Conversion of a tolerogenic to an immunogenic signal by P388AD.2 cells, a lymphoid dendritic cell-like tumor line

The lymphoid dendritic cell-like tumor P388AD.2 is capable of converting a tolerogenic signal into an immunogenic one. In the present study, adherent P388AD.2 cells were pulsed with the tolerogen, fluoresceinated (FL) sheep gamma-globulin (SGG), washed, and incubated with normal spleen cells. After 24 hr, the spleen cells were harvested and challenged in secondary cultures with immunogenic doses of FL-thymic independent (TI) antigens. The plaque-forming cell response on day 3 of secondary culture was increased as much as 400% compared with control spleen cultures exposed to untreated P388AD.2 cells. The increased response was specific for the FL-hapten and occurred only when P388AD.2 cells were pulsed with FL-Ig or FL-F(ab')2 fragments, but not with FL-synthetic tolerogens or other FL-antigens. Furthermore, the augmentation required histocompatible T cells in the primary cultures. Additional experiments showed that if cultures were devoid of Lyt-1+ cells but not Lyt-2+ cells, no augmentation occurred. A variety of other macrophage-like tumor cells, some with known antigen presenting properties, were tested for the ability to present tolerogen in an immunogenic fashion. Only an I-A+ J774 clone was able to present tolerogen in an immunogenic fashion. However, we failed to find a correlation between the presence of surface Ia antigen and tolerogen-presenting ability. The results suggest that certain types of cells may play a role in immune regulation by abrogating the tolerogenicity of Ig tolerogens via their presentation in an immunogenic mode.     

Role of self carriers in the immune response and tolerance. VIII. Suppression of the MOPC-104E idiotypic response by idiotype-modified self, but not by dextran-modified self

We have investigated the suppression of the anti-dextran B1355S immune response using our model of modified self. The anti-dextran response is idiotypically well defined in BALB/c mice. This system enables us to examine the contribution of various predominant idiotypes to the antibody response under conditions of suppression by antigen or by idiotype-specific suppressor cells. Our results demonstrate that the total anti-dextran response can be inhibited by pretreatment of animals with dextran-coupled syngeneic spleen cells; however, the representation of major idiotypes constituting this response are not reduced in percentage. In contrast, pretreatment of mice with MOPC-104E-coupled spleen cells leads to a specific suppression of the private IdI-104E idiotype. The total anti-dextran response remains unchanged, as well as proportions of other major idiotypes known (IdI-588 and IdX). This suppression is mediated by Thy-1.2+, Lyt-2.2+ T cells, as demonstrated by adoptive transfer assays. This system will allow the molecular dissection of the regulation of an idiotypically well-defined system for the suppression by either antigen- or idiotype-specific suppressor T cells.     

Role of self carriers in the immune response and tolerance. XI. Correlation of Ia expression and interleukin-1 production with delivery of immunogenic signals in vivo by hapten-modified accessory cell-like tumor lines

It was recently demonstrated that a lymphoid dendritic-like tumor, P388AD.2, presented hapten-modified self (HMS) in an immunogenic fashion even after injection via the normally "tolerogenic" intravenous (iv) route. To determine whether this property was unique to the P388AD.2 line, other hapten-modified tumors were administered iv and the result of their presentation was measured by changes in the number of splenic plaque-forming cells (PFC) following in vitro challenge with thymic-independent antigens. Of the six tumors tested, two (P388 and J774.5R) primed for augmented PFC responses, while four others (P388NA.10, P388D1, WEHI-231, and 70Z/3) did not. When these tumors were compared for Ia expression and production of interleukin-1 (IL-1), it was discovered that (1) all of the immunogenic tumors were Ia+ and IL-1 producing (IL-1+), although not all Ia+,IL-1+ tumors could elicit augmented PFC responses; (2) none of the tumors that were deficient in either Ia expression or IL-1 production could prime B-cell responses in vivo; and (3) the ability to augment PFC responses was proportional to the density of Ia on the immunogenic tumors. These results demonstrated that P388AD.2 was not the only tumor line capable of presenting HMS iv as an immunogen, and that the accessory cell phenotype is critical for the induction of an immunogenic response in vivo.     

Role of self carriers in the immune response and tolerance. IV. Active T cell suppression in the maintenance of B cell tolerance to a "T-independent" antigen.

Previous studies indicated that T cells are required for tolerance induction by hapten-modified syngeneic spleen cells (TNP-SC) in vivo. The role of T cells in the maintenance of this unresponsive state has been examined herein. By three criteria--limiting dilution precursor analysis, removal of T cells by anti-Thy-1 + C, and direct mixing experiments--we show that T cells are required for the continued suppression of the B cell response to the T-independent antigen, TNP-POL. Suppressor cells can also be induced by TNP-teratoma cells, which lack detectable H-2 antigens. Both anti-Ly-1 + C and anti-Ly-2 + C treatment reversed suppression induced by TNP-SC. These results demonstrate that normal B cell reactivity is present in the spleens of mice rendered tolerant by haptenated self, but that Ly-1,2,3 or Ly-1 + Ly-2,3 suppressor T cells prevent their responsiveness.     

Role of self carriers in the immune response and tolerance. III. B cell tolerance induced by hapten-modified-self involves both active T cell-mediated suppression and direct blockade.

The induction of B cell unresponsiveness with hapten-modified syngeneic murine lymphoid cells (hapten-modified self, HMS) can be achieved in vivo and in vitro. Tolerance in vivo in mice required a latent period of 3 to 4 days. Moreover, B cell unresponsiveness could not be induced by HMS in athymic nude mice, although their nu/+ littermates were rendered hyporesponsive by HMS. Pretreatment of normal mice with cyclophosphamide (cyclo) prevented their susceptibility to tolerance induction by haptenated lymphoid cells. Nude mice became sensitive to HMS-induced suppression if they were first reconstituted with spleen cells from normal (but not cyclo-treated) donors.Interestingly, labeling of H-2 antigens was not necessary for tolerance induction by HMS since haptenated teratoma cells (lacking H-2) were tolerogenic in normal recipients.In contrast, suppression of the in vitro response to haptenated flagellin occurred equally well with nude, nu/+ and anti-Ly 2 + C-treated spleen cells. These data suggest that cyclo-sensitive modified self-reactive (T) cells may regulate the immune response and mediate tolerance to HMS in vivo. However, the in vitro “blockade” of B cell reactivity may be directly mediated on hapten-specific PFC precursors.     

Role of self carriers in the immune response and tolerance. XII. Effect of epitope density and antigen-presenting cell phenotype on the presentation of hapten-modified self for the induction of immunity or tolerance in vitro

The data presented in the accompanying paper (J. P. Cogswell, R. P. Phipps, and D. W. Scott, Cell. Immunol. 114, 55-70, 1988) indicate that certain macrophage-like and lymphoid dendritic-like (P388AD.2) tumor lines which express major histocompatibility encoded class II (Ia) antigens and produce interleukin 1 (IL-1) are uniquely able to present hapten-modified self (HMS) in an immunogenic fashion in vivo. In the current study, the relationship between phenotype and function has been confirmed utilizing a completely in vitro system. This investigation revealed that B-cell priming required T cells restricted to P388AD.2's I-A antigens. In addition, exogenous IL-1 reconstituted the response of an IL-1-deficient tumor (P388AD.2-ILd), although it had no effect on the other nonimmunogenic Ia+ tumor lines. Unlike the in vivo system, effective B-cell tolerance was induced when P388AD.2 was modified with high concentrations (10 mM) of hapten or when highly haptenated tumor was added to 0.1 mM TNBS-modified P388AD.2. These results suggest that positive regulation of in vitro immune responses to HMS is dependent upon the phenotype of the accessory cell carrier (with lymphoid dendritic-like cells being unusually potent), while negative regulation is associated with high epitope density. This system now allows the dissection of the properties of different accessory cells and the signals required for B-cell priming or tolerance induction.     

Effect of P2X purinergic receptors in tumor progression and as a potential target for anti-tumor therapy

The development of tumors is a complex pathological process involving multiple factors, multiple steps, and multiple genes. Their prevention and treatment have always been a difficult problem at present. A large number of studies have proved that the tumor microenvironment plays an important role in the progression of tumors. The tumor microenvironment is the place where tumor cells depend for survival, and it plays an important role in regulating the growth, proliferation, apoptosis, migration, and invasion of tumor cells. P2X purinergic receptors, which depend on the ATP ion channel, can be activated by ATP in the tumor microenvironment, and by mediating tumor cells and related cells (such as immune cells) in the tumor microenvironment. They play an important regulatory role on the effects of the skeleton, membrane fluidity, and intracellular molecular metabolism of tumor cells. Therefore, here, we outlined the biological characteristics of P2X purinergic receptors, described the effect of tumor microenvironment on tumor progression, and discussed the effect of ATP on tumor. Moreover, we explored the role of P2X purinergic receptors in the development of tumors and anti-tumor therapy. These data indicate that P2X purinergic receptors may be used as another potential pharmacological target for tumor prevention and treatment.

     

A novel potent strategy for gene delivery using a single peptide vector as a carrier.

We have shown previously that a peptide, MPG, derived from the hydrophobic fusion peptide of HIV-1 gp41 and the hydrophilic nuclear localisation sequence of SV40 large T antigen, can be used as a powerful tool for the delivery of oligonucleotides into cultured cells. Now we extend the potential of MPG to the delivery of nucleic acids into cultured cells. In vitro, MPG interacts strongly with nucleic acids, most likely forming a peptide cage around them, which stabilises and protects them from degradation in cell culture media. MPG is non-cytotoxic, insensitive to serum and efficiently delivers plasmids into several different cell lines in only 1 h. Moreover, MPG enables complete expression of the gene products encoded by the plasmids it delivers into cultured cells. Finally, we have investigated the potential of MPG as an efficient delivery agent for gene therapy, by attempting to deliver antisense nucleic acids targeting an essential cell cycle gene. MPG efficiently delivered a plasmid expressing the full-length antisense cDNA of human cdc25C, which consequently successfully reduced cdc25C expression levels and promoted a block to cell cycle progression. Based on our results, we conclude that MPG is a potent delivery agent for the generalised delivery of nucleic acids as well as of oligonucleotides into cultured cells and believe that its contribution to the development of new gene therapy strategies could be of prime interest.     

Pharmacological evaluation of a novel series of urea,thiourea and guanidine derivatives as P2X7 receptor antagonists

We report on P2X₇ receptor antagonists based on a lead adamantly-cyanoguanidine-aryl moiety. We have investigated the importance of the central cyanoguanidine moiety by replacing it with urea, thiourea or guanidine moieties. We have also investigated the linker length between the central moiety and the aryl portion. All compounds were assessed for their inhibitory potency in a pore-formation dye uptake assay at the P2X₇ receptor. None of the compounds resulted in an improved potency illustrating the importance of the cyanoguanidine moiety in this chemotype.     

Identification and SAR of novel diaminopyrimidines. Part 1: The discovery of RO-4, a dual P2X3/P2X2/3 antagonist for the treatment of pain

P2X purinoceptors are ligand-gated ion channels whose endogenous ligand is ATP. Both the P2X₃ and P2X_2/3 receptor subtypes have been shown to play an important role in the regulation of sensory function and dual P2X₃/P2X_2/3 antagonists offer significant potential for the treatment of pain. A high-throughput screen of the Roche compound collection resulted in the identification of a novel series of diaminopyrimidines; subsequent optimization resulted in the discovery of RO-4, a potent, selective and drug-like dual P2X₃/P2X_2/3 antagonist.     

Identification and SAR of novel diaminopyrimidines. Part 2: The discovery of RO-51, a potent and selective,dual P2X3/P2X2/3 antagonist for the treatment of pain

The purinoceptor subtypes P2X₃ and P2X_2/3 have been shown to play a pivotal role in models of various pain conditions. Identification of a potent and selective dual P2X₃/P2X_2/3 diaminopyrimidine antagonist RO-4 prompted subsequent optimization of the template. This paper describes the SAR and optimization of the diaminopyrimidine ring and particularly the substitution of the 2-amino group. The discovery of the highly potent and drug-like dual P2X₃/P2X_2/3 antagonist RO-51 is presented.     

A novel eIF2B-dependent mechanism of translational control in yeast as a response to fusel alcohols.

Fusel alcohols are natural products of amino acid catabolism in the yeast Saccharomyces cerevisiae that cause morphological changes similar to those seen during pseudohyphal growth. We have discovered that certain of these alcohols, including butanol and isoamyl alcohol, bring about a rapid inhibition of translation at the initiation step. This inhibition is strain specific and is not explained by previously described translational control pathways. Using genetic mapping, we have identified a proline to serine allelic variation at amino acid 180 of the GCD1 gene product as the genetic locus that allows translational regulation upon butanol addition. Gcd1p forms part of the eIF2B guanine nucleotide complex that is responsible for recycling eIF2-GDP to eIF2-GTP. This represents one of the key limiting steps of translation initiation and we provide evidence that fusel alcohols target eIF2B in order to bring about translational regulation.     

Verapamil, a Ca2+ channel inhibitor acts as a local anesthetic and induces the sigma E dependent extra-cytoplasmic stress response in E. coli

Verapamil is used clinically as a Ca(2+) channel inhibitor for the treatment of various disorders such as angina, hypertension and cardiac arrhythmia. Here we study the effect of verapamil on the bacterium Escherichia coli. The drug was shown to inhibit cell division at growth sub inhibitory concentrations, independently of the SOS response. We show verapamil is a membrane active drug, with similar effects to dibucaine, a local anesthetic. Thus, both verapamil and dibucaine abolish the proton motive force and decrease the intracellular ATP concentration. This is accompanied by induction of degP expression, as a result of the activation of the RpoE (SigmaE) extra-cytoplasmic stress response, and activation of the psp operon. Such effects of verapamil, as a membrane active compound, could explain its general toxicity in eukaryotic cells.     

Production of auto-anti-idiotypic antibody during the normal immune response. XII. Enhanced auto-anti-idiotypic antibody production as a mechanism for apparent B-cell tolerance in rabbits after feeding antigen

Rabbits fed trinitrophenylated bovine serum albumin (TNP-BSA) generated fewer anti-TNP plaque-forming cells but greater numbers of hapten (TNP)-augmentable IgM and IgG PFC following immunization with TNP-Ficoll or TNP-Brucella abortus than did animals not previously fed antigen. Spleen and mesenteric and bronchial lymph nodes were similarly affected. In addition more auto-anti-idiotype (Id) antibody (anti-anti-TNP) was eluted by hapten from spleen cells of antigen-fed rabbits than from spleen cells of control rabbits not prefed antigen. Gel filtration studies ruled out the possibility that the Id binding activity in the eluates was due to immune complexes. The isotype of the anti-Id was IgG except in one rabbit where it was IgM. The results are consistent with the interpretation that the production of auto-anti-Id antibody is one of the factors responsible for the specific depression of the IgM and IgG immune responses which follows antigen feeding. In contrast the antigen feeding resulted in priming for an IgA anti-TNP response without detectable hapten-augmentable IgA PFC.     

Glutaraldehyde (GA)-hapten adducts, but without a carrier protein, for use in a specificity study on an antibody against a GA-conjugated hapten compound: histamine monoclonal antibody (AHA-2) as a model

In our recent study on monoclonal antibodies (mAbs AHA-1-5) against glutaraldehyde (GA)-conjugated histamine (HA), we identified one mAb (AHA-2) which can detect neuronal HA in the rat brain with an immunocytochemistry method (ICC) [Fujiwara et al. (1999) J. Biochem. 126, 503-509]. In the present study the specificity of AHA-2 mAb for use for ICC has been examined by means of competitive experiments involving HA and analogs, all of which had been allowed to react with GA followed by sodium borohydride, but not allowed to couple with the carrier protein. It was demonstrated that the antibody distinguished alterations in the chemical structure of the molecule, showing decreased immunoreactivity with all the GA-adducts of (R)-(-)-alpha-methylhistamine, 1- and 3-methylhistamine, L-histidine, and 1- and 3-methyl-L-histidine. On the other hand, AHA-1 mAb only reacted with GA-adducts of 3-MeHA (3-MeHA-GA) and HA (HA-GA), to almost the same degree, in relatively high concentration ranges. AHA-3, 4, and 5 mAbs reacted about 10-times more strongly with 1-MeHA-GA than with HA-GA, but reacted very little or not at all with the other analogs. These results may suggest that AHA-2 mAb recognized both the non-substituted imidazole and alpha-methine groups of a HA molecule in addition to the conjugation site of GA including the part(s) reduced with NaBH(4), and especially the imidazole group more strictly than the other mAbs. This may partly explain why AHA-2, among the five AHA mAbs, can detect neuronal HA with an ICC method. The present ELISA method for GA-hapten adducts should be applicable to other antibodies against GA-conjugated biologically active amines or amino acids, thus allowing the study of antibody specificity for ICC more easily and accurately than was previously possible with hapten-protein conjugates as antigens.     

Role of crotoxin,a phospholipase A2 isolated from Crotalus durissus terrificus snake venom,on inflammatory and immune reactions.

BACKGROUND: Crotoxin (CTX) is a potent neurotoxin from Crotalus durissus terrificus snake venom (CdtV) composed of two subunits: one without catalytic activity (crotapotin), and a basic phospolipase A2. Recent data have demonstrated that CdtV or CTX inhibit some immune and inflammatory reactions. AIM: The aim of this paper was to investigate the mechanisms involved in these impaired responses. MATERIALS AND METHODS: Male Swiss mice were bled before and at different intervals of time after subcutaneous injection of CTX or bovine serum albumin (BSA) (control animals). The effect of treatments on circulating leukocyte mobilisation and on serum levels of interleukin (IL)-6, IL-10, interferon (IFN)-gamma and corticosterone were investigated. Spleen cells from treated animals were also stimulated in vitro with concanavalin A to evaluate the profile of IL-4, IL-6, IL-10 or IFN-gamma secretion. Cytokine levels were determined by immunoenzymatic assay and corticosterone levels by radioimmunoassay. To investigate the participation of endogenous corticosteroid on the effects evoked by CTX, animals were treated with metyrapone, an inhibitor of glucocorticoid synthesis, previous to CTX treatment. RESULTS: Marked alterations on peripheral leukocyte distribution, characterised by a drop in the number of lymphocytes and monocytes and an increase in the number of neutrophils, were observed after CTX injection. No such alteration was observed in BSA-treated animals. Increased levels of IL-6, IL-10 and corticosterone were also detected in CTX-injected animals. IFN-gamma levels were not modified after treatments. In contrast, spleen cells obtained from CTX-treated animals and stimulated with concanavalin A secreted less IL-10 and IL-4 in comparison with cells obtained from control animals. Metyrapone pretreatment was effective only to reverse the neutrophilia observed after CTX administration. CONCLUSIONS: Our results suggest that CTX may contribute to the deficient inflammatory and immune responses induced by crude CdtV. CTX induces endogenous mechanisms that are responsible, at least in part, for these impaired responses.     

A novel role for human Nfs1 in the cytoplasm: Nfs1 acts as a sulfur donor for MOCS3, a protein involved in molybdenum cofactor biosynthesis

The human MOCS3 gene encodes a protein involved in activation and sulfuration of the C terminus of MOCS2A, the smaller subunit of the molybdopterin (MPT) synthase. MPT synthase catalyzes the formation of the dithiolene group of MPT that is required for the coordination of the molybdenum atom in the last step of molybdenum cofactor (Moco) biosynthesis. The two-domain protein MOCS3 catalyzes both the adenylation and the subsequent generation of a thiocarboxylate group at the C terminus of MOCS2A by its C-terminal rhodanese-like domain (RLD). The low activity of MOCS3-RLD with thiosulfate as sulfur donor and detailed mutagenesis studies showed that thiosulfate is most likely not the physiological sulfur source for Moco biosynthesis in eukaryotes. It was suggested that an l-cysteine desulfurase might be involved in the sulfuration of MOCS3 in vivo. In this report, we investigated the involvement of the human l-cysteine desulfurase Nfs1 in sulfur transfer to MOCS3-RLD. A variant of Nfs1 was purified in conjunction with Isd11 in a heterologous expression system in Escherichia coli, and the kinetic parameters of the purified protein were determined. By studying direct protein-protein interactions, we were able to show that Nfs1 interacted specifically with MOCS3-RLD and that sulfur is transferred from l-cysteine to MOCS3-RLD via an Nfs1-bound persulfide intermediate. Because MOCS3 was shown to be located in the cytosol, our results suggest that cytosolic Nfs1 has an important role in sulfur transfer for the biosynthesis of Moco.     

A direct, highly sensitive fluorometric assay for a microsomal cytochrome P450-mediated O-demethylation using a novel coumarin analog as substrate

A highly sensitive fluorometric assay for the determination of monooxygenase activity in liver microsomes is described. The assay is based on the use of 3-chloro-7-methoxy-4-methylcoumarin which is demethylated to 3-chloro-7-hydroxy-4-methylcoumarin. The rate of formation of 3-chloro-7-hydroxy-4-methylcoumarin was recorded as an increase of fluorescence (lambdaA = 380 nm, lambdaF = 480 nm) with time. When 3-chloro-7-methoxy-4-methylcoumarin was incubated in the presence of MgCl2 and NADPH with rat liver microsomes, a continuous increase of the fluorescence could be measured. The reaction proceeded linearly for about 10 min and at least up to a concentration of 0.1 mg/ml of microsomal protein. Besides 3-chloro-7-hydroxy-4-methylcoumarin a hydroxylated derivative of the substrate was formed as a second metabolite during the incubation. Using an excitation wavelength of 380 nm and a fluorescence/emission wavelength of 480 nm, the fluorescence of this substance (lambdaA = 338 nm, lambdaF = 422 nm) amounted only to about 1% of the fluorescence of the main product. The use of 3-chloro-7-methoxy-4-methylcoumarin as substrate enables the fluorometric determination of the O-dealkylation activity of a cytochrome P450-dependent monooxygenase system in rat liver which is inducible by phenobarbital but not by 3-methylcholanthrene.