Makoto Nagasaki

Capsaicin inhibits growth of adult T-cell leukemia cells

Human T-cell leukemia virus type 1 (HTLV-1)-associated adult T-cell leukemia (ATL) is resistant to conventional chemotherapy. We examined the in vitro effects of capsaicin, the principal ingredients of red pepper, on three ATL cell lines. Capsaicin treatment inhibited the growth of ATL cells both in dose- and time-dependent manner. The inhibitory effect was mainly due to the induction of cell cycle arrest and apoptosis. Capsaicin treatment also induced the degradation of Tax and up-regulation of I kappa-B alpha, resulting in the decrease of nuclear factor (NF)-kappa B/p65 DNA binding activity. In addition, the Bcl-2 level was found to be decreased. Based on these findings, capsaicin may be considered for chemoprevention of ATL.

Cathepsin B and H activities and cystatin C concentrations in cerebrospinal fluid from patients with leptomeningeal metastasis

BACKGROUND Cysteine proteases are involved in the extension of cancer into the subarachnoid space. The presence of cathepsins B and H along with their potent inhibitor cystatin C in the cerebrospinal fluid (CSF) was investigated in patients with leptomeningeal metastasis of cancer (LM). MATERIALS AND METHODS CSF samples were obtained in 16 cases of LM (10 solid tumors and 6 leukemia or lymphoma) and compared with 11 cancer cases without involvement of the central nervous system, 12 multiple sclerosis cases and 34 healthy volunteers. The activity of the enzymes was measured, their molecular forms were analyzed by the Western blotting, and the concentration of cystatin C was measured by ELISA. Immunohistochemistry of the leptomeningeal tissues was also performed in six autopsy cases of LM. RESULTS High activities of cathepsins B and H along with decreased cystatin C concentration were observed in CSF of LM as compared with three control groups. Western blot analysis revealed higher concentration of the enzyme protein as well as its active forms in samples with higher enzyme activity. Cells metastasizing leptomeningeal tissue were clearly positive in immunohistochemical staining of cathepsins, indicating active production by tumor cells. CONCLUSION Production of cathepsins B and H by tumor cells and their high activity along with concomitant decrease of their potent inhibitor, cystatin C, in the CSF might contribute in the process of metastasis and spread of the cancer cells in the leptomeningeal tissues. A high enzyme activity/cystatin C concentration ratio in the CSF could be useful when diagnosing LM in combination with other parameters.

Leukoencephalopathy with cerebral amyloid angiopathy: a semiquantitative and morphometric study.

Abstract To investigate changes in caliber of vessels in leukoencephalopathy with cerebral amyloid angiopathy (CAA) we performed a histological and morphometric study of cerebral arteries in this disease. We histologically examined changes in cortico-leptomeningeal arteries in five cases of leukoencephalopathy with CAA and compared their morphometrically determined wall-to-lumen ratio [(external diameter–internal diameter) × 0.5/internal diameter] with those of amyloid-negative arteries to estimate stenotic changes. Additionally, we compared wall-to-lumen ratios of medullary arteries in brains with CAA and white matter lesions (WML) (CAA+/WML+, n = 5), subcortical arteriosclerotic encephalopathy without CAA (CAA–/WML+, n = 7), and neither CAA nor white matter lesions (CAA–/WML–, n = 5). Amyloid-positive arteries had thinned walls and dilated lumens. The external diameter and the wall-to-lumen ratio for amyloid-positive arteries was smaller than for amyloid-negative arteries in CAA+/WML+ brains. There was no significant difference in the external diameters among the three groups. The wall-to-lumen ratio for medullary arteries in CAA–/WML+ brains was significantly greater than for CAA+/WML+ and CAA–/WML–, but there was no significant difference between CAA+/WML+ and CAA–/WML–. Amyloid deposition causes degeneration of the tunica media, resulting in thinning of the wall and dilation of the lumen. The tunica media of small arteries is important in regulation of cerebral blood flow with degeneration causing impairment of cerebrovascular autoregulation in response to blood pressure. This impairment may lead to white matter lesions.

Detection of Epstein-Barr virus transcripts in chemically or immunologically-activated cells and in a null cell-line (HLN-STL-C) by in situ hybridization with alkaline phosphatase-linked oligonucleotide probes

We report a simple procedure for the detection of Epstein-Barr virus (EBV) by in situ DNA-RNA hybridization with an alkaline phosphatase-linked oligonucleotide probe. EBV-producing cell lines P3HR-1 and Akata were treated with phorbol ester and n-butyrate, and anti-human IgG, respectively. This treatment resulted in highly increased populations of cells with EBV transcripts of the latent membrane protein 1 (LMP1) and envelop glycoprotein gp350/220, but not of EBV-encoded small nuclear RNAs (EBERs). Synthesis of the LMP1 protein, which was encoded by the induced mRNA, was mostly dependent on viral DNA synthesis, as shown by double or single labeling for in situ DNA-DNA hybridization with the oligo-nucleotide probe, and immunoperoxidase staining with a monoclonal antibody against LMP1. In situ hybridization of the null cell line HLN-STL-C established from an adult T-cell leukemia patient showed that 100% of the cells contained both EBERs and LMP1 mRNA and about 0.1% of the cells contained gp350/220 mRNA, indicating that a few of the null cells which carried the EBV genome spontaneously entered the late EBV replication cycle.

New haplotype of familial Creutzfeldt-Jakob disease with a codon 200 mutation and a codon 219 polymorphism of the prion protein gene in a Japanese family

Abstract We report a new haplotype of familial Creutzfeldt-Jakob disease (CJD) with a codon 200 mutation and a codon 219 polymorphism of the prion protein gene in a Japanese family. There were four cases diagnosed with CJD neuropathologically, one of which was identified with a codon 200 mutation (glutamic acid to lysine) and a codon 219Lys polymorphism on the same allele. Clinicopathologically, two cases had a long clinical course, whereas the others were similar to the cases with a codon 200 mutation. Three cases was diagnosed with the panencephalopathic-type CJD neuropathologically and the other was diagnosed with the subacute spongiform encephalopathy, a subtype of CJD. We consider that the clinicopathological features in familial CJD are not steadily uniform and that it is impossible to state definitely from this study whether the codon 219 polymorphism influences the clinicopathological aspects in familial CJD with a codon 200 mutation (glutamic acid to lysine).

Differential endocytotic characteristics of a novel human B/DC cell line HBM-Noda: effective macropinocytic and phagocytic function rather than scavenging function

In order to characterize a novel human B cell‐lineage dendritic cell line (B/DC line) as an antigen‐presenting cell (APC), we compared three types of endocytosis (micropinocytosis via a clathrin‐coated pit, macropinocytosis via membrane ruffling, and phagocytosis) among myeloid‐related, macrophage (Mφ) cell lines and a B/DC line. In the present examination, we used a unique human dendritic cell (DC) line, HBM‐Noda (Noda). Flow cytometric and immunocytochemical analyses revealed that Noda not only expresses some DC markers, but also it expresses some B‐cell associated markers. Noda shows strong capacities to stimulate allogenic T cells, to produce immunoglobulin G (IgG), and to perform immunoglobulin gene rearrangment. These data strongly suggest that Noda is a B‐cell lineage DC line. The endocytic differences among these cell lines were as follows. (1) The level of micropinocytosis of Noda was significantly less than that of conventional human Mφ cell lines, and the formation of a clathrin‐coated pit was not observed in Noda. (2) The level of macropinocytosis of Noda was also smaller than that of conventional Mφ cells indicating that the active membrane ruffling of Noda induces rapid recycling. (3) Phagocytosis of opsonized sheep red blood cells (SRBC) was performed more efficiently in Noda than in other Mφ cell lines. Collectively, these data suggest that in human bone marrow cells, we can identify a unique DC subtype, B/DC line, which develops through a lymphoid DC‐differentiation pathway, and DC in this lineage plays an important role in the host immune response because of its effective uptake of a variety of size of antigens by using the skilful membrane ruffling and surface receptors

Constitutive expression of granulocyte-colony stimulating factor receptor on a human B-lymphoblastoid cell line

The present study demonstrated that a human B‐ cell line derived from non‐Hodgkins lymphoma, HCF‐MLpN, constitutively expressed G‐CSF receptor on the cell surface. G‐CSF binding to the cell surface was shown by immunofluorescence staining using biotinylated G‐CSF preparation and analysed by flow cytometry. Specific binding of G‐CSF to the cells was shown by pretreatment with unlabelled G‐CSF. In the radioreceptor assay and Scatchard plot analysis using radiolabelled ligand, MLpN cells revealed a single species of binding site with an equilibrium dissociation constant of 167 (153–182) pM and a maximal binding site per cell of 1076 (1044–1116). The G‐CSF receptor mRNA transcript was exhibited in the RNA from MLpN cells by reverse transcriptase polymerase chain reaction procedure. [3H]thymidine incorporation and trypan blue exclusion showed that the G‐CSF receptor was capable of transducing the growth signal to HCF‐MLpN cells. A small fraction of fresh B blasts from six patients with B‐cell lymphoma and leukaemia displayed G‐CSF binding by two‐colour immunofluorescence staining. In contrast, a panel of seven B‐cell lines was negative for the binding to biotinylated G‐CSF preparation. These results suggest that the phenotype of G‐CSF binding may be lost during the culture. The expression of G‐CSF receptor in HCF‐MLpN cells appeared to be exceptional.

A human B-lineage dendritic cell line, HBM-noda and its potential role in human T-cell leukemia/lymphoma virus type I infection.

An Epstein–Barr virus‐transformed B‐cell line, HBM‐Noda (Noda), that has a dendritic morphology as well as several characteristic features of dendritic cells (DC) has been established. We therefore refer to Noda as B‐lineage DC. Although human T‐cell leukemia/lymphoma virus type I (HTLV‐I) exhibit substantial cellular tropism, the roles of DC in HTLV‐I infection remain unknown. To further clarify the characteristics of Noda cells, we performed infection experiments using a concentrated HTLV‐I fraction from the adult T‐cell leukemia cell line, HPB‐ATL‐2. Noda, as well as other cell lines examined, were sensitive to HTLV‐I infection as detected by proviral DNA using polymerase chain reaction, but most infected Noda cells underwent necrosis within 7 days. The most striking feature of Noda cells was the abundant expression of viral antigen (p19) on the cell surface following infection (approximately day 4), probably due to strong viral adsorption. In cocultivation experiments using Noda cells at day 1 of post‐infection and peripheral blood activated T cells, we detected a few (1.3%) viral antigen expressing T cells after 5 days of coculture by flow cytometry. These results suggest that B‐lineage DC such as Noda cells play a role in the establishment of HTLV‐I infection at an early phase.

A new monoclonal antibody (IE8) reactive with dendritically shaped cells in the human tonsil

A new monoclonal antibody (mAb), 1E8 (IgG1, k), was obtained from a hybrldoma prepared by fusion of mouse myeloma cells (NS‐1) with splenic cells of mice immunized with a human B blastic malignant lymphoma cell line, HPE‐Ret‐3 (Ret‐3). The mAb showed a reactivity unrestricted to a specific cell lineage on flow cytometrical analysis of the reacthttty with human lympho‐hematopoietic cell lines. In peripheral blood, 1E8 reacted with the cells of all lineage, that is, lymphocytes, monocytes, granulocytes and platelets, even though its intenslty was very low by immunohistochem‐Istry. Immunohistochemical examination of human tonsil with 1E8 showed a characteristic staining pattern. Positive cells scattered in follicular (mantle zone and germinal center), parafollicular (T‐dependent area), subepithelial and interstitlal connective tissue areas. These positive cells seemed to be categorized into dendritically shaped cells (DSC), including dendrltic cells (DC) and a subpopulation of macrophages in follicles, interdigitating cells (IDC) and irregularly shaped mononuclear cells. The localization of 1E8 antigen staining was similar to that of integrin CDi l c, although its distribution on hematopoietic cell lines did not coincide with that of 1E8 antigen. lmmunobiochemical studies showed that 1E8 bound two cell surface proteins with molecular size of 70000–90000 and 35000 Da each. Consequently, 1E8 antigen might be a novel marker of DSC.

Establishment of a novel cell line with T-lineage phenotype(HPB-MLp-W) from a non-Hodgkin's lymphoma patient

We report the characterization of a novel human T-cell line, HPB-MLp-W, which was established from blastic cells of a lymph node specimen from a patient with non-Hodgkins lymphoma. They demonstrated the T-cell association antigens, CD2 and CD4, but no CD3, CD8, CD1, CD5, CD7 nor T-cell antigen receptor on their cell surfaces. They were also positive for Ia and Ki-1 antigen, and negative for CD25 (Tac-1). The cell line HPB-MLp-W had the same pattern of antigen expression as the patients cells. Southern-blot analysis of DNA showed a rearrangement of the T-cell receptor-alpha and beta genes. To our knowledge, this is a novel cell line with unique T-lineage marker, to be established from a case of non-Hodgkins lymphoma.