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Biomedical subjects

A Yachie

Publications and source records attributed to A Yachie.

At least 91 records · Page 5Linked to original sources
PubMed1990

Detection by in situ hybridization and phenotypic characterization of cells expressing IL-6 mRNA in human stimulated blood.

IL-6 has manifold biologic functions in immune and inflammatory responses and is produced by a variety type of cells. In this work, we used the whole blood culture to identify the cells expressing IL-6 gene/protein after various stimulation. When the whole blood was incubated with LPS or Con A, much IL-6 activity, measured by the growth promoting assay using a murine IL-6-dependent hybridoma clone, was detected in the plasma as early as 4 h of culture and continued to increase with time, reaching a plateau around 12 h. Immunocytochemical analysis with anti-rIL-6 antiserum revealed that a proportion of mononuclear cells (MNC) contained intracytoplasmic IL-6 in LPS- or Con A-stimulated blood. Northern blot analysis for MNC from the blood stimulated with these stimuli showed that their transcripts for IL-6 peaked at 4 h, then rapidly declined and was undetectable after 24 h of stimulation. In situ hybridization technique with radiolabeled antisense RNA probe for IL-6 demonstrated that a fraction of MNC from LPS- as well as Con A-stimulated blood expressed IL-6 mRNA. With the combined use of in situ hybridization and immunofluorescence by corresponding mAb, it was confirmed that IL-6 mRNA expressing cells in stimulated blood were exclusively monocytes. In the whole blood culture, it was shown that expression of IL-6 mRNA by monocytes was inhibited by dexamethasone, but not by cyclosporin A. These results suggest that monocytes are the major cells expressing IL-6 gene/protein in the circulation after exposure to external stimuli.

Antigens, CD↗
PubMed1990

The capability of neonatal leukocytes to produce IL-6 on stimulation assessed by whole blood culture.

IL-6 is a cytokine with a wide variety of influences on the cells involved in immune and inflammatory responses. Defective production of IL-6 may be partly responsible for the impaired immune defense and inflammatory response often observed in the neonatal period. In our study, we used whole blood culture to examine the capacity of neonatal leukocytes to produce IL-6 in response to various stimuli. IL-6 activity was evaluated by growth promoting assay using an IL-6-dependent murine hybridoma clone. IL-6 activity was undetectable in fresh or unstimulated blood obtained from both newborns and adults. In contrast, incubation of whole blood with lipopolysaccharide or concanavalin A resulted in marked IL-6 activity. After stimulation, IL-6 activity was induced as early as 2 h after culture and increased with time, reaching a plateau at around 12 h. Comparative examinations suggested that the IL-6 activity induced in neonatal blood on stimulation was similar to that seen in stimulated adult blood. Neutralization experiments with anti-IL-6 anti-serum confirmed the presence of IL-6 proteins in the stimulated blood, and induction of cellular IL-6 mRNA was demonstrated in the stimulated blood as well. In addition, immunocytochemical observations suggested that the major IL-6 producing cells in the stimulated blood may be monocytes. The results suggest that the production of IL-6 in response to specified stimuli is normal at birth.

Adult↗
PubMed1990

Epstein-Barr virus-immortalized B cells produce IL-6 as an autocrine growth factor.

The continuous proliferation of Epstein-Barr virus (EBV)-immortalized B cells is enhanced by autocrine as well as paracrine growth factors. In the present study, the possibility that EBV-immortalized B cells might produce interleukin-6 (IL-6) proteins in an autocrine manner was examined. It was found that culture supernatants from EBV-transformed B cells, but not from Burkitt's lymphoma lines, augmented the proliferation of an IL-6-dependent murine hybridoma clone, MH60.BSF2. This growth-promoting activity for hybridoma cells found in culture supernatants of EBV-transformed B cells was specifically neutralized by rabbit anti-recombinant (r) IL-6 antibody. The IL-6 activity in culture supernatants of EBV-transformed B cells, though much less than that of lipopolysaccharide (LPS)-stimulated monocytes, was increased by the addition of phorbol myristate acetate. Western blot experiments using rabbit anti-rIL-6 antiserum demonstrated that supernatants from cultured EBV-transformed B cells contained the distinct forms of IL-6, with a peak of 23,000 MW. When examined by in situ hybridization analysis, it was found that IL-6 mRNA were expressed on EBV-transformed B cells. It was noted that a fraction, but not all, of these cells expressed IL-6 mRNA strongly, implying their cell cycle-dependent expression. In addition, it was shown that rIL-6 promoted the growth of EBV-transformed B cells at low cell densities. The results suggest that IL-6 serves as an autocrine growth factor in EBV-transformed B cells.

B-Lymphocytes↗
PubMed1990

Immune status in two brothers with Omenn's syndrome: no discernible chimerism on FACS analysis using a monoclonal antibody specific for a maternally restricted HLA antigen.

Sequential immunologic examinations, including lymph node biopsies, in two brothers with clinical characteristics of Omenn's syndrome are presented in this study. Although the number of circulating T cells with mature phenotype (OKT3+, TCR1+) was within normal range, the lymphocyte proliferative response to mitogens was poor. Examinations of the lymph nodes revealed marked lymphoid depletion associated with eosinophilic infiltration and reticular cell proliferation. Over the clinical course of 5 months, circulating T cells also mostly disappeared. Thymic hypoplasia was noted at autopsy. Although intrauterine graft-versus host disease (GVHD) has been hypothesized as being the pathogenetic mechanism in this syndrome, maternal lymphocytes circulating in these patients were not identified either by karyotype and HLA typing or by highly sensitive FACS analysis and immunohistochemical studies using a monoclonal antibody, HLA-A9, specific for a maternally restricted HLA antigen, Aw24. In conclusion, the familial occurrence and the absence of maternal chimerism might be the essential features of Omenn's syndrome which should be differentiated from fetal GVHD.

Antibodies, Monoclonal↗
PubMed1989

Effector and precursor phenotypes of lymphokine-activated killer cells in mice with severe combined immunodeficiency (scid) and athymic (nude) mice.

The lineage of lymphokine-activated killer (LAK) cells is poorly understood. To examine the relationship between LAK and natural killer (NK) cells we utilized two congenitally immunodeficient mice, namely severe combined immunodeficient (scid) and athymic (nude) mice that lack T cells but have normal NK cells. LAK activity was evaluated by the ability to lyze NK-resistant P815 cells. When cultured with human recombinant interleukin 2, splenocytes of scid and nude mice could generate LAK activity at levels comparable to or more than those of normal C.B-17 mice. LAK effector cells in these immunodeficient mice as well as normal mice had the phenotype resembling that of NK cells with asialo-GM1 (aGM1) expression. In vivo treatment with anti-aGM1 antiserum completely abolished the induction of LAK activity from splenocytes of normal mice. In contrast, LAK activity in splenocytes of scid and nude mice was still demonstrable even after this treatment, indicating that most LAK precursors in both mice were cells without aGM1 antigen. The aGM1- progenitors for LAK activity, probably in common with NK progenitors, appeared to be more expanded in scid and nude mice than in normal mice. The use of such congenitally immunodeficient mice should be helpful in studying the differentiation step of LAK as well as NK cells from their precursors.

Animals↗
PubMed1989

The acute phase nature of interleukin 6: studies in Kawasaki disease and other febrile illnesses.

Interleukin 6 (IL-6) has manifold biological functions involved in the immune or inflammatory responses of the host to various stimuli. Here we asked whether IL-6 might be responsible for manifestations of Kawasaki disease (KD), such as immunoglobulin hypersecretion, lymphocyte activation and systemic vasculitis. IL-6 activity in the serum was determined by a sensitive colorimetric assay using an IL-6-dependent murine hybridoma clone. Usually sera from healthy or afebrile donors contained only negligible levels of IL-6 activity below the detection threshold of the assay. Importantly it was found that serum IL-6 was markedly elevated in all patients with acute KD. Serum levels of IL-6 activity gradually diminished during the course of the disease and reached undetectable or lower levels at the convalescent phase. However, such elevated levels of serum IL-6 activity were also observed in the majority of other febrile diseases, such as bacterial or viral infections, indicating that the appearance of IL-6 in the serum could generally occur in febrile or inflammatory disease conditions. Serum IL-6 activity correlated with serum concentrations of some acute phase proteins (APP), such as C-reactive protein, haptoglobin and alpha 1-acid glycoprotein, implying its role for modulating induction of APP in vivo. IL-6 is well known to be secreted by a variety of cell types. Further studies, including immunohistochemical analysis using anti-IL-6 antibody, will be necessary to examine whether the source of serum IL-6 in KD might be different from that seen in other diseases.

Acute-Phase Proteins↗
PubMed1989

Developmental changes of double-negative (CD3+ 4-8-) T cells in human peripheral blood.

The frequency of double-negative (CD3+4-8-) T cells in the circulating pool was examined for different age groups. Adult peripheral blood contained a substantial but variable proportion of double-negative T cells. Double-negative T cells in adult peripheral blood were shown to lack the alpha/beta heterodimers of the T cell antigen receptor (TCR alpha/beta) but express the gamma chain of the TCR (TCR gamma), when examined using the corresponding monoclonal antibodies. In contrast to adult blood, double-negative, TCR alpha/beta-negative, and TCR gamma-positive T cells were very scarcely demonstrated in the cord blood of newborn infants. It was found that the percentages of double-negative T cells correlated very well with those of TCR alpha/beta negative T cells. These double-negative T cells in the peripheral blood gradually increased with advancing age and reached in frequency the adult level around one year of age. The results suggested that double-negative T cells expressing the TCR gamma/delta might play an ontogenically unique role in the development of the T cell functions.

Adolescent↗
PubMed1987

T3-T cell receptor (Ti) complex-independent activation of T cells by wheat germ agglutinin.

The T3-Ti complex appears to play a central role in the activation of T cells by antigens and mitogens. Wheat germ agglutinin (WGA) is a unique lectin which inhibits T cell proliferation induced by mitogens, but it also induces marked IL 2 production by peripheral blood T cells. The pattern of responses induced by WGA suggests that this lectin may use a different mechanism of T cell activation other than the mechanism employed by the common T cell stimulants. We first investigated the production of IL 2 by Jurkat cells (E6-1) stimulated with WGA, before and after modulation of the surface T3-Ti complex. IL 2 production was markedly reduced after modulation of the T3 antigen from the cell surface when these cells were stimulated with PHA. In contrast, little change was observed in WGA-induced IL 2 production after modulation. Furthermore, we examined the effect of WGA on a T3-mutant of E6-1 cells (T3.1) which does not produce IL 2 in response to PHA or PHA plus PMA. WGA-stimulated T3.1 cells produced a significant amount of IL 2 with or without added PMA. In addition, a small but consistent rise in intracytoplasmic free calcium was observed when these cells were stimulated with WGA. These results demonstrate the presence of an alternative mechanism of T cell activation independent of the T3-Ti complex.

Acetylglucosamine↗
PubMed1985

Immunostimulation by cytomegalovirus (CMV): helper T cell-dependent activation of immunoglobulin production in vitro by lymphocytes from CMV-immune donors.

Cytomegalovirus (CMV) is the cause of a number of different diseases ranging from self-limited benign infections in healthy adults to life threatening illnesses among immunocompromised hosts and newborns. Suppression of cell-mediated immunity is often found in cases of acute CMV infection, and in addition, the virus may also be a potent stimulant of lymphoid cells in vivo. We studied cellular proliferation and immunoglobulin (Ig) production induced by CMV to determine its effect on human lymphocytes in vitro. The CMV that was added to cultures of lymphocytes from CMV-seronegative donors failed to induce either significant cellular proliferation or Ig production. By contrast, CMV-stimulated cultures from CMV-seropositive donors induced both prominent cellular proliferation and Ig production. B cell differentiation into Ig-secreting cells required the presence of T cells, and this T cell help was sensitive to irradiation with 2000 rad and to treatment with cyclosporin A. When T cells were depleted of OKT4+ cells with monoclonal antibody and complement, the co-cultured B cells failed to produce Ig, whereas the depletion of OKT8+ cells had no effect on the Ig-secreting cell response. Inactivation of CMV before culture did not result in a reduction of either cellular proliferation or Ig production. Thus, infection of target cells is not required for in vitro lymphocyte activation by CMV. These results demonstrate that CMV is a potent activator of B cells inducing Ig production in vitro, and that this process requires the presence of virus-specific memory T cells.

Adult↗
PubMed1983

Kinetics of expression of T-cell "activation" antigens on in vivo- and in vitro-stimulated T cells.

The in vivo expression of T-cell activation antigens defined by means of an OKT series of monoclonal antibodies and the anti-Tac antibody was evaluated in peripheral T lymphocytes obtained from patients with infectious mononucleosis and in cerebrospinal fluid (CSF) cells collected from individuals with mumps meningitis by the immunoperoxidase method. Although Tac antigen was definitely expressed on about 10% of CSF cells in mumps meningitis, Tac+ T cells could not be identified on any peripheral blood T cells in infectious mononucleosis, which expressed OKIa 1+, OKT10+, and OKT9+ determinants, as a probable sign of in vivo activation. The coexpression of Ia+ antigens on a T-cell functional subset might be important for their functional properties. Our kinetic study and cell cycle analysis of the expression of these activation antigens suggested that Tac antigen might be expressed at the early stage of the G1 phase of the cell cycle; OKT9+ antigen at the late stage of the G1; and Ia+ and OKT10+ determinants might be expressed at the S phase or later on in vitro, probably one after another.

Acute Disease↗
PubMed1983

Sequential expression of T cell activation (Tac) antigen and Ia determinants on circulating human T cells after immunization with tetanus toxoid.

Recent studies have indicated that a monoclonal antibody, termed anti-Tac, may recognize the receptor sites or closely associated structures for interleukin 2 on activated human T cells. The Tac antigen, definable by anti-Tac antibody and usually found on mitogen- or alloantigen-stimulated T cells, was not expressed to any appreciable extent on normal circulating T cells. In the present study, we showed that an increase in circulating T cells expressing Tac antigen as well as Ia determinants occurred in normal individuals after immunization with tetanus toxoid. The expression of Tac antigen and Ia determinants on T cells was evaluated by the rosette method with Staphylococcal protein A-(SPA) coated bovine red blood cells (BRBC) or the indirect immunofluorescence method with monoclonal anti-Tac and anti-Ia antibodies. An increase in Tac-positive or Ia-positive T cells was more evident with the use of the rosette method with SPA-coated BRBC than with conventional immunofluorescence. The percentage of Ia-positive T cells showed a peak between 24 and 48 hr after toxoid injection, and remained at high levels until 2 wk after immunization. In contrast to Ia-positive T cells, the appearance of Tac-positive T cells was transient and at a rather early period of toxoid immunization. The maximum increase of Tac-positive T cells was apparent around 12 hr after toxoid injection, and Tac-positive T cells disappeared abruptly from circulation by 24 hr after inoculation. Ia-positive T cells were induced in both Leu-2 suppressor/cytotoxic and Leu-3 helper/inducer subsets, whereas Tac-positive T cells were generated only within the Leu-3 subset. The fact that induction of Tac-positive and Ia-positive T cells might occur at different stages of T cell activation and in different subsets of T cells seemed to be important for elucidating their roles in the in vivo T cell proliferation and differentiation.

Adult↗
PubMed1983

Cyclosporin A does not prevent expression of Tac antigen, a probable TCGF receptor molecule, on mitogen-stimulated human T cells.

Results of recent studies indicated that a monoclonal anti-Tac antibody might recognize the receptor sites or closely related structures for T cell growth factor (TCGF) on activated human T cells. In the present study, we examined the effect of cyclosporin A (CsA) on the expression of Tac antigen by mitogen-stimulated T cells. CsA inhibited the proliferative response of T cells to Con A and PHA in a dose-dependent manner. Both Con A- and PHA-induced cellular proliferation were decreased to about 10% of controls at 5 micrograms/ml of CsA. When T cells were stimulated with these mitogens, many of them expressed Tac antigen on their surfaces, assessed by the immunoperoxidase method. The appearance of Tac-positive cells occurred earlier than a rise of cellular DNA synthesis. Characteristically, CsA showed no inhibitory effect on the expression of Tac antigen by mitogen-stimulated T cells, even at a relatively high concentration of 5 micrograms/ml, whereas the expression of other "activation" antigens reactive with monoclonal anti-Ia, OKT9, or OKT10 antibodies by T cells was blocked completely by CsA. Morphologically, the majority of Tac-positive cells in culture with mitogens alone showed the characteristics of blastoid cells; Tac-positive cells in the culture containing CsA mainly consisted of medium-sized cells, indicating these cells probably accumulated at a stage of partial activation. T cells, once stimulated with Con A or PHA for 3 days whether in the presence or in the absence of CsA, were able to absorb TCGF activity from TCGF-containing media similarly. In addition, T cells, even stimulated in the presence of CsA with these mitogens for 24 hr, were capable of responding to TCGF with the same grade of proliferation as did T cells stimulated with mitogen alone. CsA showed no appreciable inhibition in a TCGF-dependent proliferation of such prestimulated cells. These functional properties of activated T cells might be correlated with their ability to express Tac antigen. These experimental findings present some evidence that CsA might not prevent the expression of probable functional receptor sites for TCGF in mitogen-dependent activation of human T cells.

Antibodies, Monoclonal↗
PubMed1982

Discrepancy in expression ability of Tac antigen and Ia determinants defined by monoclonal antibodies on activated or cultured cord blood T lymphocytes.

T lymphocytes from cord blood and adults stimulated with mitogens and alloantigen were analyzed using monoclonal antibodies that detect Ia and Tac antigens associated with lymphocyte activation. Less than 5% of T cells freshly isolated or cultured without stimuli expressed Ia and Tac antigens assessed by the indirect immunofluorescence method. When both T cells from cord blood and adults were cultured with phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM), or allogeneic cells, variable proportions of them expressed Tac antigen. The expression of Tac antigen on stimulated T cells increased in parallel with DNA synthesis, and was significantly correlated with the proliferative response of stimulated T cells. In contrast to Tac antigen, the expression of Ia antigens appeared to show some time lag behind the proliferative response. Although expression of Ia antigens was too weak to assess in PHA- or Con A-stimulated cultures, adult T cells were stimulated by PWM or allogeneic cells to express strongly Ia antigens in culture. However, the percentage of Ia+ cells in cord blood T cells stimulated with PWM or allogeneic cells was markedly lower as compared with that of adults. In addition, cord blood T cells grown in the presence of T cell growth factor showed the same degree of Tac antigen as adult ones did, whereas their expression of Ia antigens was negligible as compared with adult controls. These observations suggested that T cells in cord blood might be deficient by nature in their ability to express Ia antigens on activation. The inability to develop Ia+ T cells seemed to be characteristic of functional immaturity of T cells in early human ontogeny.

Adult↗
PubMed1982

Functional significance of Tac antigen expressed on activated human T lymphocytes: Tac antigen interacts with T cell growth factor in cellular proliferation.

Cultured human T cells (CTC), which are grown in conditioned medium containing T cell growth factor (TCGF), proliferate in response to TCGF. It has been shown that an antigen (Tac) defined by a monoclonal antibody, termed anti-Tac antibody, is expressed on human T cells activated by mitogens or antigens and CTC grown in the presence of TCGF. To elucidate the functional significance of Tac antigen expressed on activated T cells, we studied the effect of anti-Tac antibody on TCGF-dependent proliferation of CTC. The addition of anti-Tac antibody strongly inhibited the proliferation of CTC induced by TCGF. This inhibition was observed only when the antibody was added at the early phase of culture, but not when the addition of the antibody was delayed beyond 24 hr of culture. Seven-day-old PHA-induced T cell blasts, but not fresh peripheral blood lymphocytes, were able to absorb TCGF activity in conditioned medium, as assessed by the DNA synthesis of CTC. When PHA-induced blasts were treated with anti-Tac antibody before absorption, their capacity to absorb TCGF activity was almost completely eliminated. In contrast, absorption of TCGF by PHA-induced blasts was not significantly reduced even when they were pretreated with other monoclonal antibodies (anti-Ia, OKT9, or OKT10) with specificity for antigens expressed on activated T cells. Based on the view that TCGF interacts with activated T cells via specific membrane receptors, these observations suggested that anti-Tac antibody might specifically block the binding of TCGF to the corresponding membrane binding sites, resulting in the inhibition of TCGF-dependent proliferation of CTC. Tac antigen expressed on activated T cells seems to participate in responding process of activated T cells to TCGF.

Antigen-Antibody Reactions↗