Constitutive expression of parathyroid hormone-related protein gene in human T cell leukemia virus type 1 (HTLV-1) carriers and adult T cell leukemia patients that can be trans-activated by HTLV-1 tax gene.

Adult T cell leukemia (ATL) is associated with human T cell leukemia virus type 1 (HTLV-1) infection, and almost all ATL patients have the complication of hypercalcemia. To understand the mechanism of the high incidence of hypercalcemia in ATL, we studied the expression of a parathyroid hormone-related protein (PTHrP) gene that has been proposed as a causative factor of hypercalcemia in some solid tumors. The polymerase chain reaction coupled with reverse transcription of mRNA was applied to RNA from peripheral blood mononuclear cells. Cells from all 13 ATL patients examined showed abundant expression of the PTHrP gene, while cells from uninfected normal subjects did not. Significant expression of PTHrP gene was also detected in HTLV-1 carriers without any symptoms and in patients with HTLV-1-associated myelopathy or tropical spastic paraparesis. PTHrP mRNA levels correlated with the number of infected cells that were estimated by the integrated HTLV-1 DNA. These results suggest that HTLV-1-infected cells are expressing the PTHrP gene. This concept was further supported by the finding that the HTLV-1 trans-activator, the tax gene product, caused trans-activation of the PTHrP gene promoter linked to the CAT gene. These observations might explain the general expression of the PTHrP gene in ATL patients and the high incidence of hypercalcemia in ATL.

Detection of human T cell lymphotropic virus type I proviral DNA and its gene expression in synovial cells in chronic inflammatory arthropathy.

To investigate the pathogenesis of human T cell lymphotropic virus type I (HTLV-I)-associated chronic inflammatory arthropathy (HAAP), we sought to detect proviral DNA in the articular lesions. For the detection of proviral DNA, we used the polymerase chain reaction (PCR). Proviral DNA was detected not only in the peripheral blood mononuclear cells (PBMCs) and synovial fluid cells (SFCs), but also in the T lymphocyte-depleted cultured synovial cells (CSCs). These findings suggest that the infection by HTLV-I might occur in vivo in non-T cells. Furthermore, we detected HTLV-I tax1/rex1 messenger RNA in fresh synovial tissues and CSCs but not in fresh PBMCs and fresh SFCs using reverse transcription and PCR. Immunohistochemically, the CSCs from HAAP patients were also shown to express the HTLV-I antigens. These data indicate that HTLV-I in the non-T synovial cells can be transcribed and expressed. Moreover, the sequences of pXII regions in the CSCs demonstrated 97.5-99.4% homology to that in MT-2 cells, HTLV-I-infected cell line. This confirmed that the PCR-amplified bands reflect HTLV-I itself. These results suggest that this organ-specific inflammation can be attributed to non-T cell virus infection in articular lesions.

Abnormal expression of laminin suggests disturbance of sarcolemma-extracellular matrix interaction in Japanese patients with autosomal recessive muscular dystrophy deficient in adhalin.

Dystrophin is associated with several novel sarcolemmal proteins, including a laminin-binding extracellular glycoprotein of 156 kD (alpha-dystroglycan) and a transmembrane glycoprotein of 50 kD (adhalin). Deficiency of adhalin characterizes a severe autosomal recessive muscular dystrophy prevalent in Arabs. Here we report for the first time two mongoloid (Japanese) patients with autosomal recessive muscular dystrophy deficient in adhalin. Interestingly, adhalin was not completely absent and was faintly detectable in a patchy distribution along the sarcolemma in our patients. Although the M and B2 subunits of laminin were preserved, the B1 subunit was greatly reduced in the basal lamina surrounding muscle fibers. Our results raise a possibility that the deficiency of adhalin may be associated with the disturbance of sarcolemma-extracellular matrix interaction leading to sarcolemmal instability.

Mechanomyographic determination of post-activation potentiation in myopathies.

OBJECTIVE: There is a need to provide an index of muscle contractility in evaluating myopathies especially in the clinical setting. This study was conducted to investigate if the mechanomyogram post-activation potentiation (MMG-PAP) can be used as an index of muscle contractile force potentiation (force-PAP), if it differs between normal and myopathic muscles, and if it can reflect abnormalities in muscle fiber anatomy. METHODS: The correlation between MMG-PAP and force-PAP was evaluated in 12 normal subjects after maximum voluntary contraction (MVC) of the biceps brachii muscle. The same method was then applied to study MMG-PAP in 16 patients with myopathies, 16 disease and 25 normal controls. Mean fiber diameters and the proportions of type 1 and 2 fibers in biopsied biceps brachii muscle were determined and compared with MMG-PAP values. RESULTS: There was a significant positive correlation between force-PAP (197 +/- 148%) and MMG-PAP (135 +/- 68%) immediately after MVC (P < 0.05). The mean MMG-PAP in myopathies (66 +/- 53%) was significantly lower than those of the disease (128 +/- 34%; P < 0.005) and normal controls (120 +/- 56%; P < 0.005). Patients with non-dystrophic myopathies, including those with myositis, had significantly lower MMG-PAP values (38 +/- 20%; P < 0.005) than those with muscular dystrophy (148 +/- 23%). MMG-PAP did not clearly correlate with either type 2 fiber atrophy or type 2 fiber disproportion based on muscle biopsy analysis of myopathic patients. CONCLUSIONS: This study shows that MMG-PAP can be used as an index of muscle contractility and that it is significantly lower in non-dystrophic myopathies compared to normal subjects. MMG-PAP does not seem to reflect abnormal muscle fiber anatomy. SIGNIFICANCE: MMG-PAP may become a valuable non-invasive tool in augmenting routine clinical electrophysiologic studies especially in evaluating muscle contractility in myopathies.

Neuropathology of HTLV-1-associated myelopathy (HAM/TSP).

In order to clarify pathogenesis of HAM/TSP, we performed a detailed neuropathologic analysis of seven autopsy patients with HAM/TSP. Inflammatory infiltrates of mononuclear cells and degeneration of myelin and axons were noted in the middle to lower thoracic spinal cords and were continuously extended to the entire spinal cord. Horizontal distribution of inflammatory lesions was symmetric at any spinal levels. Immunohistochemical analysis demonstrated T-cell dominance. The numbers of CD4+ T cells and CD8+ T cells were equally present in patients with shorter clinical course. Apoptosis of helper/inducer T cells were observed in the presence of TIA1+ cytotoxic T cells in these active inflammatory lesions. Inflammatory infiltrates were markedly decreased and CD8+/TIA1- T cells were predominated over CD4+ cells in patients with prolonged clinical course. HTLV-1 proviral DNA amounts in the freshly frozen spinal cord measured by quantitative PCR were well correlated with the numbers of infiltrated CD4+ cells. In situ PCR of HTLV-1 proviral DNA using multi-primary pairs demonstrated the presence of HTLV-1 infected cells exclusively in the mononuclear infiltrates of perivascular areas. From these findings, it is suggested that the target of the inflammatory process seen in HAM/TSP lesions may be HTLV-1 infected CD4+ T cells infiltrating the spinal cord.

Reflux of HTLV-I infected lymphocytes from the privileged compartment(s) to peripheral blood flow in patients with HTLV-I-associated myelopathy.

To understand the mechanisms involved in the pathogenesis of human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), three in vivo phenomena which have been observed in the peripheral blood of patients and differing from that in asymptomatic HTLV-I carriers must be taken into consideration: (a) the presence of increased HTLV-I viral load, (b) a higher immune responsiveness against HTLV-I antigens, and (c) biased nucleotide substitutions in the HTLV-I pX region which indicate a decreased selection pressure for viral amino acid changes. We now propose a hypothesis which focuses on the in vivo dynamics of HTLV-I infected lymphocyte migration and which incorporates these features. In addition, the hypothesis assumes the existence of a deviation in immune surveillance for HTLV-I in the central nervous system (CNS) in spite of the presence of frequent specific immune effectors. We suggest that in the active phase of HAM/TSP, accompanied with or following autoaggressive interactions between infected lymphocytes and immunocompetent cells in the CNS, there is a consequential reflux of the infected lymphocytes to the peripheral blood. The reflux of infected cells would be expected to provide peripheral blood with tissue-derived HTLV-I proviruses which have been indulged and propagated in an immune-privileged site. This process would result in and account for the observed increase in viral load and the substitution bias in HTLV-I sequences in the peripheral blood.

First missense mutations (R388W and R425H) of AMPD1 accompanied with myopathy found in a Japanese patient.

Skeletal muscle AMP deaminase (AMPD: E.C. 3.5.4.6) deficiency is one of the most common inherited defects in the Caucasians, but not in Asians. Although a diagnosis of AMPD1 deficiency is indeed based on the reduced enzymatic activity, its clinical significance is still rather controversial since most subjects are asymptomatic. Alternative splicing of exon 2 in individuals who have inherited this defect is thought to provide a mechanism for phenotypic rescue that may explain the variability of clinical symptoms as we reported earlier. In this report we present the first case with a detectable defect of the AMPD1 gene in a Japanese patient with myopathy. Two missense mutations (R388W and R425H) in exon 9 and exon 10 of the AMPD1 gene were found. Prokaryotic expression showed a comparable amount of the AMPD1 peptides and undetectable AMPD activity in the constructs with these mutations. From this study, we have concluded that this patient is a compound heterozygote for AMPD1 mutant allele. This study also demonstrates the first reported instance of detectable dysfunction of the AMPD1 gene product, suggesting that AMPD1 indeed has a key role in muscle metabolism and function.

Cytokine expression in the spinal cord lesions in HTLV-I-associated myelopathy.

Immunocytochemical staining of spinal cords from five autopsied patients with HTLV-I-associated myelopathy/tropical spastic paraparesis was performed using a panel of monoclonal or polyclonal antibodies reactive with interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor (TNF)-alpha, interferon (IFN)-alpha, IFN-beta, IFN-gamma and transforming growth factor (TGF)-beta. In the spinal cords of patients with a shorter duration of illness, IL-1 beta, TNF-alpha, and IFN-gamma were expressed on perivascular infiltrating macrophages, astrocytes and microglia in active-chronic inflammatory lesions. In striking contrast, we rarely noted cytokine expression except for IFN-gamma in inactive-chronic lesions of patients with longer durations. In situ expression of these cytokines on microglia and astrocytes, in addition to infiltrating mononuclear cells, suggests that glial cells participate in the inflammatory process, especially in active lesions. In addition, the cytokine expression was gradually downregulated along with duration of illness.

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in HTLV-I-associated myelopathy.

Matrix metalloproteinases (MMPs) have been reported to be involved in inflammatory disorders of the central nervous system (CNS). However, little is known about the role of MMPs in the pathogenesis of HTLV-I-associated myelopathy (HAM)/Tropical spastic paraparesis (TSP). To address this issue, we examined the tissue expression and localization of MMPs and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs) in the spinal cord lesions of HAM/TSP using immunohistochemistry. In addition, the blood and cerebrospinal fluid (CSF) levels of MMPs and TIMPs of the patients with HAM/TSP were determined using sandwich enzyme immunoassays (SIA) and gelatin zymography. Immunohistochemical studies revealed that collagen IV and decorin immunoreactivity on the basement membrane of CNS parenchymal vessels was partially disrupted where inflammatory mononuclear cells infiltrated in active-chronic lesions of HAM/TSP. In these lesions, MMP-2 (gelatinase A) was immunostained mainly on the surface of foamy macrophages and lymphocytes, whereas MMP-9 (gelatinase B) expression was positive in the intravascular and perivascular mononuclear cells but not on foamy macrophages. In contrast, inactive chronic lesions of the spinal cords of the HAM/TSP contained fewer MMP-2-positive or MMP-9-positive mononuclear cells than active-chronic lesions. Many parenchymal vessels had thickened vascular walls which showed increased immunoreactivity to decorin. SIA revealed that production levels of MMP-2 and MMP-9 in both blood and CSF were higher in the patients with HAM/TSP than those in non-inflammatory other neurological disease controls (ONDs). Using zymography, proMMP-9 was detected more frequently in the CSF of patients with HAM/TSP than those in ONDs. Taken together, our data indicate that MMP-2 and MMP-9 may play an important role in the blood-brain barrier breakdown and tissue remodeling in the CNS of HAM/TSP.

Immunocytochemical analysis of the cellular infiltrate in the spinal cord lesions in HTLV-I-associated myelopathy.

Immunocytochemical staining of spinal cords from five autopsied patients with HAM/TSP was performed using a panel of monoclonal antibodies reactive with T cells. T cell subsets, B cells, macrophages, natural killer cells, IL-2 receptor-positive cells, and HLA-ABC and HLA-DR. In the spinal cords of patients with a shorter duration of illness, CD4+ cells, CD8+ cells and macrophages were evenly distributed in active-chronic inflammatory lesions. In striking contrast, we noted the predominance of CD8+ cells over CD4+ cells in the inactive-chronic inflammatory lesions of patients with longer duration of illness. Natural killer cells, IL-2 receptor-positive cells and B cells were only rarely present in both the active-chronic and inactive-chronic lesions. HLA-ABC was positive in endothelial cells and infiltrating mononuclear cells, and HLA-DR was positive in endothelial cells, microglia and infiltrating mononuclear cells. This study suggests that immune responses in the spinal cord lesions of HAM patients gradually change along with the duration of illness.

Apoptosis of T lymphocytes in the spinal cord lesions in HTLV-I-associated myelopathy: a possible mechanism to control viral infection in the central nervous system.

Immunocytochemical staining of spinal cords from five autopsied patients with HAM/TSP was performed using the monoclonal antibody TIA-1, a marker of cytotoxic T lymphocytes (CTL). Many TIA-1+, CD8+ cells are distributed in active inflammatory lesions. The number of TIA-1+ cells is related to the amount of HTLV-I proviral DNA in situ. The protein TIA-1 has been associated with the induction of apoptosis in target cells. In active inflammatory lesions, we found cells undergoing apoptosis, most of them identified as helper-inducer CD45RO T lymphocytes, which were consistent with in vivo cellular tropism of HTLV-I in patients with HAM/TSP. These findings suggest that CTL-induced apoptosis of T lymphocytes may be one of the possible mechanisms which eliminate HTLV-I-infected cells from the central nervous system. In addition, many T lymphocytes in the inflammatory lesions expressed bcl-2 oncoprotein, suggesting that infiltrated T lymphocytes may be resistant to apoptosis. Expression of bcl-2 oncoprotein may explain the longstanding inflammatory process in the central nervous system of HAM/TSP.

Clinical electrophysiologic studies of HTLV-I-associated myelopathy.

Six patients with human T-cell lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM) were studied by electrophysiologic methods. Upper-limb short-latency somatosensory evoked potentials showed slight delay of the N9-N20 interpeak latency in one of 12 limbs, while the lower-limb short-latency somatosensory evoked potentials showed prolonged N20-P40 interpeak latency in eight of 12 limbs. Frequent polyphasic potentials and occasional giant spikes were observed in the distal extremities. F-wave conduction velocity was delayed in some patients. Results of the other nerve conduction studies were unremarkable. Our data provide a valuable extension of the clinical examination of HAM and offer encouragement for a more extensive electrophysiologic study of this entity, especially in the spinal cord.

Electroencephalographic abnormalities in human T-cell lymphotropic virus type I-associated myelopathy.

Thirty-six patients with human T-cell lymphotropic virus type I (HTLV-I)-associated myelopathy were studied by electroencephalogram. Twenty-two of 36 patients showed mild to moderate electroencephalographic abnormalities, ranging from poor organization or slowing of the background activity to theta bursts and/or spikes. None of these abnormalities were considered specific for HTLV-I-associated myelopathy. These electroencephalographic abnormalities had no apparent relationship to duration or severity of illness, nor to HTLV-I antibody titers in the cerebrospinal fluid. We document electroencephalographic changes in HTLV-I-associated myelopathy. Our data are consistent with previous reports describing the fact that involvement of regions above the spinal cord may exist in HTLV-I-associated myelopathy.

Serum levels of apoptosis-related molecules in patients with multiple sclerosis and human T-lymphotropic virus Type I-associated myelopathy.

We evaluated the presence of soluble Fas (sFas), Fas ligand (sFasL), and Bcl-2 in the sera of patients with multiple sclerosis (MS) or human T-lymphotropic virus type I (HTLV-1)-associated myelopathy (HAM) using an enzyme-linked immunosorbent assay (ELISA). Patients with MS in the active phase had higher sFas and Bcl-2 levels than had controls (sFas, p < 0.005; Bcl-2, p < 0.05) or patients in the inactive phase (p < 0.05). In addition, significantly increased serum levels of sFas were found in patients with HAM (p < 0.005). Interestingly, levels of sFasL in sera from patients with HAM and MS in the active stage were higher than those from controls or from patients with MS in the inactive stage or from other inflammatory neurologic diseases (OIND), although this was not statistically significant. These results suggest that serum sFas, sFasL, and Bcl-2 may play an important role in the pathogenesis of MS and HAM.

Lymphocyte alpha-glucosidase in late-onset glycogenosis type II.

We describe the biochemical characterization of lymphocyte alpha-glucosidase in a 23-year-old man with intermediate clinical features between the childhood and adult forms of glycogenosis type II (Pompe's disease). Acid alpha-glucosidase activity was markedly reduced, but immunologic cross-reactive material against human liver acid alpha-glucosidase protein could be detected, and its amount was normal. In this patient, the disorder was induced by the catalytically inactive enzyme with a normal amount of enzyme protein.

The risk of development of HTLV-I-associated myelopathy/tropical spastic paraparesis among persons infected with HTLV-I.

Using data obtained in national surveys of human T-lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) conducted in Japan in 1987 and 1988, we estimated the yearly and lifetime risk that HAM/TSP will develop in an HTLV-I-infected person. "Definite" HAM/TSP was defined as slowly progressive myelopathy with antibodies to HTLV-I in both serum and cerebrospinal fluid. Estimates of HTLV-I infection rates in eight endemic prefectures, by age group and sex, were obtained from serologic studies of blood donors; population figures, by age group, sex, and prefecture, were obtained from the census. Of 589 definite cases of HAM/TSP reported nationally, 397 occurred in residents of the eight endemic prefectures; of these, 170 reported onset of illness during the years 1982-1988 (average incidence, 24.3 cases/year). Using the estimated HTLV-I infection rates and the 1985 census figures, we estimated the number of HTLV-I-infected persons in the eight prefectures in 1985 at 794,800. We therefore estimated the incidence of HAM/TSP among HTLV-I-infected persons at 3.1 x 10(-5) cases/year; assuming a lifetime of 75 years, the lifetime incidence is approximately one quarter of 1%. This estimate is important in counseling persons such as blood donors found to be infected with HTLV-I.

HTLV-1-associated myelopathy/tropical spastic paraparesis with pseudohypoparathyroidism.

In many short-stature patients with human T-lymphotrophic virus type I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), signs and symptoms were manifested during childhood. Successive investigations revealed 12 of 14 short-stature patients with pseudohypoparathyroidism (PHP) from the findings of short metacarpi, parathyroid hormone infusion test, immunoblotting of erythrocyte membrane, or lymphocytic Northern blotting of Gsalpha. Patients with PHP probably showed HAM/TSP based on their modified immunologic status. Human T-lymphotrophic virus type I infection did not induce PHP, but PHP may be a risk factor for the occurrence of HAM/TSP.

Freeze-fracture electronmicroscopic analysis of plasma membranes of cultured muscle cells in Duchenne dystrophy.

Depletion of intramembranous particles has been reported in the muscle fiber plasma membrane in Duchenne dystrophy. We searched for a similar abnormality in plasma membranes of cultured muscle cells obtained from six patients with Duchenne dystrophy and six controls. A precise method for phase microscopic to freeze-fracture electronmicroscopic correlation was devised to identify the cell of origin of each replicated membrane face. For both control and patient cells, P-face particle density was higher in myotubes than in mononuclear cells. However, there were no significant differences between control and dystrophic cells when comparing P faces of myotubes, E faces of myotubes, P faces of single cells, or E faces of single cells. The frequency distribution of particle diameters was similar in P faces of control and Duchenne myotubes. Other structural features of the freeze-fractured plasma membrane also were similar.

High CSF lactate and pyruvate content in Kearns-Sayre syndrome.

We studied lactate and pyruvate concentrations in CSF and blood of a patient with Kearns-Sayre syndrome (KSS), 3 patients with ocular myopathy and 11 normal control subjects. We found significant elevation of lactate and pyruvate in the CSF of the patient with KSS, suggesting a disorder of CNS lactate-pyruvate metabolism.

Werner's syndrome associated with spastic paraparesis and peripheral neuropathy.

A Werner's syndrome patient with spastic paraparesis and polyneuropathy had slowing of central and peripheral nerve conduction. Sural nerve biopsy revealed a significantly higher frequency of demyelination and remyelination and a loss of myelinated fibers. These data suggest that the central and peripheral nervous systems are affected in Werner's syndrome.