Biomedical subjects

T Gregory

Publications and source records attributed to T Gregory.

At least 37 records · Page 2Linked to original sources

PubMed1990

Soluble forms of herpes simplex virus glycoprotein D bind to a limited number of cell surface receptors and inhibit virus entry into cells.

Herpes simplex virus type 1 (HSV-1) and HSV-2 plaque production was inhibited by treating cells with soluble forms of HSV-1 glycoprotein D (gD-1t) and HSV-2 glycoprotein D (gD-2t). Both glycoproteins inhibited entry of HSV-1 and HSV-2 without affecting virus adsorption. In contrast, a soluble form of HSV-2 glycoprotein B had no effect on virus entry into cells. Specific binding of gD-1t and gD-2t to cells was saturable, and approximately 4 x 10(5) to 5 x 10(5) molecules bound per cell. Binding of gD-1t was markedly reduced by treating cells with certain proteases but was unaffected when cell surface heparan sulfate glycosaminoglycans were enzymatically removed or when the binding was carried out in the presence of heparin. Together, these results suggest that gD binds to a limited set of cell surface receptors which may be proteins and that these interactions are essential for subsequent virus entry into cells. However, binding of gD to its receptors is not required for the initial adsorption of virus to the cell surface, which involves more numerous sites (probably including heparan sulfate) than those which mediate gD binding.

Adsorption↗

PubMed1990

Effects of exercise on insulin-induced hypoglycemia.

The purpose of this study was to determine the effect of exercise on the rate of onset of hypoglycemia induced by infusion of excess insulin (0.8 mU.min-1.100 g-1). Rats were either fasted overnight (FS) or fed ad libitum (FD). FS rats were killed after 5, 10, or 15 min of infusion at rest or after running on the treadmill at 21 m/min and 15% grade. FD rats were killed after 10, 20, or 40 min of infusion at rest or after exercise. Rats were also killed 15 min postexercise for FS and 60 or 120 min postexercise for FD with continued insulin infusion. The progressive decline in blood glucose was not altered by exercise in the FS rats. FD rats showed a significant difference due to exercise only after 40 min (rest 4.2 +/- 0.3 mM, exercise 3.2 +/- 0.2 mM). A significant postexercise repletion of glycogen was observed in red vastus and soleus muscles of FD rats despite the decreasing blood glucose values. These data indicate that exercise accelerates the rate of development of hypoglycemia in FD rats. In the FS rats, where the rate of decline in blood glucose was greater, exercise had no effect on the time course of development of hypoglycemia.

Animals↗

PubMed1990

Immunoconjugates containing ricin A chain and either human anti-gp41 or CD4 kill H9 cells infected with different isolates of HIV, but do not inhibit normal T or B cell function.

We have previously reported that immunoconjugates composed of deglycosylated ricin A chain coupled to either recombinant (r) CD4 or two different monoclonal human anti-gp41 antibodies (rCD4-dgA and anti-gp41-dgA, respectively) are specifically toxic to HIV-infected lines of human T cells (H9) and monocytes (U937). In order to further evaluate these immunoconjugates as potential therapeutic reagents for killing HIV-infected cells, H9 cells infected with five different isolates of HIV were used as target cells in vitro. All three HIV-specific immunoconjugates were toxic to H9 cells infected with each HIV isolate, but were virtually nontoxic to uninfected cells. Chloroquine markedly potentiated the specific toxicity of all three conjugates, particularly the anti-gp41-dgAs. None of the conjugates affected the ability of normal peripheral blood B cells to respond to mitogen or the ability of normal T cells to respond to alloantigens.

Antibodies, Monoclonal↗

PubMed1989

Recognition of HIV glycoprotein gp120 by T cells. Role of monocyte CD4 in the presentation of gp120.

The HIV envelope glycoprotein gp120 binds with high affinity to CD4 and is responsible for the tropism of HIV for CD4+ T cells and monocytes. Efforts to develop HIV vaccines have focused on gp120 and, therefore, a detailed molecular understanding of human immune responses to gp120 is essential. In this report, we have used human T cell clones specific for gp120 to examine the processing and presentation of gp120 to T cells. In particular, we examined the role of the CD4 that is expressed at low levels on the surfaces of human monocytes in the presentation of gp120 by monocytes. The presentation of gp120 to gp120-specific human T cell clones was blocked by pretreatment of monocytes with anti-CD4 mAb. Blocking of monocyte CD4 with anti-CD4 did not inhibit presentation of other Ag or of synthetic peptides representing epitopes within gp120 recognized by gp120-specific T cell clones. These results indicated that the anti-CD4-mediated inhibition occurred at the level of the monocyte, was specific for the gp120 response, and was operative at the initial Ag uptake phase of the Ag-processing pathway. Definitive confirmation that monocyte CD4 functions in the initial uptake step of the gp120-processing pathway was obtained by using soluble CD4 to block the interaction of gp120 with monocyte CD4. These results demonstrate that gp120 expressed by human monocytes plays an important role in the initial uptake of gp120 by monocytes and that gp120 taken up via CD4 is subsequently processed to allow for exposure of epitopes recognized by gp120-specific human T cells. At limiting gp120 concentrations, uptake via CD4 is essential for the presentation of gp120.

Antigen-Presenting Cells↗

PubMed1989

Designing CD4 immunoadhesins for AIDS therapy.

A newly-constructed antibody-like molecule containing the gp120-binding domain of the receptor for human immunodeficiency virus blocks HIV-1 infection of T cells and monocytes. Its long plasma half-life, other antibody-like properties, and potential to block all HIV isolates, make it a good candidate for therapeutic use.

Acquired Immunodeficiency Syndrome↗

PubMed1989

Class II MHC molecules and the HIV gp 120 envelope protein interact with functionally distinct regions of the CD4 molecule.

A murine T cell hybridoma with a receptor specific for the class I molecule H-2 Dd was transfected with an expressible cDNA for human CD4. Expression of the human class II MHC molecule HLA-DP on Dd-positive murine fibroblasts resulted in a greatly enhanced response of the CD4-positive T cell hybridoma, measured either by lymphokine production or by rosette formation. Inhibition of these functional assays with anti-CD4 monoclonal antibodies implicated the two amino-terminal domains of CD4 in an interaction with the HLA-DP molecule. This interaction was blocked by incubation with recombinant gp120 envelope protein of HIV. In contrast, recombinant soluble CD4 did not inhibit and was able to prevent the inhibition by gp120. Anti-CD4 antibody blocking experiments clearly indicated that distinct regions of CD4 interact respectively with gp120 and with class II MHC molecules.

Animals↗

PubMed1989

Effect of dose and immunization schedule on immune response of baboons to recombinant glycoprotein 120 of HIV-1.

To evaluate the immunogenicity of purified recombinant envelope glycoprotein of HIV-1 (rgp120) as a potential vaccine for AIDS, the antibody response of 45 baboons to rgp120 was investigated using an adjuvant (alum) and route of administration (intramuscular) suitable for humans. The primary purpose was to evaluate the effects of different doses and immunization schedules on the antibody response to rgp120 in primates. A secondary objective was to evaluate possible adverse consequences of rgp120 immunization. A liquid-phase radioimmunoprecipitation (RIP) assay for detection of rgp120-reactive antibodies revealed that rgp120 doses of 30-300 micrograms per administration resulted in nearly indistinguishable serum antibody responses. However, significant enhancement of serum antibody titers was observed when the interval between the second and third administrations was increased from 4 to 20 w. Although changing the interval significantly altered the magnitude of resulting peak titers, the kinetics of antibody formation were not changed. Thus, of the three schedules of immunization tested, none resulted in a sustained humoral immune response. The significance of the RIP assay for evaluating immune responses was confirmed by analysis showing that the percentage of immunized baboons that developed in vitro HIV-1 serum neutralizing responses was greatest in groups that also exhibited high anti-rgp120 RIP titers. Immunization with rgp120 had no significant adverse effect on any clinical or laboratory parameter monitored over the course of the study.

Acquired Immunodeficiency Syndrome↗

PubMed1989

Expression and immunogenicity of the extracellular domain of the human immunodeficiency virus type 1 envelope glycoprotein, gp160.

The envelope glycoprotein of human immunodeficiency virus type 1 is synthesized as a precursor, gp160, that subsequently is cleaved to yield mature gp120 and gp41. In these studies, the gene encoding gp160 was mutagenized so as direct the synthesis of a truncated protein consisting of the extracellular domains of both gp120 and gp41. The variant protein, termed sgp160, consisted of 458 amino acids of gp120 and 172 amino acids of gp41. To facilitate protein purification, the normal polyglycoprotein processing site between gp120 and gp41 was deleted through the use of site-directed mutagenesis. This allowed for the synthesis of a molecule that could be purified by affinity chromatography, using acid elution, without dissociation of the gp120 polypeptide from the gp41 polypeptide. The conformation of the sgp160 variant appeared to be functionally relevant, as reflected by its ability to bind to CD4 with an affinity comparable to that of the variant rgp120. The structure of the sgp160-containing polypeptide differed from that of rgp120 in that it tended to form high-molecular-weight aggregates that could be dissociated to monomers and dimers in the presence of reducing agents. Antibodies against the sgp160 protein reacted with authentic virus-derived gp160, gp120, and gp41; neutralized viral infectivity; and inhibited the binding of rgp120 to CD4. Rabbit antibodies to the sgp160 protein differed from those raised against rgp120 in that they were enriched for populations that blocked CD4 binding but did not prevent human immunodeficiency virus type 1-induced syncytium formation.

Animals↗

PubMed1988

Inhibition of CD4+ T cell function by the HIV envelope protein, gp120.

The CD4 molecule is functionally involved in the class II MHC-restricted T cell response to Ag. CD4 is also the receptor for HIV-1, the major etiologic agent of AIDS. We have assessed whether the interaction of the HIV-1 envelope protein with the CD4 molecule might interfere with the normal function of CD4, thereby contributing to the immunosuppression observed after HIV infection. Using a murine T cell hybridoma which expresses the human CD4 protein and exhibits a CD4-dependent response to Ag, we demonstrate that the HIV envelope protein gp120 can specifically inhibit this response.

Animals↗

PubMed1988

HIV-infected cells are killed by rCD4-ricin A chain.

The gp120 envelope glycoprotein of the human immunodeficiency virus (HIV), which is expressed on the surface of many HIV-infected cells, binds to the cell surface molecule CD4. Soluble derivatives of recombinant CD4 (rCD4) that bind gp120 with high affinity are attractive vehicles for targeting a cytotoxic reagent to HIV-infected cells. Soluble rCD4 was conjugated to the active subunit of the toxin ricin. This conjugate killed HIV-infected H9 cells but was 1/1000 as toxic to uninfected H9 cells (which do not express gp120) and was not toxic to Daudi cells (which express major histocompatibility class II antigens, the putative natural ligand for cell surface CD4). Specific killing of infected cells can be blocked by rgp120, rCD4, or a monoclonal antibody to the gp120 binding site on CD4.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1988

Enzyme-linked immunosorbent assays (ELISAs) for the determination of contaminants resulting from the immunoaffinity purification of recombinant proteins.

Two immunoassays have been developed for the quantitation of part-per-million levels of contaminants likely to co-purify with monoclonal antibodies produced in tissue culture and purified by protein A affinity chromatography. These contaminants are bovine IgG originating from the fetal bovine serum used in cell culture, and protein A. Mouse IgG was shown not to interfere in the bovine IgG assay, where contamination levels of 0.2-0.7% bovine IgG were measured in the lots of monoclonal antibody tested. The protein A ELISA was developed with monoclonal antibody included in the standard, and in the preparations of monoclonal antibody tested, 64 parts per million (ppm) or less of protein A were demonstrated. An additional immunoassay was developed to quantitate monoclonal antibody contamination of two recombinant proteins, rHBsAg and rgp 120 from HIV, purified by affinity chromatography with such antibodies. Possible interference of monoclonal antibody quantitation by the respective antigens was examined in this ELISA, and contamination levels of less than 56 ppm of antibody were determined in the purified recombinant proteins. The three immunoassays were shown to be specific for the major protein contaminants in either monoclonal antibodies or the recombinant proteins and were necessary in demonstrating their purity.

Animals↗

PubMed1988

Analysis of host-virus interactions in AIDS with anti-gp120 T cell clones: effect of HIV sequence variation and a mechanism for CD4+ cell depletion.

The primary human T cell response to HIV was analyzed by isolating from seronegative donors T cell clones specific for HIV gp120. T cell epitopes restricted by different MHC elements were identified within gp120, and synthetic peptides were used to address the fundamental problem of how HIV sequence variability affects T cell recognition. Even one conservative substitution can drastically reduce recognition; thus the interaction of gp120 epitopes with T cell receptors and MHC is precise and poorly crossreactive. Importantly, a subset of CD4+ gp120-specific clones manifest cytolytic activity and lyse uninfected autologous CD4+Ia+ T cells in the presence of gp120 in a process that is strictly dependent upon CD4-mediated uptake of gp120 by T cells. Assuming gp120 is shed from HIV-infected cells in vivo, this novel CD4-dependent autocytolytic mechanism may contribute to the profound depletion of CD4+ cells in AIDS.

Acquired Immunodeficiency Syndrome↗

PubMed1988

T cells can present antigens such as HIV gp120 targeted to their own surface molecules.

To trigger class II-restricted T cells, antigen presenting cells have to capture antigens, process them and display their fragments in association with class II molecules. In most species, activated T cells express class II molecules; however, no evidence has been found that these cells can present soluble antigens. This failure may be due to the inefficient capture, processing or display of antigens in a stimulatory form by T-cells. The capture of a soluble antigen, which is achieved by nonspecific mechanisms in macrophages and dendritic cells, can be up to 10(3) times more efficient in the presence of surface receptors, such as surface immunoglobulin on B cells that specifically bind antigen with high affinity. We asked whether T cells would be able to present soluble antigens that bind to their own surface molecules. Here we show that such antigens can be effectively processed and presented by both CD4+- and CD8+-bearing human T cells. This indicates that T cells are fully capable of processing and displaying antigens and are mainly limited in antigen presentation by their inefficiency at antigen capture.

Antibodies, Monoclonal↗

PubMed1988

Human immunodeficiency virus type 1 challenge of chimpanzees immunized with recombinant envelope glycoprotein gp120.

The major envelope glycoprotein, gp120, of human immunodeficiency virus type 1 (HIV-1) was purified from a Chinese hamster ovary cell line transfected with a truncated form of the HIV-1 env gene. The recombinant glycoprotein (rgp120) was formulated with aluminum hydroxide adjuvant and was used to immunize chimpanzees. The recombinant preparation was effective in eliciting cellular and humoral immunity as well as immunologic memory. Anti-rgp 120 antibodies reacted with authentic viral gp120 in immunological blot assays and were able to neutralize HIV-1 infectivity in vitro. Sera from the rgp120-immunized animals were able to neutralize HIV-1 pseudotypes of vesicular stomatitis virus prepared from the IIIB isolate, from which the gene encoding rgp120 was derived, as well as two heterologous isolates, ARV-2 and RF. The immune response elicited against the rgp120 was not effective in preventing viral infection after intravenous challenge with HIV-1. The implications of these results on HIV-1 vaccine development are discussed.

Acquired Immunodeficiency Syndrome↗

PubMed1988

Efficacy of recombinant glycoprotein D subunit vaccines on the development of primary, recurrent, and latent genital infections with herpes simplex virus type 2 in guinea pigs.

Vaccination of guinea pigs with recombinant glycoprotein D (subunit vaccine) from either herpes simplex virus type 1 or type 2 (HSV-1, HSV-2), before intravaginal inoculation with HSV-2, markedly altered viral replication in the vaginal tract and reduced, or completely prevented, the development and spread of external genital lesions. Vaccination with glycoprotein D from HSV-2 prevented the establishment of latent HSV-2 infections in dorsal root ganglia and reduced the frequency of recurrent episodes. For all parameters of efficacy that we tested, vaccination with glycoprotein D from HSV-2 was more effective than was vaccination with protein derived from HSV-1. Vaccination after primary infection had no effect on the frequency or duration of recurrent HSV-2 infections.

Animals↗

PubMed1988

Epitope mapping of the human immunodeficiency virus type 1 gp120 with monoclonal antibodies.

A soluble form of recombinant gp120 of human immunodeficiency virus type 1 was used as an immunogen for production of murine monoclonal antibodies. These monoclonal antibodies were characterized for their ability to block the interaction between gp120 and the acquired immunodeficiency syndrome virus receptor, CD4. Three of the monoclonal antibodies were found to inhibit this interaction, whereas the other antibodies were found to be ineffective at blocking binding. The gp120 epitopes which are recognized by these monoclonal antibodies were mapped by using a combination of Western blot (immunoblot) analysis of gp120 proteolytic fragments, immunoaffinity purification of fragments of gp120, and antibody screening of a random gp120 gene fragment expression library produced in the lambda gt11 expression system. Two monoclonal antibodies which blocked gp120-CD4 interaction were found to map to adjacent sites in the carboxy-terminal region of the glycoprotein, suggesting that this area is important in the interaction between gp120 and CD4. One nonblocking antibody was found to map to a position that was C terminal to this CD4 blocking region. Interestingly, the other nonblocking monoclonal antibodies were found to map either to a highly conserved region in the central part of the gp120 polypeptide or to a highly conserved region near the N terminus of the glycoprotein. N-terminal deletion mutants of the soluble envelope glycoprotein which lack these highly conserved domains but maintain the C-terminal CD4 interaction sites were unable to bind tightly to the CD4 receptor. These results suggest that although the N-terminal and central conserved domains of intact gp120 do not appear to be directly required for CD4 binding, they may contain information that allows other parts of the molecule to form the appropriate structure for CD4 interaction.

Animals↗

PubMed1987

Blocking of HIV-1 infectivity by a soluble, secreted form of the CD4 antigen.

The initial event in the infection of human T lymphocytes, macrophages, and other cells by human immunodeficiency virus (HIV-1) is the attachment of the HIV-1 envelope glycoprotein gp120 to its cellular receptor, CD4. As a step toward designing antagonists of this binding event, soluble, secreted forms of CD4 were produced by transfection of mammalian cells with vectors encoding versions of CD4 lacking its transmembrane and cytoplasmic domains. The soluble CD4 so produced binds gp120 with an affinity and specificity comparable to intact CD4 and is capable of neutralizing the infectivity of HIV-1. These studies reveal that the high-affinity CD4-gp120 interaction does not require other cell or viral components and may establish a novel basis for therapeutic intervention in the acquired immune deficiency syndrome (AIDS).

Acquired Immunodeficiency Syndrome↗

PubMed1987

Delineation of a region of the human immunodeficiency virus type 1 gp120 glycoprotein critical for interaction with the CD4 receptor.

The primary event in the infection of cells by HIV is the interaction between the viral envelope glycoprotein, gp120, and its cellular receptor, CD4. A recombinant form of gp120 was found to bind to a recombinant CD4 antigen with high affinity. Two gp120-specific murine monoclonal antibodies were able to block the interaction between gp120 and CD4. The gp120 epitope of one of these antibodies was isolated by immunoaffinity chromatography of acid-cleaved gp120 and shown to be contained within amino acids 397-439. Using in vitro mutagenesis, we have found that deletion of 12 amino acids from this region of gp120 leads to a complete loss of binding. In addition, a single amino acid substitution in this region results in significantly decreased binding, suggesting that sequences within this region are directly involved in the binding of gp120 to the CD4 receptor.

Antibodies, Monoclonal↗