Host genetic factors have been demonstrated to clearly have a role in the development of HTLV-1-associated diseases, including HAM/TSP. Asymptomatic carriers from families of patients with HAM/TSP were found to have elevated proviral loads, compared to unrelated asymptomatic carriers (95), suggesting the role of inherited traits in the control of HTLV-1 proviral loads. Certain human leukocyte antigen (HLA) alleles have been associated with a lower risk of developing HAM/TSP, whereas other HLA alleles have been linked to a higher risk of developing ATLL (96–98).

HLA-A*02 and Cw*08 are associated with lower patient HTLV-1 proviral loads and a reduced risk of developing HAM/TSP, whereas HLA-B*5401 is associated with higher proviral loads and an ele- vated risk of developing HAM/TSP (96,97). Additionally, HLA-DRB1*0101 was linked to a higher risk of HAM/TSP (99), which was later demonstrated to be only in the absence of the protective effect linked to HLA-A*02 (96). Intriguingly, those individuals with major histocompatibility com- plex (MHC) class I molecules linked to a higher risk of ATLL, such as HLA-A*26, HLAB*4002, HLA-B*4006, and HLA-B*4801, have anchoring regions, unable to bind to 69 different HTLV-1 tax peptides and are thus predicted to be unable to produce efficient tax-specific cytotoxic T lympho- cytes (CTL) responses, characteristic of HAM/TSP (98). On the other hand, MHC class I molecules predisposing to persons to HAM/TSP likely have a strong affinity through anchoring regions for binding tax peptides and thus are able to generate a strong CTL response against HTLV-1-infected cells and also against resident CNS cells, leading to HAM/TSP. In support of this contention, patients with HAM/TSP exhibit elevated HTLV-1-specific CTL activity, whereas patients with ATLL have been found in some studies to have lower HTLV-1-specific CTL activity (100,101);

however, these findings and theories are not unequivocal. For example, the frequency of HLA-A*02 allele considered to be protective in HAM/TSP (96) is similar in some studies of asymptomatic car- riers and patients with HAM/TSP (98). Moreover, this allele is also found in patients with ATLL. In addition, stromal cell-derived factor 1 +801A 3′ untranslated region, and IL-15 191C alleles have been recently linked to a lower risk for developing HAM/TSP, whereas the promoter TNF-863A allele was associated with higher risk for developing HAM/TSP (102).

5.3. Strain Variations in HTLV-1

There have been several studies attempting to identify viral strains that are uniquely neu- ropathogenic or leukemogenic; however to date, unique viral strains associated with neurologic dis- ease have not been conclusively identified (89,103–107). Most of these studies focused on variation in the HTLV-1 tax gene. In addition to being recognized by HTLV-1-specific CD8

T cells, tax is a strong transactivator of many host genes, such as inflammatory cytokines and their receptors. The role of tax in the pathogenesis of HAM/TSP is not completely understood; however, it is thought that a variation of HTLV-1 tax could cause changes in host immune functions and disease progression.

Based on the high frequency of defective proviruses carrying only part of the tax gene in the CNS

of patients with HAM/TSP, tax was suggested to have a role in the development of the disease (108).

Sequence analysis of HTLV-1 tax in patients with HAM/TSP and asymptomatic carriers revealed that four nucleotide substitutions in the tax gene increase the risk of HAM/TSP (109). Another study of monozygotic twins and their infected mothers and brothers who also had HAM/TSP indicated that three infected individuals had the consensus tax sequence, whereas the asymptomatic twin car- ried sequence variations in five nucleotide positions, including four substitutions resulting in amino acid changes (110). Some patients with ATLL had cells containing HTLV-1 proviral mutants with large deletions of tax or premature stop codons in the 5´ half of tax (111). Renjifo et al. (112) also reported that amino acid substitutions in tax are associated with HAM/TSP, but Mahieux et al. (113) refuted this, reporting that HAM/TSP is simply linked to the cosmopolitan genotype.

Other viral proteins are also thought to contribute to the diversity in diseases caused by HTLV-1.

HTLV-1 accessory protein p12

, which is a product of the pX open reading frame I (ORF-I), is associated with immature forms of the MHC class I, interferes with the interaction of MHC class I with β

-microglobulin, decreases the surface expression of transfected MHC-I, directs its degra- dation in the proteasome, and is coexpressed in Hela-Tat cells (114,115). These results suggest that p12

might help the virus escape immune surveillance by downregulating MHC class I sur- face expression. Interestingly, proteasome destabilization of viral proteins is considered to be an intracellular defense mechanism against viral infection (116,117). Lysine is a known target for covalent binding of ubiquitin (118), and the metabolic instability of p12

is mediated in part by ubiquitylation at a single lysine residue at position 88 and subsequent proteasomal degradation, as well as by destabilizing residues at its amino terminus (119).

Interestingly, earlier analysis of p12

ORF in 21 HTLV-1 strains from different geographical areas (120) had demonstrated that the p12

amino acid sequence is highly conserved and that an arginine residue is found more frequently at position 88, whereas the less frequent lysine-carrying allele was found only in some HAM/TSP cases. Trovato et al. (119) extended this information by studying an additional 32 ex vivo samples from healthy carriers, patients with HAM/TSP or ATLL, and families in which both diseases occur, and confirmed that the lysine residue is found only in patients with TSP/HAM, regardless of geographical locations, suggesting that a selective pressure over p12

might occur in the host; however, lysine residue at position 88 in p12

was not found in all patients with HAM/TSP. It is hypothesized that the reduced stability of p12

in patients with HAM/TSP caused by this sequence variation may facilitate generation of a viral-specific CTL response, since degradation of p12

would alleviate the reduction of MHC class I molecules at the cell surface.

This lysine residue at position 88 of p12

did not appear to be a universal diagnostic marker for HTLV-1-associated neurological disease, since this phenotype was found not only in patients with HAM/TSP patients but also in asymptomatic HTLV-1 carriers who did not develop neurologic signs (121). A limitation of most studies is the analysis of samples from individuals who were born in the same geographic region, and thus, these findings might simply represent a particular HTLV-1 carrier population in which the selective pressure on the p12

sequence would not occur. Therefore, the significance of natural p12

alleles is unclear, and it is possible that the lysine at position 88 of p12

might have a significant effect on the biological effects of the protein in the host, including giv- ing a possible selective advantage in individuals with a certain MHC class I. Thus, to date the role of HTLV-1 sequence variation does not appear to be a significant factor in the pathogenesis of HAM/TSP, and most evidence indicates host immunologic factors are more important determinants of HTLV-1-associated neurological disease.

5.4. Route of Infection

HTLV-1 and HTLV-2 are highly cell-associated viruses and are transmitted by routes that favor

preservation of infected cells. Consequently, these viruses are transmitted by exposure to whole

blood or blood products that contain intact cells, intravenous drug use, sexual transmission (pre-

dominantly from male seminal fluid), and breastfeeding (90,122). Exposure to HTLV-1 via blood

transfusion has been associated with the development of HAM/TSP (123), whereas mucosal exposure

has been linked to the development of ATLL (122,124). In a rat model of HTLV-1 infection, oral inoculation of HTLV-1-infected cells resulted in immune unresponsiveness in spite of persistent HTLV-1 infection (125). Oral vs intravenous routes of infection in rats has revealed differences in levels of anti-HTLV-1 antibodies and proliferative T-cell response against the virus, indicating that oral exposure may result in a reduced antiviral immune response (73). From these data, investigators have hypothesized that the absence of an effective T-cell response in subjects exposed via mucosal or oral routes might lead to survival and subsequent transformation of the infected cell population, resulting in ATLL. On the other hand, intravenous inoculation may result in a vigorous immune response that, in the process of elimination of cells infected with HTLV-1, may promote inflammation in the CNS typical of HAM/TSP (33). Collectively, these studies suggest that the route of infection, in particular intravenous inoculation of HTLV-1-infected cells, influences the immune response gener- ated against the virus and contributes to the incidence of HAM/TSP in patients exposed by this route.

5.5. The Host Immune Response to HTLV-1

Patients with HAM/TSP exhibit 10 to 100-fold increases in proviral DNA in their peripheral blood, compared to asymptomatic, but infected, subjects (90,126). Furthermore, when an infected subject’s proviral load is more than 1% of PBMCs, there is higher risk of HAM/TSP (11), although HAM/TSP occurs in some patients with a low proviral DNA load, indicating that a high proviral DNA load by itself is not a conclusive predictor for the development of HAM/TSP (11).

Most studies support the hypothesis that the progression to HAM/TSP is influenced by an interac- tion of host factors, including the immune response, viral factors, and high proviral DNA load.

Typically, T-cell transformation is a feature of ATLL, whereas cellular destruction and inflamma- tion are a feature of HAM/TSP. These distinctive characteristics might account for the fact that there are very few patients with both ATLL and HAM/TSP. Similarly, these diseases differ in various immunologic parameters related to HTLV-1 infection. Although CD4

T cells, CD8

T cells, and activated macrophages are present within white-matter lesions of patients with HAM/ TSP (127), leukemic cells in ATLL are mostly CD4

T cells and occasionally are CD4/CD8 double-positive (17,18). Peripheral blood and the CSF of patients with HAM/TSP have high levels of proinflam- matory cytokines, such as IFN- γ, TNF-α, IL-1, and IL-6, suggesting the importance of these proinflammatory cytokines in HAM/TSP (128–131). Additionally, CSF of these patients contains activated lymphocytes, suggesting that activated lymphocytes and macrophages also have a role in the development of HAM/TSP (127).

Patients with HAM/TSP develop a vigorous expansion of CD8

T cells, compared to asymp- tomatic carriers (132) and HTLV-1 tax-specific CTLs are readily demonstrated in the CSF of these patients (133). In fact, patients with HAM/TSP have about 40 to 280-fold more tax-specific precur- sor CTLs than do asymptomatic carriers, consistent with an increase in virus-specific CD8

CTL response in these patients. Additionally, patients with HAM/TSP have high levels of anti-HTLV-1 antibodies in serum and CSF (134). Studies of peripheral blood and CSF from the same patient with HAM/TSP have revealed tax-specific CTLs to be about twofold higher in the CSF, suggesting the possible role of tax-specific CTLs in the cellular destruction and inflammation in the CNS of these patients (133). Activated CTLs are present in active inflammatory lesions in the spinal cords of patients with HAM/TSP (92,135). The mechanism of cell destruction by tax-specific CTLs is not elucidated; however, it is proposed that CTLs could directly kill not only HTLV-1-infected cells expressing tax but also CNS cells expressing a crossreactive cellular determinant and neighboring uninfected neuronal cells by apoptosis mediated via the toxic inflammatory cytokines (18).

6. VIRAL ENTRY INTO THE CENTRAL NERVOUS SYSTEM

HTLV-1-infected cells are present in the CNS of patients with HAM/TSP. These patients also

have higher HTLV-1 proviral loads in the CSF than in their PBMCs (136). The HTLV-1 integration

site in the cellular DNA of HTLV-1-infected lymphocytes was the same in the CSF and peripheral

blood of these patients, indicating that HTLV-1-infected cells in peripheral blood migrate to the CNS and accumulate in lesions in patients with HAM/TSP (137,138). The mechanism of selective migration of HTLV-1-infected cells to the CNS of these patients is not clear. CD4

T cells from patients with HAM/TSP have an enhanced ability to attach and transmigrate through reconstituted basement membranes in vitro and endothelial cells, compared with CD4

T cells from asymp- tomatic carriers (139,140), indicating that these cells likely have more ability to migrate into the CNS across the blood–brain barrier (BBB). Additionally, close contact between brain endothelial cells and HTLV-1-infected CD4

T cells causes increased production of TNF- α, likely increasing the paracellular permeability of the brain endothelial monolayer and transmigration of activated T cells and monocytes across the BBB. Romero et al. (141) demonstrated that viral particles were taken up into vesicles by brain endothelial cells and released from the basolateral surface, whereas HTLV-1-infected T cells fuse to brain endothelial cells. Based on these observations, mechanisms proposed for the entry of HTLV-1 into the CNS of patients with HAM/TSP include paracellular transmigration of virus-infected cells through the BBB, absorption and release of viral particles from vesicles within brain endothelial cells, and direct infection of brain endothelial cells (127).

Adhesion molecules are considered critical for this migration, correlating with the increased expression of vascular cell adhesion molecule-1 expression in spinal cord lesions (142) and very late antigen-4 (VLA-4), as well as monocyte chemoattractant protein-1 in perivascular inflamma- tory infiltrates and matrix metalloproteinase (MMP)-2 and MMP-9 in infiltrating mononuclear cells of HAM/TSP patients (143). In addition, α4β1 and α5β1 integrins appear to be overexpressed in HTLV-1-infected T cells of patients with HAM/TSP (144).

After entering the CNS, HTLV-1-infected CD4

and CD8

T cells are believed to participate in a number of events, resulting in infection of resident CNS cell populations, activation of astro- cytes and microglial cells, induction of proinflammatory cytokine and chemokine synthesis, recruitment of inflammatory infiltrates into the CNS, BBB disruption, dysregulation of oligoden- drocyte homeostasis, demyelination, and axonal degradation (145). Within the CNS, astrocytes and microglial cells are susceptible to HTLV-1 infection (43,146,147), presumably serving to pro- mote increased production of proinflammatory cytokines and chemokines and to recruit of antigen- specific CD4

and CD8

T cells from the circulation.

7. MECHANISM OF VIRAL PERSISTENCE, DESPITE A STRONG IMMUNE RESPONSE

Among the many characteristics of HTLV-1 that influence viral persistence, one important pecu-

liarity of this virus is its low rate of evolution and low sequence diversity, in contrast to human

immunodeficiency virus-1. A number of mechanisms have been postulated to explain how this

retrovirus maintains persistence yet elicits a prominent immune response in infected individuals. In

this regard, it is important to understand that the virus may be spread either by infectiously (primarily

from infected cells) or by mitotic division of infected cells (replication of the integrated provirus

during cell division) (148), although based on the relatively lower level of variability in sequence,

HTLV-1 is thought to be maintained mainly by mitotic transmission and proliferation of cells har-

boring the provirus, since this route is associated with a much lower rate of mutation (17) than infec-

tious transmission. In support of this hypothesis, clonal expansion of infected CD4

and CD8

T-cell

populations have been observed in patients with HAM/TSP (137,148,149). In contrast and perhaps

not mutually exclusive are observations that some patients form a persistent and vigorous CTL

response against HTLV-1 tax, and this accounts for reduced proviral loads and protection against

HAM/TSP; these studies suggest the existence of active viral gene expression in vivo (89). Based upon

positive responses to treatments with the nucleoside analogue lamivudine (3TC), a reverse transcrip-

tase inhibitor, Taylor et al. (150) has speculated that HTLV-1 must undergo reverse transcription and

maintain tax expression to elicit a high frequency of tax-specific CD8

T cells. Recent studies suggest

that an elevated proportion of HTLV-1-infected cells (10–80%) express tax within 12 h of infection

and thus stimulate a high frequency of tax-specific CTLs (17,89). These in turn are stimulated to divide rapidly but are made highly susceptible to tax-specific CTL-mediated lysis. The virus cannot complete the replication cycle because of limited availability of activated susceptible host cells and the rapid CTL-mediated lysis; thus, mitotic spread might be responsible for the maintenance of the proviral load, possibly accounting for the low rate of mutation and evolution (17). Furthermore, this sequence of events would also account for the immunodominance of tax as a target antigen for CTLs, since tax-expressing cells appear to be susceptible to CTL lysis before expressing other viral proteins. Since tax is essential for the maintenance of proviral load, generation of strong CTL response to tax possibly lowers proviral load and susceptibility to HAM/TSP (89). Thus, host factors may determine the strength or efficiency of CTL response to HTLV-1 and an individual’s proviral load and their risk of HAM/TSP. Equally likely in this balance is the tenet that CTLs might con- tribute to the inflammation in HAM/TSP (90,151). The concentration of antigen necessary to elicit IFN- γ secretion by a CD8

T cell is greater than for target cell lysis. Based upon this immunological principal, it has been suggested that a patients balance between an active CTL response and control of virus replication vs an overactive immune response with IFN- γ secretion that promotes inflammation is a key determinant of whether a patient develops HAM/TSP (89).

8. PROPOSED MECHANISMS OF PATHOGENESIS

Immunologic mechanisms are generally considered to have a significant role in the pathogenesis of HAM/TSP. This belief is strongly supported by studies indicating that patients with HAM/TSP have elevated cellular and humoral anti-HTLV-1 immune responses in comparison to asymptomatic carriers and seronegative controls. The virus predominantly targets CD4

T cells (152–155), which have increased adhesion activity to endothelial cells, promoting transmigrating activity through base- ment membranes (156) and secreting proinflammatory cytokines, such as IFN- γ and TNF-α (157).

Both the peripheral blood and CSF of patients with HAM/TSP contain HTLV-1-specific HLA class I-restricted CD8

CTLs (100), with activity mostly against pX gene products, especially the Tax 11–19 peptide (LLFGYPVYV) (158). With spinal cord lesions, the presence of CD8

T cells, presumably in response to CD4

T cells expressing HTLV-1 proteins, suggests a direct involvement of these cells in lesion development (37,41). These HTLV-1-specific CD8

CTLs most likely mediate target cell destruction through perforin-dependent mechanisms or the production of inflammatory cytokines and chemoattractants (159,160), although the mechanism of maintaining these CD8

T cells during the prolonged course of infection is not certain. Although IL-15 may have a role in the disease (161), it is also thought that viral antigens continuously drive these cells in vivo (91).

An autoimmune mechanism has also been proposed to be involved in the pathogenesis of HAM/TSP, as in inflammatory neurological diseases, such as multiple sclerosis (MS) and Guillain- Barre syndrome. In support of this hypothesis, researchers have demonstrated that infiltrating lymphocytes in the spinal cord lesions of patients with HAM/TSP have a unique T-cell receptor CDR3 motif, which is also found in brain lesions in patients with MS and experimental autoimmune encephalomyelitis (162). An HTLV-1-infected CD4

T-cell clone derived from the peripheral blood of a patient with HAM/TSP showed a proliferating response to crude protein extracted from seronegative spinal cord samples but not to proteins from lymph nodes (11), whereas the HTLV-1 tax-specific CD8

CTL clones recognized and lysed self-peptide-pulsed target cells in vitro (163).

Another hypothesis couples infection with autoimmune disease. Molecular mimicry has also

been implicated in the pathogenesis of diseases, such as diabetes, lupus, and MS, in which there is

little direct evidence linking causative agents to pathogenic immune reactions. Molecular

mimicry is characterized by an immune response to an environmental agent that crossreacts with

a host antigen, resulting in disease. The clinical presentation of HAM/TSP is often difficult to dis-

tinguish from MS. In this regard, serum immunoglobulin (IgG) from HAM/TSP patients reacted

to neurons in human CNS not infected with HTLV-1 but not to cells in the peripheral nervous sys-

tem or other organs, and more importantly, the reactivity could be abrogated by pretreatment with

recombinant tax protein (164). Interestingly, IgG from patients with HAM/TSP identified hetero- geneous nuclear ribonuclear protein-A1 as the autoantigen (164). It was hypothesized that axons are preferentially damaged by such autoantibodies, which inhibits neuronal firing.

9. TREATMENT AND PREVENTION

To date, effective treatments against the devastating neurological complications of HAM/TSP or preventive measures to stop the development of the disease in HTLV-1-infected patients have had limited success. Interventions with corticosteroids, plasmapheresis, IFN, and antiretroviral drugs have been used as treatments in patients with HAM/TSP with poor or inconsistent results, although virological and clinical improvement has been obtained patients with HAM/TSP who were treated with zidovudine and/or lamivudine (150). Inhibition of HTLV reverse transcriptase, along with the cytostatic effect of some nucleoside analogues, including zidovudine, is thought to reduce the virus’ replication, as well as the proviral load; however, the clinical consequences of this need further examination (165). Based on the finding that MMPs, specifically MMP-2 and MMP-9, have important roles in the breakdown of the BBB in the CNS of patients with HAM/TSP, N-biphenyl sulphonyl-phenylalanine hydroxamic acid, a selective inhibitor of MMP-2 and MMP-9, was recently used tested as an inhibitor of the migrating activity of CD4

T cells in patients with HAM/TSP and was found effective, suggesting its therapeutic potential against HAM/TSP (166). In the past, some cases of TSP have responded to steroids, such as prednisone and danazol. These are generally considered useful in the clinical management of patients, partic- ularly females, with HAM/TSP (167). Prednisolone, IFN- α, fosfomycin, high-dose vitamin C, blood purification therapy, heparin, sulfapyridine, thyrotropin-releasing hormone, erythromycin, and mizoribine have been found useful in improving motor disability in patients with HAM/TSP, although success rates vary (168,169). Rolipram, a phosphodiesterase type IV inhibitor was demonstrated to inhibit TNF- α production in HTLV-1-infected cells lines and peripheral blood cells of patients with HAM/TSP, to variable degrees (168,170). Overall, an effective and long-term therapeutic protocol or specific drug that treats HAM/TSP is lacking.

Multiple factors favor development of a vaccine against HTLV-1: the virus displays relatively low antigenic variability, natural immunity occurs in humans, and experimental vaccination with the envelope antigen has been successful in animal models (171–173). There have been sugges- tions that gene therapy to control virus replication may be useful against HAM/TSP (174), although, thus far, there is no successful gene therapy or vaccine for HAM/TSP, owing in part to the lack of animal models that can be used for testing. In addition, the low incidence and unpre- dictability of onset of clinical signs of the disease among those infected with the virus makes clinical trial accrual problematic.

10. CONCLUSION AND FUTURE DIRECTIONS

It is critical that future studies focus on therapeutic and prevention strategies of HTLV-1- associated diseases. Although the clinical symptoms of these diseases are well characterized, the pathogenesis is not clearly understood. Our understanding of the risk factors for HAM/TSP has increased tremendously, and many theories have been proposed concerning pathogenic mecha- nisms of the disease. Many of these theories on pathogenesis and immune response are contradictory, and additional studies are necessary to elucidate this apparent dichotomy in our understanding.

Nevertheless, the knowledge of risk factors and possible pathogenesis mechanisms should enable us, in the near future, to predict the risk of progression from asymptomatic carrier stage to HAM/TSP. Future studies directed toward understanding the role of cell-mediated immunity in HTLV-1 infection will likely lead to further clarification of the pathogenesis of HAM/TSP.

This knowledge, in turn, will provide valuable directions to develop efficient therapeutic and pre-

ventive strategies against HAM/TSP. Recent studies have provided new information about repli-

cation strategies used by the virus to regulate its gene expression and will undoubtedly help in the

effort to understand the role of viral-mediated events vs immune-mediated damage in HAM/TSP.

Additional studies of the transcriptional regulation of the long terminal repeat during cell activa- tion should facilitate the design of therapeutic strategies to prevent the initiation of viral gene expression within the peripheral blood and CNS. Studies of proviral DNA and viral gene expres- sion in the peripheral blood and CNS during clinical latency and HAM/TSP would also provide important information.

There has been great progress in the development of new animal models that exhibit many of the pathological features associated with HAM/TSP and ATLL, and future studies to refine these animal models would clarify the early events that take place after initial exposure of the host to HTLV-1 and the kinetics of viral gene expression in various cell types and stages of disease development.

Gene array studies to identify patterns of gene expression in various stages in asymptomatic carriers and patients with ATLL or HAM/TSP would provide valuable insight into the pathogene- sis of these diseases. The ultimate goal of these studies is to prevent the onset of the devastating neurological diseases associated with HTLV-1 infection.

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