Acetate Promotes T Cell Effector Function during Glucose Restriction.

Competition for nutrients like glucose can metabolically restrict T cells and contribute to their hyporesponsiveness during cancer. Metabolic adaptation to the surrounding microenvironment is therefore key for maintaining appropriate cell function. For instance, cancer cells use acetate as a substrate alternative to glucose to fuel metabolism and growth. Here, we show that acetate rescues effector function in glucose-restricted CD8+ T cells. Mechanistically, acetate promotes histone acetylation and chromatin accessibility and enhances IFN-γ gene transcription and cytokine production in an acetyl-CoA synthetase (ACSS)-dependent manner. Ex vivo acetate treatment increases IFN-γ production by exhausted T cells, whereas reducing ACSS expression in T cells impairs IFN-γ production by tumor-infiltrating lymphocytes and tumor clearance. Thus, hyporesponsive T cells can be epigenetically remodeled and reactivated by acetate, suggesting that pathways regulating the use of substrates alternative to glucose could be therapeutically targeted to promote T cell function during cancer.

Ethylene Perception Is Associated with Methyl-Jasmonate-Mediated Immune Response against Botrytis cinerea in Tomato Fruit.

Jasmonic acid (JA)- and ethylene-mediated signaling pathways are reported to have synergistic effects on inhibiting gray mold. The present study aimed to explain the role of ethylene perception in methyl jasmonate (MeJA)-mediated immune responses. Results showed that exogenous MeJA enhanced disease resistance, accompanied by the induction of endogenous JA biosynthesis and ethylene production, which led to the activation of the phenolic metabolism pathway. Blocking ethylene perception using 1-methylcyclopropene (1-MCP) either before or after MeJA treatment could differently weaken the disease responses induced by MeJA, including suppressing the induction of ethylene production and JA contents and reducing activities of lipoxygenase and allene oxide synthase compared to MeJA treatment alone. Consequently, MeJA-induced elevations in the total phenolic content and the activities of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, 4-coumarate:coenzyme A ligase, and peroxidase were impaired by 1-MCP. These results suggested that ethylene perception participated in MeJA-mediated immune responses in tomato fruit.

Antimodified protein antibody response pattern influences the risk for disease relapse in patients with rheumatoid arthritis tapering disease modifying antirheumatic drugs.

OBJECTIVE: To perform a detailed analysis of the autoantibody response against post-translationally modified proteins in patients with rheumatoid arthritis (RA) in sustained remission and to explore whether its composition influences the risk for disease relapse when tapering disease modifying antirheumatic drug (DMARD) therapy. METHODS: Immune responses against 10 citrullinated, homocitrullinated/carbamylated and acetylated peptides, as well as unmodified vimentin (control) and cyclic citrullinated peptide 2 (CCP2) were tested in baseline serum samples from 94 patients of the RETRO study. Patients were classified according to the number of autoantibody reactivities (0-1/10, 2-5/10 and >5/10) or specificity groups (citrullination, carbamylation and acetylation; 0-3) and tested for their risk to develop relapses after DMARD tapering. Demographic and disease-specific parameters were included in multivariate logistic regression analysis for defining the role of autoantibodies in predicting relapse. RESULTS: Patients varied in their antimodified protein antibody response with the extremes from recognition of no (0/10) to all antigens (10/10). Antibodies against citrullinated vimentin (51%), acetylated ornithine (46%) and acetylated lysine (37%) were the most frequently observed subspecificities. Relapse risk significantly (p=0.011) increased from 18% (0-1/10 reactivities) to 34% (2-5/10) and 55% (>5/10). With respect to specificity groups (0-3), relapse risk significantly (p=0.021) increased from 18% (no reactivity) to 28%, 36% and finally to 52% with one, two or three antibody specificity groups, respectively. CONCLUSIONS: The data suggest that the pattern of antimodified protein antibody response determines the risk of disease relapse in patients with RA tapering DMARD therapy. TRIAL REGISTRATION NUMBER: 2009-015740-42; Results.

Acetylated Trivalent Mannobioses: Chemical Modification, Structural Elucidation, and Biological Evaluation.

People suffering from allergies can be treated with repeated injections of increasing amounts of a specific allergen. This type of specific immunotherapy is currently the only way to treat the underlying pathological immune response associated with an allergy. The approach can afford long-lasting protection, but the process takes 3-5 years, can produce allergic reactions, and in severe cases treatment is often aborted due to anaphylaxis. However, treatment can be optimized with the use of specific adjuvants that modify the immune response, its duration, and that increase the production of the correct type of antibodies. In the pursuit of such adjuvants, two new trivalent acetylated ß-(1→2)-linked mannobioses based on a previously discovered lead molecule were prepared. The new molecules, along with the previously developed lead, were investigated by rigorous NMR and molecular modeling experiments in order to elucidate their behavior and preferred conformations in solution. Furthermore, the molecules were subjected to a biological investigation in which their immunostimulatory properties were evaluated by assessing their effect on the production of TH 2-type cytokine interleukin-4 (IL-4) and Treg pro-inflammatory cytokine tumor necrosis factor (TNF). Treatment of peripheral mononuclear blood cell cultures from patients suffering from birch allergy with birch allergen Bet v induced a strong IL-4 response, whereas the same treatment together with the trivalent acetylated mannobioses caused significant suppression of the induced IL-4.

Preparation of novel monoclonal antibodies against chelated cadmium ions.

The detection of cadmium ions using enzyme-linked immunosorbent assays (ELISA) has been reported by several research groups. Because cadmium ions are too small to stimulate the immune system, high molecular weight immunogens of cadmium are constructed using bifunctional chelators. At present, the most commonly used bifunctional chelator for the preparation of antigens for heavy metal ions is 1-(4-isothiocyanobenzyl) ethylenediamine N,N,N',N'-tetraacetic acid (ITCBE). However, the price of ITCBE is high. So we are interested in a cheaper bifunctional chelator, 1-(4-aminobenzyl) ethylenediamine N,N,N',N'-tetraacetic acid (aminobenzyl-EDTA). Here, cadmium ions were conjugated to carrier proteins using aminobenzyl-EDTA to make artificial antigens. Then, several mice were immunized with the antigen. And monoclonal antibodies (MAbs) against cadmium were produced. Spleen cells of immunized mice were fused with myeloma cells. The resulting hybridomas were screened using protein conjugates which were covalently bound to metal-free EDTA or cadmium. Three hybridoma cell lines (A3, E4 and B5) that produced MAbs with high selectivity and sensitivity were expanded for further study. Cross-reactivities with other metals were below 1 %. These antibodies were used to construct competitive ELISAs. The IC50 for A3 was 8.4 µg/l. The detection range and the lowest detection limit using the antibody A3 was 0.394-64.39 and 0.051 µg/l, respectively. Spike-recovery studies in tap water showed that the antibody A3 could be used for cadmium detection in drinking water.

Assessing acetylation of NF-kappaB.

To achieve its full biological activity, NF-kappaB must undergo a variety of post-translational modifications, including acetylation. Acetylation plays a prominent role in regulating the nuclear action of NF-kappaB. The RelA subunit of NF-kappaB forms the major target of acetylation at several different sites. Acetylation of discrete lysine residues in RelA modulates distinct functions of NF-kappaB, including transcriptional activation, DNA binding, and assembly with its inhibitor IkappaBalpha. Here, we describe the experimental methods that have allowed the detection and functional analysis of acetylated forms of NF-kappaB. Acetylation of NF-kappaB can be studied both in vivo and in vitro. In vivo [3H]acetate labeling assays provides a useful, albeit rather insensitive, method for initial verification of acetylation of either over-expressed or endogenous subunits of NF-kappaB. A second valuable in vivo approach involves the use of anti-acetylated lysine antibodies for immunoblotting. However, the success of this approach varies with the specific antibody employed and the target protein studied. In vitro acetylation assays provide a rapid and sensitive method to validate the involvement of candidate histone acetyltransferases and to map the sites of acetylation. Anti-RelA antibodies that selectively react with site-specific acetylated forms of RelA are a singularly powerful tool for the study of NF-kappaB acetylation both in vivo and in vitro.

Effects of a cysteinyl leukotriene receptor antagonist on eosinophil recruitment in experimental allergic rhinitis.

The cysteinyl leukotrienes (cysLTs) are potent lipid mediators in allergic disease, acting through a receptor (cysLT1-R) which can be targeted in rhinitis and asthma. We investigated the effects of cysLT1-R antagonism in experimental allergic rhinitis, focusing on bone marrow eosinophil progenitor responses. BALB/c mice were sensitized, then given daily intranasal ovalbumin for 2 weeks, with montelukast sodium (5 mg/kg or 2.5 mg/kg) or placebo by gavage. Bone marrow eosinophil/basophil colonies were enumerated, and colony cells were morphologically assessed as indices of eosinophil differentiation and maturation. Montelukast treatment resulted in a significant decrease of eosinophils in the nasal mucosa, and in either bone marrow interleukin (IL)-5-, but not IL-3-, or granulocyte-macrophage colony-stimulating factor-responsive eosinophil/basophil colony-forming units, and IL-5-stimulated eosinophil maturation. These results indicate that cysLT1-R antagonism in vivo limits both IL-5-responsive eosinophilopoiesis, acting at several stages of eosinophil differentiation and maturation. The anti-allergic effects of cysLT1-R antagonists are consistent with the concept that cysLTs and IL-5 act together in the recruitment of eosinophils and eosinophil progenitors from the marrow during upper airway allergic inflammation.

The effects of butterbur on the histamine and allergen cutaneous response.

BACKGROUND: Butterbur or Petasites hybridus is an herbal remedy that exhibits antihistamine and antileukotriene activity and has been shown to attenuate the response to adenosine monophosphate challenge in patients with allergic rhinitis and asthma. However, no data are available regarding its effects on the histamine and allergen cutaneous response. OBJECTIVE: To evaluate the effects of butterbur compared with fexofenadine and montelukast on the histamine and allergen wheal and flare cutaneous responses. METHODS: Atopic patients were randomized into a double-blind, double-dummy, crossover study to receive for 1 week butterbur, 50 mg twice daily (8 AM and 10 PM); fexofenadine, 180 mg once daily (10 PM), and placebo once daily (8 AM); montelukast, 10 mg once daily (10 PM), and placebo once daily (8 AM); or placebo twice daily (8 AM and 10 PM). Patients attended the department at 10 AM and had measurements of the cutaneous wheal and flare responses to histamine, allergen, and saline control at 10-minute intervals for 60 minutes. RESULTS: Twenty patients completed the study. The mean +/- SE histamine wheal and flare responses, respectively, were significantly attenuated (P < .05) by fexofenadine (9.4 +/- 1.8 mm2 and 13.5 +/- 3.2 mm2) compared with placebo (15.5 +/- 3.3 mm2 and 179.8 +/- 74.3 mm2) but not by butterbur (16.4 +/- 2.1 mm2 and 297.7 +/- 121.2 mm2) or montelukast (19 +/- 1.9 mm2 and 240.2 +/- 66.6 mm2). The allergen wheal and flare responses, respectively, were also significantly attenuated (P < .05) by fexofenadine (31.1 +/- 6.3 mm2 and 256.9 +/- 86.5 mm2) compared with placebo (65.4 +/- 15.2 mm2 and 1,014.5 +/- 250.0 mm2) but not by butterbur (50.4 +/- 9.2 mm2 and 1,110.3 +/- 256.1 mm2) or montelukast (58.8 +/- 9.1 mm2 and 1,463.6 +/- 295.6 mm2). CONCLUSIONS: Butterbur did not produce any significant effects on the histamine and allergen cutaneous response compared with placebo, whereas mediator antagonism with fexofenadine but not montelukast produced significant attenuation. This finding would suggest that butterbur may not be effective in allergic skin disease.

[Preparation of a monoclonal antibody against methyl jasmonate and quantification of jasmonic acid in florets of wheat and Italian ryegrass].

A monoclonal antibody (McAb) against methyl jasmonate (MeJA) was prepared and characterized. The McAb, J2-4B, was derived from an immunogen in which the C1-COOH of jasmonic acid (JA) was conjugated to the -NH2 of keyhole limpet hemocyanin (KLH). The McAb showed a higher recognition ability to methyl esters of JA than to its free acids. The integrity of a pentenyl in JA molecule was necessary for the recognition of McAb. Hydrogenation at C-9 and C-10 (dihydrojasmonic acid, 2H-JA) or eliminating the methyl group at C-12 (JAS-25) significantly abolished the binding force of JA molecule with the McAb. Some structural or functional analogues or precursor of JA, such as cucurbic acid, theobroxide, coronatine, and linolenic acid, could not be recognized by the McAb. The McAb has been used to set up a competitive enzyme-linked immunosorbent assay (ELISA) with a linearity range from 2.06 to 500 pmol of MeJA. Using this method, the fluctuations of JA content in florets during anthesis of wheat and Italian ryegrass were analyzed. Results showed that JA level increased obviously as the florets approaching to opening, arrived at a "peak" value at full opening and decreased sharply afterwards.

Effects of montelukast on surrogate inflammatory markers in corticosteroid-treated patients with asthma.

We evaluated whether montelukast conferred additive effects in patients with asthma receiving fluticasone/salmeterol (FP/SM) combination and FP alone. Twenty-two patients with mild to moderate asthma completed a double-blind, placebo-controlled study. After a 2-week run-in using FP 250 microg/SM 50 microg 1 puff twice daily, patients entered a randomized crossover period to receive additional montelukast 10 mg daily or placebo for 3 weeks each. For the first 2 weeks, they received FP/SM 1 puff BID, and then they received FP 250 microg 1 puff BID for the 3rd week. The primary outcome was adenosine monophosphate challenge threshold and recovery time; secondary outcomes included surrogate inflammatory markers and lung function. Compared with FP/SM run-in, adding montelukast to FP/SM was better (p < 0.05) than placebo for inflammatory markers but not for lung function. For adenosine monophosphate threshold, recovery, exhaled nitric oxide, and blood eosinophils, there were 1.4 (95% confidence interval, 1.1-1.8) geometric mean fold, 10 minutes (3-17 minutes), 2.1 parts per billion (0.2-3.9 parts per billion), and 88 (34-172) x 10(6)/L differences, respectively. The combination of FP plus montelukast was superior to FP/SM for inflammatory markers but was inferior for lung function. Thus, in patients taking FP/SM or FP, montelukast conferred complimentary effects on surrogate inflammatory markers, which were dissociated from lung function. Further studies are required to evaluate whether these effects of montelukast translate into clinical benefits.

Construction and characterization of two anti-sweetener single chain antibodies using radioligand binding, fluorescence and circular dichroism spectroscopy.

Two single-chain antibodies (scFv) that bind the superpotent sweetener ligand, NC-174, were generated from mouse monoclonal antibodies (mAb) NC6.8 (IgG, kappa) and NC10.14 (IgG, lambda). These scFv were constructed by cloning the variable region sequences of the mAb, connecting them in tandem with a 25-amino-acid polypeptide linker, and expressing them in E. coli using the pET-11a system. The recombinant proteins were purified using Ni(2+)-NTA-agarose by virtue of a hexahistidine sequence introduced to the C-terminus of the heavy chain variable region during the cloning process. The secondary structure and ligand binding properties of the two scFv, the parent mAbs and proteolytically derived Fab fragments were examined using radioligand binding, circular dichroism (CD) and fluorescence spectroscopy. The far-UV CD spectra of both scFv possessed predominantly beta character, as did those of the Fab, and the near-UV CD spectral data for scFvNC10.14, NC6.8 and NC10.14 Fab indicated that chromophore perturbation occurred upon ligand binding. The affinity constants determined for the two scFv, Fab and mAb were nearly equivalent.

Histocompatibility of polyvinyl acetate, an ingredient of chewing gum.

The histocompatibility of polyvinyl acetate (PVA), an ingredient of chewing gum, was evaluated from both a direct irritant (toxic reaction) and an allergic (antigen-antibody) standpoint. Subcutaneous injections in rats and topical application of hamster pouches were utilized. Polyvinyl acetate was shown to cause a moderate to severe inflammatory reaction when injected subcutaneously, which peaked at day 7 and was minimal at day 42. The histologic appearance of the hamster cheek pouch was not significantly altered by the PVA. Although PVA has been used as a plasticizer in chewing gum, it was not possible to induce any histologic changes in tissues which resembled the specific changes present in atypical gingivostomatitis (plasma-cell gingivostomatitis).

Studies of surface immunoglobulins on human B lymphocytes. I. Dissociation of cell-bound immunoglobulins with acid pH or at 37 degrees C.

Lymphocyte preparations isolated from the human peripheral blood were exposed to different acid pH or incubated at 37 degrees C and the presence of immunoglobulin (Ig) on the cell surface was examined by immunofluorescence (IF) tests. Subsequently, such treated cells were incubated in the autologous serum or in the purified IgG, IgA or IgM proteins and their ability to bind each class of Ig was examined. The results showed that IgG molecules dissociated from large proportions of IgG-positive cells upon exposure to pH 4 at 1 degrees C for 1 min or upon incubation at 37 degrees C for 20 min. The cells from which IgG had been dissociated could again combine with IgG, whereupon the number of positive cells increased, being restored to the number of equivalent to or higher than those before acid or 37 degrees C treatment. These results indicated that the treatment could elute the cell-bound IgG present on the cell and that the receptor sites were not degraded by the treatment and could combine with IgG. These cell-bound IgG were observed not only on the monocytes, but also on the small lymphocytes. It was also found that certain proportions of mononuclear cells carried the cell-bound IgA that could be dissociated with acid pH or 37 degrees C. No cell-bound IgM was observed on any mononuclear cells. Microscopic observations before and after acid or 37 degrees C treatment revealed that the staining distribution of the cell-bound IgG and IgA on the cell was granular, appearing as a discontinuous fluorescence ring and forming multiple aggregates but no typical polar caps on warming. In contrast, IgG, IgA, and IgM stable to acid or 37 degrees C treatment were found on the lymphocytes but not on the monocytes, and their staining distribution was uniformaly diffuse, appearing as a continuous ring and forming a typical cap on warming. Exposure of the cells to pH 4 or 37 degrees C could also elute the cell-bound IgG passively adsorbed to the human lymphoid cells in a culture, but did not affect the intrinsic S.Ig on the lymphoid cells in a culture or on the lymphoma cells. These results indicate that the exposure of the cells to acid pH or to 37 degrees C may enable us to detect unfailingly S.Ig lymphocytes by removing the cell-bound IgG and IgA present on the monocytes and/or lymphocytes. Thus, an average value of approximately 10% was obtained for the S.Ig lymphocyte in the lymphocyte preparations from 11 healthy individuals. In addition, the results provided the evidence that, even in normal peripheral blood lymphocytes, there may be a population of B lymphocytes which lack the S.Ig but carry the cell-bound Ig.