BJ-1108, a 6-Amino-2,4,5-trimethylpyridin-3-ol analogue, regulates differentiation of Th1 and Th17 cells to ameliorate experimental autoimmune encephalomyelitis.

BACKGROUND: CD4+ T cells play an important role in the initiation of an immune response by providing help to other cells. Among the helper T subsets, interferon-γ (IFN-γ)-secreting T helper 1 (Th1) and IL-17-secreting T helper 17 (Th17) cells are indispensable for clearance of intracellular as well as extracellular pathogens. However, Th1 and Th17 cells are also associated with pathogenesis and contribute to the progression of multiple inflammatory conditions and autoimmune diseases. RESULTS: In the current study, we found that BJ-1108, a 6-aminopyridin-3-ol analogue, significantly inhibited Th1 and Th17 differentiation in vitro in a concentration-dependent manner, with no effect on proliferation or apoptosis of activated T cells. Moreover, BJ-1108 inhibited differentiation of Th1 and Th17 cells in ovalbumin (OVA)-specific OT II mice. A complete Freund's adjuvant (CFA)/OVA-induced inflammatory model revealed that BJ-1108 can reduce generation of proinflammatory Th1 and Th17 cells. Furthermore, in vivo studies showed that BJ-1108 delayed onset of disease and suppressed experimental autoimmune encephalomyelitis (EAE) disease progression by inhibiting differentiation of Th1 and Th17 cells. CONCLUSIONS: BJ-1108 treatment ameliorates inflammation and EAE by inhibiting Th1 and Th17 cells differentiation. Our findings suggest that BJ-1108 is a promising novel therapeutic agent for the treatment of inflammation and autoimmune disease.

BJ-1108, a 6-Amino-2,4,5-trimethylpyridin-3-ol analogue, regulates differentiation of Th1 and Th17 cells to ameliorate experimental autoimmune encephalomyelitis

BACKGROUND: CD4+ T cells play an important role in the initiation of an immune response by providing help to other cells. Among the helper T subsets, interferon-γ (IFN-γ)-secreting T helper 1 (Th1) and IL-17-secreting T helper 17 (Th17) cells are indispensable for clearance of intracellular as well as extracellular pathogens. However, Th1 and Th17 cells are also associated with pathogenesis and contribute to the progression of multiple inflammatory conditions and autoimmune diseases. RESULTS: In the current study, we found that BJ-1108, a 6-aminopyridin-3-ol analogue, significantly inhibited Th1 and Th17 differentiation in vitro in a concentration-dependent manner, with no effect on proliferation or apoptosis of activated T cells. Moreover, BJ-1108 inhibited differentiation of Th1 and Th17 cells in ovalbumin (OVA)-specific OT II mice. A complete Freund's adjuvant (CFA)/OVA-induced inflammatory model revealed that BJ-1108 can reduce generation of proinflammatory Th1 and Th17 cells. Furthermore, in vivo studies showed that BJ-1108 delayed onset of disease and suppressed experimental autoimmune encephalomyelitis (EAE) disease progression by inhibiting differentiation of Th1 and Th17 cells. CONCLUSIONS: BJ-1108 treatment ameliorates inflammation and EAE by inhibiting Th1 and Th17 cells differentiation. Our findings suggest that BJ-1108 is a promising novel therapeutic agent for the treatment of inflammation and autoimmune disease.

Anti-inflammatory and immunosuppressive effects of the enaminone E121.

Asthma is a chronic inflammatory disease of the airways. The treatment of asthma is far from optimal and hence the need for novel therapeutic agents exists. The purpose of this study was to assess the anti-asthma effects of an enaminone, E121, and also its effects on human peripheral blood mononuclear cell proliferation and cytokine release. The effects of E121 were assessed in an ovalbumin-induced model of airway inflammation and airway hyperresponsiveness. In addition, the effects of E121 on phytohemagglutinin (PHA), anti-CD3 monoclonal antibody and lipopolysaccharide (LPS)-induced human peripheral blood mononuclear cell proliferation and cytokine release, respectively, were assessed. Treatment of mice with E121 significantly decreased the ovalbumin-induced increase in airway total cell influx and eosinophil infiltration and this was associated with an inhibition of ovalbumin-induced airway hyperresponsiveness. Moreover, E121 reduced PHA and anti-CD3-induced human peripheral blood mononuclear cell proliferation in vitro. E121 also inhibited PHA, anti-CD3 monoclonal antibody and LPS-induced cytokine release from human peripheral blood mononuclear cell cultures. These findings indicate that E121 exhibits anti-inflammatory and immunosuppressive activities.

Production of antibodies for selective detection of malachite green and the related triphenylmethane dyes in fish and fishpond water.

This study provides a practical method for production of the antibodies against malachite green (MG) and its primary metabolite leucomalachite green (LMG). Two ELISA kits are constructed with the MG and LMG antibodies for detection of the residual MG and LMG in fish muscle and fishpond water. The detection limit is established at the level of 0.05 microg/L for both MG and LMG. Our ELISA kits show the advantages of good specificity, high sensitivity, and convenience in rapid screening of MG and LMG residues. The sample of fishpond water, without extraction or prior preparation, is directly assayed by the ELISA kit. More then 80 fish samples can be simultaneously tested in a kit. The toxic crystal violet and its metabolite leucocrystal violet of illegal use in aquaculture are detected by our prepared MG and LMG antibodies, whereas the antibodies do not cross-react with common antibiotics, sulfonamides, and benzene derivatives.

T cells ignore aniline, a prohapten, but respond to its reactive metabolites generated by phagocytes: possible implications for the pathogenesis of toxic oil syndrome.

The most basic arylamine, aniline, belongs to a class of compounds notorious for inducing allergic and autoimmune reactions. In 1981 in Spain, many people succumbed to toxic oil syndrome (TOS), a disease caused by ingestion of cooking oil contaminated with aniline. Indirect evidence points toward an immune pathogenesis of TOS driven by T lymphocytes, but it is unclear to which antigens these cells could react. Here, using the popliteal lymph node (PLN) assay in mice, we analyzed the sensitizing potential of aniline, its metabolites, and some of the aniline-coupled lipids detected in the contaminated cooking oil. Whereas aniline itself and its non-protein-reactive metabolites nitrobenzene, p-aminophenol and N-acetyl-p-aminophenol, failed to elicit PLN responses, its reactive metabolites nitrosobenzene and N-hydroxylaniline did. The aniline-coupled lipids, namely, linoleic anilide and linolenic anilide, and a mixture of fatty acid esters of 3-(N-phenylamino)-1,2-propanediol, all implicated in TOS, induced significant PLN responses, whereas the respective aniline-free lipids, linoleic acid, linolenic acid, and triolein, did not. Hence, the aniline moiety plays a crucial role in the immunogenicity of the aniline-coupled lipids of TOS. PLN responses to the reactive aniline metabolites and the one aniline-coupled lipid that was tested, linolenic anilide, were T-cell-dependent. Secondary PLN responses to nitrosobenzene were detectable not only after priming with nitrosobenzene but, in some experiments, also after priming with linolenic anilide. This suggests that the aniline moiety was cleaved from the aniline-coupled lipid and metabolized to the intermediate nitrosobenzene that generated the prospective neoantigens. Consistent with this, in lymphocyte proliferation tests in vitro, T cells primed to nitrosobenzene reacted in anamnestic fashion to white bone marrow cells (WBMCs) pulsed with aniline. Hence, we propose that aniline is a prohapten that can be metabolized by WBMCs, which form neoantigens that are recognized by T cells. The possible significance of these findings for the pathogenesis of TOS is discussed.

Comparison of the immunotoxicity of propanil and its metabolite, 3,4-dichloroaniline, in C57Bl/6 mice.

Propanil (3,4-dichloropropionaniline), used extensively as a postemergence herbicide in rice and wheat, has as its major metabolite, 3,4-dichloroaniline (DCA). Propanil has previously been shown to affect the T cell-dependent antibody response. To determine the immunotoxicity of DCA, as well as extend the previous immunotoxicity studies, several T cell-dependent and -independent immune responses were determined after DCA or propanil exposure. Unlike propanil, DCA caused a significant reduction in T-dependent antibody production (anti-SRBC response) only at a high dose (150 mg/kg). DCA or propanil at 150 or 200 mg/kg, respectively, caused a significant reduction in the number of anti-DNP antibody producing cells. However, doses of 37 or 50 mg/kg of DCA or propanil, respectively, caused an increase in the number of anti-DNP antibody producing cells. These data indicate that both propanil and DCA have a differential effect on the T-independent antibody response depending on the dose. Similar to propanil, DCA (at 150 mg/kg) caused a significant increase in spleen weight and cellularity. The effect of DCA or propanil on selected cellular immune functions was also determined. DCA caused a significant decrease in the natural killer (NK) cell activity at doses of 75 or 150 mg/kg, and propanil caused a significant decrease at 100 or 200 mg/kg. Cytotoxic T lymphocyte activity, however, was unaffected even at 150 or 200 mg/kg DCA or propanil, respectively. Thus, it appears that T cells are relatively resistant to the effects of propanil and DCA, whereas, other immune cell types, e.g., NK cells are sensitive to its effects.

The immune response to a schistosomacide, amoscanate. I. Serum antibody responses.

A potent antischistosomal drug, Amoscanate, was found to be immunogenic to mice and Cebus apella monkeys. The drug was readily haptenated to proteins under relatively mild conditions. The Amoscanate-protein conjugates were observed to be immunogenic when injected into the footpads of several strains of mice. However, such protein conjugates were not found to raise IgE antibody to the drug in high-responder strains using several procedures. When the formulated drug (dissolved in oil) was fed to CD 1 mice, a rise in serum antibody against the drug was noted 1 week following the primary dose. This is preliminary evidence that the drug, or a cross-reactive metabolite, becomes covalently bound to proteins in vivo. Cebus apella monkeys fed the drug exhibited a rise in anti-Amoscanate antibody one month after a second oral dose. These data suggest that the immunogenicity of Amoscanate is readily detected; furthermore, since there is no lasting immunity to schistosomiasis, thus necessitating multiple administration of the drug, the possibility that serum antibody titers to Amoscanate may interfere with its therapeutic efficacy cannot be overlooked.

The immune response to a schistosomacide, amoscanate. II. Cell-mediated immune responses.

A potent antischistosomal drug, Amoscanate, was found to induce vigorous serum antibody responses when either fed or administered parenterally as a drug-protein conjugate. Because of preliminary evidence that the drug could bind covalently to proteins in vivo, we decided to investigate the possibility that the drug could act as a contact sensitizing agent like DNCB. It was found that Amoscanate could induce a delayed-type hypersensitivity (DTH) response when painted on the shaved skin of guinea pigs. Moreover, the type of DTH response elicited was found to be cutaneous basophilic hypersensitivity (CBH). The significance of these findings are discussed.

Sensitivity to the weed killer DNA-nitralin and cross-sensitivity to dinitrochlorobenzene.

A man, with a dermatitis acquired while working in a factory producing a weed killer, showed sensitivity to 4-methylsulfonyl 2,6-dinitro-N,N-dipropylaniline (DNA-nitralin) and its precursor, 4-chloro 3,-5-dinitrophenylmethyl sulfone (DNC), and cross-sensitivity to dinitrochlorobenzene (DNCB). Sensitization capacities of DNA-nitralin and DNC compared with DNCB, and cross-sensitizations among 11 dinitrobenzene derivatives, including DNA-nitralin, DNC, and DNCB, were studied in guinea pigs. We found that the order of potency was DNCB, DNC, and DNA-nitralin for the sensitization capacity, and that cross-sensitizations may occur among DNCB, DNC, DNA-nitralin, and dinitrofluorobenzene, in comparatively high incidence.

Liposomes as immunological carriers for the preparation of antimannosyl antibodies.

Antiserum was raised against an aminophenyl derivative of D-mannose grafted on to a liposomal surface. As characterized by immunodiffusion, quantitative precipitation and hapten inhibition, the antiserum was found to contain mannose specific antibodies in addition to antibodies against the aromatic phenyl group.

Immunogenicity of p-phenetidine, 2-hydroxy-p-phenetidine and their protein conjugates in guinea pigs, rabbits and man.

Guinea pigs were sensitized by p-phenetidine (PT), 2-hydroxy-p-phenetidine (HPT) as well as by conjugates prepared by reacting PT and HPT with proteins in vitro. Sensitization was evaluated by delayed skin reactivity and in vitro antigen-induced lymphocyte proleferation. HPT and HPT-protein conjugates were found to be the most effective sensitizing agents. Reaginic antibodies could be raised in both guinea pigs and rabbits by immunizing with PT- and HPT-protein conjugates but not by PT and HPT alone: these PCA antibodies showed strong cross-reactivity and could be elicited equally well with either the PT- or HPT-protein derivatives. By contrast, no precipitating antibodies could be raised in either species even after repeated immunizations over a period of 4 months. Peripheral blood lymphocytes, from a few patients who gave a positive patch test with PT, could be stiumlated in vitro with phenacetin and to a lesser degree with PT and with a HPT-derivative of human serum albumin.

Hapten-specific lymphocyte transformation in humans sensitized with NDMA or DNCB.

The primary immune response to a contact sensitizing dose of para-N-dimethylnitrosaniline (NDMA) and dinitrochlorobenzene (DNCB) was obtained in humans and measured in vitro by increased thymidine incorporation into sensitized lymphocytes. No cross-reaction was found between these two haptens, and it is thus possible on two separate occasions to quantify and follow the primary cellular immune response in man.

Detection of liver-bound metabolites of azocarcinogens by the use of anti-hapten antibodies.

The presence of the azocompounds, p-dimethylaminoazobenzene and 3'-methyl-p-dimethylaminoazobenzene, and p-amino-N-acetyl-N-methylaniline (or their metabolites) bound to components of the liver cells of rats fed a single large dose of each compound has been detected using rabbit antibodies raised against the p-azo-N-acetyl-N-methylaniline hapten in the indirect fluorescent antibody technique. Binding of these antibodies was seen on liver sections from rats fed any one of these compounds. When the anti-p-azo-N-acetyl-N-methylaniline antiserum was absorbed with either liver sediments or cytosol fractions from rats fed p-amino-N-acetyl-N-methylaniline, the antibodies reacting with the liver-bound compounds were removed from the antiserum. Also, absorption of the antiserum with liver sediments or cytosol fractions of rats fed either one of the azocompounds selectively removed all of the antibodies reacting with the livers of rats fed that compound but did not remove other antibodies that were still capable of reacting with liver cells of rats fed the other azocompound or p-amino-N-acetyl-N-methylaniline. Thus this antiserum appears to contain several different anti-p-azo-N-acetyl-N-methylaniline antibodies with different structural requirements for reaction. Some can react with the azocompounds or certain of their metabolites, while others require more of the p-azo-N-acetyl-N-methylaniline structure for reaction. Some of the antibodies appear to react with liver-bound p-dimethylaminoazobenzene but not with liver-bound 3'-methyl-p-dimethylaminoazobenzene, while still others react with 3'-methyl-p-dimethylaminoazobenzene but not with p-dimethylaminoazobenzene.

Specific acquired immune unresponsiveness to contact allergens with cyclophosphamide in the mouse.

Treatment with cyclophosphamide (Cy) can modulate the acquisition of allergic contact dermatitis in the mouse. We compared the effect of a single dose of Cy given at different times before or after allergen. Cy one or more days prior to allergen intensified, and Cy several days after allergen inhibited the acquisition of sensitivity. Mice whose immunological response to an allergen had been suppressed by Cy were specifically immunologically tolerant to that allergen, but not to an unrelated allergen. This tolerance probably represents a combination of clone deletion and inhibition; almost certainly it does not depend on the generation of enhancing ('blocking') antibody.