Cystatin C, alias post-gamma-globulin: a marker for multiple sclerosis?

Cystatin C, alias post-gamma-globulin or gamma-trace protein, has been shown to be a potent inhibitor of cysteine proteinases; this protein is normally present in different biological fluids, but particularly so in cerebrospinal fluid. The concentration of cystatin C was determined by radial immunodiffusion in cerebrospinal fluid from patients affected with multiple sclerosis, patients affected with various neurological diseases and in controls; it was also determined in brain tissue from 2 patients affected with multiple sclerosis and 3 control brains. Cystatin C cerebrospinal fluid levels were undetectable or depressed in many multiple sclerosis cases and the median value differed significantly from the control one. Its low concentration in multiple sclerosis suggests that the regulation of cysteine proteinases is impaired in this disease; hence enhanced activity of cysteine proteinases could initiate, or increase the breakdown of myelin. Although it is perhaps a little premature to consider cystatin C as a marker for multiple sclerosis, this protein is nevertheless associated to demyelination; consequently its biochemical assay in cerebrospinal fluid is recommended as a complementary diagnostic tool.     

HTLV-I/II prevalence in different geographic locations

Human T-cell lymphotropic virus (HTLV) type I (HTLV-I) is the etiological agent of adult T-cell leukemia and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-II is a closely related virus, and this infection is not clearly associated with clinical disease, although neurologic disorders are observed resembling HAM/TSP. Prevalence rates for HTLV-I infection in the general population are greater than 1% in the Caribbean Basin, Central Africa, and South Japan. In most other areas in the world, as far as we know, HTLV-I/II infections are mainly found in high-risk groups (ie, immigrants from endemic areas, their offspring, their sexual contacts and in patients and intravenous injection users attending sexually transmitted disease clinics). Also, a high rate of infection for both HTLV-I and HTLV-II infection was observed in the native Amerindian population in North America as well as South America. Blood donors are routinely screened for HTLV-I/II in North America, several countries in Europe, Japan, and Taiwan.     

Clonal expansion of CD8+ cytotoxic T lymphocytes against human T cell lymphotropic virus type I (HTLV-I) genome products in HTLV-I-associated myelopathy/tropical spastic paraparesis patients.

Short-term culture of peripheral blood mononuclear cells (PBMC) derived from patients with human T cell lymphotropic virus type I-associated myelopathy (HAM)/tropical spastic paraparesis resulted in dominance by DR+ activated CD8+ T cells. Variations in the T cell receptor (TCR) V alpha and V beta chains in these cells were analyzed, and in all 10 patients examined, 2-3 V gene families were dominant in both TCR V alpha and V beta. In five patients we examined, cultured lymphocytes contained cytotoxic lymphocytes for p40tax (patients HAM2, 3, 7, and 8) or env protein (patient HAM4) of human T lymphotropic virus type I. In patients HAM2 and HAM8, cultured lymphocytes contained a large proportion of V beta 8+ CD8+ and/or V beta 12+ CD8+ cells. The sequence of V beta 8+ and V beta 12+ cDNA revealed that they were oligoclonal with identical or similar sequences in each patient. Elimination experiments with monoclonal antibodies for TCR V beta 8 and V beta 12 showed that they were CD8+ cytotoxic T lymphocytes (CTL) for p40tax. In addition, flow cytometry and sequencing analysis of uncultured PBMC revealed that in HAM2, V beta 8+ CTL and their precursors account for 7% and V beta 12+ CTL and their precursors account for 18% of total CD8+ cells. This indicates the presence of two markedly expanded clones in vivo. No common dominant TCR V alpha or V beta were observed among 10 HAM patients analyzed.     

Candidate Polyanionic Microbicides Inhibit Human T-Cell Lymphotropic Virus Type 1 Receptor Interactions, Cell-Free Infection, and Cell-Cell Spread

The human T-cell lymphotropic virus type 1 (HTLV-1) is the cause of adult T-cell leukemia and inflammatory diseases including HTLV-1-associated myelopathy/tropical spastic paraparesis. HTLV-1 can be transmitted through sexual contact, mother-to-child transmission, and exposure to contaminated blood. Microbicides are agents that interfere with microbial infectivity at mucous membranes, and candidates are under development for use against sexually transmitted viruses such as human immunodeficiency virus type 1. We previously demonstrated that cell surface polyanionic heparan sulfate proteoglycans bind the HTLV-1 envelope glycoprotein surface subunit gp46, facilitating cell-cell and cell-free virus spread in vitro. We now show, using assays for Env-receptor binding inhibition, Env-induced cell-cell fusion, cell-cell virus spread, and pseudotype HTLV-1 infectivity, that the soluble polyanions PRO 2000 and dextran sulfate are potent inhibitors of HTLV-1 spread in vitro, with PRO 2000 being the more promising candidate. The results of these studies suggest that candidate topical microbicides may be of use in reducing HTLV-1 sexual transmission.     

Human T-cell lymphotrophic virus type-I (HTLV-I) retrovirus and human disease

Human T-cell lymphotrophic virus type-I (HTLV-I) was the first pathogenic retrovirus identified in humans. HTLV-I is now linked to a number of clinical diseases, most notably adult T-cell leukemia/lymphoma and the syndrome known as HTLV-associated myelopathy or tropical spastic paraparesis (HAM/TSP). For the emergency physician practicing among patients from high-risk groups, HTLV-I infection and its associated diseases are presenting an increasing challenge. This report describes its transmission, seroprevalence, associated diseases, and methods to control the spread of this retrovirus.     

A tax on luxury: HTLV-I infection of CD4+CD25+ Tregs

Almost a quarter of a century ago, Oldstone and colleagues proposed that infection of cells by noncytopathic viruses may lead to an alteration of the cells' ability to produce certain products or perform certain tasks, i.e., inhibition of "luxury function." In this issue of the JCI, this topic has been revisited by Yamano et al., who demonstrate that human T cell lymphotropic virus type I (HTLV-I) infection of CD4(+)CD25(+) Tregs in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) results in a decrease in FOXP3 mRNA and protein expression. This leads to the inability of HTLV-I-infected CD4(+)CD25(+) Tregs to inhibit the proliferation of CD4(+)CD25(-) Tregs, due to the effect of the HTLV-I tax gene. Defects in the Treg population could be responsible for the large numbers of virus-specific T cells and occurrence of lymphoproliferation and inflammatory autoimmune disease in HAM/TSP patients.     

Global epidemic of human T-cell lymphotropic virus type-I (HTLV-I)

Infection with human T-cell lymphotrophic virus-I (HTLV-I) is now a global epidemic, affecting 10 million to 20 million people. This virus has been linked to life-threatening, incurable diseases: adult T-cell leukemia/lymphoma (ATLL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The cumulative lifetime risk of developing these incurable diseases is approximately 5% in asymptomatic patients. For the emergency physician practicing among patients from high-risk groups, HTLV-I and its associated diseases are presenting an increasing challenge. This report describes its transmission, seroprevalence, treatment, and methods of controlling spread of this retrovirus. Coinfection with HTLV-I and HIV has been shown to accelerate the progression of acquired immune deficiency syndrome (AIDS).     

Human T-cell leukemia virus type I (HTLV-I) and blood transfusion

Human T-cell leukemia (or T-lymphotropic) virus type I (HTLV-I) is a human exogenous infectious retrovirus of the family Retroviridae. This virus has been associated with adult T-cell leukemia and endemic myelopathies (tropical spastic paraparesis and HTLV-I associated myelopathy). HTLV-I is transmitted by sexual contact, from mother to child, by intravenous drug abuse, and by blood transfusion. The estimated lifetime risk of developing disease in antibody-positive patients is 1 in 80, and a latency period as long as 20 years can intervene. No case of transfusion-transmitted disease has been reported to date. Currently, no testing of blood donors for HTLV-I is required in the United States, and no such test has been approved by the Food and Drug Administration. Because data on the natural history of this virus may take years to accumulate, it is probably wise to begin excluding anti-HTLV-I-positive units from the blood supply in the United States as soon as a licensed test is available.     

Myelin basic protein, oligoclonal bands, and IgG in cerebrospinal fluid as indicators of multiple sclerosis

There currently are three clinical laboratory procedures for use with cerebrospinal fluid that assist in the diagnosis of multiple sclerosis: measurement of myelin basic protein and IgG, and demonstration of an oligoclonal band. We compared characteristics of these procedures, using CSF samples from 166 patients identified as having (54 patients) or not having (112 patients) multiple sclerosis. We find that oligoclonal band demonstration is the most useful single test in helping to establish the presence of multiple sclerosis; IgG quantitation is the least helpful. Myelin basic protein should be quantitated for following the activity of multiple sclerosis; it may be applied only selectively in the context of screening. The incidence of false-positive results reinforces the view that the diagnosis of multiple sclerosis must be made in clinical context. These laboratory procedures are not suitable for use as screening tests.     

An antibody to lymphotoxin and tumor necrosis factor prevents transfer of experimental allergic encephalomyelitis

Uncertainty regarding pathogenic mechanisms has been a major impediment to effective prevention and treatment for human neurologic diseases such as multiple sclerosis, tropical spastic paraparesis, and AIDS demyelinating disease. Here, we implicate lymphotoxin (LT) (tumor necrosis factor beta [TNF-beta]) and TNF-alpha in experimental allergic encephalomyelitis (EAE), a murine model of an autoimmune demyelinating disease. In this communication, we report that treatment of recipient mice with an antibody that neutralizes LT and TNF-alpha prevents transfer of clone-mediated EAE. LNC-8, a myelin basic protein-specific T cell line, produces high levels of LT and TNF-alpha after activation by concanavalin A, antibody to the CD-3 epsilon component of the T cell receptor, or myelin basic protein presented in the context of syngeneic spleen cells. LNC-8 cells transfer clinical signs of EAE. When LNC-8 recipient mice were also treated with TN3.19.12, a monoclonal antibody that neutralizes LT and TNF-alpha, the severity of the transferred EAE was reduced, while control antibodies did not alter the disease. The effect of anti-LT/TNF-alpha treatment was long lived and has been sustained for 5 mo. These findings suggest that LT and TNF-alpha and the T cells that produce them play an important role in EAE.     

Induction of pro-inflammatory cytokines by human T-cell leukemia virus type-1 Tax protein as determined by multiplexed cytokine protein array analyses of human dendritic cells.

Human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is characterized by a hyperstimulated immune response, including elevated levels of inflammatory cytokines/chemokines and oligoclonal expansion of virus-specific CD8(+) T cells in the cerebrospinal fluid. Studies have shown that the HTLV-1 transactivator protein Tax is available for immune recognition by antigen presenting cells (APCs), such as dendritic cells (DCs). DCs are relevant to the pathogenesis of HAM/TSP because the presentation of Tax peptides by activated DCs to na?ve CD8(+) T cells may play an important role in the induction of the Tax-specific immune response that is observed in HAM/TSP. In this study, a human cytokine protein array was used to study the secretion of cytokines by monocyte-derived DCs (MDDCs) exposed to Tax. Of the 16 cytokines analyzed, 6 cytokines were secreted in significantly high amounts (> or =2-fold), including Th1 cytokines (IFN-gamma, IL-12, and TNF-alpha) and C-C chemokines (Eotaxin, MCP-1, and MCP-3). Selected cytokines were further examined at two concentrations of Tax and at two time periods. Furthermore, a transient exposure to Tax did not result in any cytokine production when examined at three different time points after exposure, indicating that a prolonged presence of Tax is required for its activity. Finally, inhibition of the NF-kappaB signaling pathway by specific inhibitors, abrogated Tax-mediated cytokine secretion. Collectively, these findings suggest a role for Tax-induced cytokine secretion from MDDCs, which may be critical for the cellular activation and tissue damage that has been observed in HAM/TSP.     

Clinical and analytical evaluation of an enzyme immunoassay for myelin basic protein in cerebrospinal fluid

BACKGROUND: RIA of myelin basic protein (MBP) in cerebrospinal fluid (CSF) is commonly used as a biochemical marker of demyelination in patients with multiple sclerosis (MS). Our aim was to develop a sufficiently sensitive ELISA for MBP and evaluate it clinically in patients with MS. METHODS: The ELISA used anti-bovine MBP antibody coated on plates and biotinylated anti-MBP antibody. The bound antibody complex was quantified with streptavidin-horseradish peroxidase. MBP was determined in CSF from 84 MS patients and 55 patients with other neurological diseases. RESULTS: The respective within- and between-assay CVs were 4.7% and 7.2% at 200 ng/L, and 6. 3% and 8.8% at 2000 ng/L. The detection limit was 30 ng/L. Most of the MS patients with acute exacerbations had markedly increased MBP in the CSF. Longitudinal studies of six MS patients with recurrent exacerbation confirmed this observation. MBP concentrations from 78 MS patients, as tested with our ELISA, correlated well with those obtained by RIA (r = 0.9; P: <0.01), but the detection limit of the ELISA was much lower than that of the RIA. CONCLUSIONS: This convenient ELISA with higher sensitivity than the existing assays is a suitable routine assay that provides a diagnostic indicator of myelin breakdown in the central nervous system; moreover, it is an excellent indicator of MS disease activity.     

Paraplegia and quadriplegia after intrathecal chemotherapy

Transient or permanent paraplegia after the use of intrathecal (IT) methotrexate (MTX) or cytosine arabinoside (Ara-C) for treatment or prophylaxis of patients with meningeal leukemia is an unusual complication, with an incidence of less than 3% among such patients. Only 15 cases involving IT MTX have been documented and even fewer with IT Ara-C. Three patients were studied who developed permanent or ascending myelopathy from treatment of their leukemia or rhabdomyosarcoma with IT chemotherapy. The patients' ages ranged from 7 to 62 years. Two of the three patients had electromyographic examinations. These revealed a primary motor neuron degeneration or a polyradiculopathy, superimposed on a mild axonal peripheral neuropathy associated with vincristine therapy. This is consistent with other electromyographic studies. Two of the patients showed an elevation of the cerebral spinal fluid (CSF) protein before development of paraplegia; one also showed a rise in myelin basic protein associated with his myelopathy. Neuropathologic findings suggest demyelination as the primary process leading to myelopathy. Increasing evidence has shown that total CSF protein, or more specifically, the myelin basic protein, may be elevated before development of paraplegia. Routine serial testing of the CSF for total protein could be used as a screening test during therapy.     

Detection of human T lymphotrophic virus type I (HTLV-I) proviral DNA and analysis of T cell receptor V beta CDR3 sequences in spinal cord lesions of HTLV-I-associated myelopathy/tropical spastic paraparesis

Identification of the localization of human T lymphotrophic virus type I (HTLV-I) proviral DNA in the central nervous system (CNS) is crucial to the understanding of the pathogenesis of HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) pathogenesis. We have developed a sensitive detection method, called two-step polymerase chain reaction (PCR) in situ hybridization, which enabled us to detect the HTLV-I proviral DNA in paraffin-embedded spinal cord tissue sections from HAM/TSP patients. HTLV-I proviral DNA was detected only in the nucleus of lymphocytes that had infiltrated into the spinal cord. However, no proviral DNA was amplified in any neuronal cells, including neurons and glial cells. This indicates that the demyelination of the spinal cord by HTLV-I as a result of viral infection of oligodendrocytes or neuronal cells is unlikely. The T cell receptor V beta gene sequence from lymphocytes in the spinal cord lesions taken from the same HAM/TSP autopsy cases revealed unique and restricted CDR3 motifs, CASSLXG(G) (one-letter amino acid. X is any amino acid), CASSPT(G), and CASSGRL which are similar to those described in T cells from brain lesions of multiple sclerosis (MS) and in a rat T cell clone derived from experimental allergic encephalomyelitis (EAE) lesions. The present results suggest that T cells containing restricted V beta CDR3 motifs, which are also found in MS and EAE, become activated upon HTLV-I infection and infiltrate into the spinal cord lesions of HAM/TSP patients.     

Silica gel radioimmunoassay for myelin basic protein.

1. A radioimmunoassay which relies upon the capacity of silica gel to absorb free human basic protein of myelin (BP) is described. 2. Reagents involved in the assay include unlabelled BP, I-BP, and rabbit anti-BP. The assay is performed in excess normal rabbit serum, and uses 3-4% silica gel for absorption of free I-BP. 3. The assay is simple, reproducible and not particularly time-consuming. It is capable of detecting BP at a level of 1-10 or more nanograms in samples containing varying amounts of serum. The assay is thus highly suitable as a routine test in clinical laboratories interested in detecting BP in cerebrospinal fluid in patients with suspected or proven demyelination.     

Infectious agents as the triggers for the pathogenesis of the neuroimmunological disorders

The development of the neuroimmunological disorders is regulated by the environmental factors in connection with the genetic predisposition. Of the environmental factors, infectious agents play a crucial role as the triggers for the pathogenesis of these disorders. Although specific infectious agents are identified in only HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and some parts of Guillain-Barré syndrome (GBS), both the epidemiological and the experimental evidences with regard to infectious agents as the triggers are accumulated even in another disorders, such as multiple sclerosis (MS) and inflammatory myopathies (IM). Several hypotheses, such as bystander mechanism and molecular mimicry of some neuromuscular components, are proposed in the pathogenesis of these diseases. We, herein, discuss about the relationship between infectious agents and these diseases.     

Autoreactive T lymphocytes in multiple sclerosis determined by antigen-induced secretion of interferon-gamma.

Multiple sclerosis (MS) is a disease with unknown cause characterized by inflammation and demyelination in the central nervous system. Although an autoimmune pathogenesis has been suggested, there are no conclusive data on the number of T cells autoreactive with myelin antigens in MS compared to controls. We showed that T lymphocytes secreting interferon-gamma in response to possible target autoantigens are severalfold more common among PBL mononuclear cells in patients with MS than in patients with aseptic meningitis and tension headache. On average T cells reactive with myelin basic protein (MBP), two different MBP peptides, or with proteolipid protein amounted to 2.7-5.2/10(5) PBL from MS patients. MBP-reactive T cells were still more frequent among mononuclear cells isolated from the cerebrospinal fluid (CSF; 185/10(5) CSF cells). We concluded that T cells reactive with myelin autoantigens are strongly increased in MS. This approach to detect them could allow definition of immunodominant T cell epitopes in individual MS patients, and thereby enable further development towards specific immunotherapy.     

Efficacy of donor screening for HTLV-I and the natural history of transfusion-transmitted infection

BACKGROUND: It has been 10 years since the implementation in Japan of donor blood screening for human T-cell lymphotropic virus type I (HTLV-I). This report reviews the effectiveness of screening in preventing transmission of HTLV-I through blood transfusion and the current status of patients with confirmed seroconversion due to transfusions given before the implementation of screening. STUDY DESIGN AND METHODS: Patients who received blood at Kyushu University Hospital from 1990 to 1997 were followed. Serum samples were collected before transfusion and 60 days or more after transfusion. Seroconversion was determined by a second-generation particle agglutination test. Confirmation tests were an immunofluorescence assay, enzyme-linked immunosorbent assay, and immunoblotting. Confirmed seroconverted patients were followed by a search of hospital records. RESULTS: Seroconversion was found in one of 4672 transfused patients, but the donor was identified and confirmed to be negative for anti-HTLV-I and virus genome by nested polymerase chain reaction. A total of 23,323 red cell concentrates and 17,237 platelet concentrates were transfused to these 4672 patients. Therefore, the anti-HTLV-I prevalence in blood for transfusion after screening was estimated at 1 in 45,560 (0.0022%; the upper 95% CI was 0.0080%). One hundred two seroconverted patients who were transfused before donor screening for HTLV-I were followed. One patient developed HTLV-I-associated myelopathy, diagnosed 18 weeks after seroconversion, and another patient developed uveitis 1 month after seroconversion. No patients developed adult T-cell lymphoma, and the survival rate of seroconverted patients was 92.5 percent 15 years after transfusion. CONCLUSION: This study confirmed that the present donor screening program for HTLV-I by the new particle agglutination test can almost completely prevent virus transmission by transfusion. Complications of HTLV-I transmission were at lower rates than expected.     

The current and future approaches to the treatment of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP)

Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that is the causative agent of a progressive neurological disease termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HAM/TSP is a chronic neuroinflammatory disease characterized by spastic paraparesis, lower limb sensory disturbance and bladder/bowel dysfunction. Over the twenty-five years since the discovery of this disease, significant advancements have been made in the pathogenic mechanisms associated with HAM/TSP, however, little progress has been made in the treatment of this disorder. This review highlights the natural history of HAM/TSP, informative results of clinical trials, and discusses the current and future approaches to the treatment of HAM/TSP within the context of our understanding of the underlying pathogenic mechanisms.