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K Teshigawara

Publications and source records attributed to K Teshigawara.

At least 19 recordsLinked to original sources
PubMed2001

Identification of a novel GH isoform: a possible link between GH and melanocortin systems in the developing chicken eye.

GH is considered to play a role in the pathogenesis of diabetic retinopathy, causing neovascularization in the retina. The present study was conducted to assess the possibility that GH may play a role in ocular development by determining whether GH is expressed in the eye of the chicken during development. In the 17-d-old embryo, immunocytochemistry detected immunoreactive GH in retinal pigment epithelial (RPE) cells. Characterization of GH mRNA expressed in the eye by RT-PCR and rapid amplification of cDNA 5'-ends revealed it to be a novel GH mRNA transcribed from the middle of the intron 3 of the chicken GH (cGH) gene. The deduced protein, designated small GH isoform (s-cGH), was a cytosolic protein of 16.5 kDa with 140 amino acid (aa) residues, lacking the signal peptide and the N-terminal 71 aa residues of 22-kDa cGH, replacing them with 20 aberrant aa residues, and identical to 22-kDa cGH for the C-terminal 120-aa residue portion. Western blotting determined the molecular size of immunoreactive GH in RPE cells to be 80-84 kDa, similar to the computed molecular mass of s-cGH/GH receptor complex. Furthermore, RT-PCR demonstrated that GH receptor mRNA, but not s-cGH mRNA, was expressed in RPE cells. These results suggest that RPE cell is one of the target cells of s-cGH in the eye. During embryonic development, the immunoreactivity for s-cGH in RPE cells was initially observed on embryonic d 10, and the staining intensity increased and peaked on embryonic d 17. By hatching, s-cGH immunoreactivity in RPE cells was gradually decreased, and it was not detectable after hatching. This ontogenetic staining pattern correlates well with the pattern of the production of alpha MSH in RPE cells. The cell type expressing s-cGH remains to be identified; however, our findings imply a possible involvement of GH in the regulation of ocular development by acting on the intraocular melanocortin system in the chicken.

Amino Acid Sequence↗
PubMed2001

Identification of avian alpha-melanocyte-stimulating hormone in the eye: temporal and spatial regulation of expression in the developing chicken.

The presence and possible physiological roles of alpha-melanocyte-stimulating hormone (alpha-MSH) in the peripheral tissues of birds have not been established. By a combination of RT-PCR, immunocytochemistry and in situ hybridization, we have examined alpha-MSH expression in the eye of the chicken during development. In the 1-day-old chick, alpha-MSH was expressed in the retinal pigment epithelial (RPE) cells, and also at a lower level in the cone cells. The melanocortin receptor subtypes, CMC1, CMC4 and CMC5, were expressed in the layers of the choroid and the neural retina, but not in the RPE cells. It is probable that the RPE cells secrete alpha-MSH to exert paracrine effects on the choroid and neural retina. During embryonic development, alpha-MSH immunoreactivity in the RPE cells was initially detected at embryonic day 10, and increased in intensity as development proceeded. No cone cells were stained with anti-alpha-MSH antiserum in any of the embryonic stages tested. The immunoreactivities for two prohormone convertases, PC1 and PC2, were co-localized to the RPE cells with a pattern of staining similar to that of alpha-MSH. Despite containing alpha-MSH immunoreactivity, the RPE cells in 1-day-old chicks expressed no immunoreactivity for the endoproteases. Furthermore, in a 3-day-old chick, pro-opiomelanocortin mRNA was detectable by in situ hybridization only in the photoreceptor layer and not in the RPE cells. These results suggest that the RPE cells and the cone cells are intraocular sources of alpha-MSH in the embryonic and postnatal life of the chicken respectively. Embryonic expression of alpha-MSH in the RPE cells implies a possible role for the peptide in ocular development.

Animals↗
PubMed2000

Widespread expression of Agouti-related protein (AGRP) in the chicken: a possible involvement of AGRP in regulating peripheral melanocortin systems in the chicken.

Agouti-related protein (AGRP) is a naturally occurring antagonist of melanocortin action. It is expressed mainly in the arcuate nucleus where it plays an important role in the hypothalamic control of feeding and energy homeostasis by antagonism of central melanocortin 4 receptors in mammals. Besides in the brain, the melanocortin 4 receptor is expressed in numerous peripheral tissues in the chicken. To examine whether or not the peripheral melanocortin 4 receptor signaling could be regulated by AGRP, we cloned and localized the expression of the AGRP gene in the chicken. The chicken AGRP gene was found to encode a 154 or 165 amino acid protein, depending on the usage of two alternative translation initiation sites. The coding sequence consisted of three exons, like that of mammalian species. The C-terminal cysteine-rich region of the predicted AGRP displayed high levels of identity to mammalian counterparts (78-84%) and all 10 cysteine residues conferring functional conformation of AGRP were conserved; however, other regions showed apparently no homology, suggesting that biological activities of AGRP are located in its C-terminal region. RT-PCR analysis detected the AGRP mRNA in all tissues examined: the brain, adrenal gland, heart, liver, spleen, gonads, kidney, uropygial gland, skeletal muscle and adipose tissues. Interestingly, the skin also expressed the AGRP mRNA, where Agouti, another melanocortin receptor antagonist regulating hair pigmentation, is expressed in rodents. Most of those AGRP-expressing tissues have been demonstrated to express melanocortin 4 receptors and/or other subtypes of melanocortin receptor whose mammalian counterparts can bind AGRP. These results imply the possibility that some peripheral melanocortin systems could be regulated by the functional interaction between melanocortins and AGRP at melanocortin receptors in the chicken.

Adrenal Glands↗
PubMed1999

Molecular cloning and characterization of the chicken pro-opiomelanocortin (POMC) gene.

The gene for pro-opiomelanocortin (POMC), a common precursor of melanocortins, lipotropins and beta-endorphin, was isolated in the chicken first among avian species. The chicken POMC gene was found to be a single copy gene and appeared to show the same structural organization as that of other species of different classes. The predicted POMC displayed the highest identity to Xenopus POMC(A) (60. 1%), and consisted of 251 amino acid residues with nine proteolytic cleavage sites, suggesting that it could be processed to give rise to all members of the melanocortin family, including adrenocorticotropic hormone and alpha-, beta- and gamma-melanocyte-stimulating hormones, as well as the other POMC-derived peptides. RT-PCR analysis detected the POMC mRNA in the brain, adrenal gland, gonads, kidney, uropygial gland and adipose tissues, each of which has been demonstrated to express melanocortin receptors. These results suggest that melanocortins act in a paracrine and/or autocrine manner to control a variety of functions both in the brain and in the peripheral tissues in the chicken.

Adrenal Glands↗
PubMed1999

Redox control of Epstein-Barr virus replication by human thioredoxin/ATL-derived factor: differential regulation of lytic and latent infection.

Human thioredoxin (hTRX)/adult T-cell leukemia (ATL)-derived factor (ADF) was originally reported as an interleukin-2 (IL-2) receptor-alpha-inducing factor produced by human T-cell lymphotropic virus-1-positive (HTLV-1+) cell lines. Growing evidence indicates that hTRX/ADF plays important roles in cellular responses against oxidative stress and is involved in a variety of cellular functions. A high level of hTRX/ADF expression is also observed in other human virus-infected cell lines including those of Epstein-Barr virus (EBV) and human papillomavirus. In this report, we analyzed the effect of hTRX/ADF on lytic amplification and persistent replication of EBV as a model for lytic versus latent phase of viral replication in host cells. Addition of hTRX/ADF clearly suppressed lytic replication of EBV in Raji cells and B95-8 cells induced to the lytic phase of 12-O-tetradecanoylphorbol-13-acetate (TPA), and it prevented the death of these cells evoked by the lytic induction. In contrast, hTRX/ADF did not have any effect on persistent replication in the latent phase. These data indicated that hTRX/ADF prevents EBV-transformed cells from proceeding into the lytic phase and regulates cohabitation of EBV and its host cells.

Carcinogens↗
PubMed1997

Induction of the 2B9 antigen/dipeptidyl peptidase IV/CD26 on human natural killer cells by IL-2, IL-12 or IL-15.

Activation of human natural killer (NK) cells involves sequential events including cytokine production and induction of cell surface molecules, resulting in the enhancement of cytolytic activity. To delineate the activation process of NK cells, we generated murine monoclonal antibodies (mAbs) against YT, a human large granular lymphocyte/natural killer (LGL/NK) cell line. Among the mAbs reactive with YT cells, one mAb, termed 2B9, was noted because of the lack of reactivity with most of the human T- and B-cell lines tested. In fresh peripheral blood mononuclear cells (PBMC), however, the majority of cells expressing this antigen (Ag) were T cells but not CD16+ nor CD56+ NK cells. Since YT cells showed an activated phenotype expressing interleukin-2 (IL-2) receptor alpha chain, we examined whether 2B9 Ag could be induced on normal human peripheral blood NK cells by cytokines known to activate NK cells. The 2B9 Ag was induced on NK cells by IL-2, IL-12 or IL-15 while no induction was observed by interferon-gamma (IFN-gamma). Biochemical analysis showed that anti-2B9 mAb recognized a 115 kDa molecule in YT cells. A cDNA clone encoding the 2B9 Ag was isolated from a cDNA expression library of YT cells and its sequence was identical to CD26 cDNA although it was not of full length. Transient expression of the 2B9 cDNA on COS-7 cells revealed that this cDNA encodes the antigenic epitope(s) recognized by anti-2B9 mAb as well as Ta1, an anti-CD26 mAb. These results showed that the 2B9 Ag is identical to CD26, and demonstrated that CD26 is an activation antigen on CD16+ CD56+ NK cells inducible by IL-2, IL-12 or IL-15.

Antibodies, Monoclonal↗
PubMed1996

Induction of autologous tumor killing by heat treatment of fresh human tumor cells: involvement of gamma delta T cells and heat shock protein 70.

Autologous tumor killing (ATK) has been implicated as an important prognostic factor in cancer patients since the ability of blood lymphocytes to kill freshly isolated autologous tumor cells was strongly associated with good prognosis of the patients. The present study was designed to induce or enhance ATK sensitivity of fresh human tumor cells by heat stress. Brief exposure of fresh human tumor cells to elevated temperature increased their susceptibility to lysis by autologous blood lymphocytes in a short-term (51)Cr release assay. In addition, the heat-elevated ATK sensitivity was confirmed by clonogenic assays. An increase in ATK was observed with unstimulated lymphocytes in 42% of the cases and OK432 (streptococcal preparation)-activated lymphocytes in 80% of the cases. Stimulation of blood lymphocytes with autologous, heat-stressed tumor cells and OK432 resulted in an increase in number of gamma delta T cells, which was associated with elevated ATK activity against the stressed tumor cells. At the clonal level, three gamma delta T-cell clones (V gamma 9/V delta 2+) proliferated in response to autologous, heat stressed tumor cells and/or OK432 and exhibited elevated cytotoxicity against the tumor cells. Western blot analysis revealed an increased expression of heat shock protein (HSP) 70 in heat- treated tumor cells. Some of them expressed HSP70 on their surfaces. The elevated cytoxicity against heat-stressed tumor cells was inhibited by treatment of targets with anti-HSP70 monoclonal antibody (mAb) or of effector cells with anti-V delta2 mAb. Reactivity of gamma delta T cells to autologous, heat- stressed tumor cells was also inhibited by anti-HSP70 mAb. These results indicate that exposure to heat of tumor cells induces ATK susceptibility, especially to OK432-activated effector cells, and suggest that gamma delta T cells may be involved in ATK against stressed tumor cells through recognition of HSP70 on the target cells.

Adult↗
PubMed1996

BCL-2 inhibits B7-induced MHC-unrestricted cytolysis mediated by a human NK cell line.

Recent work demonstrated that B7 expression by tumor cells can enhance antitumor immune responses. However, the B7 molecule is expressed abundantly on most non-Hodgkin's B-cell lymphomas and solid lymphoid tumors. How these tumor cells escape from immune surveillance mechanisms remains unclear. Lately, it has become clear that bcl-2 oncogene is overexpressed in a wide variety of human cancers and renders tumor cells more resistant to cytolytic T-cells (CTL) mediated cytotoxicity. We cloned B7 and B7/Bcl-2 transfectants and compared their susceptibilities to a human natural killer (NK) cell line and normal NK cells. The results demonstrate that Bcl-2 oncoprotein in tumor cells blocks B7-induced cytolysis mediated by a NK cell line and NK cells. Thus, they suggest that Bcl-2 oncoprotein plays a role in tumor avoidance of effective antitumor cytotoxicity.

B7-1 Antigen↗
PubMed1995

Inhibition of proliferation and induction of apoptosis by abrogation of heat-shock protein (HSP) 70 expression in tumor cells.

Tumor cells often express elevated levels of heat-shock protein (HSP) 70. The present study was designed to investigate the role of HSP70 in the proliferation and survival of tumor cells in the human system. When Molt-4 and other tumor cells were treated in vitro with HSP70 antisense oligomer, they displayed propidium-iodide-stained condensed nuclei (intact or fragmented). A ladder-like pattern of DNA fragments was observed with HSP70 antisense-oligomer-treated tumor cells in agrose gel electrophoresis, which was consistent with internucleosomal DNA fragmentation. Flow cytometry analysis revealed the hypodiploid DNA peak of propidium-iodide-stained nuclei in the antisense-oligomer-treated cells. The apoptosis induced by HSP antisense oligomer was dose- and time-dependent. The antisense oligomer induced apoptosis mainly in tumor cells at G1 and S phase, resulting in an inhibition of cell proliferation. HSP70 antisense oligomer caused DNA-sequence-specific inhibition of HSP70 expression, which preceded apparent apoptosis. These results indicate that HSP70 antisense treatment inhibits the expression of HSP70, which in turn inhibits cell proliferation and induces apoptosis in tumor cells and suggest that HSP70 is required for tumor cells to proliferate and survive under normal condition.

Apoptosis↗
PubMed1995

Activation of natural killer cells by the mAb YTA-1 that recognizes leukocyte function-associated antigen-1.

The mAb YTA-1, which brightly stains CD3-CD16+ large granular lymphocytes (LGL)/natural killer (NK) cells and CD8+ T cells by immunofluorescence, is specific for leukocyte function-associated antigen (LFA)-1. Some mAbs recognizing the LFA-1 alpha chain (CD11a) or LFA-1 beta chain (CD18) inhibited the binding of YTA-1 to peripheral blood mononuclear cells. YTA-1 mAb could be chemically cross-linked to 170 and 96 kDa molecules, whose molecular weights correspond to those of LFA-1 alpha and beta respectively. YTA-1 bound to COS-7 cells co-transfected with CD11a and CD18 cDNAs, but not to untransfected cells. Reactivities of YTA-1 to K562 cells transfected with LFA-1 alpha and beta (CD11a/CD18) cDNAs and to CHO cells transfected with Mac-1 (CD11b/CD18) or p150, 95 (CD11c/CD18) cDNAs strongly suggest that YTA-1 recognizes either LFA-1 alpha or an epitope formed by a combination of LFA-1 alpha and beta. Treatment of fresh CD3-CD16+ LGL with YTA-1 augmented cytolytic activity and induced proliferation. F(ab')2 fragments of YTA-1 augmented NK cytotoxicity, indicating that the NK activating signal was transmitted through LFA-1 without involvement of Fc gamma receptor III. In contrast, the other mAbs against LFA-1 could not activate NK cells. These results collectively indicate that YTA-1 recognizes a unique epitope of LFA-1, which is involved in activation of fresh NK cells.

Animals↗
PubMed1995

Accumulation of gamma/delta T cells in human dysgerminoma and seminoma: roles in autologous tumor killing and granuloma formation.

The precise biological function of a subset of T cells bearing gamma/delta T cell receptor (TCR) remains poorly understood. The present study demonstrated the presence of gamma/delta T cells in tumor-infiltrating lymphocytes (TIL) and peripheral blood lymphocytes (PBL) of human patients with dysgerminoma and seminoma when determined by flow cytometry and in situ immunohistochemical staining. TIL contained a high percentage of gamma/delta T cells, ranging from 17.3 to 35.1%. gamma/delta T cells often accumulated within the granulomatous inflammation of tumor tissues. The majority of gamma/delta T cells were V gamma 9/V delta 2+ cells. Freshly isolated PBL, TIL and purified gamma/delta T cells showed autologous tumor killing (ATK) activity, which could be inhibited by monoclonal antibodies (mAb) against V delta 2. Furthermore, two gamma/delta T cell clones established from TIL showed cytotoxicity against autologous and allogeneic dysgerminoma, while they had low or no lytic effects on other cell types including carcinomas of ovary and tumor cell lines such as K562, Daudi and Molt-4. Lysis of autologous tumor cells by the clone was inhibited completely by anti-V delta 2 mAb and partially by mAb against CD3, LFA-1 alpha and ICAM-1 molecules, while it was resistant to anti-CD8, anti-HLA-ABC and anti-HLA-DR mAb. Supernatants produced by gamma/delta T cell clones induced adhesion, aggregation and increased DNA synthesis of monocytes and some characteristics of activated macrophages or epithelioid cells. Tumor necrosis factor (TNF)-alpha, granulocyte-macrophage colony stimulating factor (GM-CSF) and interferon (IFN)-gamma were detected in the supernatants of gamma/delta T cell clone. These results suggest that gamma/delta T cells accumulating in dysgerminoma and seminoma exhibit ATK activity through V gamma 9/delta 2 TCR and these gamma/delta T cells also play a role in the formation of granulomatous inflammation, which is associated with human dysgerminoma and seminoma.

Adult↗
PubMed1994

Induction of apoptosis by quercetin: involvement of heat shock protein.

Quercetin, a widely distributed bioflavonoid, inhibits the growth of tumor cells. The present study was designed to investigate the possible involvement of apoptosis and heat shock protein in the antitumor activity of quercetin. Treatment with quercetin of K562, Molt-4, Raji, and MCAS tumor cell lines resulted in morphological changes, including propidium iodide-stained condensed nuclei (intact or fragmented), condensation of nuclear chromatin, and nuclear fragmentation. Agarose gel electrophoresis of quercetin-treated tumor cells demonstrated a typical ladder-like pattern of DNA fragments. In addition, the hypodiploid DNA peak of propidium iodide-stained nuclei was revealed by flow cytometry. Quercetin induced apoptosis in cells at G1 and S in a dose- and time-dependent manner. The apoptosis-inducing activity of quercetin was enhanced by cycloheximide and actinomycin D. A nuclease inhibitor, aurintricarboxylic acid, inhibited quercetin-induced apoptosis, whereas deprivation of intracellular calcium by EGTA had no effect. 12-O-Tetradecanoylphorbol-13-acetate and H-7 did not affect the induction of apoptosis by quercetin. The synthesis of HSP70 was inhibited by quercetin when determined by immunocytochemistry, Western blot analysis, and Northern blot analysis. Quercetin-treated tumor cells were not induced to show aggregation of HSP70 in the nuclei and nucleolus in response to heat shock, resulting in apoptosis. By contrast, when tumor cells were first exposed to heat shock, no apoptosis was induced by quercetin. In addition, pretreatment of tumor cells with HSP70 antisense oligomer that specifically inhibited the synthesis of HSP70 enhanced the subsequent induction of apoptosis by quercetin. These results suggest that quercetin displays antitumor activity by triggering apoptosis and that HSP70 may affect quercetin-induced apoptosis.

Apoptosis↗
PubMed1994

A reporter system using a flow cytometer to detect promoter/enhancer activity in lymphoid cell lines.

We have devised an experimental system using a flow cytometer to examine the promoter/enhancer activity of DNA fragments in human lymphoid cell lines. Murine CD8 alpha gene cDNA used as a reporter gene was inserted in the reporter constructs under the control of various promoter/enhancers. Furthermore, the Epstein-Barr virus (EBV) OriP, which supports a high transient expression, was also included in the reporter constructs. Cell lines expressing EBV nuclear antigen-1 (EBNA-1) were transfected with the reporter constructs by electroporation. The expression of the reporter gene was measured by a flow cytometric analysis. This experimental system is quite simple and may be especially useful for the analysis of transcriptional elements functioning in lymphocytes.

B-Lymphocytes↗
PubMed1994

Lineage determination of CD7+ CD5- CD2- and CD7+ CD5+ CD2- lymphoblasts: studies on phenotype, genotype, and gene expression of myeloperoxidase, CD3 epsilon, and CD3 delta.

The gene expression of myeloperoxidase (MPO), CD3 epsilon, and CD3 delta molecules, the gene rearrangement of T-cell receptor (TCR) delta, gamma, and beta and immunoglobulin heavy (IgH) chain, and the expression of cell-surface antigens were investigated in seven cases of CD7+ CD5- CD2- and four cases of CD7+ CD5+ CD2- acute lymphoblastic leukemia or lymphoblastic lymphoma (ALL/LBL) blasts, which were negative for cytochemical myeloperoxidase (cyMPO). More mature T-lineage blasts were also investigated in a comparative manner. In conclusion, the CD7+ CD5- CD2- blasts included four categories: undifferentiated blasts without lineage commitment, T-lineage blasts, T-/myeloid lineage blasts, and cyMPO-negative myeloblasts. The CD7+ CD5+ CD2- blasts included two categories; T-lineage and T-/myeloid lineage blasts. The 11 cases were of the germ-line gene (G) for TCR beta and IgH. Four cases were G for TCR delta and TCR gamma. The others were of the monoclonally rearranged gene (R) for TCR delta and G for TCR gamma or R for both TCR delta and TCR gamma. The expression or in vitro induction of CD13 and/or CD33 antigens correlated with the immaturity of these neoplastic T cells, since it was observed in all 11 CD7+ CD5- CD2- and CD7+ CD5+ CD2-, and some CD7+ CD5+ CD2+ (CD3- CD4- CD8-) cases, but not in CD3 +/- CD4+ CD8+ or CD3+ CD4+ CD8- cases. CD3 epsilon mRNA, but not CD3 delta mRNA, was detected in two CD7+ CD5- CD2- cases, while mRNA of neither of the two CD3 molecules was detected in the other tested CD7+ CD5- CD2- cases. In contrast, mRNA of both CD3 epsilon and CD3 delta were detected in all CD7+ CD5+ CD2- cases, indicating that CD7+ CD5- CD2- blasts at least belong to T-lineage. The blasts of two CD7+ CD5- CD2- cases with entire germ-line genes and without mRNA of the three molecules (MPO, CD3 epsilon, and CD3 delta) were regarded as being at an undifferentiated stage prior to their commitment to either T- or myeloid-lineage. The co-expression of the genes of MPO and CD3 epsilon in a CD7+ CD5- CD2- case MPO, CD3 epsilon, and CD3 delta in a CD7+ CD5+ CD2- case suggested the presence of some overlapping phase for T- and myeloid-lineage commitment during immature stages of differentiation. This helps understand the conversion of some T-ALL/LBL cases to acute myeloblastic leukemia (AML).(ABSTRACT TRUNCATED AT 400 WORDS)

Acute Disease↗
PubMed1992

Detection of Epstein-Barr virus genome in natural-killer-like cell line, YT.

YT cells, originally reported as a natural-killer-like (NK-like) lymphoid cell line, were investigated for Epstein-Barr virus (EBV) genome, gene rearrangement for T-cell receptor (TCR), phenotype and function. The YT cells of the original batch (YT-0) and two subclones (YT2C2 and YTC3) expressed EBV-associated nuclear antigen, and the BamHI-digested DNA showed the 3.4 kb hybridizing band with the BamHIW probe of EBV DNA in Southern blot analysis. When tested with latent-infection membrane protein probe, an identical hybridizing band was shared, indicating that all three sources of YT cells were of monoclonal derivation in terms of the terminal repeat junctional structure of EBV DNA, and that the original YT cells had been infected with EBV before the isolation of the two subclones. The cell-surface antigen analysis revealed the expression of CD7, CD28, CD30, CD45R0, TLiSA and S6F1 antigen besides the originally recorded CD25, CD56 and HLA-DR antigen. Gene rearrangement analysis showed the germ-line genotype, including TCR gamma and delta as well as beta chain. The Northern blot study using the CD3 epsilon and CD3 delta chain gene probes revealed CD3 epsilon, but not CD3 delta RNA. The YT-0 cells exhibited NK and antibody-dependent cellular cytotoxicity activity, but the YT2C2 and YTC3 cells did not. It was not resolved whether the fresh neoplastic NK-like cells of the YT-cell donor carry EBV genome, but YT cells, the first lymphoid cell line found to have EBV genome and non-B lymphoid properties, are valuable for investigating the relationship between EBV and human non-B lymphoid hematopoietic cells.

Antigens, Surface↗
PubMed1991

Inhibition of human immunodeficiency virus replication in a human T cell line by antisense RNA expressed in the cell.

The effect of the expression of antisense RNA against the human immunodeficiency virus (HIV) genome in a human T-cell line CEM on HIV replication was investigated. A 2.7 kilobase (kb) fragment of the HIV genome, including tat and a part of rev and env, was cloned into the retroviral vector WB in the antisense orientation under the SV40 or H-2K promoter. CEM cells transduced with this antisense gene via recombinant retrovirus expressed the RNAs of three different molecular sizes containing the antisense construct. CEM cells and these transduced cells were infected with HIV. HIV replication was evaluated 4-10 days later by an immunofluorescence assay and by determining the reverse transcriptase activity in the culture supernatant. The results indicate that although the recombinant retrovirus WB strongly enhanced the HIV replication in CEM cells, the expression of antisense RNA in the cells was highly effective in impeding the replication of HIV. The inhibitory effect was especially high in CEM cells transduced with the antisense gene under the control of SV40 promoter. In this case, HIV antigen-positive cells and reverse transcriptase activity in the culture supernatant of transduced cells were reduced to 30-50% and 5-10% of those in CEM cells and in the CEM cells transduced with WB, respectively.

Blotting, Northern↗
PubMed1990

Detection of Epstein-Barr virus genome in benign polyclonal proliferative T cells of a young male patient.

Epstein-Barr virus (EBV) DNA was detected in polyclonal T cells that proliferated transiently in a 21-year-old male (referred to as H.J.) who underwent an apparently benign lymphocytosis (white blood cells, 31 x 10(6)/microL; lymphocyte, 79%) with fever, tonsillar swelling, lymphadenopathy, and hepatosplenomegaly. The symptoms and signs subsided mostly within a month of hospitalization. The major population of the lymphocytes at admission was positive for CD3, CD8 (4/8 ratio, 0.16), WT31, and DR antigen. Eight percent of the leukocytes were too blastoid to be classified as atypical lymphocytes of infectious mononucleosis (IM). The blastoid lymphocytes and the duration and degree of the lymphocytosis and hypergammaglobulinemia appeared inconsistent with IM, whereas the EBV serology indicated either EBV primary infection or a secondary alteration of normal seropositive EBV immunity. The genomic analysis of T-cell receptor beta chain in the peripheral blood mononuclear cells (PBMC) at admission with a C beta probe did not show a monoclonal rearrangement. EBV genome was detected in these cells, using the BamHI W and K probe, but not in the cells after discharge. Analysis of the EBV terminal repeat junctional sequence, using Xho I fragment of the latent membrane protein (LMP) probe binding with the terminus, did not show monoclonal or oligoclonal populations. EBV-associated nuclear antigen (EBNA) was detected in 36% of the PBMC at admission, but not in the later cells. These EBNA-positive cells were found to form rosette with sheep erythrocytes. The PBMC of six acute IM patients contained neither EBV DNA nor EBNA-positive cells. The observations in this case show a unique type of EBV infection in T cells that has not been previously reported.

Adult↗