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We investigated the expression and function of Fas and Fas ligand (FasL) on peripheral blood lymphocytes (PBLs). The cells were stimulated with various cytokines or 12-0-tetradecanoyl phorbol 13-acetate (PMA) plus ionomycin. About 30% of unstimulated PBLs expressed Fas, and the expression was augmented by interleukin-1beta (IL-1beta), IL-2, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), or PMA plus ionomycin. Although only minimal FasL expression was detected on unstimulated PBLs, FasL expression was markedly induced by IL-2 or PMA plus ionomycin, suggesting that Fas and FasL were both expressed on IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs. Although IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs were positive for both Fas and FasL, no significant increase in apoptosis was demonstrated in these activated PBLs. In addition, treatment of PBLs with IL-2 or PMA plus ionomycin did not change anti-Fas-induced apoptosis, although these activated PBLs expressed Fas strongly when compared with unstimulated PBLs. Only IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs killed Fas+ target cells efficiently via the interaction of Fas on target cells with FasL of PBLs. Bcl-2 was constitutively expressed on unstimulated PBLs, but its expression was significantly augmented by IL-2 or PMA plus ionomycin. The expression of Bax was clearly induced only on IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs and that of other Bcl-2 family proteins such as Bcl-x and Bad could not be detected on human PBLs, including IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs. Our results suggest that PBLs activated by IL-2 or PMA plus ionomycin express both Fas and FasL and that they kill Fas+ target cells by using FasL on the surface. The resistance of these activated PBLs to Fas-mediated apoptosis may be due to the augmented Bcl-2 expression or the presence of Bcl-2:Bax heterodimers on these cells.

Several lipid second messengers are important mediators of extracellular signals. Among them ceramide, which is formed by cell membrane sphingomyelin, influences the apoptotic signal pathway through Fas antigen. We examined the apoptotic effect of cell-permeable C2-ceramide on rheumatoid synovial fibroblasts in vitro and in vivo. Exposure of cultured human rheumatoid synovial fibroblasts to C2-ceramide for 24 hours produced internucleosomal DNA fragmentation and morphologic changes characteristic of apoptosis. This C2-ceramide-mediated apoptosis was dose dependent as confirmed by analysis of cytosolic oligonucleosome-bound DNA of treated synovial fibroblasts. We also demonstrated that intra-articular administration of C2-ceramide into Fas-deficient MRL lpr/lpr mice produced a profound reduction of synovial hyperplasia within 24 hours. In situ nick end labeling analysis confirmed the induction of apoptosis in synovial lining cells. Our results indicate that C2-ceramide may function as a potent inducer of apoptosis in the synovium and suggest that pharmacologically-induced apoptosis may be useful as a new therapeutic modality for rheumatoid arthritis.

We compared soluble E-selectin (sE-selectin) and L-selectin (sL- selectin) levels in sera and cerebrospinal fluid (CSF) of 30 patients with human T-lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM), with those of 10 patients with the relapsing-remitting form of multiple sclerosis (MS), and 16 patients with other neurological diseases (OND). Serum levels of both sE-selectin and sL-selectin, as measured by enzyme-linked immunosorbent assay, were significantly elevated in patients with HAM, compared to patients with OND. In addition, serum levels of sL-selectin were significantly elevated in HAM patients compared to MS patients. No significant difference was found in CSF levels of sL-selectin between HAM patients and controls. However, HAM patients who had received blood transfusions had significantly higher CSF levels of sL-selectin than HAM patients without a past history of transfusions, suggesting that HAM patients with past history of transfusion have a more active immunological state in the central nervous system. sE-selectin was not detected in CSF of HAM patients and controls. This finding might be based on exaggerated inflammatory conditions following increased attachment of lymphocytes to activated endothelial cells in HAM patients.

We investigated the mRNA expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) in human T cell lymphotropic virus type I (HTLV-I) p40tax-transfected U937 cells, a human monoblast cell line. Transfection of HTLV-I p40tax U937 cells induced up-regulation of iNOS mRNA expression and subsequent NO production. Furthermore, interferon gamma (IFN-gamma) stimulation of HTLV-I p40tax-transfected U937 cells enhanced iNOS mRNA expression and NO production. The kinetics of iNOS mRNA expression and NO production indicated maximal effect at 24 and 48 hours, respectively, after culture with or without IFN-gamma. These findings suggest that HTLV-I p40tax can act as a transactivator of NO production in cells of Mo/M phi lineage. To what extent this mechanism may be involved in the pathogenesis of HTLV-I-associated diseases warrants further investigation.

beta 2-Microglobulin (beta 2-m) is a major constituent of amyloid fibrils in hemodialysis-associated amyloidosis (HAA), a serious complication in patients on long-term hemodialysis. The most distinctive pathological feature of HAA is the deposition of amyloid fibrils with subsequent articular inflammation and destruction. However, the pathological role of beta 2-m is not well known at present. We investigated the effects of beta 2-m on the production of proteinases from synovial fibroblasts isolated from patients with rheumatoid arthritis. beta 2-m stimulated synovial fibroblasts to produce stromelysin, a neutral matrix metalloproteinase (MMP-3). The production of MMP-2 and of a tissue inhibitor of metalloproteinase-1 (TIMP-1) were not enhanced by beta 2-m-treated synovial fibroblasts. Stromelysin is capable of degrading several components of the extracellular matrix and believed to be the key enzyme causing articular destruction in inflammatory joint diseases. Our results suggest a novel role for beta 2-m in articular inflammation and destruction mediated by stromelysin in HAA., (Copyright 1997 Academic Press.)

Fifteen patients with human T-cell lymphotropic virus type-I (HTLV-I)-associated myelopathy (HAM) were treated in an uncontrolled preliminary trial by oral administration of pentoxifylline (PTX). Motor function, neurological evaluation, immunological markers and parameters were evaluated after four weeks. In 13 of the 15 patients, motor disability, especially spasticity, improved substantially. PTX suppressed spontaneous proliferation of peripheral blood mononuclear cells in 14 of the 15 patients at four weeks. No adverse effect was observed. We concluded that PTX may be a safe and beneficial agent for the treatment of HAM.

We investigated whether the reduced intramembranous particles (IMP) in the muscle plasma membrane in mdx mice reflects a preferential depletion of a particular size of the IMP. The experiments were performed using the freeze-fracture method to analyze the frequency distribution of the size of IMP, the density of orthogonal array and caveolae in the extensor digitorum longus (EDL) and soleus (SOL) muscles obtained from mdx and control mice. We detected a reduced density of IMP and orthogonal array, and the increased density of caveolae in EDL muscle but not in SOL in the mdx mouse compared with those of the same muscles in control animals. The reduction of IMP was, however, not limited to any specific size of IMP. Our results suggest that the dystrophin associated glycoprotein present in the membrane does not reflect a specific size of IMP. Therefore, our findings indicate that the mechanism of reduced IMP in dystrophinopathy may be different from that of diminished dystrophin binding glycoprotein associated with dystrophin deficiency in Duchenne muscular dystrophy.

Superantigens activate a large number of T cells in a V beta-restricted manner after binding to MHC class II molecules on the antigen presenting cells (APC). Superantigens also activate APC directly by interacting with their ligands, MHC class II molecules. In the present study, we examined the effects of superantigens on matrix metalloproteinases (MMPs) secretion from rheumatoid synovial fibroblasts. We demonstrated that stimulation of interferon (IFN)-gamma-treated synovial fibroblasts with staphylococcal enterotoxin A (SEA) selectively induced the secretion of stromelysin, a neutral MMP, from synovial fibroblasts. Pretreatment of synovial fibroblasts with cycloheximide, an inhibitor of protein synthesis, prevented MMP-3 secretion from rheumatoid synovial cells suggesting that protein synthesis was required for SEA-induced MMP-3 secretion after SEA binding to MHC class II molecules. Our data suggest that in the synovium, bacterial superantigens are potent inducers of stromelysin which plays a critical role in articular destruction observed in inflammatory joint disease.

Alteration in mesangial cell function induced by high glucose levels is implicated in the development of diabetic nephropathy. The aim of this study was to investigate the mechanism by which high glucose attenuates mesangial cell proliferation. Thymidine incorporation in cultured mesangial cells decreased in the presence of high glucose concentrations in a dose dependent manner, with the maximum decrease of 25% occurring at a glucose concentration of 55.5 mM. Phosphorylation of mitogen-activated protein (MAP) kinase was abolished when the cells were treated with 55.5 mM glucose compared with 11.1 mM glucose. The concentration of intracellular cAMP doubled in the presence of 55.5 mM glucose. The addition of 8Br-cAMP (1.0 mM) to the culture media containing 11.1 mM glucose decreased thymidine incorporation by 34%, similar to the effect of high glucose. In order to clarify the contribution of protein kinase A (PKA) to the MAP kinase cascade, we used PKA inhibitor (H-8). The addition of H-8(10 microM) recovered MAP kinase phosphorylation in the presence of 55.5 mM glucose. Our data indicated that the inhibition of this mitogenic pathway mediated by activation of PKA, which is probably induced by high glucose levels, may play an important role in the perturbation of mesangial cells.

Background/methods: The cause of fulminant hepatitis is still not fully understood. We studied 23 patients with fulminant hepatitis, using polymerase chain reaction to detect hepatitis virus genomes. Tests for HBsAg and IgM anti-HAV and IgM anti-HBc were performed in all patients. Serum samples were stored at -70 degrees C for later analysis of anti-HCV and hepatitis virus genomes such as hepatitis B virus, hepatitis C virus and hepatitis D virus., Results: Of 23 patients, 17 (74%) had HBV-DNA and two (9%) had HCV-RNA. No patient was positive for both viruses or positive for HDV-RNA. Serological tests indicated that two patients, negative for HBV-DNA and HCV-RNA, were positive for IgM anti-HAV. In contrast, 8 of 17 (47%) HBV-DNA positive patients were negative for both IgM anti-HBc and HBsAg in conjunction with low levels of viremia. Four patients were positive for anti-HCV, but only one was positive for HCV-RNA. One patient, positive for HCV-RNA, was negative for anti-HCV., Conclusions: Our results indicate that analysis of hepatitis virus genomes using polymerase chain reaction allows accurate identification of the virus causing fulminant hepatitis.

Recent reports revealed that Fas antigen is functionally expressed on human synovial cells and apoptosis can be induced in these cells by anti-Fas antibody. We examined the effect of interleukin 1 beta (IL-1 beta) on Fas antigen-mediated apoptosis on human synovial cells in vitro. Using flowcytometric analysis, IL-1 beta inhibited Fas antigen expression on synovial cells in a dose-dependent fashion. No significant difference of Fas antigen gene expression between IL-1 beta-treated and untreated synovial cells was observed by RT-PCR analysis, suggesting that the inhibitory effect of Fas antigen expression by IL-1 beta is at posttranscriptional level. Apoptosis of synovial cells was easily induced by treatment of these cells with anti-Fas antibody. In contrast, pretreatment of synovial cells with IL-1 beta protected these cells against Fas antigen-mediated apoptosis. The expression of bcl-2 on synovial cells, known to interfere with the apoptotic process mediated by the Fas antigen, was not influenced by IL-1 beta. Our results suggest that IL-1 beta inhibits Fas antigen-mediated apoptosis of synovial cells and may perpetuate the hyperplasia of the synovium in patients with rheumatoid arthritis.

We investigated the surface markers as well as the expression of beta 2-integrin (LFA-1 beta/CD 18), in T cells migrating through human umbilical vein endothelial cells (EC) in patients with HTLV-I-associated myelopathy (HAM). No significant differences were found in the percentages of both HLA-DR+ T cells and total CD4+ cells and in the expression of beta 2-integrin between the EC-transmigrated and the EC adherent T cells. However, the percentages of HLA-DR+CD4+ cells in the transmigrated cells were significantly higher than those in the adherent cells. These results suggest that activated or HLA-DR+CD4+ T cells play an important role as the first trigger in the immunopathogenesis of HAM.

Several proteins have been postulated as possible targets of immune attack in Lambert-Eaton myasthenic syndrome (LEMS). Heterogeneity of autoantibodies in sera from 20 LEMS patients was studied by comparing their reactivity to synaptotagmin, a synaptic vesicle protein, and voltage-gated calcium channels (VGCCs). Six patients' sera (1 with small cell lung carcinoma (SCLC)) contained antibodies specifically recognizing the recombinant synaptotagmin on immunoblots. Thirteen (11 with SCLC) and 16 (11 with SCLC and 1 with poorly differentiated cell carcinoma in the lung) patients' sera immunoprecipitated omega-conotoxin GVIA-labeled N-type and omega-conotoxin MVIIC-labeled Q-type VGCCs, respectively. Three of 6 synaptotagmin-positive sera had cross-reactivity with N and/or Q subtypes of VGCC; the remaining 3 showed no cross-reactivity with VGCCs. Results indicate that LEMS sera are heterogeneous in the spectrum of containing antibodies, and suggest that this heterogeneity reflects the immune response to various synaptic proteins including not only multiple VGCCs but also synaptosecretory complex proteins.

Neonatal rats born of and nursed by mothers immunized with Narke japonica acetylcholine receptor protein had elevated serum anti-acetylcholine receptor antibodies that reached the mother's level on day 10 after delivery and decreased rapidly after weaning. IgG was present at the motor end-plates up to day 170, and the motor end-plate fine structure remained abnormal up to day 80. Miniature end-plate potential amplitudes in the diaphragm were at the control levels within 10 days of birth, but were lower than those of the controls up to day 80 after birth. We could not obtain the direct evidence that transient synthesis of antibodies occurs in experimental autoimmune myasthenia gravis pups. This model can serve as an experimental model of transient neonatal myasthenia gravis in humans, exception for the route of antibody transfer and the time of the onset of illness.

The histological features of rheumatoid arthritis (RA) consist of overgrowth of synovial cells. Several growth factors that cause synovial hyperplasia have been identified in RA synovium. The basic-fibroblast growth factor (b-FGF), representing one of these growth factors, may play an important role in the pathogenesis of RA. We examined the b-FGF-mediated intracellular signal pathway involved in synovial cell growth. b-FGF-induced synovial cell growth was inhibited by protein tyrosine kinase (PTK) inhibitors, herbimycin A and genistein, but not by H7 that inhibits protein kinase C (PKC). Stimulation of synovial cells with b-FGF resulted in tyrosine phosphorylation of cellular proteins and MAP kinase activation. Our results also demonstrated that b-FGF-mediated activation of MAP kinase was inhibited by herbimycin A indicating that protein tyrosine kinase may be involved in the activation of MAP kinase in human synovial cells. However, inhibition of b-FGF-mediated MAP kinase activation by PKC downregulation did not occur.

Transforming growth factor beta 1 (TGF-beta 1) is a potent growth inhibitor of human umbilical vein endothelial cells (HUVEC). We investigated whether apoptosis is involved in the TGF-beta 1 mediated HUVEC growth inhibition. Using immunofluorescence microscopy and staining by propidium iodide, we showed HUVEC treated with TGF-beta 1 contained characteristic apoptotic nuclei with condensation of chromatin. FACS analysis demonstrated that the number of cells with hypodiploid DNA contents were increased in HUVEC treated with TGF-beta 1. The DNA fragmentation assay exhibited typical ladder formation in TGF-beta 1-treated HUVEC. Using Western blot analysis, the expression of bcl-2 on HUVEC was down-regulated by TGF-beta 1-treatment. These findings suggest that TGF-beta 1 regulates the growth of HUVEC by inducing apoptosis accompanied with down-regulation of bcl-2 expression.

The developmental change of both GLUT1 and GLUT3 protein in rat embryonal and fetal brain was examined using Western blot analysis and immunohistochemistry. The brains were collected from fetuses (gestational days 10 to 20), newborn, and adult rats. On day 10, the levels of GLUT1 and GLUT3 expressions were twofold higher than those of adult levels, but thereafter decreased rapidly as the gestation progressed. The tissue distribution of GLUT1 and GLUT3 in embryo was apparently distinct. On day 10, GLUT1 was expressed in the neural tube, gut, heart and optic vesicle, while GLUT3 was expressed in the surface ectoderm and gut. Thus, high affinity glucose transporters may be required in the early organogenesis period because their energy requirement is completely dependent upon anaerobic glycolysis. GLUT3 may facilitate glucose transfer from amniotic fluid to the embryo and GLUT1 may supply glucose for use as an embryonal fuel.

There is accumulating evidence that human T-lymphotropic virus-1 (HTLV-1) infection contributes to the development of various inflammatory disorders. To elucidate the relation between the infection and Sjögren's syndrome, seroepidemiological and virological studies were conducted on patients with this syndrome in Nagasaki Prefecture, Japan, an area heavily endemic for HTLV-1. The HTLV-1 seroprevalence rate among the patients with Sjögren's syndrome (17/74, 23%) was significantly higher than that among blood donors (916/27,284, 3%), whereas the difference between patients with systemic lupus erythematosus and blood donors was insignificant. Moreover, among Sjögren's syndrome patients the seroprevalence was high irrespective of age, unlike that among blood donors, which rose with age. Titres of serum antibodies in the HTLV-1 seropositive patients with Sjögren's syndrome were similar to those among patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and significantly higher than those among healthy carriers. IgM class antibodies were commonly detected in the serum of patients with Sjögren's syndrome. However, unlike that in HAM/TSP patients, the viral load in peripheral-blood mononuclear cells was not necessarily high in the seropositive Sjögren syndrome group. Salivary IgA antibodies to HTLV-1 were common among seropositive patients with Sjögren's syndrome (5/7), which might be due to increased viral activity in the salivary glands. These antibodies were barely detectable in HAM/TSP patients (prevalence 1/10) or in healthy carriers (0/11). The findings strongly suggest that HTLV-1 is involved in the pathogenesis of the disease in a subset of patients with Sjögren's syndrome in endemic areas.

Interleukin-6 (IL-6) is a multifunctional cytokine that regulates both humoral and cellular immune responses. Accumulating evidence suggests that the infection of T cells and other cell types with human T-lymphotropic virus type 1 (HTLV-1) results in the constitutive expression of IL-6. However, the underlying molecular mechanisms are little understood. When a reporter plasmid, pIL6-CAT-E3, in which the human IL-6 enhancer/promoter region from -630 to +14 was linked to the bacterial chloramphenicol acetyltransferase (CAT) gene, was transfected, HTLV-1-infected but not -uninfected T-cell lines activated the IL-6 promoter. This indicated the presence of a factor transactivating the IL-6 gene in the infected cells. To evaluate the involvement of the HTLV-1-encoded transacting factor (Tax) in this transactivation, we examined the effect of transient cotransfection with the Tax-expression plasmid, pMAX-Neo, on the transcription from the IL-6 promoter by use of COS1 cells. The cotransfected COS1 has about six-times greater the CAT activity than that transfected with pIL6-CAT-E3 alone. The analysis of a series of deletions of the IL-6 promoter suggested that the region (-105/-47) containing a NF kappa B site was crucial for the Tax responsiveness. We further examined the effect of Tax on endogenous IL-6 gene expression using the Jurkat clone, JPX-9, stably transfected with pMAX-Neo. JPX-9 accumulated steady state transcripts of the endogenous IL-6 gene in response to the induction of Tax expression. Our findings indicate an important role of the Tax protein in the expression of IL-6 in cells infected with HTLV-1.

To clarify the phenomenon of increased adherence of T cells to endothelial cells (EC) in patients with HTLV-I-associated myelopathy (HAM), we determined the surface markers and expression of lymphocyte function antigen-1 (LFA-1) in T cells adherent or nonadherent to EC. The percentage of activated or HLA-DR+ T cells and the expression of LFA-1 in the adherent cell population were significantly higher than those in the nonadherent cell population. Moreover, the CD4 to CD8 ratio of the HLA-DR+ cells in the EC-adherent T cells was significantly higher than that in the nonadherent cells. Collectively, these results indicate that increased adherence of T cells to EC in HAM patients is based on the increase of activated T cells with high density LFA-1 expression in the peripheral blood. Moreover, CD4+ HLA-DR+ cells exhibited more adhesive activity to EC than CD8+ HLA-DR+ cells, suggesting that activated CD4+ cells, rather than activated CD8+ cells, may be important as the first trigger for T cell-infiltration to the central nervous system in the immunopathogenesis of HAM.

We investigated culture supernatants of peripheral blood mononuclear cells (MNC) derived from patients with human T-lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM) for antiproliferative activity against the human glioblastoma cell line T98G. When T98G cells were cultured with condition medium containing culture supernatants of MNC from patients with HAM, the proliferation of T98G cells was significantly suppressed, compared with that of supernatants from HTLV-I seropositive carriers or seronegative controls. To clarify which population of MNC produced the antiproliferative humoral factor for T98G cells, we separated MNC into macrophage-depleted or B cell depleted populations, and further to both CD4+ and CD8+ T cells by using the panning method or plastic adherence. These studies demonstrated that the antiproliferative activity was mediated by a humoral factor produced by T cells, specifically CD4+ cells. This activity was blocked by a neutralizing monoclonal antibody against interferon-gamma (IFN-gamma). Moreover, IFN-gamma levels were elevated in the culture supernatants of CD4+ cells from HAM patients. Thus, the antiproliferative activity against T98G cells is mainly due to IFN-gamma derived from CD4+ cells of patients with HAM.

We have cloned a full length protein-coding sequence of human platelet-type phosphofructokinase (PFK) from pancreatic islet cDNA library. The platelet-type PFK was composed of 784 amino acids and had a deduced molecular weight of 85,590. Homologies in the primary structure with muscle- and liver-type PFK were 71 and 67%. Clear similarities of the amino and carboxyl halves with a prokaryotic PFK indicated an evolutionary event that duplicated genes of a prototype PFK fused into larger genes of eukaryotic PFKs. Amino acid residues constituting the binding sites for various allosteric modulators were well conserved, while a couple of different residues at the inhibitory ATP sites among three isozymes may partly explain their varied degree of sensitivities to ATP. Considerable amount of platelet-type PFK expression was demonstrated in brain, heart, kidney, colon and testis.

We investigated the production of granulocyte-macrophage colony stimulating factor (GM-CSF) by human T-lymphotropic virus type I (HTLV-I)-infected human glioma cells (KG-1-C and T98G). When glioma cells were co-cultured with HTLV-I-producing T cell lines (HCT-1 and MT-2), GM-CSF was detected in the culture supernatant. GM-CSF was produced in all the co-cultures even after several passages. In co-cultures of KG-1-C and HCT-1 cells with Millicell, the amount of GM-CSF produced in the supernatant was almost as low as in the culture of HCT-1 alone. Moreover, for co-cultures of KG-1-C and HCT-1 or MT-2 cells, the production of GM-CSF was significantly suppressed in the presence of IgG from patients with HAM. Double-label immunostaining showed that GM-CSF-producing glioma cells always were stained by a monoclonal antibody against HTLV-I p19, indicating that HTLV-I infection of glioma cells caused GM-CSF production. These data suggest that human glial cells infected with HTLV-I gain the ability to produce cytokines.

Cell-to-cell contact is usually essential for syncytium formation by HTLV-I-infected cell lines. The present study was undertaken to determine the inhibitory effect of polyanionic compounds, dextran sulfate and heparin, on HTLV-I-induced syncytium formation, as demonstrated by the fusion of HTLV-I-infected cells with target cells. These two compounds almost completely blocked syncytium formation in the early phase of the reaction at a concentration of 125 micrograms/ml, but dextran, as a control, did not inhibit it at concentrations up to 625 micrograms/ml. 50% inhibition of syncytium formation was detected at a concentration of 2 micrograms/ml of dextran sulfate 5000, 3 micrograms/ml of dextran sulfate 8000 and 8 micrograms/ml of heparin. The binding of radiolabeled HTLV-I-infected cells (HCT-1) to the target cells was inhibited by addition of dextran sulfate and heparin, and the inhibitory effects were concentration-dependent. No marked changes were detected in the expression of adhesion molecules on the virus-infected cells and target cells, and in the expression of envelope proteins on the virus-infected cells after exposing them to the polyanionic compounds. These results suggest that the blocking of cell-to-cell contact by polyanionic compounds, probably independent of surface adhesion molecules, is important for their inhibitory effect on HTLV-I-induced syncytium formation.

Ten patients with HTLV-I-associated myelopathy (HAM) were treated in an uncontrolled preliminary trial of heparin. In 7 patients, motor dysfunction improved substantially and the effect continued for more than a month after the discontinuation of therapy. Sensory and urinary disturbances also improved in 3 of 4 and in 2 of 10 patients, respectively. Heparin did not alter the subsets of peripheral blood lymphocytes nor the titers of anti-HTLV-I antibodies in serum and cerebrospinal fluid. Spontaneous proliferation of peripheral blood lymphocytes, however, was depressed significantly (P < 0.05) in all cases. Heparin therapy has some advantages in cost, ease of administration and fewer side effects compared to other therapies such as plasmapheresis and interferon-alpha. We conclude that heparin can be administered safely to HAM, and that a double-blind placebo-controlled trial is warranted to determine its efficacy in HAM.

A novel variant of antithrombin III (AT III) that lacks affinity for heparin was found in a 33-year-old man who suffered from recurrent cerebral infarction. The propositus had a history of recurrent ischemic attacks from the age of 28. He was obese and had a smoking habit (30 to 40 cigarettes/day), low high-density lipoprotein cholesterol, and a mild glucose intolerance as the possible risk factors for thrombosis. No family history of thromboembolic episodes was observed. Coagulation studies found low heparin cofactor activity (55%) of AT III, with a normal immunoreactive level (109.7%) and progressive antithrombin activity (117%). Other factors capable of predisposing him to hereditary thrombophilia were within normal ranges. Analysis by crossed immunoelectrophoresis in the presence of heparin and affinity chromatography on heparin-Sepharose demonstrated that the propositus' AT III was composed of two populations, one having no affinity for heparin and the other binding heparin normally. Nucleotide sequencing of 7 exons of the propositus' AT III gene using polymerase chain reaction and subcloning disclosed a transition of thymine to cytosine in exon 3a (codon 116) of the AT III gene leading to a Ser116-Pro conversion. Allele-specific oligonucleotide hybridization procedures confirmed the presence of the mutation in the propositus' genomic DNA. Using the same technique, the mutation was also found in his father's genomic DNA, but not in that of his mother. These findings indicate that Ser116 is an important amino acid residue in heparin binding and that the propositus is heterozygous for the abnormality. Furthermore, the fact that the propositus suffered from recurrent cerebral infarction suggested that being heterozygous for a heparin-binding defect would lead to a predisposition to thrombosis when associated with various risk factors. The name AT III Nagasaki is proposed for this variant AT III containing a novel Ser116-Pro mutation.

Administration of epidermal growth factor (EGF) stimulates DNA synthesis in gut epithelial cells and inhibits gastric acid secretion. A physiological role of EGF in cell kinetics of gastric epithelium, however, has not been fully understood. In mature male mice, large amounts of EGF are produced in the submandibular glands, and sialoadenectomy (removal of the submandibular glands) causes a marked reduction of plasma EGF levels. For the evaluation of a biophysical function of EGF, sialoadenectomized mice and sham-operated mice were injected with 3H-thymidine to compare the proliferative activity and the cell-turnover of gastric epithelium between the two groups using the autoradiographic analysis. When mice were killed 90 min after a single injection of 3H-thymidine, the percentages of fundic gland mucosal cells radiolabeled in sialoadenectomized and sham-operated mice were 27.3 +/- 5.0% and 26.3 +/- 5.8% (mean +/- SD), respectively. The difference was not significant (p > 0.05). Similarly, the labeling indices of pyloric gland mucosal cells were not different between the two groups (26.7 +/- 4.3% vs 27.8 +/- 3.7%, p > 0.05). In contrast, when mice were given 17 repeated injections of 3H-thymidine at 6 hr intervals and killed 48 hr after the last injection, labeling indices in sialoadenectomized mice were significantly lower than those in sham-operated mice (35.3 +/- 4.3% vs 52.8 +/- 1.1% in the fundic gland area; 41.0 +/- 6.2% vs 55.1 +/- 5.9% in the pyloric gland area, p < 0.001, respectively). Treatment of sialoadenectomized mice with EGF (5 mg/mouse per day) completely restored the percentages of the radiolabeled cells to control levels. These findings suggest that endogenous EGF plays a major role in maintaining biological cell-turnover of the mouse gastric epithelium.

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes; however, in certain human hepatoma cell lines, the growth is inhibited by HGF. In the present study, the effect of HGF on the alpha-fetoprotein (AFP) gene expression was analyzed in PLC/PRF/5 human hepatoma cells. HGF did not inhibit cell proliferation, but dose-dependently suppressed AFP secretion at the concentrations of 10 ng/ml or less. By Northern blot analysis, the levels of AFP mRNA were suppressed by HGF, whereas the levels of beta-actin mRNA used as a control did not show any significant changes. In the transient chloramphenicol acetyltransferase plasmid transfection assays, the AFP promoter activity was repressed by HGF, in contrast, the AFP enhancer activity was not affected by HGF. These results suggest that the AFP gene expression is down-regulated by HGF through the suppression of its promoter activity in human hepatoma cells.

In the present study, we report the molecular cloning and sequencing of an alternatively spliced form of the human thyrotropin receptor (hTSHR) mRNA transcript, which has previously been detected on Northern blot analysis of human thyroid cells. The smaller hTSHR cDNA, designated hTSHR cDNA-I, is approximately 1 kb in size and encodes a protein of 253 amino acids. Comparison of the nucleotide sequence of hTSHR cDNA-I with available hTSHR genomic sequence data reveals that the cDNA-I contains exons 1-8 and unidentified DNA tract, presumably an intron. Thus, the hTSHR cDNA-I encodes for the N-terminal half of the extracellular domain of the hTSHR (approximately 60%). The truncated TSHR-I may be secreted and function as a TSH binding protein.

The interaction of IFN-alpha with IL-1 beta or TNF-alpha on hepatitis B surface antigen (HBsAg) expression was analysed in hepatitis B virus (HBV)-DNA integrated PLC/PRF/5 and non-integrated HuH-7 human hepatoma cells. Secretion of HBsAg in PLC/PRF/5 cells was reduced by IFN-alpha, IL-1 beta or TNF-alpha, and synergistically depressed when IFN-alpha was used in combination with IL-1 beta or TNF-alpha. By Northern blot analysis, the levels of HBsAg mRNA were suppressed by IFN-alpha in combination with IL-1 beta or TNF-alpha. In the chloramphenicol acetyltransferase plasmid transfection assay, IFN-alpha in combination with IL-1 beta or TNF-alpha caused a much greater suppression of HBV enhancer activity than IFN-alpha, IL-1 beta or TNF-alpha alone in both hepatoma cells. These findings suggest that the interaction of IFN-alpha with IL-1 beta or TNF-alpha synergistically represses HBV enhancer activity, resulting in depressed expression of HBsAg.

We treated 17 patients with HTLV-I-associated myelopathy (HAM) with interferon-alpha (IFN-alpha) in an open, nonrandomized, uncontrolled study. They were administered 1.5-9.0 x 10(6) international units of IFN-alpha daily for 4 weeks, and 4 patients showed a marked clinical response, 7 showed a moderate response and the others did not show any clinical response. Increased unstimulated (spontaneous) peripheral blood lymphocyte (PBL) proliferation was observed in all patients who were measured lymphocyte transformation. Spontaneous PBL proliferation was significantly inhibited by IFN-alpha treatment (P less than 0.01), whereas anti-HTLV-I-antibodies did not show any significant changes. Likewise, decreased stimulation indices to phytohemagglutinin (SI) due to increased spontaneous PBL proliferation were significantly increased after IFN-alpha treatment (P less than 0.01). In the patients who showed marked clinical responses, the changes of SI in IFN-alpha treatment were higher than those in other patients. These data indicate that further studies are warranted for evaluation of IFN-alpha treatment of HAM in a randomized, controlled study.

We investigated serum IgM antibodies against human T-lymphotropic virus type I (HTLV-I) in 29 HTLV-I associated myelopathy (HAM) patients and 34 HTLV-I carriers, using western blot analysis. Anti-HTLV-I IgM was detected in all 6 post-transfusional HAM patients and in 19 of 23 (83%) HAM patients with no history of blood transfusion, but in only 4 of 21 (19%) HTLV-I carriers. In HAM patients, HTLV-I proviral DNA integrated into peripheral blood lymphocyte (PBL) was detected by Southern blot analysis in all of the 6 (100%) and 18 of the 23 (78%). In contrast, it was detected in only 2 of 25 (8%) HTLV-I carriers. For the serum anti-HTLV-I IgM and HTLV-I provirus in PBL, the differences between the HAM and HTLV-I carriers were statistically significant (P less than 0.01). Our data indicate that the increased HTLV-I proviral DNA in PBL is produced by the persistent active replication of HTLV-I in HAM. Furthermore, Southern blot analysis showed intense bands in HAM patients with histories of blood transfusion, in whom the progression of the disease had been rapid. We conclude that the persistent active replication of HTLV-I is an important factor in the pathogenesis of HAM.

This study was undertaken to investigate whether target cell class I HLA antigen expression induced by phorbol ester and interferon-alpha (IFN-alpha) was associated with resistance to natural killer (NK) cells and lymphokine-activated killer (LAK) cell-mediated cytotoxicity. Class I antigen expression on the surface of the K562 erythroleukemia cell line was enhanced by either IFN-alpha or phorbol ester (PDBu). Addition of PDBu together with IFN-alpha had a synergistic effect on class I antigen expression on the cells. Furthermore, synergism between IFN-alpha and PDBu was also found in class I antigen expression by MOLT-3 cells. This synergistic effect on class I antigen expression was blocked by the protein synthesis inhibitor (cycloheximide). Pretreatment of K562 cells with PDBu and IFN-alpha made them more resistant to lysis by NK and LAK cells than did either PDBu or IFN-alpha. In contrast to PDBu, 4 alpha PDD, a biologically inactive phorbol analogue, alone or combination with IFN-alpha, had no effect on class I antigen expression and susceptibility to lysis by NK and LAK cells. Kinetic experiments showed an inverse relationship between the expression of class I antigens and susceptibility to NK cell-mediated cytolysis. Using cold target competition analysis, target cells pretreated with PDBu and IFN-alpha clearly competed less effectively than did untreated cells for lysis of untreated target cells. These results demonstrate that target cells pretreated with PDBu and IFN-alpha decrease their sensitivity to natural killer and lymphokine-activated killer cells inversely with target cell class I HLA antigen expression.

Studies have been undertaken to clarify the localizing properties of radioactively labeled antibody to AFP using ascitic hepatoma cells which produce AFP. In the in vitro studies, radioantibodies to AFP were incorporated into the AFP-producing cells, even though excess antigen existed in the medium and the antibodies remained on the cell surface for a long time. In the in vivo studies, the radioantibodies to AFP bound to the AFP-producing cells, especially those producing high levels of AFP (AH66). Tumor localization by scintigraphy was clearer with the AH66 hepatoma than with the AH41C hepatoma, which produces less AFP. The presence of large amounts of AFP in the circulation did not prevent the tumor radioimmunodetection. It is suggested that radioantibodies to AFP bind to the AFP-producing cells despite the presence of large amounts of AFP in the circulation or in ascitic fluid.

The rate of aqueous formation was measured with fluorophotometry in a group of 113 normal human subjects who ranged in age from 20 to 83 years. A slight decrease in the rate of aqueous humor formation with aging was detected, but the rate was remarkably stable. The rate of aqueous humor formation was more stable than intraocular pressure or anterior chamber volume in this group and more stable than the tonographic C value reported by other investigators.

In 11 of 18 patients with human T-lymphotropic virus type-I (HTLV-I) associated myelopathy (HAM) gait, sensory, and/or sphincter disturbance improved with plasmapheresis (4 to 6 sessions in 2 weeks), and the effects were maintained for 2 to 4 weeks. Plasmapheresis lowered the titre of HTLV-I antibody in serum but not in cerebrospinal fluid, and change of HTLV-I antibody titres did not correlate with the effects of plasmapheresis. These results suggest that plasmapheresis is useful treatment, at least in producing a temporary improvement, in patients with HAM, and that some humoral factor(s), but not HTLV-I antibody, may be important in the pathogenesis of HAM.

The present study was undertaken to investigate degradation of thyroxine (T4) mediated by thyroid peroxidase in man. A particulate fraction (1,000-100,000 x g) of normal human thyroid tissue was prepared and used as crude enzyme. 125I-T4 and unlabeled T4 were incubated with the particulate fraction in buffer containing glucose and glucose oxidase for generation of H2O2. After incubation, iodoamino acids were extracted with ethanol and the products of T4 degradation were analyzed by thin layer chromatography. In this system, T4 was degraded in time-, temperature- and pH-dependent manners, but not in the absence of the H2O2-generating system. The rate of degradation was related to concentration of the particulate fraction. The reaction was inhibited by methimazole, propylthiouracil and catalase. When [3',5'-125I] T4 was used as a tracer, major labeled products of T4 degradation were inorganic iodide and ethanol-unextracted fraction and no detectable labeled 3,5,3'-triiodothyronine (T3) or 3,3',5'-triiodothyronine (rT3) was generated. From a kinetic study by adding various doses of unlabeled T4, the apparent Km value for T4 was 30 microM and the Vmax value was 230 pmol/mg protein/min. When [3,5-125I] T4 was incubated with enzyme preparation, one third of degraded T4 was recovered as diiodotyrosine (DIT) and half of 125I-DIT was degraded in parallel incubation. No formation of radiolabeled DIT was observed in incubation with Na- 125I done in tandem. These findings suggest that thyroid hormones can be metabolized by peroxidase in human thyroid by pathways that include cleavage of ether linkage.

Sera from patients with collagen diseases, chronic liver diseases and hepatocellular carcinoma were analysed for alphafetoprotein (AFP)-binding protein. The method used was a 125I-labeled AFP-binding assay with goat anti-human immunoglobulin G (IgG) as the precipitating antibody. AFP-binding IgG was found in the sera from one of 34 patients with systemic lupus erythematosus (SLE) and one of nine patients with hepatocellular carcinoma. This protein may interfere with the conventional radioimmunoassay of human AFP, and may be of interest because of its usefulness as a carrier of radioactivity or therapeutic agents to AFP-producing tumors.

To clarify the role of Ia antigen positive macrophages which invade motor end-plates in the induction of the chronic phase, experimental autoimmune myasthenia gravis (EAMG) rats were injected intraperitoneally with silica dust on day 6 after immunization. Silica injection partially inhibited the invasion of motor end-plates with macrophages as compared with a saline injection. The titer of antibodies to the Narke acetylcholine receptor (AChR) in the chronic phase did not differ in either the saline or the silica groups, while the titer of antibodies to rat-AChR (non-crossreactive antibodies) was lower in the silica group than in the saline group. The silica group survived longer than the saline group. These results suggest that Ia antigen positive macrophages in the acute phase act as antigen-presenting cells and play an important role in the production of antibodies to self-AChR in the chronic phase.