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PubMed1999

Stabilization of an antibody conjugated with enzyme by 2-methacryloyloxyethyl phosphorylcholine copolymer in enzyme-linked immunosorbent assay.

The purpose of this study was to develop a novel synthetic stabilizer of enzyme-linked antibody in the enzyme-linked immunosorbent assay (ELISA). The water-soluble amphiphilic phospholipid polymer, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-styrene (St)] was synthesized, and its stabilizing functions for the antibody were compared with conventional stabilizers of the antibody conjugated with enzyme (enzyme-antibody conjugate), such as bovine serum albumin (BSA) and casein. In the absence of the stabilizer, the remaining immunologic activity decreased to about 10% of its initial value after 37 days. The same tendency was observed even when the enzyme-antibody conjugate in 1.0 wt % BSA solution was used as a stabilizer. In 1.0 wt % casein solution, the immunologic activity decreased to 29% of the initial value after 37 days. On the other hand, in 0.1 wt % and 1.0 wt % poly(MPC-co-St) solution, the activity remained 74% and 92% of the initial value, respectively. The effects of poly(MPC-co-St) on the stabilization of the enzyme-antibody conjugate depended on the concentration of poly(MPC-co-St). During the ELISA procedure, not only did poly(MPC-co-St) have no effect on the reaction between the antigen and the antibody, but it also had no effect on the reaction between the enzyme and the substrate. These results indicate that poly(MPC-co-St) has the ability to suppress the denaturation of protein, enzyme, and antibody. We concluded that water-soluble poly(MPC-co-St) is an effective synthetic stabilizer in the ELISA.

Animals↗
PubMedDate not supplied

Affinity liposomes in vivo: factors influencing target accumulation.

To make universal and efficient liposome-based drug carriers, liposomes should be able to recognize and bind other targets beyond their natural targets, the cells of the reticuloendothial system. To make liposomes targeted, numerous methods to couple active substances, primarily, monoclonal antibodies, to the liposome surface have been developed. Resulting immunoliposomes (or affinity liposomes) demonstrate good targeting to cells and organs both in vitro and in vivo. However, the short circulation time of immunoliposomes prevented them from accumulating in targets with diminished blood flow or low antigen concentration. Long-circulating liposomes were prepared by coupling soluble and flexible polymers, such as polyethylene glycol, to the liposome surface. The mechanism of liposome steric protection with flexible polymers is based on the formation of dense "conformational cloud' by a grafted polymer over the liposome surface, and might be analyzed in terms of a statistical model of polymer solutions. By co-immobilization of specific antibodies and protecting polymers on the liposome surface, liposomes can be prepared combining both targetability and prolonged circulation in vivo. A biological model (experimental myocardial infarction in rabbit) was used to estimate the relative importance of different factors (liposome size and coating with protective polymer and/or specific antibody) for effective accumulation of liposomes in the target. Statistical analysis demonstrated that different types of liposomes have to be used in order to reach maximum absolute delivery of liposomes to the target, or maximum target-to-non-target ratio (relative delivery). Therefore, different liposomes should be used as carriers of diagnostic and therapeutic agents.

Animals↗
PubMedDate not supplied

Autoantibody negative new onset type 1 diabetic patients lacking high risk HLA alleles in a caucasian population: are these type 1b diabetes cases?

BACKGROUND: In Caucasians, a small number of Type 1 diabetic patients do not show evidence of humoral islet autoimmunity at disease onset, at least with common screening procedures. In African- and Hispanic-American diabetic children at time of diagnosis, many show no evidence of autoimmunity but have an atypical clinical form of the disease. According to the recent American Diabetes Association classification, this subgroup of autoantibody negative patients is referred to as Type 1b diabetic subjects. In the present study, a homogeneous Caucasian Type 1 diabetic clinic-based cohort has been evaluated at diagnosis using a large panel of diabetes-related antibodies and then characterized for various genetic features in order to identify newly diagnosed Type 1 diabetics who are potentially autoantibody negative, i.e. possibly referrable to as idiopathic Type 1b diabetes. METHODS: Newly diagnosed Type 1 diabetic patients of Italian origin (n=141, mean age 12.0+/-7.6 years) were tested for anti-islet cell, anti-insulin, anti-65 kDa isoform of glutamic acid decarboxylase and anti-amino acid residues 256-979 of the tyrosine-phosphatase IA-2 molecule autoantibodies (Step 1). Only those patients found to be autoantibody negative were tested for anti-disialo-ganglioside GD3, anti-thyroid peroxidase, anti-thyroglobulin, anti-21-OH hydroxylase, anti-gastric parietal cell and anti-transglutaminase antibodies (Step 2). Sera negative for the presence of these six autoantibodies as well were characterized in terms of HLA DRB1, DQB1 and CTLA-4. RESULTS: Six out of 141 subjects (3.5%) were autoantibody negative in the first step of the study and five out of six in the second. These five autoantibody negative patients underwent genetic analysis. Three of them had at least one Type 1 diabetes-related high risk HLA haplotype (3/141, 2.1%) while the remaining two cases showed neutral (DR5-DQB1*0301/DR5-DQB1*0301) or strongly protective (DR2-DQB1*0602/DR2-DQB1*0602) HLA genotypes, respectively (2/141, 1. 4%). CONCLUSIONS: Clinically defined Type 1 diabetic patients with no sign of autoimmunity do exist in a Caucasian population. These patients (2 out of 141) that cannot be classified as Type 1a diabetic patients lack clinical characteristics of Type 1b diabetes and have to be reconsidered for a more appropriate ADA classification. These data suggest the need of further large population-based studies to understand if Type 1b diabetes really occurs in a Caucasian population. The patient with a strongly protective HLA genotype is particularly interesting considering that among Caucasians only a few sporadic cases with Type 1 diabetes and DQB1*0602, have been reported, none of whom was homozygous at DQB1 locus.

Abatacept↗
PubMed1998

Absence of B7.1-CD28/CTLA-4-mediated co-stimulation in human NK cells.

Recent studies have suggested that B7-CD28 interactions provide co-stimulatory signals for activation of NK cells. Transduction of the B7.1 (CD80) gene into tumor cells has been shown to trigger proliferation and cytotoxicity of murine NK cells and a human NK cell line, YT2C2. Therefore, transduction of the B7.1 gene into CD80-negative human squamous cell carcinomas of the head and neck (SCCHN) and its stable expression was expected to upregulate proliferation and cytotoxic activities of human NK cells. However, expression of the B7.1 receptors, CD28 and CTLA-4, could not be demonstrated on the surface or in the cytoplasm of normal human NK cells, irrespective of the state of their activation. In proliferation experiments or various cytotoxicity assays, utilizing highly purified human NK cells as responder or effector cells, no enhancement of NK cell generation or activity, respectively, by B7.1+ SCCHN was observed relative to non-transduced or LacZ gene-transduced SCCHN. In contrast, co-incubation of B7.1+ SCCHN targets with human NK cells induced significant inhibition of NK cell growth. Thus, the B7.1-CD28/CTLA-4 pathway is not involved in triggering of human adult NK cells.

Abatacept↗
PubMed1998

Secondary but not primary T cell responses are enhanced in CTLA-4-deficient CD8+ T cells.

Negative as well as positive co-stimulation appears to play an important role in controlling T cell activation. CTLA-4 has been proposed to negatively regulate T cell responses. CTLA-4-deficient mice develop a lymphoproliferative disorder, initiated by the activation and expansion of CD4+ T cells. To assess the function of CTLA-4 on CD8+ T cells, CTLA-4(-/-) animals were crossed to an MHC class I-restricted 2C TCR transgenic mouse line. We demonstrate that although the primary T cell responses were similar, the CTLA-4-deficient 2C TCR+ CD8+ T cells displayed a greater proliferative response upon secondary stimulation than the 2C TCR+ CD8+ T cells from CTLA-4 wild-type mice. These results suggest that CTLA-4 regulates antigen-specific memory CD8+ T cell responses.

Abatacept↗
PubMed1998

MAdCAM-1 costimulates T cell proliferation exclusively through integrin alpha4beta7, whereas VCAM-1 and CS-1 peptide use alpha4beta1: evidence for "remote" costimulation and induction of hyperresponsiveness to B7 molecules.

We have analyzed the effects of the alpha4 integrin ligands mucosal addressin cell adhesion molecule-1 (MAdCAM-1), vascular cell adhesion molecule-1 (VCAM-1), and the fibronectin CS-1 splice variant on T cell activation. Immobilized MAdCAM-1 and VCAM-1 IgG-Fc chimeras and a fibronectin CS-1 peptide efficiently costimulate T cell proliferation when antigen presentation is mimicked by anti-CD3 antibody. VCAM-1-Fc and fibronectin CS-1, which are adhesive ligands for both the alpha4beta1 and alpha4beta7 integrins, medicate T cell costimulation exclusively through integrin alpha4beta1, but not through alpha4beta7. The inability of VCAM-1-Fc to costimulate via alpha4beta7 suggests that cell adhesion per se is insufficient, and that exquisite recognition and activation events must be triggered. MAdCAM-1-Fc mediates costimulation exclusively via alpha4beta7, and can both synergize with and induce hyperresponsiveness to the classical costimulator B7-2. MAdCAM-1-Fc and VCAM-1-Fc, but not B7-2, effectively costimulate when immobilized on sites spatially distant from the anti-CD3 antibody ("remote" costimulation). In vitro, the relative potencies of the CAM were VCAM-1-Fc> ICAM-1-Fc> MAdCAM-1-Fc > B7-Fc, except at high concentrations where ICAM-1 was the most potent. Features of costimulatory CAM revealed by this study have important implications for the design of immunotherapeutic vaccine strategies to combat cancer and infection.

Abatacept↗
PubMed1998

Local gene therapy with CTLA4-immunoglobulin fusion protein in experimental allergic encephalomyelitis.

It has been reported previously that the induction phase of experimental allergic encephalomyelitis (EAE) is highly sensitive to systemic blockade of stimulation via MHC class II molecules and co-stimulation via the CD28:CD80/CD86 pathways. In contrast, the effector phases of EAE were relatively unaffected by similar treatments using MHC class II antigen (Ag)-specific mAb and cytotoxic T lymphocyte antigen (CTLA)4-Ig fusion proteins in some studies. This has been attributed to different sensitivities of effector cell function or the poor penetrance of inhibitory proteins into the central nervous system (CNS). To examine this question further, MHC class II Ag-specific mAb and CTLA4-Ig were delivered directly into the CNS following EAE induction, and both were found to inhibit disease. While it was found that systemic administration of mouse CTLA4-Ig could also inhibit the progression of effector immune responses when administered shortly before or during clinical disease, these were significantly more active when delivered directly into the CNS, which probably involved an action on both CD28 ligands, CD80 and CD86. Although mouse CTLA4-human Ig was therapeutically less efficient than mouse CTLA4-mouse Ig protein, probably due to the enhanced immunogenicity and lower functional activity, gene delivery of CTLA4-human Ig into the CNS using a non-replicating adenoviral vector was more effective than a single injection of CTLA4-human Ig protein. Gene delivery significantly ameliorated the development of EAE, without necessarily inhibiting unrelated peripheral immune responsiveness. Local gene delivery of CTLA4-Ig may thus be an important target for immunotherapy of human autoimmune conditions such as multiple sclerosis.

Abatacept↗
PubMed1998

CTLA4 (CD152) modulates the Th subset response and alters the course of experimental Leishmania major infection.

Since both the nature and the amplitude of an antigen-specific T cell response are dependent on co-stimulatory signals, we have investigated the role of CD28/CD152-mediated T cell co-stimulation in the regulation of experimental cutaneous leishmaniasis. CD28-deficient mice and their wild-type littermates are equally susceptible to Leishmania major infection. Whole anti-CD152 antibody significantly exacerbates the disease while anti-CD152 Fab ameliorates the disease in genetically susceptible BALB/c mice but not in C57BL/6, a resistant strain. The anti-CD152-induced exacerbation of the disease is accompanied by increased IL-4-secreting cell number, diminished parasite-specific delayed-type hypersensitivity (DTH) response and augmented anti-2,4,6-trinitrophenyl (TNP) IgG1 in response to TNP-leishmanial antigen crude soluble antigen (CSA), suggesting an exaggerated Th2 type of response. Anti-CD152 Fab-mediated amelioration of the disease is associated with increased IFN-gamma-secreting cell number, increased parasite-specific DTH response and enhanced IgG2a isotype in response to TNP-CSA suggesting a Th1 type of response. Unlike TNP-CSA, TNP-keyhole limpet hemocyanin does not induce the change in Ig isotype, indicating that the immunomodulatory effect of anti-CD152 is antigen specific. Anti-CD152 antibody-induced early change in Th subsets suggests an important role for CD152 in determining the course of L. major infection, perhaps by alteration of Th subset differentiation.

Abatacept↗
PubMed1998

Deaggregated homologous immunoglobulin-peptide conjugates induce peptide-specific T cell nonresponsiveness in vivo.

Previous studies have proven the efficacy of intravenous injection of deaggregated protein as a means of inducing tolerance. In the present study, the immunodominant peptide 70-86 of myelin basic protein (MBP) was covalently linked to either mouse Ig or Lewis rat IgG. Lewis rats immunized with MBP in complete Freund's adjuvant were completely protected from development of experimental allergic encephalomyelitis (EAE) by their injection with as little as 40 microg of peptide conjugate on days 0 and 10 after immunization. Peptide-specific proliferative and cytokine responses by T cells from treated rats in vitro were severely depressed compared with controls, while responses to whole MBP were unaffected. Significantly, injections of 100 microg of peptide conjugate on days 0 and 4 after adoptive transfer of peptide-specific T lines protected rats from passive EAE while a single injection of 100 microg of conjugate at the onset of active EAE prevented any further disease progression. Both results suggest that primed effector cells as well as naive T cells are prone to tolerance induction by this means. The ability to intervene in ongoing immune responses with such specificity may be useful therapeutically in control of autoimmunity or allergic responses to environmental antigens.

Animals↗
PubMed1999

CTLA4-Ig inhibits optimal T helper 2 cell development but not protective immunity or memory response to Nippostrongylus brasiliensis.

The B7 co-stimulatory pathway is critical to T cell activation, however its role in the generation of Th2 cells in vivo remains controversial. We have studied the role of B7 co-stimulation in the development of a Th2 immune response to the nematode parasite Nippostrongylus brasiliensis. Blockade of B7 co-stimulation with murine CTLA4-Ig (mCTLA4-Ig) resulted in decreased Th2 cell development as determined by IL-4 and IL-5 cytokine production in vitro. It also resulted in lowered Th2 cell effector function in vivo, with marked reductions in IgE production. Blood eosinophilia was variably affected by mCTLA4-Ig treatment, which resulted in both slight and very severe inhibition in different experiments. However, an effective immune response was still evident as demonstrated by the further reduction of cytokine production, IgE titers, and blood eosinophilia in mice treated with a combination of mCTLA4-Ig and anti-CD4 mAb, and by the ability of mCTLA4-Ig-treated mice to expel adult worms. In addition, mCTLA4-Ig treatment did not alter the development of a memory response following secondary infection with N. brasiliensis, with the exception of IgE production. We conclude from these results that B7 co-stimulation is required in this experimental model for optimal Th2 cell development and effector function in vivo but is not necessary for protective immunity.

Abatacept↗
PubMed1999

CTLA4 ligation attenuates AP-1, NFAT and NF-kappaB activity in activated T cells.

CTLA4 is currently viewed as a late-appearing T cell surface receptor which is able to inhibit the proliferation of activated T cells. We sought to identify how CTLA4 ligation exerts these anti-proliferative effects by studying its influence on the activities of the relevant nuclear transcription factors AP-1, NFAT and NF-kappaB. We found that cross-linking CTLA4 on activated T cells completely blocks AP-1 and NFAT transcription factor activity before any effects on T cell proliferation can be observed, with NF-kappaB activity affected to a lesser degree. The suppression of AP-1 and NFAT transcriptional activity correlates with reduced levels of AP-1 and NFAT DNA binding as early as 10 h after T cell activation, prior to detectable up-regulation of CTLA4 on the T cell surface. Additionally, inhibitory effects on T cell proliferation only occurred when CTLA4 molecules were ligated in proximity to signaling TCR complexes, and inhibition of transcription factor DNA binding and activity was observed in the absence of CD28 stimulation. CTLA4 can thus act early during T cell activation to reduce the activity of several key nuclear transcription factors important for continued T cell proliferation and differentiation.

Abatacept↗
PubMed1999

Cytotoxic T lymphocyte antigen 4 (CTLA-4) inhibits CD28-induced IkappaBalpha degradation and RelA activation.

Purified CD4+ cells from the spleens of C57BL/6 mice were stimulated with anti-CD3, anti-CD28 and anti-cytotoxic T lymphocyte antigen (CTLA)-4 monoclonal antibodies. The results show that CTLA-4 stimulation inhibits IL-2 production induced by CD3-CD28 co-stimulation. Since CD3-CD28 co-stimulation induces IkappaBalpha degradation and consequently activates RelA, an NFkappaB family member relevant for the induction of IL-2 mRNA transcription, we tested whether the inhibitory effect of CTLA-4 stimulation interferes with this mechanism. CD3-CD28 co-stimulation was found to induce a drastic decrease in cytoplasmic IkappaBalpha and increase in nuclear RelA. CTLA-4 stimulation abrogates this effect of co-stimulation by increasing the level of cytoplasmic IkappaBalpha and decreasing the nuclear RelA level and DNA-binding activity. In conclusion, our results indicate that the inhibitory effect of CTLA-4 engagement on cytokine production correlates with prevention of IkappaBalpha degradation and inhibition of RelA nuclear translocation.

Abatacept↗
PubMed1999

Blocking CD40 - CD154 and CD80/CD86 - CD28 interactions during primary allogeneic stimulation results in T cell anergy and high IL-10 production.

Although CD28 triggering provides an important co-stimulatory signal to T cells, blocking the CD80/CD86 - CD28 interaction with CTLA-4lg fusion protein is not sufficient for tolerance induction in vivo or in vitro. According to more recent data, interruption of the CD40 - CD154 interaction might complement the effect of CTLA-4lg and induce graft acceptance. We studied the effects of a blocking anti-CD40 monoclonal antibody (mAb) and/or blocking anti-CD80/anti-CD86 mAb in cultures of human peripheral blood mononuclear cells (PBMC) stimulated with allogeneic PBMC. T cells activated by alloantigens in the presence of anti-CD80, anti-CD86 and anti-CD40 entered a state of alloantigen-specific non-responsiveness as evidenced upon restimulation by lack of proliferation, cytotoxic activity, and IL-2, IL-5 and IL-13 production. IFN-gamma production during restimulation was less than in the control cultures, while the production of IL-10 was enhanced. Addition of recombinant IL-2 during the restimulation rescued alloantigen-specific activity. We conclude that the simultaneous blocking of the CD40 - CD154 and CD80/CD86 - CD28 interaction during allogeneic T cell activation induces T cell anergy. Since anergic cells induced by this treatment still produce high levels of IL-10, the latter could contribute to modulation of antigen-presenting cell activity and to bystander suppression of residual reactive T cells.

Abatacept↗
PubMed1999

CD28 co-stimulation results in down-regulation of lymphotactin expression in human CD4(+) but not CD8(+) T cells via an IL-2-dependent mechanism.

Chemokines are key molecules in promoting leukocyte migration and, for some of them, T cell adhesion and activation. Lymphotactin, which is the unique known member of the C class of chemokines, is produced by and acts on T lymphocytes, but the requirement of co-stimulatory pathways such as CD28 for its expression is largely unknown. CD28 plays a dominant role in the amplification of T cell proliferation, survival and cytokine production. In this report, we demonstrate that human lymphotactin expression, at both the mRNA and protein levels, is optimally induced by CD3/TCR activation alone, whereas CD28 co-stimulation turns off this expression. This down-regulation is not attributable to secondary activation via CTLA-4, the alternative counter-receptor of B7 ligands. Only the CD4(+) and not the CD8(+) subset is directly affected by this negative regulation. Transcript destabilization can be ruled out as a mechanism by which CD28 down-regulates lymphotactin expression. However, such down-regulation can be partly induced by IL-2 and abrogated by blocking IL-2/IL-2 receptor interaction. This particular profile of lymphotactin expression is not in line with the prevailing dogma of up-regulation of cytokine gene expression by CD28 co-stimulation, and represents a new CD28-mediated regulatory mechanism for lymphotactin expression.

Abatacept↗
PubMed1999

Enhancement of the T cell response to a mycobacterial peptide by conjugation to synthetic branched polypeptide.

A peptide-based approach towards improving the immunodiagnosis of, and vaccination against, tuberculosis faces the problems of MHC restriction of T cell recognition and the poor immunogenicity of peptides in the absence of adjuvant. We sought to compensate this by the use of synthetic branched polypeptides of the poly[Lys-(Xi-DL-Alam)] type, containing a glutamic acid residue (EAK), and further modified either by succinylation (SucEAK) or acetylation (AcEAK). These carriers were conjugated to two permissively recognized peptides of Mycobacterium tuberculosis. The 38p350 - 369-SucEAK conjugate enhanced IFN-gamma production more than 13-fold (from 22.6 to 294 pg / ml, p = 0.001) in peripheral blood mononuclear cells from healthy subjects, and 8.7-fold (p = 0. 012) in cells from tuberculosis patients. The effect was dependent on the carrier used and on covalent linkage of SucEAK to 38p350 - 369. An increased response occurred best in cells from subjects bearing at least one HLA-DR allele for which 38p350 - 369 had high binding affinity and required cellular processing of the conjugate as inhibitors (chloroquine and wortmannin) blocked the IFN-gamma response. SucEAK conjugation of peptide 16p91 - 110 did not significantly increase IFN-gamma production, indicating that the ability of conjugation to enhance the response was peptide structure dependent. These data indicate that the use of SucEAK polymer coupled with permissively recognized peptides could contribute to the development of an improved immunodiagnostic or vaccine reagent for tuberculosis.

Adult↗
PubMed1999

T cells are the main cell type expressing B7-1 and B7-2 in the central nervous system during acute, relapsing and chronic experimental autoimmune encephalomyelitis.

T cell co-stimulation through the CD28 receptor on T cells is critical to the induction of experimental autoimmune encephalomyelitis (EAE). In this study, expression of the co-stimulatory ligands B7-1 (CD80) and B7-2 (CD86), as well as the receptors CD28 and CTLA-4, were quantitated in central nervous system (CNS) tissues from mice at various stages of EAE. Immunohistochemistry and flow cytometry of CNS-infiltrating cells revealed a high percentage of infiltrating T cells expressing B7-1 and B7-2 during acute, chronic and relapsing EAE. Of the infiltrating cells 10-20% were CTLA-4(+), most of which were CD4(+) T cells. B7-1 and B7-2 expression within the CNS during active EAE might increase the potential for local activation of autoimmune T cells; however, the high level of expression of B7 molecules may also provide a mechanism for the autoregulation of activated CTLA-4(+) T cells.

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