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R E Cone

Publications and source records attributed to R E Cone.

At least 37 records · Page 2Linked to original sources
PubMed1995

High-dose cyclophosphamide inhibits anterior chamber-associated immune deviation (ACAID) and the production of extracellular antigen-specific T cell proteins induced by trinitrophenylated (TNP) spleen cells.

Injection of antigen into the anterior chamber (AC) of the eye induces the production of IgM and IgG1 antibodies and potentiates the appearance in serum of extracellular antigen-specific T cell proteins (TABM) that are specific for the AC-injected antigen. In contrast, delayed-type hypersensitivity (DTH) to the injected antigen is suppressed. This manifestation of anterior chamber-associated immune deviation (ACAID) is believed to be a key part of the basis of the immune privilege of the eye. Because cyclophosphamide (CY) exerts selective effects on immunoregulatory T cells and macrophages, we sought to determine its effects on the appearance in serum of TNP-specific TABM in mice following intracameral injection with TNP murine spleen cells followed by epicutaneous sensitization and challenge with picrylchloride. Injection of 200 mg/kg CY 2 days before AC injection of TNP spleen cells, sensitization, and challenge prevented the suppression of DTH and the production of TNP-specific TABM. The production of TNP-specific immunoglobulins was not affected. Injection of a low dose of 20 mg/kg CY enhanced DTH 100-300% in control animals, but did not prevent either ACAID or the production of TABM. These results provide further evidence that serum TABM may be a serologic indicator of T lymphocyte activity in ACAID and that ACAID is mediated by cells sensitive to high-dose CY.

Animals↗
PubMed1995

Extravascular secretion of t-PA by the intact superfused choroid.

PURPOSE: Demonstrate the continuous extravascular secretion of enzymatically active t-PA by the intact choroid. METHODS: Enucleated rat eyes were dissected under microscopic control to prepare eyecups for superfusion in a small organ culture system. Individual preparations provided access to intact retinal, retinal pigment epithelial, or choroidal surfaces in situ. Cups were superfused with oxygenated Kreb's buffer (100 microliters/minute) at 37 degrees C and pH 7.4 while suspended in a microincubator. Released t-PA activity was measured by an amidolytic assay in 1-minute overflow samples and in cup media after flow interruptions. RESULTS: Minimal basal secretion was detected in the overflow with the retina in place. Interruption of flow (10 minutes) produced a static accumulation of 0.10 +/- 0.01 IU/ml. With the retinal pigment epithelium in place after retinal removal, t-PA release was moderately accelerated. Removal of the retinal pigment epithelium to expose the Bruch's-choroidal surface produced the greatest acceleration. The 10-minute static level was 0.48 +/- 0.13 IU/ml. Release from the scleral shell after choroid removal was negligible. An acute burst release of t-PA activity (1.3 +/- 0.18 IU/ml) followed the infusion of bradykinin (5 to 20 microM) into choroidal cups. CONCLUSION: Choroid appears to be the dominant source of a continuously secreted t-PA in superfused rat eyecup organ cultures. This enzymatically active t-PA is able to pass through an apparently intact Bruch's membrane but is largely impeded by an intact retinal pigment epithelium. It is also possible that retinal pigment epithelium contains inhibitors that could reduce the released t-PA activity. The authors propose that this secretory function of the uveal tissue, which has thus far not been recognized, may be a principal mechanism for the regulation of intraocular fluid circulation and matrix remodeling by plasmin in vivo.

Animals↗
PubMed1995

Intracameral injection of antigen potentiates the production of antigen-specific T cell proteins in serum after the induction of delayed-type hypersensitivity.

PURPOSE: To determine whether the introduction of antigen into the anterior chamber induces the production of extracellular antigen-specific T cell proteins (T cell antigen-binding molecules [TABM] specific for the antigen. METHODS: Balb/c mice received an intracameral or subconjunctival injection of trinitrophenylated spleen cells (TNP spleen cells) before skin sensitization and challenge with picrychloride. The production of TNP-specific TABM in serum was quantified by enzyme-linked immunosorbent assay-based antigen binding and immunoblotting using a rabbit antiserum raised against a monoclonal antigen-specific T cell protein that induces suppressor T cells. RESULTS: Intracameral, but not subconjunctival, injection of TNP spleen cells before contact sensitization increased the level of TNP-specific TABM in serum. At 2 days, TABM levels began to rise and peaked at 5 days after contact sensitization. Anterior chamber injection of TNP spleen cells alone into nonimmunized mice did not induce a detectable increase of TNP-specific TABM in serum. Mice that did not receive an intracameral TNP spleen cell injection but were made contact sensitive to TNP showed a diminished TNP-specific TABM response. Specificity of the TABM induced by intracameral-injection and sensitization for the TNP hapten was documented by showing that the induced TABM binds TNP-bovine serum antigen (BSA) but does not bind either BSA alone or azobenzenearsonate-ovalbumin and that immune sera raised against ovalbumin did not contain an increase in TNP-specific TABM. TNP-specific TABM in the sera of mice receiving an intracameral injection of TNP spleen cells, followed by contact sensitization with picrylchloride (PCl), were purified by affinity for TNP and were resolved by polyacrylamide gel electrophoresis and immunoblotting as M(r) 110,000 polypeptides. CONCLUSIONS: Serum levels of TNP-specific TABM induced by contact sensitization alone are enhanced fourfold to fivefold when sensitization is preceded by the intracameral, but not the subconjunctival, injection of TNP spleen cells. The authors propose that besides suppression of systemic delayed type hypersensitivity, the intracameral injection of antigen into nonimmune mice may prime an increased production of TABM, which may indicate systemic activation of the immunoregulatory T cell circuit.

Animals↗
PubMed1994

T cell-derived antigen-specific humoral immune response. II. Further characterization of the response and the antigen binding T cell immunoproteins.

Murine T lymphocyte-derived proteins which bind nominal antigen specifically (TABM) were detected in serum during a humoral immune response to bovine serum albumin. The TABM response was observed only when the adjuvants polyadenylic:polyuridylic acid complex (poly(A: U)), Freund's complete adjuvant or Titermax were used concurrently with antigen to immunize. When poly(A:U) was used, higher doses of antigen were optimal to stimulate TABM production than those required to stimulate immunoglobulin production. The binding of TABM in the serum of BSA-immunized mice to BSA in solid phase was inhibited specifically by 10-fold more BSA than that required to inhibit BSA-specific immunoglobulins suggesting that TABM have much less affinity for antigen than immunoglobulins. Immunoblotting of TABM in serum from BSA-immune mice, which bind BSA, demonstrated that these serum TABM are comprised of M(r) 110,000 polypeptides linked by disulfide bonds. Antigen-specific proteins in a lysate of an NP-specific T cell hybrid inhibited the recognition of serum TABM by anti-TABM antiserum while a lysate of a B cell hybridoma was not inhibitory, and serum TABM were adsorbed by monoclonal antibodies to TCR-C beta. These results provide more evidence that serum TABM may be a soluble analogue of the T cell receptor for antigen.

Adjuvants, Immunologic↗
PubMed1994

Increased basal levels of free plasminogen activator activity found in human aqueous humor.

PURPOSE: To quantify the basal free t-PA activity in human aqueous humor. METHODS: Human aqueous humor obtained by simplified pipette paracentesis at cataract surgery was tested for free t-PA activity in a revised 15-hour amidolytic assay using a t-PA standard curve (n = 15). Total antigenic levels of PAI-1, the principal PA inhibitor, were determined using an ELISA kit. The available PAI-1 activity was tested indirectly using anti-human PAI-1 antibody blocking before t-PA activity assay (n = 11). Plasminogen activator type was determined by anti-human t-PA and urokinase (u-PA) antibody blocking before activity assay (n = 7). RESULTS: Free PA activity ranged widely (0.072 to 0.47 IU/ml; mean, 0.20 +/- 0.10 IU/ml) and was almost completed inactivated (> or = 89%) by antibody against human t-PA but not by the u-PA antibody. PAI-1 total antigen also ranged widely between (0.25 to 8.0 ng/ml; mean, 2.25 +/- 2.54 ng/ml). However, pretreatment of samples with PAI-1 antibody or by acidification (pH 3.2) to inactivate inhibitors did not increase t-PA activity levels. CONCLUSIONS: A basal-free t-PA activity, which is predominantly t-PA, is present in human aqueous humor at approximately 20 times higher levels than previously found with an earlier assay. This activity is estimated to represent roughly 10% of total released t-PA antigen. PAI-1 is present in aqueous humor at levels considerably lower than reported values for plasma in a predominantly PA-complexed or inactive form.

Aged↗
PubMed1994

Tissue plasminogen activator activity in human aqueous humor.

PURPOSE: To determine the levels of free plasminogen activator activity in human aqueous humor and to identify the type of activity (i.e., tissue-type t-PA or urokinase-type u-PA) that is responsible. METHODS: Aqueous humor was obtained by a simplified pipette paracentesis before cataract surgery in 31 subjects, ages 57 to 93 years. Levels of plasminogen activator activity were determined using a modified 17-hour specific amidolytic assay. The type of plasminogen activator was investigated in selected samples based on its dependence on soluble fibrin, inhibition by amiloride, and specific antibody blocking. Activity-antigen ratios were compared in seven samples. RESULTS: Plasminogen activator (PA) activity was present in all samples tested. PA activity ranged widely between 0.54 and 26.7 mIU/ml, with a mean value of 10.8 +/- 8.1 mIU/ml. Soluble fibrin, a known stimulator of tissue-type plasminogen activator (t-PA), was required in the assay system. Its absence decreased the measured activity by more than 90%. Amiloride, a known inhibitor of urokinase-type PA, had little or no effect in selected samples tested. The activity was blocked by anti-human t-PA antibodies but not by antibodies against human u-PA, further defining the type of PA responsible for the detected activity. t-PA antigen levels showed less variation among individuals than did activity levels. Antigen-activity ratios ranged between 89 and 552. CONCLUSION: Plasminogen activator activity is present in the human aqueous humor in measurable quantities. The type of PA activity present is almost exclusively t-PA. t-PA activity varies more widely than antigen, as is the case in plasma.

Aged↗
PubMed1993

Regulatory effect of interferon-gamma and phorbol esters on the surface expression and biosynthesis of MHC class I antigens by human leukemia cells.

We have used cell surface radioiodination, biosynthetic incorporation of [35S]methionine, and flow cytometry to analyze the effects of interferon gamma (IFN-gamma) and/or phorbol esters (PMA) on the turnover and expression of class I antigens of a human leukemia B cell line. Our results demonstrated that although both IFN-gamma and PMA enhance HLA expression, they act synergistically to increase by eightfold the amount of HLA polypeptides synthesized by the acute lymphoblastic leukemia cells and acted additively to augment the cell surface expression of HLA as quantified by flow cytometry. We observed a cyclic increase or decrease in the expression of class I antigens as a function of time in cell culture. IFN-gamma and/or PMA modulated this effect inducing more cells to express HLA maximally. These results suggest that there is a physiologic limit for the expression of major histocompatibility complex class I antigens.

B-Lymphocytes↗
PubMed1993

Partial amino acid sequence of monoclonal extracellular antigen-specific T cell proteins.

Antigen-specific molecules secreted by murine T cell hybrids (TABM) specific for 4-hydroxy 3-nitrophenyl (NP) or azobenzenearsonate (ABA) were purified from ascitic fluid by ion exchange chromatography and/or affinity for antigen. Partial amino acid sequence of reduced Mr 72,000 NP-specific polypeptides and Mr 20,000 peptides prepared by treatment of the ABA-specific immunoprotein with cyanogen bromide was obtained and a septapeptide of the NP-specific TABM shared 3/7 residues with the ABA-specific TABM. Both TABM shared residues present in 95% T cell receptor for antigen (TCR) V alpha subgroup I and 83-96% murine immunoglobulin V kappa Fr3. These results provide evidence that extracellular antigen-specific T cell proteins are soluble analogues of TCR alpha chains and belong to the immunoglobulin supergene family.

Amino Acid Sequence↗
PubMed1992

Appearance of T lymphocyte-derived proteins specific for the immunizing antigen in serum during a humoral immune response.

Some T lymphocytes produce extracellular proteins that bind nominal antigen specifically (TABM), and these proteins exhibit potent immunoregulatory activity. We have utilized an ELISA for Ag binding by Ag-specific TABM to detect and quantitate the appearance of Ag-specific TABM in murine serum during a humoral Ir to protein Ag. The TABM response was specific for the inducing Ag, stronger and more rapid during a secondary response, and temporally distinct from the appearance of Ig. The non-Ig serum TABM were bound by mAb specific for TCR-alpha chains and isolated by affinity for Ag were Mr 110,000 polypeptides. The TABM response did not occur in scid/scid mice unless the mice were reconstituted with thymocytes and thymocyte-reconstituted scid/scid mice produced TABM, but did not produce Ig. The results suggest that soluble TABM are an Ag-specific humoral manifestation of the Ir of some T lymphocytes.

Animals↗
PubMed1992

Specific antigen binding by proteins secreted by an antigen-specific T cell hybrid.

Antigen-specific molecules secreted by a murine T cell hybrid specific for azobenzene arsonate (ABA) were purified from ascites fluid by ion exchange chromatography and affinity for antigen. The antigen-specific proteins were purified 250 fold and were resolved predominantly as Mr 110,000 polypeptides by reduction and SDS-polyacrylamide gel electrophoresis. The ability of these molecules to bind antigen was analyzed by an ELISA using antigen-coated microtiter trays. Binding of the T cell proteins to antigen was detected with antisera specific for the proteins. Antigen binding to ABA-ovalbumin but not ovalbumin was optimal at 37 degrees C and protein derived from another T cell hybrid did not bind ABA-ovalbumin. Solid phase antigen binding was inhibited specifically by soluble ABA-ovalbumin, indicating that these T cell-derived proteins bind nominal antigen in the solid or liquid phase. It is suggested that these proteins represent a soluble, antigen specific manifestation of some T cell function.

Animals↗
PubMed1991

T cell non-MHC-restricted antigen-binding molecules secreted or associated with the cell membrane are antigenically distinct.

Some T cells produce membrane-associated or soluble molecules which bind nominal antigen specifically (TABM) and effect immunoregulation or events similar to cell-mediated hypersensitivity. We have used polyclonal antisera raised against an azobenzene arsonate (ABA)-specific TABM secreted by an ABA-specific T cell hybrid or against TNP-specific polypeptides produced by immunoregulatory T cells to identify the expression of soluble (secreted) or membrane-associated TABM. Ascites fluid or culture medium containing a T cell hybrid or T cell lines, respectively, contain TABM recognized only by an antiserum specific for the secreted T cell hybrid (ABA-specific) derived TABM. Conversely, an antiserum that recognized the TNP-specific polypeptides detected cell-membrane associated TABM but did not bind TABM secreted by the T cell hybrid or cell lines.

Animals↗
PubMed1991

IFN-gamma retards the turnover of H-2Dd antigens by splenic lymphocytes.

The effect of IFN-gamma on the rate of shedding and biosynthesis of H-2Dd was determined by culture of cell surface-radioiodinated BALB/c spleen cells with rIFN-gamma or spleen cells metabolically labeled with 35S-methionine in the presence of IFN-gamma. Radioiodinated or 35S-labeled H-2Dd was quantitated by immunoprecipitation of H-2Dd from detergent lysates of radiolabeled cells taken at different culture intervals. The loss of 125I-labeled H-2Dd was retarded 75 to 90% by IFN-gamma whereas the biosynthetic rate was unaffected during the first 10-h culture. The net result was a ninefold increase in newly synthesized cell-associated H-2Dd. The results were consistent with determination of the kinetics of increased expression of H-2Dd determined by immunofluorescence and suggest that an early effect of IFN-gamma on the expression of class I Ag is a retardation of catabolism leading to an increase of newly synthesized class I Ag.

Animals↗
PubMed1991

T cell derived proteins from normal human sera and their relationship to T cell antigen binding molecules.

We have used procedures which have been developed to isolate murine T cell antigen binding molecules (TABM) in order to isolate TABM from normal human sera. To begin purification, ammonium sulfate (NH4)2SO4 was added to human serum and precipitated protein was dissolved in low salt buffer and resolved by ion-exchange chromatography on carboxymethylcellulose (CM). The most strongly CM nonadherent fraction was absorbed with anti-human albumin and anti-human immunoglobulin (Ig) antibodies conjugated to Sepharose beads. The resulting nonadsorbed 110,000, 70,000 and 45,000 Mr polypeptides were reactive in ELISA with a rabbit antiserum produced against non-Ig, anti-specific molecules of rhesus monkeys. These proteins possess alpha mobility upon immunoelectrophoresis and represent 0.02 to 0.05% of total serum protein. In addition, these proteins are bound by an antiserum made against a synthetic peptide corresponding to the J region of the TcR beta chain. We have made R28, a rabbit antiserum against these serum proteins which binds specifically to tetanus-specific polypeptides obtained from the culture supernatant of human T cell lines specific for tetanus. This antiserum also binds to proteins isolated from T cell but not B cell lines, and T cell proteins are able to inhibit the binding of R28 to the human serum polypeptides. The results suggest that the proteins isolated from normal human sera are T cell antigen binding molecules.

Animals↗
PubMed1991

Inhibition of major histocompatibility complex class I antigen shedding up-regulates the surface expression of class I antigens on the lymphocyte cell surface.

Interferon-gamma induces the expression of major histocompatibility complex class I and class II gene products. Moreover, the density of MHC antigens present on the lymphocyte surface is regulated by exfoliation of the plasma membrane. To probe the cellular mechanisms involved in IFN-gamma-induced alteration of MHC antigen expression, we measured the effects of IFN-gamma on the rate of MHC antigen shedding and the biosynthesis of H-2Dd. Balb/c splenic lymphocytes were surface-iodinated with 125I and incubated in the presence and absence of up to 1000 U/ml IFN-gamma, or they were metabolically labeled with [35S]methionine with or without 500 U/ml IFN-gamma. Radioiodinated or 35S-labeled H-2Dd was quantified by immunoprecipitation of H-2Dd from detergent lysates of radiolabeled cells that were incubated with the appropriate antibody for 4-20 h at 37 degrees C. Monoclonal antibody 34-5-8 was employed as a specific probe for H-2Dd. Loss of radioiodinated H-2Dd from the cell surface was diminished by 75-90% at 12 h in tests of lymphocytes continuously cultured with IFN-gamma (compared to control, p less than 0.05). In contrast, the biosynthetic rate was unaffected during the initial 10 h of incubation. The net result of these changes was the early appearance of an increase in H-2Dd on the cell surface. This result was in accordance with data obtained by phenotyping the untreated and treated cells using double-antibody staining methods and fluorescence-activated cell sorter analysis. Our results suggest that IFN-gamma induces MHC expression by initially retarding the exfoliation of MHC antigens from the lymphocyte surface. Delayed effects on MHC expression may be, on the other hand, mediated by increased antigen biosynthesis.

Animals↗
PubMed1990

Ontogeny and expression of non-MHC-restricted T cell antigen-binding molecules by thymocytes and peripheral T cell subsets.

The ontogeny of T cells which express major histocompatibility complex (MHC)-unrestricted T cell antigen-binding molecules (TABM) on the cell membrane was investigated. We used a rabbit anti-mouse TABM antiserum to investigate the expression of TABM by subsets of adult thymocytes, peripheral T cells, and thymocytes during gestation. TABM are expressed by CD4+, CD8-, CD4+, CD8+ thymocytes and single-positive thymocytes. During gestation, TABM are expressed as early as Day 16, and at birth the expression of TABM on thymocytes has reached adult levels. Data are also presented which suggest that the expression of membrane TABM (mTABM) on peripheral T cells can be upregulated during T cell activation. The results suggest that TABM are expressed by different T cell subsets and that TABM+ cells may utilize the same intrathymic developmental pathway as that of T cells which express the alpha/beta T cell receptor.

Age Factors↗
PubMed1989

Expression of non-MHC-restricted T cell antigen-binding molecules by thymic lymphocytes.

Heterologous antisera which recognize non-major histocompatibility complex (MHC)-restricted T cell antigen-binding molecules (TABM) were used to characterize the expression and structure of TABM on thymic lymphocytes. Approximately 70% of thymocytes express membrane molecules bound by anti-TABM antibodies (mTABM). Antibody activity for thymocyte TABM could be removed by adsorption to splenic T cells, but not by adsorption to splenic B cells. Similarly, adsorption of the antiserum to thymocytes or splenic T cells removed antibody activity to a purified TABM whereas adsorption with B cells had no effect. Radioiodinated thymic and splenic T cell mTABM were resolved by 2D-polyacrylamide gel electrophoresis and when reduced, both populations of mTABM migrated primarily as Mr 23,000 proteins with an isoelectric point range of 6.8-7.8. Multimers of this protein were also observed at Mr 85-97,000 and 130-150,000 on both thymocytes and splenic T cells. These data indicate that MHC-unrestricted antigen-binding molecules are expressed by a majority of thymocytes and these thymic TABM are structurally and antigenically similar to mTABM on peripheral cells. This suggests an ontogenic relationship between thymic TABM and peripheral TABM.

Animals↗