Biomedical subjects

B T Rouse

Publications and source records attributed to B T Rouse.

At least 145 records · Page 8Linked to original sources

PubMed1988

Kinetic and ultrastructural studies of interactions of target-sensitive immunoliposomes with herpes simplex virus.

The bilayer phase of dioleoylphosphatidylethanolamine (PE) can be stabilized with palmitoyl-IgG monoclonal antibody to the glycoprotein gD of the herpes simplex virus (HSV). Interactions of PE immunoliposomes with the target virions were characterized by analyzing the kinetics of lipid mixing, by liposomal content release, and by ultrastructural studies. As revealed by a resonance energy transfer assay, lipid mixing between PE immunoliposomes and virions was very rapid, with a second-order rate constant (kapp) of 0.173 (min)-1 (microgram/mL virus)-1. In comparison, content release from PE immunoliposomes was much slower and exhibited multiple-phase, mixed-order kinetics, indicating that liposome destabilization involved fusion of liposomes with HSV. The extent and the apparent rate of liposome destabilization were strongly dependent on liposome concentration. This was evident by the fact that only one to two liposomes were destabilized by each virus particle at low liposome concentration (0.1 microM). For higher liposome concentrations (1-10 microM), this value was 35-104. This finding implies that collision among the virus-bound liposomes is essential for the eventual collapse of PE immunoliposomes to form the hexagonal (HII) equilibrium phase which was observed using freeze-fracture electron microscopy. Studies employing soluble gD, immobilized on latex beads, indicated that a multivalent antigen source is essential for PE immunoliposome destabilization. Immediately after liposome-virus binding, fusion of liposome with the viral membrane then follows. Upon growth of the fusion complexes, which increase to 35-104 liposomes for each virus, an eventual collapse of the structure results, driving PE to its equilibrium structure of HII phase.

Antibodies, Monoclonal↗

PubMed1988

A tissue-specific endothelial cell molecule involved in lymphocyte homing.

An endothelial cell surface molecule that is selectively expressed in mucosal organs is required for lymphocyte homing to mucosal lymphoid tissues. This 'vascular addressin' appears to function as a tissue-specific marker or address signal for recognition by lymphocytes circulating in the blood.

Animals↗

PubMed1988

Functional T cell recognition of synthetic peptides corresponding to continuous antibody epitopes of herpes simplex virus type 1 glycoprotein D.

Four synthetic peptides which correspond to continuous antibody epitopes of herpes simplex virus (HSV) type 1 glycoprotein D (gD) within amino acid residues 1-23 (8-23), 268-287 and 340-356 were evaluated for in vitro stimulating activity on HSV-primed murine T lymphocytes. All peptides stimulated lymphoproliferative responses and interleukin 2 (IL2) production from draining lymph node (LN) cell populations taken 5 days after footpad immunization with live HSV. Similar responses were elicited from splenic memory T cells only if these T cells were restimulated with HSV in vitro and rested prior to peptide stimulation. Furthermore, peptide stimulated memory T cell populations released soluble factor(s) into the culture supernates which modulated the induced lymphoproliferative and cytotoxic T lymphocyte (CTL) activities of HSV-stimulated, HSV-immune splenocytes (indicator cultures). Memory T cell supernates suppressed lymphoproliferation of indicator cultures, while CTL activity of indicator cultures was either enhanced or suppressed, depending on the peptide and concentration. In contrast, supernates generated by peptide stimulation of draining LN cells had no effect on CTL activity of indicator cultures. However, the lymphoproliferative responses were augmented with three of the four peptides at the highest concentration of peptides tested. Our experiments indicate T helper (Th) and T suppressor (Ts) lymphocyte recognition of four synthetic peptides which encompass continuous antibody epitopes of HSV gD. Immunization with one of these peptides (1-23) induces virus neutralizing antibodies and protection against lethal viral challenge. Th lymphocyte recognition of this peptide in particular, together with its observed function in the induction of protection against HSV infection, indicates that this peptide is a promising candidate as a synthetic vaccine against HSV infection.

Antigens, Viral↗

PubMed1988

Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes.

The tissue localization or "homing" of circulating lymphocytes is directed in part by specialized vessels that define sites of lymphocyte exit from the blood. In peripheral lymph nodes, mucosal lymphoid tissues (Peyer's patches and appendix), and sites of chronic inflammation, for example, lymphocytes leave the blood by adhering to and migrating between those endothelial cells lining postcapillary high endothelial venules (HEV). Functional analyses of lymphocyte interactions with HEV have shown the lymphocytes can discriminate between HEV in different tissues, indicating that HEV express tissue-specific determinants or address signals for lymphocyte recognition. We recently described such a tissue-specific "vascular addressin" that is selectively expressed by endothelial cells supporting lymphocyte extravasation into mucosal tissues and that appears to be required for mucosa-specific lymphocyte homing (Streeter, P. R., E. L. Berg, B. N. Rouse, R. F. Bargatze, and E. C. Butcher. 1988. Nature (Lond.). 331:41-46). Here we document the existence and tissue-specific distribution of a distinct HEV differentiation antigen. Defined by monoclonal antibody MECA-79, this antigen is expressed at high levels on the lumenal surface and in the cytoplasm of HEV in peripheral lymph nodes. By contrast, although MECA-79 stains many HEV in the mucosal Peyer's patches, expression in most cases is restricted to the perivascular or ablumenal aspect of these venules. In the small intestine lamina propria, a mucosa-associated site that supports the extravasation of lymphocytes, venules do not stain with MECA-79. Finally, we demonstrate that MECA-79 blocks binding of both normal lymphocytes and a peripheral lymph node-specific lymphoma to peripheral lymph node HEV in vitro and that it also inhibits normal lymphocyte homing to peripheral lymph nodes in vivo without significantly influencing lymphocyte interactions with Peyer's patch HEV in vitro or in vivo. Thus, MECA-79 defines a novel vascular addressin involved in directing lymphocyte homing to peripheral lymph nodes.

Animals↗

PubMed1988

Cytotoxic T lymphocytes. Their relevance in herpesvirus infections.

We have used recombinant vaccinia viruses expressing the cloned genes coding for glycoprotein B (gB) or glycoprotein D (gD) of HSV-1 to analyze the role of HSV-1--specific cytotoxic T lymphocytes (CTL) in antiviral immunity. Various studies in mice revealed that either vector could stimulate some aspects of HSV-1--specific immunity, but surprisingly, HSV-specific CTL were not induced. Even though gD appeared to be a target antigen for class II-MHC-restricted CTL, neither the gB or the gD vector was capable of forming a target-cell complex that was recognized by class I-MHC-restricted HSV-specific CTL. The inability of these major extracellular glycoproteins to act as CTL-target antigens was even more unusual in light of the ability of CTL to apparently recognize the immediate early genes of HSV, none of which are considered to be expressed on the surface of infected cells. The selective failure of either the gB or gD vector to induce numerous aspect of anti-HSV immunity in the absence of a CTL response allowed us to assess the consequence of this failure in terms of the level of protective immunity against HSV challenge seen in vector-immunized mice. These studies suggest that this failure to induce HSV-specific CTL appears to minimize the protective response to only efficiently protecting against low-challenge doses of HSV-1. These findings are discussed with relevance to the role of CTL in the control of herpesvirus infections.

Animals↗

PubMed1988

Herpes simplex virus type 1-specific cytotoxic T lymphocytes recognize virus nonstructural proteins.

The specificity of herpes simplex virus type 1-specific cytotoxic T cells was examined with target cells expressing either input viral structural antigens or antigens resulting from permissive infection or cells from an interrupted infection in which they expressed predominantly nonstructural immediate-early proteins. These studies indicated that only an insignificant minority of cytotoxic T cells recognized the input viral antigens, whereas a significant proportion (20 to 35%) recognized target cells that expressed the immediate-early proteins despite the absence of serologically detectable viral antigens upon the infected cell surface. The finding that a significant proportion of cytotoxic T-cell populations obtained from the draining lymph nodes of mice acutely infected with herpes simplex virus type 1 also recognized immediately-early gene-expressing target cells indicates the importance of nonstructural herpes simplex virus proteins to antiviral immunity in vivo.

Cytotoxicity, Immunologic↗

PubMed1988

Molecular cloning of complementary DNA from a pneumopathic strain of bovine viral diarrhea virus and its diagnostic application.

A pneumopathic strain of bovine viral diarrhea virus was grown in cell culture and purified. Genomic ribonucleic acid was extracted, polyadenylated at the 3' end, and copied into complementary DNA after oligo-dT priming. Complementary DNA was male double stranded and cloned into the pUC9 plasmid. Approximately 200 complementary DNA clones varying in length from 0.5 to 2.5 kilobases were obtained. Hybridization assays indicated that the sequences isolated were specific for bovine viral diarrhea virus and that at least 5.5 kilobases of bovine viral diarrhea virus genome was represented in the library of complementary DNA clones, the majority of which may have originated from the 3' end of the virus genome. One cloned complementary DNA sequence was used as a 32P-labelled hybridization probe for bovine viral diarrhea virus detection. The probe hybridized with all cytopathic and noncytopathic strains of bovine viral diarrhea virus tested and was 100 times more sensitive than infectivity assays for the detection of bovine viral diarrhea virus. Hybridization did not occur with nucleic acids from bovine coronavirus, bluetongue virus, bovine adenovirus or uninfected cell cultures. Native plasmid DNA sequences, labelled with 32P, did not hybridize with bovine viral diarrhea virus ribonucleic acid.

Animals↗

PubMed1987

Target-sensitive immunoliposomes as an efficient drug carrier for antiviral activity.

We have shown previously that target-sensitive immunoliposomes composed of palmitoyl antibody stabilized phosphatidylethanolamine bilayers could be destabilized by binding to the target cells (Ho, R. J. Y., Rouse, B. T., and Huang, L., Biochemistry (1986) 25, 5500-5506). Target-sensitive immunoliposome-encapsulated and free cytotoxic drugs of nucleoside analogs cytosine-beta-D-arabinoside (AraC) or acycloguanosine (acyclovir, ACV) were compared for their antiviral efficacy and cell cytotoxicity. Target-insensitive immunoliposomes and nontargeted liposomes were also investigated. When the mouse fibroblast L929 cells were infected at low multiplicity with herpes simplex virus, AraC encapsulated in target-sensitive immunoliposomes composed of transphosphatidylated egg phosphatidylethanolamine effectively inhibited virus replication and had far less cell cytotoxicity than free drug. As a measure of cytotoxicity, the drug concentration required to inhibit 50% of [3H]thymidine incorporation from 6 to 42 h (CD50) was determined. For free AraC, this value was 0.3 ng/ml, whereas for target-sensitive immunoliposome-encapsulated AraC, the CD50 exceeded 1 microgram/ml. However, target-sensitive immunoliposome-encapsulated AraC was virus inhibitory (50% effective dose = ED50) at 1.8 ng/ml. A free drug concentration of at least 1000-fold greater was required for comparable antiviral activity. A similar phenomenon was observed when ACV was administered via target-sensitive immunoliposomes. The CD50 values of the free and target-sensitive immunoliposome-encapsulated ACV were 12.5 ng/ml and 1.4 micrograms/ml, respectively, whereas the ED50 values of the free and target-sensitive immunoliposome-encapsulated ACV were 1.1 and 125 ng/ml, respectively. Consequently, our results indicated the superiority of target-sensitive immunoliposomes at drug delivery, especially when drugs were cytotoxic to cells. The use of liposomes of the target-insensitive variety provided some enhancement of activity, but this was several-fold less than that observed with target-sensitive immunoliposomes. In addition, the nucleoside transport inhibitors, p-nitrothiobenzylinosine and dipyridamole, were shown to inhibit the liposome-mediated antiviral activity of AraC. This finding indicated that site-specific cytosolic delivery of nucleoside analogs by target-sensitive immunoliposomes involved a cellular nucleoside transport system. A mechanism of action is proposed.

Animals↗

PubMed1987

Interactions of target-sensitive immunoliposomes with herpes simplex virus. The foundation of a sensitive immunoliposome assay for the virus.

Interactions between target-sensitive (TS) immunoliposomes and herpes simplex virus (HSV) were investigated. Target sensitivity of phosphatidylethanolamine (PE) immunoliposomes is a result of the ability of acylated monoclonal anti-HSV glycoprotein D (gD) to stabilize the bilayer phase of PE, whereas by itself, PE does not form stable liposomes (Ho, R. J. Y., Rouse, B. T., and Huang, L. (1986) Biochemistry 25, 5500-5506). Upon binding of these immunoliposomes to HSV antigen-containing gD, destabilization of PE immunoliposomes was observed. By encapsulating either a self-quenching fluorescent dye, calcein, or alkaline phosphatase inside the liposomal compartment, the HSV-induced destabilization of TS immunoliposomes was shown to be target-specific. Neither Sendai, Semliki Forest, nor Sindbis virus could significantly destabilize the TS immunoliposomes. Moreover, HSV-induced liposome destabilization could be inhibited by free anti-gD (the same antibody used in TS immunoliposomes) but not by monoclonal anti-HSV glycoprotein B, indicating that the interaction was antigen-specific. Destabilization could also be induced by binding to truncated gD (tgD), but only when in a multivalent form immobilized on latex beads. Truncated gD is a cloned, 312-amino acid fragment of HSV-gD that lacks the transmembrane segment. Preincubation of soluble tgD with the TS immunoliposomes failed to induce destabilization and, in addition, abolished the tgD-bead-induced destabilization. This finding strongly indicated that multivalent binding is essential for TS immunoliposome destabilization. Using alkaline phosphatase encapsulated in the liposomes, TS immunoliposomes could be used to detect HSV in fluid phase with 50% signal recorded at 5 microliters of 3.2 x 10(3) pfu/ml; at least 10-fold more sensitive than the standard double-antibody sandwich enzyme-linked immunosorbent assay. The interactions described here may be useful in designing a homogeneous and sensitive immunoliposome assay.

Animals↗

PubMed1987

The mechanisms of antiviral immunity induced by a vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D: clearance of local infection.

We have shown that immunization of mice with a vaccinia virus recombinant expressing glycoprotein D of Herpes simplex virus (HSV)-1 will induce a variety of L3T4+ T cell responses. These included a HSV-specific delayed-type hypersensitivity response, T cell help for the induction of antiviral antibodies, and the ability to eliminate a challenge dose of HSV from the pinna. This protection against a subcutaneous virus challenge was not mediated by the delayed-type hypersensitivity response because intravenous inoculation of the vaccinia virus recombinant expressing HSV-1-gD induced a state of split tolerance. Thus, mice could still clear a HSV challenge inoculum from the pinna yet were unable to mount a HSV-specific delayed-type hypersensitivity response. Evidence is presented that suggests the protective response was, at least, in part mediated by a T cell-dependent induction of virus-neutralizing antibodies. Evidence is also presented that may suggest the failure of a vaccinia virus recombinant expressing HSV-1-gD to induce HSV-specific cytotoxic T cell responses appears to minimize the protective response to only efficiently clearing low 10(4) 50% tissue culture infective dose) challenge populations of virus. These findings are discussed with relevance to the immune control of HSV infections and to the future development of anti-HSV vaccines.

Animals↗

PubMed1987

Production, binding characteristics, and immunogenicity of heterologous anti-idiotypic antibody to herpes simplex virus glycoprotein C.

A monoclonal antibody specific for glycoprotein C (gC) of herpes simplex virus type 1 (HSV-1) was used to prepare a heterologous anti-idiotypic antibody in rabbits. After absorption with normal mouse immunoglobulin (NMS) the anti-idiotypic (anti-id) antibody retained binding activity for MoAb D4.1, the immunogen. The anti-id (anti-id C) also demonstrated a cross-reactive binding activity, as shown by ELISA, for MoAb D4.2 and MoAb D4.8 which was specific for glycoprotein D (gD) and glycoprotein B (gB) of HSV-1, respectively. Also, anti-id C bound to and eluted from MoAb D4.2 and MoAb D4.8 affinity columns retained the ability to bind all three monoclonal antibodies. This cross-reactive anti-id could inhibit the binding of each of the three monoclonal antibodies to their respective proteins, suggesting an antigen combining site specificity. Subsequently, the idiotope on MoAb D4.8 was shown to be outside the antigen combining site, since anti-id C recognized MoAb D4.8 complexed with gB. The anti-id, however, did not bind MoAb D4.1 or MoAb D4.2, if these monoclonals were bound to gC or gD, respectively, suggesting the cross-reactive determinant was paratopic on those two monoclonals. Immunization of mice with anti-id C could prime splenocytes in vivo to proliferate in response to HSV antigen stimulation in vitro. Thus, spleen cells involved in the HSV immune response in vitro recognized the anti-idiotypic antibody in vivo.

Animals↗

PubMed1987

The regulation of herpes simplex virus-specific CTL induction by suppressor cells.

The incubation of herpes simplex virus (HSV) immune murine splenocytes with HSV antigens induced suppressor cells which inhibited HSV-specific cytotoxic T lymphocyte (CTL) induction. The cell mediating the suppression was identified as a Thy 1+ Lyt 2+ I-J+ cell. The induction of this suppressor cell required the participation of at least three leukocyte populations. That is, depleting the cultures of either Lyt 1+ or Lyt 2+ splenocytes resulted in a failure to induce suppressor cell activity. Likewise the removal of macrophage-like antigen-presenting cells (APC), in particular I-A- I-J+ APC, abolished suppressor-cell induction. Though the Lyt 2+ I-J+ cells had to be provided by HSV-immune mice, both the APC and the Lyt 1+ cells could be provided by HSV-naive mice. Though the induction of the suppressor cell was virus specific, its action was nonspecific as evidenced by the suppression of influenza-specific CTL induction. The implication of our results for the understanding and manipulation of herpesvirus disease is briefly discussed.

Animals↗

PubMed1987

Suppression of delayed type hypersensitivity to herpes simplex virus type 1 following immunization with anti-idiotypic antibody: an example of split tolerance.

Intraperitoneal (i.p.) immunization with herpes simplex virus type 1 (HSV-1) or an anti-idiotypic antibody (anti-id C) prepared against a monoclonal antibody specific for glycoprotein C of HSV-1, tolerizes mice for an HSV-1 delayed type hypersensitivity (DTH) response. This tolerization could be adoptively transferred to naive X-irradiated mice by splenic T cells, and was specific for HSV-1 DTH. Thus, DTH-tolerized mice responded to vaccinia virus or HSV-2 challenge, while remaining tolerized for HSV-1 DTH. In addition, these animals demonstrated a form of split tolerance such that HSV antibody, cytotoxic T cell and lymphoproliferative responses were detected in vitro. Thus, anti-id C induced a T cell population capable of specifically suppressing the HSV-1 DTH response, mimicking the effect of i.p. and intravenous immunization with HSV-1 found previously.

Animals↗

PubMed1987

Mechanisms of antiviral immunity induced by a vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D: cytotoxic T cells.

We used a transfected L cell and a vaccinia vector carrying the herpes simplex virus type 1 (HSV-1) gene coding for glycoprotein D (gD) to characterize HSV-specific T-cell responses. Various studies with mice revealed that the vectors could stimulate some HSV-specific T-cell responses. Although the majority of the T cells contributing to the HSV-1 gD-specific proliferative response were of the Lyt-2.1+ phenotype, cytotoxic T cells (Tc), surprisingly, were not induced by these gD vectors. Even though gD appeared to be a target for a class II major histocompatibility complex (MHC)-restricted killer cell, neither gD vector was capable of forming a target cell complex which could be recognized by class I MHC-restricted HSV-specific Tc. Further investigation of the gD-specific responses revealed the presence of potent suppressor cells and factors capable of inhibiting HSV-specific Tc induction in in vitro assays. One interpretation of these data is that class I MHC-restricted HSV- and gD-specific Tc do not develop during HSV infection because of active suppression.

Antigens, Viral↗

PubMed1987

Inhibition of herpes simplex virus replication in the mouse cornea by drug containing immunoliposomes.

Monoclonal antibody to HSV glycoprotein D was derivatized with palmitic acid and incorporated into liposomes. These immunoliposomes bound specifically to intact mouse corneas infected with HSV-1 in vitro. Furthermore, in yield reduction assays, anti-gD immunoliposomes loaded with acyclovir proved far more effective at inhibiting viral replication in the cornea than free drug or drug delivered in untargeted liposomes. Site-specific sustained release immunoliposomes of this type are potentially an improved vehicle for drug delivery in the treatment of ocular HSV.

Acyclovir↗

PubMed1986

Target-sensitive immunoliposomes: preparation and characterization.

A novel target-sensitive immunoliposome was prepared and characterized. In this design, target-specific binding of antibody-coated liposomes was sufficient to induce bilayer destabilization, resulting in a site-specific release of liposome contents. Unilamellar liposomes were prepared by using a small quantity of palmitoyl-immunoglobulin G (pIgG) to stabilize the bilayer phase of the unsaturated dioleoylphosphatidylethanolamine (PE) which by itself does not form stable liposomes. A mouse monoclonal IgG antibody to the glycoprotein D of Herpes simplex virus (HSV) and PE were used in this study. A minimal coupling stoichiometry of 2.2 palmitic acids per IgG was essential for the stabilization activity of pIgG. In addition, the minimal pIgG to PE molar ratio for stable liposomes was 2.5 X 10(-4). PE immunoliposomes bound with HSV-infected mouse L929 cells with an apparent Kd of 1.00 X 10(-8) M which was approximately the same as that of the native antibody. When 50 mM calcein was encapsulated in the PE immunoliposomes as an aqueous marker, binding of the liposomes to HSV-infected cells resulted in a cell concentration dependent lysis of the liposomes as detected by the release of the encapsulated calcein. Neither uninfected nor Sendai virus infected cells caused a significant amount of calcein release. Therefore, the release of calcein from PE immunoliposomes was target specific. Dioleoylphosphatidylcholine immunoliposomes were not lysed upon contact with infected cells under the same conditions, indicating that PE was essential for the target-specific liposome destabilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Destabilization of target-sensitive immunoliposomes by antigen binding--a rapid assay for virus.

Interactions of antibody stabilized phosphatidylethanolamine (PE) immunoliposomes with Herpes Simplex virus (HSV) and virus infected cells were studied by detecting the immune-dependent lysis of liposomes. Employing PE immunoliposomes bearing anti-HSV glycoprotein D (gD) IgG, immune-specificity of these liposomes were documented by the sole ability of HSV and the HSV-infected L cells to induce immunoliposome lysis. In addition, inhibition of PE immunoliposome lysis by free anti-gD IgG, but not anti-HSV glycoprotein B IgG, indicated the target antigen specificity of these immunoliposomes. Based on these observations, alkaline phosphate encapsulated PE liposomes were used to directly detect HSV in fluid phase. This immunoliposome assay which does not require washing was shown to be very rapid and sensitive: 35pfu of HSV-1 in 5ul could be detected within 1.5hr.

Animals↗

PubMed1986

Characterization of a two-signal-dependent, Ia+ mononuclear phagocyte progenitor subpopulation that is sensitive to inhibition by ferritin.

Evidence is presented that the ferritin-inhibitable, Ia+ monocyte progenitor in murine marrow requires two signals for stimulation of clonal proliferation. Escherichia coli K235 lipopolysaccharide (LPS) at 0.1 ng/ml enhanced macrophage colony formation by 25 to 70% in murine marrow cultures stimulated with colony-stimulating factor (CSF-1). The progenitors which responded to LPS and CSF-1 represented a distinct subpopulation. Pretreatment of marrow cells with complement plus anti-Ia, anti-H2, anti-asialo GM1, and anti-Mac-1 antibodies specifically depleted the two-signal-requiring progenitors. In addition, the same progenitors were depleted by preincubation with hydroxyurea, indicating that these cells were in cell cycle when removed from the marrow. When compared with the quiescent progenitors, the Ia+, cycling cells were more sensitive to the antiproliferative effects of interferon alpha/beta but were more resistant to inhibition by E prostaglandins. Pretreatment with T cell-specific antibodies and complement specifically enhanced cloning of quiescent progenitors without affecting cloning of the Ia+, cycling subpopulation. Moreover, rat liver ferritin at 10(-8) to 10(-10) M specifically inhibited clonal proliferation of the Ia+ progenitors. Finally, the requirement for LPS as the additional stimulant could be replaced by the addition of haplotype-specific anti-Ia antibody to CSF-stimulated cultures. In contrast to LPS, anti-IA was competitive with inhibitory ferritin in clonal proliferation of the Ia+ progenitors. The significance of these observations in regulation of monocytopoiesis is discussed.

Animals↗