Design, engineering and production of functional single-chain T cell receptor ligands.

Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers on the surface of antigen presenting cells (APCs) that bind the T cell receptor, initiating a cascade of interactions that results in antigen-specific activation of clonal populations of T cells. The peptide binding/T cell recognition domains of rat MHC class II (alpha-1 and beta-1 domains) were expressed as a single exon for structural and functional characterization. These recombinant single-chain T cell receptor ligands (termed 'beta1alpha1' molecules) of approximately 200 amino acid residues were designed using the structural backbone of MHC class II molecules as template, and have been produced in Escherichia coli with and without N-terminal extensions containing antigenic peptides. Structural characterization using circular dichroism predicted that these molecules retained the antiparallel beta-sheet platform and antiparallel alpha-helices observed in the native MHC class II heterodimer. The proteins exhibited a cooperative two-state thermal folding-unfolding transition. Beta1alpha1 molecules with a covalently linked MBP-72-89 peptide showed increased stability to thermal unfolding relative to the empty beta1alpha1 molecules. This new class of small soluble polypeptide provides a template for designing and refining human homologues useful in detecting and regulating pathogenic T cells.     

Dissociation of oxidant production by peroxisome proliferator-activated receptor ligands from cell death in human cell lines

Ligands of peroxisome proliferator-activated receptors (PPARs) come from a diverse group of chemicals that include pharmaceutical drugs, phthalate plasticizers, steroids, and pesticides. PPAR ligands exhibit a number of effects, including an ability to induce apoptosis in some systems. The mechanism(s) underlying the induction of apoptosis is not known. The current study examined the ability of Wy14643, a fibrate and PPARalpha agonist, and ciglitazone, a thiazolidinedione and PPARgamma agonist, to induce apoptosis as well as the production of oxidants in human Jurkat T cells that express all PPAR isoforms. Treatment with increasing doses of Wy14643 caused a substantial time-dependent increase in the overall oxidant status (as reflected by increased dichlorofluorescein fluorescence) of Jurkat cells without any change in viability except at the highest dose and longest time. Ciglitazone also caused a dose- and time-dependent increase in oxidant production. However, although the extent of this production was less than that seen with Wy14643, ciglitazone caused a dose- and time-dependent increase in apoptosis that could not be inhibited by antioxidants. Confocal micrographs of Jurkat cells loaded with dichlorofluorescein diacetate or dihydrorhodamine 123 and treated with Wy14643 or ciglitazone revealed a punctate pattern of fluorescence at early time points suggestive of a mitochondrial origin for these oxidants. Rotenone and antimycin A prevented Wy14643- but not ciglitazone-induced oxidant production. Other relatively specific PPARgamma agonists (15delta-PGJ2, and troglitazone), but not nonspecific agonists (bezafibrate and conjugated linoleic acid), were also able to induce oxidant production in Jurkat cells. These data, as well as the findings that oxidant production could be induced by Wy14643 in A549 cells that lack PPARalpha, and could not be blocked in Jurkat cells by the PPARalpha inhibitor MK886, indicate oxidant formation is unrelated to PPARalpha. These data also suggest that oxidant production induced by PPARalpha ligands originates in the mitochondria.     

MHC class II derived recombinant T cell receptor ligands protect DBA/1LacJ mice from collagen-induced arthritis

We previously demonstrated the therapeutic effects of MHC class II derived recombinant T cell receptor ligands (RTL), single-chain two domain complexes of the alpha1 and beta1 domains of MHC class II molecules genetically linked with an immunodominant peptide, in experimental autoimmune encephalomyelitis. In the current study, we produced a monomeric murine I-Aq-derived RTL construct covalently linked with bovine collagen type II peptide (bCII257-270) suitable for use in DBA/1LacJ mice that develop collagen-induced arthritis (CIA), an animal model of human rheumatoid arthritis, after immunization with bCII protein in CFA. In this study, we demonstrate that the I-Aq-derived RTLs reduced the incidence of the disease, suppressed the clinical and histological signs of CIA and induced long-term modulation of T cells specific for arthritogenic Ags. Our results showed that the I-Aq/bCII257-270 molecule could systemically reduce proinflammatory IL-17 and IFN-gamma production and significantly increase anti-inflammatory IL-10, IL-13, and FoxP3 gene expression in splenocytes. Moreover, I-Aq/bCII257-270 molecule could also selectively inhibit IL-1beta, IL-6, and IL-23 expression in local joint tissue. This is the first report demonstrating effective prevention of joint inflammation and clinical signs of CIA with an I-Aq-derived RTL, thus supporting the possible clinical use of this approach for treating rheumatoid arthritis in humans.     

Bioactive recombinant human lactoferrin,derived from rice,stimulates mammalian cell growth

Today there is a concern about the use of animal source proteins and peptides in cell culture applications due to potential contamination by adventitious infectious pathogens. Recombinant production of these proteins using a plant host provides a safe and cost effective alternative. In this paper, we tested the effect of rice-derived recombinant human lactoferrin (rhLF) on mammalian cell growth. The purified rhLF was partially (about 50%) iron-saturated (pis-rhLF). Chemical modification of pis-rhLF generated apo-rhLF (<10% iron saturation) or holo-rhLF (>90% iron saturation). All three forms of rhLF (pis, apo, holo) promoted growth of intestinal cells (HT-29) measured as [³H]-thymidine incorporation or viable cell count, but holo-rhLF was most effective. Holo-rhLF was further tested on hybridoma, osteoblast, and human embryonic kidney cells. Results showed that holo-rhLF promoted cell growth and reduced cell doubling time. The concentration of holo-rhLF in media was critical in promoting cell growth and each cell line had different concentration dependence with the most effective range from 5 to 200 mg/L. The effect of rhLF on antibody production was determined using a hybridoma cell line. Significantly, more antibodies were produced by cells grown with holo-rhLF than cells grown without holo-rhLF. We also compared the effect of holo-rhLF to that of human transferrin, a component commonly used in cell culture media as an iron source. Holo-rhLF was as effective as human transferrin in promoting cell growth and antibody production. Considering all the data obtained, we conclude that rhLF from rice is effective in promoting mammalian cell growth and increasing cell productivity.     

Antibodies to common leukocyte antigen p220 influence human T cell proliferation by modifying IL 2 receptor expression

The p220 antigen is found on the high m.w. form of the T200 common leukocyte antigen family. Although T200 monoclonal antibodies (MAb) react with all hematopoietic cells, p220 MAb react only with B cells, NK cells, about 50% of CD4+ T helper cells, and about 90% of CD8+ T suppressor cells. The p220 antigen appears to play an important role in T cell activation, because anti-p220 MAb at doses as low as 5 ng/ml accelerate the proliferation kinetics of PHA-stimulated T cells and augment anti-CD3-driven proliferation when IL 2 is in excess. Fab fragments have no effect on PHA-stimulated cells but partially block proliferation in response to anti-CD3. Our data suggest that p220 is functionally related to expression of the IL 2 receptor. Anti-p220 MAb cause an increase in the number of T cells that express the IL 2 receptor early after activation. In addition, T cells begin to turn off expression of p220 after activation, and two-color immunofluorescence shows that by day 3 after activation the cells expressing the most IL 2 receptor have the least p220. The loss of p220 on T cells may reflect a post-thymic differentiation process related to cell activation. Our data are consistent with a model where the p220- T cells in peripheral blood are a more activated population of T cells that have lost p220 and its ability to regulate their IL 2 receptor expression.     

Novel cell lines derived from transgenic mice expressing recombinant human proteins

We have used transgenic mouse technology to establish immortalized hepatoma cell lines stably secreting heterologous proteins, such as human α₁-antitrypsin and human factor IX. Hepatocyte-specific regulatory DNA sequences were used to target both the expression of anonc gene and the gene coding for the human protein to the liver of transgenic mice which eventually developed hepatocellular carcinomas. Tumour cells were subsequently established as permanent cell lines, which maintained a differentiated phenotype under specific culture conditions, being capable of producing biologically active and correctly processed human α₁-antitrypsin and factor IX. Moreover, a preliminary analysis has shown that certain cell lines express elevated total cytochrome P450 activity. These cells could therefore represent a useful alternative to the use of animals or primary cultures in drug safety testing.     

Biophysical studies of a transmembrane peptide derived from the T cell antigen receptor

Core peptide (CP) is a unique peptide derived from thetransmembrane sequence of T cell antigen receptor (TCR)-alpha chain and is capable of inhibiting the immuneresponse both in vitro and in animal models of Tcell mediated inflammation. The structure of CP, withsequence GLRILLLKV, is similar to the amphipathic regionof many peptides. Unlike antimicrobial peptides,however, which damage cell membranes, electron microscopyand propidium iodide exclusion assays on cell membranessuggest that CP does not create pores and may act byinterfering with signal transduction at the membranelevel. To investigate this effect further we report theresults of ³¹P and ²H solid-state NMRspectroscopy of CP on model membranes. As predicted,even at high concentrations of CP, the structure of modelmembranes was not significantly perturbed. Only at thevery high peptide-to-lipid molar ratio of 1:10significant effects on the model membranes were observed. We conclude that CP does not destroy the integrity of thelipid bilayer.     

Biophysical studies of a transmembrane peptide derived from the T cell antigen receptor

Summary Core peptide (CP) is a unique peptide derived from the transmembrane sequence of T cell antigen receptor (TCR)-alpha chain and is capable of inhibiting the immune response both invitro and in animal models of T cell mediated inflammation. The structure of CP, with sequence GLRILLLKV, is similar to the amphipathic region of many peptides. Unlike antimicrobial peptides, however, which damage cell membranes, electron microscopy and propidium iodide exclusion assays on cell membranes suggest that CP does not create pores and may act by interfering with signal transduction at the membrane level. To investigate this effect further we report the results of³¹P and²H solid-state NMR spectroscopy of CP on model membranes. As predicted, even at high concentrations of CP, the structure of model membranes was not significantly perturbed. Only at the very high peptide-to-lipid molar ratio of 1∶10 significant effects on the model membranes were observed. We conclude that CP does not destroy the integrity of the lipid bilayer.     

Design,synthesis and biological evaluation of CB1 cannabinoid receptor ligands derived from the 1,5-diarylpyrazole scaffold

The CB1 receptor belongs to the G-protein-coupled receptor superfamily. CB1 antagonism has been considered as a new therapeutic target for the treatment of obesity. In this study, we report the synthesis and in vitro binding affinity assay of some 1,5-diarylpyrazole scaffold compounds. The binding results showed that some of the target compounds had an excellent potency toward the CB1 receptor with IC₅₀ values lying at the nanomole level.     

The direct effects of Toll-like receptor ligands on human NK cell cytokine production and cytotoxicity

Toll-like receptor (TLR) ligands are potent inducers of the innate immune system, of which NK and NKT cells play an important role. We examined the direct activation of highly purified human NK and/or NKT cells with known TLR ligands. NK/NKT cells were positive for all known TLR mRNA (TLR1-10). Ligands for TLR2-5 induced production of significant amounts of IFN-gamma by purified NK cells. However, a TLR9 ligand failed to induce significant levels of the cytokine. NK cells were depleted from PBMCs to confirm that they were the main source of IFN-gamma following treatment with TLR ligands, which resulted in a significant decrease in cytokines. The direct effects of TLR ligands on NK cytotoxicity were determined using 51Cr-labeled K562 target cells. Ligands for TLR2-5 were potent inducers of NK cell cytotoxicity, a TLR9 ligand was not. Our results suggest that TLR ligands can directly stimulate and enhance NK cell cytokine production and induce cytotoxic activities.     

The effect of beta-estradiol, progesterone and testosterone on the production of human leukocyte derived interferons

Sex hormones including estrogens, progesterone and testosterones are known to have adverse effects on the immune system and particularly on the proliferative response. Since cytokine production is known to be dissociable from the proliferation of lymphocytes and since other steroid hormones profoundly affect cytokine production, we felt it would be important to know the effect of sex steroids on the production of interferons (IFN), particularly since the latter are known to be key substances in the immune response. We have shown estradiol can slightly reduce gamma IFN yields with certain inducers (Con A, SEA) but only in pharmacologic concentrations. Similarly, progesterone had a modest effect in the same concentrations but only when Con A was the inducer. Testosterone did not effect IFN titers at any concentration. None of the sex steroids affected alpha IFN production and none of them influenced the bioactivity of either IFN species. In all cases these hormones diminished proliferative responses as has been previously noted.     

Novel sulfated gangliosides, high-affinity ligands for neural siglecs, inhibit NADase activity of leukocyte cell surface antigen CD38

Three kinds of novel sulfated gangliosides structurally related to the Chol-1 (alpha-series) ganglioside GQ1balpha were synthesized. These sulfated gangliosides were potent inhibitors of NADase activity of leukocyte cell surface antigen CD38. Among the synthetic gangliosides, GSC-338 (II(3)III(6)-disulfate of iso-GM1b) was surprisingly found to be the most potent structure in both the NADase inhibition and MAG-binding activity. The present study indicates that the sulfated gangliosides are useful to study the recognition of the internal tandem sialic acid residues alpha2-3-linked to Gal(II(3)) as well as the siglec-dependent recognition including a terminal sialic acid residue.     

Synthesis and evaluation of isoleucine derived angiotensin II AT2 receptor ligands

Sixteen new C-terminally modified analogues of 2, a previously described potent and selective AT₂R ligand, were designed, synthesized and evaluated for their affinity to the AT₂R receptor. The introduction of large, hydrophobic substituents was shown to be beneficial and the most active compound (17, K_i = 8.5 μM) was over 12-times more potent than the lead compound 2.     

Improved serodiagnosis of cystic echinococcosis using the new recombinant 2B2t antigen

A standardized test for the serodiagnosis of human cystic echinococcosis (CE) is still needed, because of the low specificity and sensitivity of the currently available commercial tools and the lack of proper evaluation of the existing recombinant antigens. In a previous work, we defined the new ELISA-B2t diagnostic tool for the detection of specific IgGs in CE patients, which showed high sensitivity and specificity, and was useful in monitoring the clinical evolution of surgically treated CE patients. Nevertheless, this recombinant antigen gave rise to false-negative results in a percentage of CE patients. Therefore, in an attempt to improve its sensitivity, we constructed B2t-derived recombinant antigens with two, four and eight tandem repeat of B2t units, and tested them by ELISA on serum samples of CE patients and patients with related parasites. The best diagnostic values were obtained with the two tandem repeat 2B2t antigen. The influence of several clinical variables on the performance of the tests was also evaluated. Finally, the diagnostic performance of the 2B2t-ELISA was compared with that of an indirect haemagglutination commercial test. The 2B2t recombinant antigen performed better than the HF and B2t antigens, and the IHA commercial kit. Therefore, this new 2B2t-ELISA is a promising candidate test for the serodiagnosis of CE in clinical settings.     

Occurrence in human bone marrows of an antigen released from continuous cell cultures derived from human leukemia.

Fluorescent antibodies prepared against extracellular particles from a continuous culture of cells derived from a monocytic leukemia stained JIII cells but not cells infected with Rauscher leukemia virus or simian sarcoma virus. These antibodies reacted with 38% of bone marrow preparations from patients with lymphoma, 26% of preparations from patients with nonmalignant blood disorders and 6% of preparations from patients with leukemia. Bone marrow films from patients with lymphoma over the age of 50 stained less frequently than those from patients under 50. These particles released from JIII cells are not antigenically related to two of the commonly studied oncornaviruses, but may be indicative of the etiology or disease process of lymphoma in young patients.     

Design, synthesis and pharmacological profile of novel dopamine D2 receptor ligands

The present study describes the synthesis and pharmacological profile of three novel heterocyclic compounds originally designed, on the basis of bioisosterism, as dopamine D2 receptor ligands: 1-[1-(4-chlorophenyl)-1H-pyrazol-4-ylmethyl]-4-phenyl-piperazine (LASSBio-579), 1-phenyl-4-(1-phenyl-1H-[1,2,3]triazol-4-ylmethyl)-piperazine (LASSBio-580) and 1-[1-(4-chlorophenyl)-1H-[1,2,3]triazol-4-ylmethyl]-4-phenyl-piperazine (LASSBio-581). Binding studies performed on brain homogenate indicated that all three compounds bind selectively to D2 receptors. In addition, electrophysiological studies carried out in cultured hippocampal neurons suggested that LASSBio-579 and 581 act as D2 agonists, whereas LASSBio-580 acts as a D2 antagonist.     

Design of soluble recombinant T cell receptors for antigen targeting and T cell inhibition

The use of recombinant T cell receptors (TCRs) to target therapeutic interventions has been hindered by the naturally low affinity of TCR interactions with peptide major histocompatibility complex ligands. Here, we use multimeric forms of soluble heterodimeric alphabeta TCRs for specific detection of target cells pulsed with cognate peptide, discrimination of quantitative changes in antigen display at the cell surface, identification of virus-infected cells, inhibition of antigen-specific cytotoxic T lymphocyte activation, and identification of cross-reactive peptides. Notably, the A6 TCR specific for the immunodominant HLA A2-restricted human T cell leukemia virus type 1 Tax(11-19) epitope bound to HLA A2-HuD(87-95) (K(D) 120 microm by surface plasmon resonance), an epitope implicated as a causal antigen in the paraneoplastic neurological degenerative disorder anti-Hu syndrome. A mutant A6 TCR that exhibited dramatically increased affinity for cognate antigen (K(D) 2.5 nm) without enhanced cross-reactivity was generated; this TCR demonstrated potent biological activity even as a monomeric molecule. These data provide insights into TCR repertoire selection and delineate a framework for the selective modification of TCRs in vitro that could enable specific therapeutic intervention in vivo.     

Monovalent ligands of complement receptor 2 inhibit whereas polyvalent ligands enhance anti-Ig-induced human B cell intracytoplasmic free calcium concentration

We have performed experiments to investigate the role of ligands for complement receptor 2 (CR2) in human B cell activation. Flow microfluorimetry was used to assess changes in free intracytoplasmic calcium concentration [Ca2+] in indo-loaded B cells, immediately after exposure to anti-mu antibody and to monovalent or polyvalent CR2 ligands. As monovalent ligands we used the C3d fragment and synthetic C3 peptides (peptides P14, residues 1201-1214, and P28, residues 1187-1214). As polyvalent ligands we used i) an intact monoclonal mouse anti-CR2 antibody (HB5) and its F(ab')2 fragment, ii) tetravalent P13 [residues 1202-1214) 4-template), and iii) P28 conjugated to BSA (molar ratio 5/1). Anti-CR2 antibody HB5, tetravalent P13, and P28 conjugated to BSA, enhanced the ability of F(ab')2 fragments of the IgG fraction of goat anti-human mu antibody to increase human B cell [Ca2+]i. In contrast, the monomeric CR2 ligands C3d and P28 inhibited the anti-mu-induced increase in human B cell [Ca2+]i. Multivalent P13, P28, and the HB5, by themselves, did not affect B cell [Ca2+]i. These experiments suggest that the valence of the CR2 ligands is crucial for the nature (synergistic vs antagonistic) of the message transmitted through the CR2.