Elaine F Lizzio - Academia.edu (original) (raw)
Papers by Elaine F Lizzio
Journal of Aquatic Animal Health, 1989
Abstract Immunosuppression was demonstrated in sections of rainbow trout Oncorhynchus mykiss (for... more Abstract Immunosuppression was demonstrated in sections of rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri) spleens immunized in vitro and exposed in culture to different concentrations of copper chloride. The sections were immunized with ...
Clinical Immunology and Immunopathology, Mar 31, 1991
Human peripheral blood monocytes exposed to MK-886 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopro... more Human peripheral blood monocytes exposed to MK-886 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2- dimethylpropanoic acid) at doses which abolish formation of 5-lipoxygenase metabolites showed unaltered interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha) levels in response to phorbol ester, concanavalin A, serum-treated zymosan, or lipopolysaccharide. Indomethacin (10 microM), alone or in combination with MK-886, also failed to modulate monokine production in response to any stimulus. Exogenous arachidonate (3-30 microM) which augmented the formation of PGE2 and LTB4 in the absence of stimulation, also had no effect on monokine production. LPS-induced IL-1 and TNF production occurred despite stimulation of PGE2 synthesis. The results make a role for endogenous prostaglandins and leukotrienes in the regulation of monocyte IL-1 beta and TNF-alpha production unlikely. These data also indicate that MK-886, a novel inhibitor of 5-lipoxygenase product formation, is a potentially useful leukotriene inhibitor which does not affect monokine production.
Inflammation, 1992
Human monocytes released superoxide anion, prostaglandin E2, leukotriene B4, IL-1, and TNF when e... more Human monocytes released superoxide anion, prostaglandin E2, leukotriene B4, IL-1, and TNF when exposed to plastic surfaces coated with murine anti-CD3 monoclonal antibody, OKT 3. Stimulation of mediator release by OKT 3 was dependent on the amount of antibody immobilized onto wells of plastic tissue culture plates. Soluble antibody or antibody adsorbed to monocytes and reacted with an aggregating ("cross-linking") second antibody failed to induce mediator release. Monocytes "armed" with OKT 3 formed rosettes with T cells in a fashion indistinguishable from that seen between monocytes and T cells sensitized with OKT 3. Monocytes with adsorbed OKT 3 antibodies released IL-1 beta and TNF-alpha when exposed to unsensitized T cells, although increased superoxide release could not be detected. OKT 4a, a murine IgG2a antibody that reacts with a different T cell epitope (CD4), failed to induce cytokine release from monocytes when cross-linked by T cells or a CD4+ T cell line, even in the presence of IL-2 or IFN-gamma. These data indicate that certain antibodies bound to Fc receptors (FcR) of monocytes may trigger monocyte function when reacting with cells bearing the appropriate target antigens. FcR-mediated signaling resulting in mediator release may be involved in initiating or regulating the immune response. Furthermore, systemically administered monoclonal antibodies may induce inflammatory responses and their attendant symptomatologies via their interaction with FcR-bearing inflammatory cells.
Transplantation, 1986
The effect of prior administration of anti-DNP (N-[2,4-dinitrophenyl]-B-alanylglycylglycine) mono... more The effect of prior administration of anti-DNP (N-[2,4-dinitrophenyl]-B-alanylglycylglycine) monoclonal antibodies on the humoral immune response of BALB/c mice was examined. One-time administration of a "cocktail" of two anti-DNP monoclonals resulted in suppression of the IgM anti-DNP response for one week after challenge but not longer. Maximal suppression of anti-DNP IgM plaque-forming cells was achieved by administration of antibody 1-2 weeks before challenge with DNP. Maximal suppression of serum IgM antibody was seen by administration of antibody 2-3 weeks before challenge with antigen. Following one-time administration of suppressive monoclonal antibody, the serum IgG antibody response to DNP was suppressed beginning 2 weeks after immunization and remained so for up to 241 days despite continual booster injections of antigen. Although most effective suppression of the humoral anti-DNP response was seen in animals receiving their single dose of suppressive antibody 2 weeks before first exposure to antigen, suppression of the IgG response was evident at all intervals examined up to 232 days in mice given monoclonal antibody between 0.1 day and 30 days before antigen, but not at earlier times. These findings suggest that regulatory networks, rather than the masking of antigenic determinants by passively administered antibody, play a role in antibody-mediated immunoregulation. They may be of use in designing strategies for optimizing immunosuppression protocols in clinical studies.
![Research paper thumbnail of Absence of modulation of monokine production via endogenous cyclooxygenase or 5-lipoxygenase metabolites: MK-886 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid), indomethacin, or arachidonate fail to alter immunoreactive interleukin-1β, or TNF-α production...](https://attachments.academia-assets.com/66356227/thumbnails/1.jpg)
](Clinical Immunology and Immunopathology, 1991
Human peripheral blood monocytes exposed to MK-886 (3-[l-(4-chlorobenzyl)-3t-butyl-thio-5-isoprop... more Human peripheral blood monocytes exposed to MK-886 (3-[l-(4-chlorobenzyl)-3t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylprop~oic acid) at doses which abolish formation of 5-lipoxygenase metabolites showed unaltered interleukin-ll3 (IL-1B) or tumor necrosis factor-a (TNF-a) levels in response to phorbol ester, concanavalin A, serum-treated zymosan, or lipopolysaccharide. Indomethacin (10 t&f), alone or in combination with MK-886, also failed to modulate monokine production in response to any stimulus. Exogenous arachidonate (3-30 l&) which augmented the formation of PGEz and LTB, in the absence of stimulation, also had no effect on monokine production. LPS-induced IL-l and TNF production occurred despite stimulation of PGE, synthesis. The results make a role for endogenous prostaglandins and leukotrienes in the regulation of monocyte IL-lp and TNF-a production unlikely. These data also indicate that MK-886, a novel inhibitor of Slipoxygenase product formation, is a potentially useful leukotriene inhibitor which does not affect monokine production.
Clinical Immunology and Immunopathology, 1987
The simultaneous production of prostaglandins, leukotrienes, and hydroxyeicosatetraenoic acids wa... more The simultaneous production of prostaglandins, leukotrienes, and hydroxyeicosatetraenoic acids was studied in human peripheral blood monocytes obtained by counter-current centrifugal elutriation. Monocytes prelabeled for 4 hr with [3H]arachidonic acid (AA) released label into the surrounding medium in response to treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) or calcium ionophore (A23187). High-performance liquid chromatography of monocyte supernatants demonstrated that labeled compounds included those which eluted with authentic standards for thromboxane B2, 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT), 6-keto-prostaglandin F alpha, prostaglandin E2, and 15-hydroxyeicosatetraenoic acid (HETE). 5-HETE and leukotriene B4 (LtB4) were detected only in response to ionophore treatment. Highly purified lymphocytes did not convert AA to autocoids, despite the release of free arachidonate in response to either stimulus. Pretreatment of monocytes with recombinant human interferon (IFN)-gamma or IFN-alpha for 18 hr resulted in enhanced release of labeled arachidonic acid and increased conversion to autocoids after TPA or ionophore stimulation. Absolute amounts of prostaglandin E2 produced in response to TPA or ionophore treatment were increased as well. These results demonstrate the autocoid profile released by stimulated human monocytes and illustrate the effects of IFN treatment on the production of lipoxygenase metabolites of arachidonic acid as well as cyclooxygenase products.
Advances in Experimental Medicine and Biology, 1990
Human monocytes release arachidonic acid upon stimulation with a variety of soluble or particulat... more Human monocytes release arachidonic acid upon stimulation with a variety of soluble or particulate agents. These include: phorbol esters (i.e., 12-O-tetradecanoate phorbol-13-acetate, TPA), calcium ionophores (ionomycin), serum-treated zymosan (STZ) concanavalin A (Con A), and, to a minor degree, lipopolysaccharides (LPS). Protein Kinase C activation or increased intracellular Ca2+ are common features of the actions of most, if not all, of these stimuli. Prevention of PKC activation by the use of staurosporine or chelation of extracellular calcium by EGTA selectively impaired AA release, indicating that PLA2 may be regulated by either pathway concurrently. The generation of inositol phosphates and diacylglycerol by the action of phospholipase C, notably upon interaction with opsonized particles during phagocytosis, apparently constitutes the physiological correlate of stimulation via these agents. Release of arachidonic acid by the action of PLA2 or other phospholipid hydrolyzing enzymes leads directly to the formation of cyclooxygenase products. In the presence of markedly elevated calcium concentrations, 5-lipoxygenase (LO) is activated as well, leading to the formation and release of leukotrienes. Agents which stimulate AA release also initiate other monocyte functions, including generation of reactive oxygen intermediates and lymphokine release. This observation makes it tempting to implicate PLA2 activation in many aspects of monocyte physiology. However, no correlation with PLA2 activation and either superoxide or lymphokine release was found when multiple stimuli, including TPA, ionomycin, serum-treated zymosan, concanavalin A, or LPS, were compared simultaneously. Instead, our results indicate that PLA2 activation is regulated by the same mechanisms, including PKC activation and increased Ca2+, as are other enzymes which determine expression of monocyte function. Phospholipase A2 (PLA2) hydrolyzes fatty acid from the sn-2 position of a wide variety of phospholipids. Substrates for this (these) enzyme(s) include species which contain a variety of polar head groups (choline, serine, ethanolamine, etc.) and some phospholipids with either linkages in sn-1. In many cell types, including human monocytes, phospholipase A2 commonly acts on substrates containing arachidonic acid (AA). The liberation of free arachidonate is a first step in the metabolism of prostaglandins, hydroxyeicosatetraeinoic acids, (HETE'S), and leukotrienes (Lt's). Monocytes and macrophages have been shown to be rich sources of arachidonate and its metabolites. Some biologic properties of monocytes, notably their role as immunomodulating cells, have been attributed to eicosanoid production and release. Accordingly, much of the interest regarding PLA2 in human monocytes centers on this aspect of their function.(ABSTRACT TRUNCATED AT 400 WORDS)
Transplantation, 1986
The effect of prior administration of anti-DNP (N-[2,4-dinitrophenyl]-B-alanylglycylglycine) mono... more The effect of prior administration of anti-DNP (N-[2,4-dinitrophenyl]-B-alanylglycylglycine) monoclonal antibodies on the humoral immune response of BALB/c mice was examined. One-time administration of a "cocktail" of two anti-DNP monoclonals resulted in suppression of the IgM anti-DNP response for one week after challenge but not longer. Maximal suppression of anti-DNP IgM plaque-forming cells was achieved by administration of antibody 1-2 weeks before challenge with DNP. Maximal suppression of serum IgM antibody was seen by administration of antibody 2-3 weeks before challenge with antigen. Following one-time administration of suppressive monoclonal antibody, the serum IgG antibody response to DNP was suppressed beginning 2 weeks after immunization and remained so for up to 241 days despite continual booster injections of antigen. Although most effective suppression of the humoral anti-DNP response was seen in animals receiving their single dose of suppressive antibody 2 weeks before first exposure to antigen, suppression of the IgG response was evident at all intervals examined up to 232 days in mice given monoclonal antibody between 0.1 day and 30 days before antigen, but not at earlier times. These findings suggest that regulatory networks, rather than the masking of antigenic determinants by passively administered antibody, play a role in antibody-mediated immunoregulation. They may be of use in designing strategies for optimizing immunosuppression protocols in clinical studies.
Journal of Immunological Methods, 1988
Peripheral blood monocytes release superoxide (02) and arachidonic acid (AA) when stimulated with... more Peripheral blood monocytes release superoxide (02) and arachidonic acid (AA) when stimulated with 12-O-tetradecanoate phorbol-13-acetate (TPA) or calcium ionophores (A23187 or ionomycin). In vitro assays of AA or 0 2 release performed in the presence of albumin failed to detect superoxide production when ionophore was used as the stimulating agent. Raising the concentration of ionophore or reducing the BSA concentration balanced one another in terms of detection of superoxide release. Binding of ionophore by albumin, which reduced the effective concentration of the stimulating agent, most likely accounted for this effect, but a small component could be attributed to calcium binding by albumin as well. These results indicate differing susceptibilities of monocyte functions to stimulation by increased intracellular Ca 2÷. Under strictly defined conditions, superoxide and arachidonate release may be assayed simultaneously. Binding of ionophores (and, by analogy, other agents) to albumin must be taken into account when determining stimulating doses for use in monocyte function assays.
Journal of Clinical Apheresis, 1991
Transfusion of peripheral blood monocytes may be of benefit as adjuvant treatment of leukopenic p... more Transfusion of peripheral blood monocytes may be of benefit as adjuvant treatment of leukopenic patients with serious infections. To study the feasibility of this approach, a large-scale monocyte separation procedure employing leukapheresis, density gradient centrifugation, and counterflow centrifugal elutriation was established. By processing 5 to 6 liters of normal donor blood, it was possible to obtain a mean of 1.1 x 10(9) (range: 0.5-1.7 x 10(9) cells) of mononuclear cells, of which 89% (range: 82-94%) were monocytes by Wright's stain morphology. When the elutriation was performed in XVIVO-10, a commercially available, serum-free medium developed for adoptive immunotherapy, spontaneous secretion of superoxide by the monocytes was significantly higher than for monocytes elutriated in Hanks' balanced salt solution without calcium and magnesium or non-elutriated peripheral blood mononuclear cells. This stimulated state of the monocytes was observed both immediately after elutriation and after overnight storage at 4 degrees C, and it was not affected by the type of storage vessel used. Overnight storage of the monocytes at 37 degrees C resulted in a reversal of the stimulated state of the cells. Monocytes elutriated in XVIVO-10 and kept overnight at 4 degrees C released high amounts of arachidonic acid. A subsequent decrease in this release was seen after additional storage at 37 degrees C for 18 hours. These observations demonstrate that separation and storage variables have important effects on the state of stimulation of monocytes. Further investigations of such variables may suggest improved procedures for preparation and storage of these cells, as well as possible ways to stimulate monocytes prior to transfusion.
Journal of Aquatic Animal Health, 1989
Abstract Immunosuppression was demonstrated in sections of rainbow trout Oncorhynchus mykiss (for... more Abstract Immunosuppression was demonstrated in sections of rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri) spleens immunized in vitro and exposed in culture to different concentrations of copper chloride. The sections were immunized with ...
European Journal of Immunogenetics, 1981
Inflammation, 1992
Human monocytes released superoxide anion, prostaglandin E2, leukotriene B4, IL-1, and TNF when e... more Human monocytes released superoxide anion, prostaglandin E2, leukotriene B4, IL-1, and TNF when exposed to plastic surfaces coated with murine anti-CD3 monoclonal antibody, OKT 3. Stimulation of mediator release by OKT 3 was dependent on the amount of antibody immobilized onto wells of plastic tissue culture plates. Soluble antibody or antibody adsorbed to monocytes and reacted with an aggregating ("cross-linking") second antibody failed to induce mediator release. Monocytes "armed" with OKT 3 formed rosettes with T cells in a fashion indistinguishable from that seen between monocytes and T cells sensitized with OKT 3. Monocytes with adsorbed OKT 3 antibodies released IL-1 beta and TNF-alpha when exposed to unsensitized T cells, although increased superoxide release could not be detected. OKT 4a, a murine IgG2a antibody that reacts with a different T cell epitope (CD4), failed to induce cytokine release from monocytes when cross-linked by T cells or a CD4+ T cell line, even in the presence of IL-2 or IFN-gamma. These data indicate that certain antibodies bound to Fc receptors (FcR) of monocytes may trigger monocyte function when reacting with cells bearing the appropriate target antigens. FcR-mediated signaling resulting in mediator release may be involved in initiating or regulating the immune response. Furthermore, systemically administered monoclonal antibodies may induce inflammatory responses and their attendant symptomatologies via their interaction with FcR-bearing inflammatory cells.
European Journal of Immunology, 2005
Using adoptive transfer of TCR-transgenic T cells, we examined the homing of transgenic T cells t... more Using adoptive transfer of TCR-transgenic T cells, we examined the homing of transgenic T cells to splenic compartments in situ. After systemic immunization with peptide or protein antigen, the location of clonotypic T cells, cytokine production, cell surface markers, and apoptosis were assessed. There were distinct differences in the splenic homing of CD4(+) TCR-transgenic T cells in mice immunized with peptide as compared to mice immunized with whole-protein antigen. T cells in peptide-immunized mice were found almost exclusively in the splenic red pulp, but not in the T and B cell zones (white pulp), while the majority of T cells immunized with whole protein were found in the white pulp. Many more Fas ligand-expressing and apoptotic cells were present after peptide immunization than after whole-protein immunization. Localization of IL-4-, IL-2- and IFN-gamma-producing cells to the lymphocyte-containing splenic white pulp was only observed with whole-protein immunization. The unique homing and increased apoptosis of immune cells post peptide immunization may help explain the ineffectiveness of many peptide vaccines. Linkage of the same peptide epitope to a carrier protein increased white pulp T cell localization and decreased apoptosis, suggesting a strategy to enhance peptide vaccine responses.
Blood, 2002
Movement of T-lymphocyte cell surface CD43 is associated with both antigen activation of T-cell c... more Movement of T-lymphocyte cell surface CD43 is associated with both antigen activation of T-cell clones and chemokine induction of T-lymphocyte motility. Here, we demonstrate that CD43 movement away from the site of T-cell receptor ligation occurs in unprimed CD4+ T cells as well as T-cell clones. The T-cell receptor (TCR)-dependent movement of CD43 in unprimed T cells is associated with a polarized morphology and CD43 accumulation at the uropods of the cells, unlike that reported for primed T cells. The polarization of CD43 has a requirement for Src kinases and occurs in conjunction with lipid raft coalescence. Thymocytes and T-cell hybridomas, cells that have altered responses to TCR activation and lack lipid raft coalescence, do not polarize CD43 as readily as unprimed T cells. The movement of CD43 depends on the cholesterol biosynthetic pathway enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. Blockade of this enzyme can specifically prevent CD43 redistribution wi...
Biotechnology Progress, 2001
Murine hybridoma cells used in the production of monoclonal antibodies (mAb's) produce endogenous... more Murine hybridoma cells used in the production of monoclonal antibodies (mAb's) produce endogenous type C retrovirus particles. Regulatory agencies require a demonstration that mAb's intended for human use are free of retrovirus with an adequate margin of safety. This is usually achieved by validation studies, performed at small scale, to demonstrate that the manufacturing process is capable of removing or inactivating several different model viruses, including a murine retrovirus. In this report, we assess the utility of the TaqMan fluorogenic 5'-nuclease Product-Enhanced Reverse Transcriptase (TM-PERT) assay for measuring reverse transcriptase (RT) activity in cell-culture samples and RT removal by models of processing steps. The levels of RT activity contained in laboratory-scale cell-culture harvests (10 8 -10 13 pU/ mL) were substantially above the detection limit of the TM-PERT assay (∼10 6 pU/ mL). The nature of the RT activity from cell culture was complex, but the bulk of RT activity in clarified mAb harvests appears to be contained in large molecular weight viral particles. In laboratory-scale chromatographic runs, sufficient RT activity was present in mAb-containing eluates to accurately calculate its log 10 reduction value (LRV), typically between 2 and 4 log 10 per step. Monoclonal antibody purified using a model purification scheme consisting of three serial columns contained some residual RT activity near the limit of detection. The data indicate that the TM-PERT assay, because it is quantitative and highly sensitive and can be used to analyze a large number of samples in a short period, is ideally suited to investigate and optimize retrovirus clearance in purification processes.
AIDS Research and Human Retroviruses, 1991
In the present study inactivated human immunodeficiency virus type 1 (HIV-1) was conjugated to Br... more In the present study inactivated human immunodeficiency virus type 1 (HIV-1) was conjugated to Brucella abortus and tested for immunogenicity in normal and anti-L3T4-treated BALB/c mice. HIV-BA was more immunogenic than uncoupled HIV in normal mice, since 6-fold less virus in HIV-BA preparations elicited higher titer responses than HIV-1 alone. Furthermore, the HIV-BA antibody response reached higher levels before the HIV-1 response. Immunoblot analysis showed that most of the HIV-1 antigens were recognized by antibodies induced by either HIV-1 or HIV-BA. Isotype analysis revealed that HIV-1 induced similar levels of IgG1 and IgG2a antibodies, whereas the IgG2a responses to HIV-BA were more pronounced than the IgG1 response. These different IgG subclass patterns suggest that conjugation of HIV-1 to BA changed the immunogenic nature of HIV-1. The requirement for helper T cells was examined by immunizing mice that were depleted of CD4+ T cells by in vivo anti-L3T4 treatment. Under these conditions the IgG responses to HIV-1 were completely eliminated. Although HIV-BA antibody responses were markedly reduced in anti-L3T4-treated mice, anti-HIV-1 antibodies, mainly of the IgG2a isotype, were produced. The antibodies generated by HIV-1 and HIV-BA immunization were also tested for their ability to inhibit syncytia formed by infecting CD4 + CEM cells with gp160 vaccinia. Sera from normal mice, immunized with either HIV-1 or HIV-BA were capable of inhibiting syncytia. In contrast, following anti-L3T4 treatment, only mice immunized with HIV-BA, but not HIV-1, produced antibodies capable of inhibiting syncytia.
Blood, 2002
Movement of T-lymphocyte cell surface CD43 is associated with both antigen activation of T-cell c... more Movement of T-lymphocyte cell surface CD43 is associated with both antigen activation of T-cell clones and chemokine induction of T-lymphocyte motility. Here, we demonstrate that CD43 movement away from the site of T-cell receptor ligation occurs in unprimed CD4+ T cells as well as T-cell clones. The T-cell receptor (TCR)-dependent movement of CD43 in unprimed T cells is associated with a polarized morphology and CD43 accumulation at the uropods of the cells, unlike that reported for primed T cells. The polarization of CD43 has a requirement for Src kinases and occurs in conjunction with lipid raft coalescence. Thymocytes and T-cell hybridomas, cells that have altered responses to TCR activation and lack lipid raft coalescence, do not polarize CD43 as readily as unprimed T cells. The movement of CD43 depends on the cholesterol biosynthetic pathway enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. Blockade of this enzyme can specifically prevent CD43 redistribution wi...
Immunology, 1984
CBA/N mice harbour an X-linked B cell defect which is transmitted by CBA/N female mice to their h... more CBA/N mice harbour an X-linked B cell defect which is transmitted by CBA/N female mice to their hybrid male progeny. These mice mount normal responses to thymus-dependent (TD) and some thymus-independent (TI-1) antigens, while the response to TI-2 antigens is absent. Hapten-specific plaque-forming cell (PFC) responses to TD antigens can be blockaded by concomitant exposure of these mice to TI-2 antigens bearing the same hapten. This paper investigates in defective mice the blockade of their response to TNP3-LPS (trinitrophenylated lipopolysaccharide, a TI-1 antigen), imposed by DNP59-Ficoll (dinitrophenylated Ficoll, a TI-2 antigen). The effectiveness of the blocking agent, DNP59-Ficoll, differed in various inbred mouse strains: CBA/N X C3H/HeN F1 male greater than CBA/N female greater than CBA/N X C3H/HeN F1 female. The role of T cells in the observed hapten-specific blockade phenomenon was investigated using athymic CBA/N nude mice and a B cell tolerogen. Our findings indicate tha...
Journal of Aquatic Animal Health, 1989
Abstract Immunosuppression was demonstrated in sections of rainbow trout Oncorhynchus mykiss (for... more Abstract Immunosuppression was demonstrated in sections of rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri) spleens immunized in vitro and exposed in culture to different concentrations of copper chloride. The sections were immunized with ...
Clinical Immunology and Immunopathology, Mar 31, 1991
Human peripheral blood monocytes exposed to MK-886 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopro... more Human peripheral blood monocytes exposed to MK-886 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2- dimethylpropanoic acid) at doses which abolish formation of 5-lipoxygenase metabolites showed unaltered interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha) levels in response to phorbol ester, concanavalin A, serum-treated zymosan, or lipopolysaccharide. Indomethacin (10 microM), alone or in combination with MK-886, also failed to modulate monokine production in response to any stimulus. Exogenous arachidonate (3-30 microM) which augmented the formation of PGE2 and LTB4 in the absence of stimulation, also had no effect on monokine production. LPS-induced IL-1 and TNF production occurred despite stimulation of PGE2 synthesis. The results make a role for endogenous prostaglandins and leukotrienes in the regulation of monocyte IL-1 beta and TNF-alpha production unlikely. These data also indicate that MK-886, a novel inhibitor of 5-lipoxygenase product formation, is a potentially useful leukotriene inhibitor which does not affect monokine production.
Inflammation, 1992
Human monocytes released superoxide anion, prostaglandin E2, leukotriene B4, IL-1, and TNF when e... more Human monocytes released superoxide anion, prostaglandin E2, leukotriene B4, IL-1, and TNF when exposed to plastic surfaces coated with murine anti-CD3 monoclonal antibody, OKT 3. Stimulation of mediator release by OKT 3 was dependent on the amount of antibody immobilized onto wells of plastic tissue culture plates. Soluble antibody or antibody adsorbed to monocytes and reacted with an aggregating ("cross-linking") second antibody failed to induce mediator release. Monocytes "armed" with OKT 3 formed rosettes with T cells in a fashion indistinguishable from that seen between monocytes and T cells sensitized with OKT 3. Monocytes with adsorbed OKT 3 antibodies released IL-1 beta and TNF-alpha when exposed to unsensitized T cells, although increased superoxide release could not be detected. OKT 4a, a murine IgG2a antibody that reacts with a different T cell epitope (CD4), failed to induce cytokine release from monocytes when cross-linked by T cells or a CD4+ T cell line, even in the presence of IL-2 or IFN-gamma. These data indicate that certain antibodies bound to Fc receptors (FcR) of monocytes may trigger monocyte function when reacting with cells bearing the appropriate target antigens. FcR-mediated signaling resulting in mediator release may be involved in initiating or regulating the immune response. Furthermore, systemically administered monoclonal antibodies may induce inflammatory responses and their attendant symptomatologies via their interaction with FcR-bearing inflammatory cells.
Transplantation, 1986
The effect of prior administration of anti-DNP (N-[2,4-dinitrophenyl]-B-alanylglycylglycine) mono... more The effect of prior administration of anti-DNP (N-[2,4-dinitrophenyl]-B-alanylglycylglycine) monoclonal antibodies on the humoral immune response of BALB/c mice was examined. One-time administration of a "cocktail" of two anti-DNP monoclonals resulted in suppression of the IgM anti-DNP response for one week after challenge but not longer. Maximal suppression of anti-DNP IgM plaque-forming cells was achieved by administration of antibody 1-2 weeks before challenge with DNP. Maximal suppression of serum IgM antibody was seen by administration of antibody 2-3 weeks before challenge with antigen. Following one-time administration of suppressive monoclonal antibody, the serum IgG antibody response to DNP was suppressed beginning 2 weeks after immunization and remained so for up to 241 days despite continual booster injections of antigen. Although most effective suppression of the humoral anti-DNP response was seen in animals receiving their single dose of suppressive antibody 2 weeks before first exposure to antigen, suppression of the IgG response was evident at all intervals examined up to 232 days in mice given monoclonal antibody between 0.1 day and 30 days before antigen, but not at earlier times. These findings suggest that regulatory networks, rather than the masking of antigenic determinants by passively administered antibody, play a role in antibody-mediated immunoregulation. They may be of use in designing strategies for optimizing immunosuppression protocols in clinical studies.
Clinical Immunology and Immunopathology, 1991
Human peripheral blood monocytes exposed to MK-886 (3-[l-(4-chlorobenzyl)-3t-butyl-thio-5-isoprop... more Human peripheral blood monocytes exposed to MK-886 (3-[l-(4-chlorobenzyl)-3t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylprop~oic acid) at doses which abolish formation of 5-lipoxygenase metabolites showed unaltered interleukin-ll3 (IL-1B) or tumor necrosis factor-a (TNF-a) levels in response to phorbol ester, concanavalin A, serum-treated zymosan, or lipopolysaccharide. Indomethacin (10 t&f), alone or in combination with MK-886, also failed to modulate monokine production in response to any stimulus. Exogenous arachidonate (3-30 l&) which augmented the formation of PGEz and LTB, in the absence of stimulation, also had no effect on monokine production. LPS-induced IL-l and TNF production occurred despite stimulation of PGE, synthesis. The results make a role for endogenous prostaglandins and leukotrienes in the regulation of monocyte IL-lp and TNF-a production unlikely. These data also indicate that MK-886, a novel inhibitor of Slipoxygenase product formation, is a potentially useful leukotriene inhibitor which does not affect monokine production.
Clinical Immunology and Immunopathology, 1987
The simultaneous production of prostaglandins, leukotrienes, and hydroxyeicosatetraenoic acids wa... more The simultaneous production of prostaglandins, leukotrienes, and hydroxyeicosatetraenoic acids was studied in human peripheral blood monocytes obtained by counter-current centrifugal elutriation. Monocytes prelabeled for 4 hr with [3H]arachidonic acid (AA) released label into the surrounding medium in response to treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) or calcium ionophore (A23187). High-performance liquid chromatography of monocyte supernatants demonstrated that labeled compounds included those which eluted with authentic standards for thromboxane B2, 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT), 6-keto-prostaglandin F alpha, prostaglandin E2, and 15-hydroxyeicosatetraenoic acid (HETE). 5-HETE and leukotriene B4 (LtB4) were detected only in response to ionophore treatment. Highly purified lymphocytes did not convert AA to autocoids, despite the release of free arachidonate in response to either stimulus. Pretreatment of monocytes with recombinant human interferon (IFN)-gamma or IFN-alpha for 18 hr resulted in enhanced release of labeled arachidonic acid and increased conversion to autocoids after TPA or ionophore stimulation. Absolute amounts of prostaglandin E2 produced in response to TPA or ionophore treatment were increased as well. These results demonstrate the autocoid profile released by stimulated human monocytes and illustrate the effects of IFN treatment on the production of lipoxygenase metabolites of arachidonic acid as well as cyclooxygenase products.
Advances in Experimental Medicine and Biology, 1990
Human monocytes release arachidonic acid upon stimulation with a variety of soluble or particulat... more Human monocytes release arachidonic acid upon stimulation with a variety of soluble or particulate agents. These include: phorbol esters (i.e., 12-O-tetradecanoate phorbol-13-acetate, TPA), calcium ionophores (ionomycin), serum-treated zymosan (STZ) concanavalin A (Con A), and, to a minor degree, lipopolysaccharides (LPS). Protein Kinase C activation or increased intracellular Ca2+ are common features of the actions of most, if not all, of these stimuli. Prevention of PKC activation by the use of staurosporine or chelation of extracellular calcium by EGTA selectively impaired AA release, indicating that PLA2 may be regulated by either pathway concurrently. The generation of inositol phosphates and diacylglycerol by the action of phospholipase C, notably upon interaction with opsonized particles during phagocytosis, apparently constitutes the physiological correlate of stimulation via these agents. Release of arachidonic acid by the action of PLA2 or other phospholipid hydrolyzing enzymes leads directly to the formation of cyclooxygenase products. In the presence of markedly elevated calcium concentrations, 5-lipoxygenase (LO) is activated as well, leading to the formation and release of leukotrienes. Agents which stimulate AA release also initiate other monocyte functions, including generation of reactive oxygen intermediates and lymphokine release. This observation makes it tempting to implicate PLA2 activation in many aspects of monocyte physiology. However, no correlation with PLA2 activation and either superoxide or lymphokine release was found when multiple stimuli, including TPA, ionomycin, serum-treated zymosan, concanavalin A, or LPS, were compared simultaneously. Instead, our results indicate that PLA2 activation is regulated by the same mechanisms, including PKC activation and increased Ca2+, as are other enzymes which determine expression of monocyte function. Phospholipase A2 (PLA2) hydrolyzes fatty acid from the sn-2 position of a wide variety of phospholipids. Substrates for this (these) enzyme(s) include species which contain a variety of polar head groups (choline, serine, ethanolamine, etc.) and some phospholipids with either linkages in sn-1. In many cell types, including human monocytes, phospholipase A2 commonly acts on substrates containing arachidonic acid (AA). The liberation of free arachidonate is a first step in the metabolism of prostaglandins, hydroxyeicosatetraeinoic acids, (HETE'S), and leukotrienes (Lt's). Monocytes and macrophages have been shown to be rich sources of arachidonate and its metabolites. Some biologic properties of monocytes, notably their role as immunomodulating cells, have been attributed to eicosanoid production and release. Accordingly, much of the interest regarding PLA2 in human monocytes centers on this aspect of their function.(ABSTRACT TRUNCATED AT 400 WORDS)
Transplantation, 1986
The effect of prior administration of anti-DNP (N-[2,4-dinitrophenyl]-B-alanylglycylglycine) mono... more The effect of prior administration of anti-DNP (N-[2,4-dinitrophenyl]-B-alanylglycylglycine) monoclonal antibodies on the humoral immune response of BALB/c mice was examined. One-time administration of a "cocktail" of two anti-DNP monoclonals resulted in suppression of the IgM anti-DNP response for one week after challenge but not longer. Maximal suppression of anti-DNP IgM plaque-forming cells was achieved by administration of antibody 1-2 weeks before challenge with DNP. Maximal suppression of serum IgM antibody was seen by administration of antibody 2-3 weeks before challenge with antigen. Following one-time administration of suppressive monoclonal antibody, the serum IgG antibody response to DNP was suppressed beginning 2 weeks after immunization and remained so for up to 241 days despite continual booster injections of antigen. Although most effective suppression of the humoral anti-DNP response was seen in animals receiving their single dose of suppressive antibody 2 weeks before first exposure to antigen, suppression of the IgG response was evident at all intervals examined up to 232 days in mice given monoclonal antibody between 0.1 day and 30 days before antigen, but not at earlier times. These findings suggest that regulatory networks, rather than the masking of antigenic determinants by passively administered antibody, play a role in antibody-mediated immunoregulation. They may be of use in designing strategies for optimizing immunosuppression protocols in clinical studies.
Journal of Immunological Methods, 1988
Peripheral blood monocytes release superoxide (02) and arachidonic acid (AA) when stimulated with... more Peripheral blood monocytes release superoxide (02) and arachidonic acid (AA) when stimulated with 12-O-tetradecanoate phorbol-13-acetate (TPA) or calcium ionophores (A23187 or ionomycin). In vitro assays of AA or 0 2 release performed in the presence of albumin failed to detect superoxide production when ionophore was used as the stimulating agent. Raising the concentration of ionophore or reducing the BSA concentration balanced one another in terms of detection of superoxide release. Binding of ionophore by albumin, which reduced the effective concentration of the stimulating agent, most likely accounted for this effect, but a small component could be attributed to calcium binding by albumin as well. These results indicate differing susceptibilities of monocyte functions to stimulation by increased intracellular Ca 2÷. Under strictly defined conditions, superoxide and arachidonate release may be assayed simultaneously. Binding of ionophores (and, by analogy, other agents) to albumin must be taken into account when determining stimulating doses for use in monocyte function assays.
Journal of Clinical Apheresis, 1991
Transfusion of peripheral blood monocytes may be of benefit as adjuvant treatment of leukopenic p... more Transfusion of peripheral blood monocytes may be of benefit as adjuvant treatment of leukopenic patients with serious infections. To study the feasibility of this approach, a large-scale monocyte separation procedure employing leukapheresis, density gradient centrifugation, and counterflow centrifugal elutriation was established. By processing 5 to 6 liters of normal donor blood, it was possible to obtain a mean of 1.1 x 10(9) (range: 0.5-1.7 x 10(9) cells) of mononuclear cells, of which 89% (range: 82-94%) were monocytes by Wright's stain morphology. When the elutriation was performed in XVIVO-10, a commercially available, serum-free medium developed for adoptive immunotherapy, spontaneous secretion of superoxide by the monocytes was significantly higher than for monocytes elutriated in Hanks' balanced salt solution without calcium and magnesium or non-elutriated peripheral blood mononuclear cells. This stimulated state of the monocytes was observed both immediately after elutriation and after overnight storage at 4 degrees C, and it was not affected by the type of storage vessel used. Overnight storage of the monocytes at 37 degrees C resulted in a reversal of the stimulated state of the cells. Monocytes elutriated in XVIVO-10 and kept overnight at 4 degrees C released high amounts of arachidonic acid. A subsequent decrease in this release was seen after additional storage at 37 degrees C for 18 hours. These observations demonstrate that separation and storage variables have important effects on the state of stimulation of monocytes. Further investigations of such variables may suggest improved procedures for preparation and storage of these cells, as well as possible ways to stimulate monocytes prior to transfusion.
Journal of Aquatic Animal Health, 1989
Abstract Immunosuppression was demonstrated in sections of rainbow trout Oncorhynchus mykiss (for... more Abstract Immunosuppression was demonstrated in sections of rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri) spleens immunized in vitro and exposed in culture to different concentrations of copper chloride. The sections were immunized with ...
European Journal of Immunogenetics, 1981
Inflammation, 1992
Human monocytes released superoxide anion, prostaglandin E2, leukotriene B4, IL-1, and TNF when e... more Human monocytes released superoxide anion, prostaglandin E2, leukotriene B4, IL-1, and TNF when exposed to plastic surfaces coated with murine anti-CD3 monoclonal antibody, OKT 3. Stimulation of mediator release by OKT 3 was dependent on the amount of antibody immobilized onto wells of plastic tissue culture plates. Soluble antibody or antibody adsorbed to monocytes and reacted with an aggregating ("cross-linking") second antibody failed to induce mediator release. Monocytes "armed" with OKT 3 formed rosettes with T cells in a fashion indistinguishable from that seen between monocytes and T cells sensitized with OKT 3. Monocytes with adsorbed OKT 3 antibodies released IL-1 beta and TNF-alpha when exposed to unsensitized T cells, although increased superoxide release could not be detected. OKT 4a, a murine IgG2a antibody that reacts with a different T cell epitope (CD4), failed to induce cytokine release from monocytes when cross-linked by T cells or a CD4+ T cell line, even in the presence of IL-2 or IFN-gamma. These data indicate that certain antibodies bound to Fc receptors (FcR) of monocytes may trigger monocyte function when reacting with cells bearing the appropriate target antigens. FcR-mediated signaling resulting in mediator release may be involved in initiating or regulating the immune response. Furthermore, systemically administered monoclonal antibodies may induce inflammatory responses and their attendant symptomatologies via their interaction with FcR-bearing inflammatory cells.
European Journal of Immunology, 2005
Using adoptive transfer of TCR-transgenic T cells, we examined the homing of transgenic T cells t... more Using adoptive transfer of TCR-transgenic T cells, we examined the homing of transgenic T cells to splenic compartments in situ. After systemic immunization with peptide or protein antigen, the location of clonotypic T cells, cytokine production, cell surface markers, and apoptosis were assessed. There were distinct differences in the splenic homing of CD4(+) TCR-transgenic T cells in mice immunized with peptide as compared to mice immunized with whole-protein antigen. T cells in peptide-immunized mice were found almost exclusively in the splenic red pulp, but not in the T and B cell zones (white pulp), while the majority of T cells immunized with whole protein were found in the white pulp. Many more Fas ligand-expressing and apoptotic cells were present after peptide immunization than after whole-protein immunization. Localization of IL-4-, IL-2- and IFN-gamma-producing cells to the lymphocyte-containing splenic white pulp was only observed with whole-protein immunization. The unique homing and increased apoptosis of immune cells post peptide immunization may help explain the ineffectiveness of many peptide vaccines. Linkage of the same peptide epitope to a carrier protein increased white pulp T cell localization and decreased apoptosis, suggesting a strategy to enhance peptide vaccine responses.
Blood, 2002
Movement of T-lymphocyte cell surface CD43 is associated with both antigen activation of T-cell c... more Movement of T-lymphocyte cell surface CD43 is associated with both antigen activation of T-cell clones and chemokine induction of T-lymphocyte motility. Here, we demonstrate that CD43 movement away from the site of T-cell receptor ligation occurs in unprimed CD4+ T cells as well as T-cell clones. The T-cell receptor (TCR)-dependent movement of CD43 in unprimed T cells is associated with a polarized morphology and CD43 accumulation at the uropods of the cells, unlike that reported for primed T cells. The polarization of CD43 has a requirement for Src kinases and occurs in conjunction with lipid raft coalescence. Thymocytes and T-cell hybridomas, cells that have altered responses to TCR activation and lack lipid raft coalescence, do not polarize CD43 as readily as unprimed T cells. The movement of CD43 depends on the cholesterol biosynthetic pathway enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. Blockade of this enzyme can specifically prevent CD43 redistribution wi...
Biotechnology Progress, 2001
Murine hybridoma cells used in the production of monoclonal antibodies (mAb's) produce endogenous... more Murine hybridoma cells used in the production of monoclonal antibodies (mAb's) produce endogenous type C retrovirus particles. Regulatory agencies require a demonstration that mAb's intended for human use are free of retrovirus with an adequate margin of safety. This is usually achieved by validation studies, performed at small scale, to demonstrate that the manufacturing process is capable of removing or inactivating several different model viruses, including a murine retrovirus. In this report, we assess the utility of the TaqMan fluorogenic 5'-nuclease Product-Enhanced Reverse Transcriptase (TM-PERT) assay for measuring reverse transcriptase (RT) activity in cell-culture samples and RT removal by models of processing steps. The levels of RT activity contained in laboratory-scale cell-culture harvests (10 8 -10 13 pU/ mL) were substantially above the detection limit of the TM-PERT assay (∼10 6 pU/ mL). The nature of the RT activity from cell culture was complex, but the bulk of RT activity in clarified mAb harvests appears to be contained in large molecular weight viral particles. In laboratory-scale chromatographic runs, sufficient RT activity was present in mAb-containing eluates to accurately calculate its log 10 reduction value (LRV), typically between 2 and 4 log 10 per step. Monoclonal antibody purified using a model purification scheme consisting of three serial columns contained some residual RT activity near the limit of detection. The data indicate that the TM-PERT assay, because it is quantitative and highly sensitive and can be used to analyze a large number of samples in a short period, is ideally suited to investigate and optimize retrovirus clearance in purification processes.
AIDS Research and Human Retroviruses, 1991
In the present study inactivated human immunodeficiency virus type 1 (HIV-1) was conjugated to Br... more In the present study inactivated human immunodeficiency virus type 1 (HIV-1) was conjugated to Brucella abortus and tested for immunogenicity in normal and anti-L3T4-treated BALB/c mice. HIV-BA was more immunogenic than uncoupled HIV in normal mice, since 6-fold less virus in HIV-BA preparations elicited higher titer responses than HIV-1 alone. Furthermore, the HIV-BA antibody response reached higher levels before the HIV-1 response. Immunoblot analysis showed that most of the HIV-1 antigens were recognized by antibodies induced by either HIV-1 or HIV-BA. Isotype analysis revealed that HIV-1 induced similar levels of IgG1 and IgG2a antibodies, whereas the IgG2a responses to HIV-BA were more pronounced than the IgG1 response. These different IgG subclass patterns suggest that conjugation of HIV-1 to BA changed the immunogenic nature of HIV-1. The requirement for helper T cells was examined by immunizing mice that were depleted of CD4+ T cells by in vivo anti-L3T4 treatment. Under these conditions the IgG responses to HIV-1 were completely eliminated. Although HIV-BA antibody responses were markedly reduced in anti-L3T4-treated mice, anti-HIV-1 antibodies, mainly of the IgG2a isotype, were produced. The antibodies generated by HIV-1 and HIV-BA immunization were also tested for their ability to inhibit syncytia formed by infecting CD4 + CEM cells with gp160 vaccinia. Sera from normal mice, immunized with either HIV-1 or HIV-BA were capable of inhibiting syncytia. In contrast, following anti-L3T4 treatment, only mice immunized with HIV-BA, but not HIV-1, produced antibodies capable of inhibiting syncytia.
Blood, 2002
Movement of T-lymphocyte cell surface CD43 is associated with both antigen activation of T-cell c... more Movement of T-lymphocyte cell surface CD43 is associated with both antigen activation of T-cell clones and chemokine induction of T-lymphocyte motility. Here, we demonstrate that CD43 movement away from the site of T-cell receptor ligation occurs in unprimed CD4+ T cells as well as T-cell clones. The T-cell receptor (TCR)-dependent movement of CD43 in unprimed T cells is associated with a polarized morphology and CD43 accumulation at the uropods of the cells, unlike that reported for primed T cells. The polarization of CD43 has a requirement for Src kinases and occurs in conjunction with lipid raft coalescence. Thymocytes and T-cell hybridomas, cells that have altered responses to TCR activation and lack lipid raft coalescence, do not polarize CD43 as readily as unprimed T cells. The movement of CD43 depends on the cholesterol biosynthetic pathway enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. Blockade of this enzyme can specifically prevent CD43 redistribution wi...
Immunology, 1984
CBA/N mice harbour an X-linked B cell defect which is transmitted by CBA/N female mice to their h... more CBA/N mice harbour an X-linked B cell defect which is transmitted by CBA/N female mice to their hybrid male progeny. These mice mount normal responses to thymus-dependent (TD) and some thymus-independent (TI-1) antigens, while the response to TI-2 antigens is absent. Hapten-specific plaque-forming cell (PFC) responses to TD antigens can be blockaded by concomitant exposure of these mice to TI-2 antigens bearing the same hapten. This paper investigates in defective mice the blockade of their response to TNP3-LPS (trinitrophenylated lipopolysaccharide, a TI-1 antigen), imposed by DNP59-Ficoll (dinitrophenylated Ficoll, a TI-2 antigen). The effectiveness of the blocking agent, DNP59-Ficoll, differed in various inbred mouse strains: CBA/N X C3H/HeN F1 male greater than CBA/N female greater than CBA/N X C3H/HeN F1 female. The role of T cells in the observed hapten-specific blockade phenomenon was investigated using athymic CBA/N nude mice and a B cell tolerogen. Our findings indicate tha...