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Inhibition of helper function with anti-Lyt-2 or anti-L3T4 monoclonal antibodies depending on stimulating antigens.

The role of L3T4+ and Lyt-2+ cells in the induction of cytotoxic T cells (CTL) after stimulation with trinitrobenzene sulfonate (TNBS)-treated syngeneic cells, or with cells differing for the entire major histocompatibility complex or for class I molecules alone was investigated using anti-L3T4 and anti-Lyt-2 monoclonal antibodies (mAb). Anti-L3T4 mAb inhibited both induction of CTL and interleukin 2 (IL2) production by T cells stimulated with allogeneic (class I + class II differences) or by TNBS-treated syngeneic cells. In the same culture conditions, anti-Lyt-2 mAb inhibited CTL induction but had no effect on IL2 production. By contrast, only anti-Lyt-2 mAb inhibited both CTL induction and IL2 production when T cells were stimulated by allogeneic cells differing only for class I antigens. Anti-L3T4 mAb had no inhibitory effect in this situation. These results indicate that two helper pathways can be activated, depending on the type of T cell stimulation: one, implicating L3T4+ cells, is used when stimulation involves class I and class II alloantigens or hapten on syngeneic cells; the other, involving Lyt-2+ T cells, is preferentially stimulated when responding and stimulating cells differ only for class I major histocompatibility complex antigen.

Animals↗

Cross-reactive trinitrophenylated peptides as antigens for class II major histocompatibility complex-restricted T cells and inducers of contact sensitivity in mice. Limited T cell receptor repertoire.

The induction of contact sensitivity in mice by hapten reagents such as trinitrochlorobenzene (TNCB) involves the activation of class II major histocompatibility complex (MHC)-restricted, hapten-specific, CD4+ T cells. Reports from different laboratories have indicated that the relevant antigenic epitopes in such reactions might include hapten-conjugated, MHC class II-associated peptides. This study for the first time directly demonstrates that hapten-peptides account for the majority of determinants recognized by trinitrophenyl (TNP)-specific CD4+ T lymphocytes. The sequences of those TNP carrier peptides do not have to be related to mouse proteins. Thus, we show that TNP-modified peptides derived from mouse IgG, pigeon cytochrome c or staphylococcal nuclease known to bind to I-Ab or from lambda repressor with specificity to I-Ad as well as TNP-proteins such as bovine serum albumin, ovalbumin or keyhole limpet hemocyanin all create class II-restricted hapten determinants for a number of TNP-specific T cell clones and hybridomas. All of these cells were induced with cells modified by trinitrobenzene sulfonic acid (TNBS). In addition, we present arguments indicating that individual TNP-specific helper T cells may cross-react with different TNP-peptides bound to identical class II molecules. Chemical treatment of antigen-presenting cells with TNCB or TNBS may thus result in a limited number of particularly repetitive immunodominant hapten epitopes. Immunodominant epitopes were also indicated by an overrepresentation of the TCR elements V beta 2 and V alpha 10 in I-Ab/TNP-specific T cells. Most importantly, however, we demonstrate that TNP attached to lysine 97 in the staphylococcal nuclease peptide 93-105 (i.e. a clearly "non-self" sequence) is able to prime mice for subsequent elicitation of contact sensitivity by TNCB in the absence of foreign protein. We take this to indicate that those TNP-peptide determinants defined by us as immuno-dominant are responsible for the induction of contact sensitivity to haptens.

Amino Acid Sequence↗

Two-dimensional electrophoresis of precision-cut testis slices: toxicologic application.

Advances in tissue slice technology and a recent novel application of this technique to reproductive toxicology using bovine testis have demonstrated the remarkable utility of this approach. The objective of the present study was to combine this in vitro toxicity test system with large-scale two-dimensional polyacrylamide gel electrophoresis (2-DE) to detect and study alterations in testicular-slice protein patterns as molecular correlates of 1,3,5-trinitrobenzene (TNB) and 1,3-dinitrobenzene (DNB) toxicity. Previous studies have shown that testicular slices remain viable for > 24 h and, as measured by protein synthesis inhibition, TNB causes dose-related injury. Tissue-slices were prepared from bovine testicles incubated for 2, 4 or 6 h and exposed to either 100 microM, 500 microM or 1 mM DNB or TNB in the incubation medium. Slices were collected, solubilized, and separated by large scale 2-DE. Resulting protein patterns were then examined by image analysis, which revealed coefficients of variation in protein spot abundance comparable to patterns from fresh rodent tissue samples. Furthermore, specific protein alterations indicated dose-related inductions and declines in protein abundance, some progressive over time. The results of this investigation demonstrate the potential toxicologic utility of combining in vitro tissue-slice technology with high-resolution 2-DE protein mapping. The consolidation of these methods offers a novel approach for toxicity screening and testing, reduces experimental cost, and reduces the use of laboratory animals.

Animals↗

Ghrelin stimulates interleukin-8 gene expression through protein kinase C-mediated NF-kappaB pathway in human colonic epithelial cells.

Ghrelin, a newly identified gastric peptide, is known for its potent activity in growth hormone (GH) release and appetite. Although ghrelin is involved in several other responses such as stress and intestinal motility, its potential role in intestinal inflammation is not clear. Here, we show that expression of ghrelin and its receptor mRNA is significantly increased during acute experimental colitis in mice injected intracolonically with trinitrobenzene sulfate (TNBS). We found by PCR that ghrelin receptor mRNA is expressed in non-transformed human colonic epithelial NCM460 cells. Exposure of NCM460 cells stably transfected with ghrelin receptor mRNA to ghrelin, increased IkappaBalpha phosphorylation and its subsequent degradation. In addition, ghrelin stimulated NF-kappaB-binding activity and NF-kappaB p65 subunit phosphorylation, and induced IL-8 promoter activity and IL-8 protein secretion. Furthermore, our data show that ghrelin-induced IkappaBalpha and p65 phosphorylation was markedly reduced by pharmacological inhibitors of intracellular calcium mobilization (BAPTA/AM) and protein kinase C (GF 109203X). Pretreatment with BAPTA/AM or GF109203X also significantly attenuated ghrelin-induced IL-8 production. Together, our results strongly suggest that ghrelin may be a proinflammatory peptide in the colon. Ghrelin may participate in the pathophysiology of colonic inflammation by inducing PKC-dependent NF-kappaB activation and IL-8 production at the colonocyte level.

Animals↗

Polynitro aromatic compounds in analytical chemistry I: reaction with ouabain and digitoxin.

By the use of NMR spectroscopy, the highly colored reaction products formed by ouabain or digitoxin in with 1,3,5-trinitrobenzene or 2,4,6-trinitroanisole in the presence of alkali (as used for the determination of these glycosides) are shown to be Meisenheimer complexes. The complexes are produced by attachment of a carbon of the butenolide ring to an aromatic carbon of the nitro compound with formation of a charge-delocalized cyclohexadienate anion.

Anisoles↗

Role of the N-terminal region of the A chain in beta 1-bungarotoxin from the venom of Bungarus multicinctus (Taiwan-banded krait).

The N-terminal alpha-amino groups of beta 1-bungarotoxin (beta 1-Bgt) from Bungarus multicinctus venom were modified with trinitrobenzene sulfonic acid and the modified derivative was separated by high performance liquid chromatography. The trinitrophenylated (TNP) derivative contained two TNP groups at the alpha-amino groups of A chain and B chain and showed a marked decrease in enzymatic activity. Methionine residues at positions 6 and 8 of the A chain were oxidized with chloramine T or cleaved with cyanogen bromide to remove the N-terminal octapeptide. Oxidation of methionine residues and removal of the N-terminal octapeptide caused a precipitous decrease in enzymatic activity, whereas antigenicity remained unchanged. The presence of dihexanoyllecithin influenced the interaction between beta 1-Bgt and 8-anilinonaphthalene sulfonate (ANS) and revealed that beta 1-Bgt consists of two types of ANS-binding sites, one at the substrate binding site of the A chain and the other might be at the B chain. The modified derivatives still retained their affinity for Ca2+ and ANS, indicating that the N-terminal region is not involved in Ca2+ and substrate binding. A fluorescence study revealed that the alpha-amino group of the A chain was in the vicinity of substrate binding site and that the TNP alpha-amino groups were in proximity to Trp-19 of the A chain. In addition, the study showed that the N-terminal region is important for stabilizing the architectural environment of Trp-19. The results, together with the proposal that Trp-19 of the A chain is involved in substrate binding, suggest that the N-terminal region of the A chain plays a crucial role in maintaining a functional active site for beta 1-Bgt.

Amino Acids↗

Comparative and mixture sediment toxicity of trinitrotoluene and its major transformation products to a freshwater midge.

The explosive trinitrotoluene (TNT) is a prevalent contaminant in many military installations worldwide. Limited knowledge of the comparative toxicity of sediment-associated TNT and related compounds contributes to uncertainty when assessing ecological risks in contaminated sites. Trinitrotoluene undergoes transformation when associated with soils and sediments and typically occurs as a mixture dominated by its reduction products. The objective of this study was to comparatively evaluate the single-compound toxicity of TNT and its major transformation products to the freshwater midge Chironomus tentans in 10-day exposures to sediment spiked with TNT, 2-aminodinitrotoluene (2-ADNT), 2,4-diaminonitrotoluene (2,4-DANT), or trinitrobenzene (TNB). In addition, the nature of the toxicological interactions of the latter compounds in a mixture was evaluated. Upon spiking to sediment, TNT and TNB rapidly degraded to reduced products, and disappearance of extractable compounds suggested irreversible binding to sediment particles. The high degree of transformation and reactivity occurring during 10 days at spiking concentrations as high as 4000 micromol/kg dry weight suggests that TNT and related compounds are unlikely to be encountered in fine-grained sediments at contaminated sites. Similar to previous investigations, the high reactivity of the spiked compound hampered determination of accurate toxic concentrations of TNT and related compounds, and of the nature of toxicological interaction of compounds in a mixture in this study. Sediment concentrations associated with decreased survival were similar for all four compounds, with the 10-d median lethal concentrations (LC50s) determined using initial concentrations ranging from 175 (2-ADNT) to 605 (2,4-DANT) micromol/kg dry weight. Sublethal decrease in growth was not observed for any compound. Results from the mixture experiment suggest additive interaction among TNT and related compounds in sediment exposures.

Animals↗

Metabolism of explosive compounds by sulfate-reducing bacteria.

The metabolism of various explosive compounds-1,3,5-trinitrobenzene (TNB), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine (HMX)-by a sulfate-reducing bacterial consortium, Desulfovibrio spp., was studied. The results indicated that the Desulfovibrio spp. used all of the explosive compounds studied as their sole source of nitrogen for growth. The concentrations of TNB, RDX, and HMX in the culture media dropped to below the detection limit (<0.5 ppm) within 18 days of incubation. We also observed the production of ammonia from the nitro groups of the explosive compounds in the culture media. This ammonia served as a nitrogen source for the bacterial growth, and the concentration of ammonia later dropped to <0.5 mg/L. The sulfate-reducing bacteria may be useful in the anaerobic treatment of explosives-contaminated soil.

Ammonia↗

Abnormal expression of surface immunoglobulin isotypes on antigen-binding B lymphocytes from mice tolerant to trinitrophenyl determinants.

Temporary B-cell tolerance to the trinitrophenyl (TNP) hapten can be produced in BDF1 mice by intraperitoneal injection of trinitrobenzene sulfonic acid (TNBS). Antigen-binding cells (ABC) specific to TNP, measured as TNP donkey erythrocyte rosettes, are found in tolerant mice as well as in immune mice. We have studied the surface immunoglobulin isotype profile of these TNP-binding lymphocytes (TNP-ABC) in four groups of animals: nonimmune, immune, tolerant, and tolerant-challenged. Immune mice received intravenous TNP sheep erythrocytes (TNP-SRC), whereas tolerant-challenged mice received TNP-SRC and TNBS on Day 0. TNP-ABC from mice immunized with TNP-SRC exhibit increased expression of surface IgG and decreased expression of surface IgD, compared to the ABC from nonimmune mice. Tolerant mice have a higher proportion of ABC with surface IgG, and a lower proportion with surface IgD, than nonimmune mice. Tolerant-challenged mice have a lower proportion of ABC with surface IgG, and a higher proportion with surface IgD, than immune mice. Thus, B-cell tolerance in this model entails an attenuation of the surface immunoglobulin isotype switch (loss of IgD and gain of IgG) on ABC seen in the normal immune response. For most TNP-ABC, tolerogen exposure prevents the switch in surface isotypes normally induced by exposure to TNP antigen; i.e., the tolerance lesion precedes the surface isotype switch. However, a minority of the TNP-ABC appear to switch surface isotypes in response to the tolerogen itself.

Animals↗

Shark cytotoxic macrophages interact with target membrane amino groups.

The types of target structures recognized by cytotoxic macrophages have been described for various microorganisms, but have not been defined for tumor cells. Tumoricidal macrophages are selective in their destructive mechanisms, sparing normal cells while directing their lytic machinery toward neoplastic targets. The cytotoxic activity of macrophages from a primitive vertebrate, the nurse shark, closely resembles the activity of mammalian tumoricidal macrophages. Host defense mechanisms of these animals appear to rely on antigen nonspecific cellular effector systems, and it has been postulated that macrophage-mediated cytotoxicity plays a dominant role in protection during periods of decreased environmental temperatures when lymphocyte responses of poikilothermic vertebrates are compromised. Similar to mammalian tumoricidal macrophages shark macrophages display selective recognition of target cells. Previous studies showed that TNP modification of targets was protective, preventing recognition by the shark spontaneously cytotoxic macrophage. Additionally, it was shown that cytotoxic activity was inhibited in a dose dependent fashion by the addition of excess unlabeled targets. In the present study, similar inhibition experiments with hapten-modified targets have been used to determine the nature of the target structures recognized by the shark cytotoxic macrophage. Cold targets modified with haptens which react covalently with free amino groups on cell membranes, trinitrobenzene sulfonic acid (TNBS) and flourescein isothiocyanate (FITC), are not recognized by the cytotoxic macrophage. The relative amount of membrane bound TNP was correlated with inhibition of cytotoxicity. Conversely, target cells modified with sulfhydryl reacting reagents, N-iodoacetyl-N'-(5-sulfonic-1-naphthyl) ethylene diamine and dithionicotinic acid, are recognized similarly to untreated targets. Moreover, TNP-containing lipids, permitted to diffuse into target membranes without covalent binding, do not alter target recognition, indicating that TNP itself has no effect on macrophage:target interaction. From these data, it is concluded that the shark cytotoxic macrophage interacts with membrane bound amino, but not sulfhydryl groups. The ability to distinguish between membrane structures may have appeared early in evolution as a means of preserving self cells while retaining protective nonspecific cytotoxic mechanisms.

Amines↗

The development of peripheral TNP-tolerance and suppressor function in Xenopus laevis, the South African clawed toad.

In adult Xenopus laevis, inducer- and effector-suppressor functions are located in the spleen. These peripheral suppressor functions must be established at this location near the end of metamorphosis, since both functions are in the thymus in premetamorphic and in developmentally-blocked metamorphosing larvae. This study examined whether TNP-conjugated self-antigens resulting from exposure to trinitrobenzene sulfonic acid (TNBS), will stimulate TNP-tolerance in premetamorphic, metamorphic, and in developmentally-blocked metamorphosing larvae. Premetamorphic and developmentally-blocked larvae produce little TNP-tolerance or peripheral suppressor function. However, when TNBS exposure includes the late stages of the metamorphic period, both TNP-tolerance and splenic anti-hapten suppressor function are demonstrable. Removal of suppressor function with cyclophosphamide prevents expression of tolerance, thus, they are functionally related. Suppressor function and tolerance both differentiate during the late metamorphic stages when new adult antigens are being expressed and incorporated into a library of self.

Animals↗

Differential variations of sensitivities of TNP-modified lymphoma cells to lysis by cytotoxic lymphocytes and by antibodies.

Cultured RDM4 cells were modified with 1 mM trinitrobenzene sulphonate (TNBS) and assayed for lysis by either in vitro generated cytotoxic T-lymphocytes (CTL) or complement-mediated humoral immunity. It was observed that cells harvested from the exponentially growing phase culture (as assessed by an important incorporation of thymidine) were more sensitive to CTL, but less sensitive to humoral immunity, than cells harvested from resting phase culture. Radiolabelled [14C]TNBS permitted to show that exponentially growing cells bound about 5 times less TNBS than cells at the resting phase. It is therefore concluded that the efficiency of the CTL on TNP-modified cells is not proportional to the density of TNP-groups on the targets, and that CTL on the one hand, and complement-mediated humoral immunity on the other hand, must have very different cell surface requirements to be able to lyze the targets.

Animals↗

Anti-TNP antibody localization of the reactive lysine residues in myosin.

Myosin contains reactive lysine residues which are trinitrophenylated by 2,4,6-trinitrobenzene sulfonate much faster than the rest of the lysines. Here we find the location of these residues in the primary and spatial structure of myosin with the help of an anti-trinitrophenyl antibody. This antibody was raised against trinitrophenyl hemocyanin in rabbits. It reacted with trinitrophenylated myosin, and with some of the tryptic fragments of trinitrophenylated myosin. By analyzing the reaction with Western blots, it was found that the antibody preferentially reacts with the 27 kDa N-terminal fragment of the myosin head, and more weakly with the light meromyosin region of the myosin rod. The 27 kDa fragment contains the most reactive lysine residue, while the intermediate lysine residue is located in the light meromyosin region. The locations of the epitopes of the antibody were visualized on electron microscope images of rotary-shadowed trinitrophenylated myosin-antibody complexes. The distances of the epitopes to the head-rod junction of myosin were measured as 13 and 113 nm for the epitope on the head (reactive lysine residue) and for that on the rod (intermediary reactive lysine residue), respectively.

Antibodies↗

A new assay system detecting antibody production and delayed-type hypersensitivity responses to trinitrophenyl hapten in an individual mouse.

A new assay system detecting antibody production and delayed-type hypersensitivity (DTH) responses to trinitrophenyl hapten in an individual mouse (AS-DAD) was established. BALB/c mice were immunized intraperitoneally with varying amounts of 2,4,6-trinitrophenylated sheep red blood cells (TNP-SRBC) on day 0. Venous blood was collected on days 2, 4, 6, 8 and 10. Levels of anti-TNP IgM and IgG serum were assayed by enzyme-linked immunosorbent assay (ELISA). After series of bleeding the mice were challenged with 2,4,6-trinitrobenzene sulfonic acid (TNBS) solution in the footpad on day 14. Footpad swelling was measured 24 or 48 h after the challenge. Peak responses of the anti-TNP IgM and IgG production were detected 4 or 6 days after the immunization with 10(9) TNP-SRBC. Maximum DTH response was also observed with 10(9) TNP-SRBC 24 h after the challenge on day 14. The antibody and DTH responses were also induced in other normal inbred strains such as C3H/He and DBA/1 but not BALB/c nu/nu mice. To evaluate AS-DAD in immunopharmacological studies, various immunomodulating agents were examined in BALB/c mice by subcutaneous administration on days 0, 1, 2 and 3. Cyclosporin or cyclophosphamide at 100 mg/kg/day completely inhibited not only the anti-TNP IgM and IgG production but also the TNP-specific DTH response. Prednisolone at 0.5 mg/kg/day had no significant effect on the IgM and IgG production, whereas it inhibited the TNP-specific DTH response. Interestingly, histamine-added mouse gamma-globulin at 150 MG/kg/day clearly enhanced the anti-TNP IgM and IgG production, while it showed a suppressive effect on the TNP-specific DTH response. Levamisole at 5.0 mg/KG/day showed suppressive effects on the anti-TNP IgG production without affecting the IgM production and the DTH response. These results suggest that AS-DAD is useful for evaluating the immunopharmacological action of various agents.

Animals↗

Inhibition of anti-hapten antibody plaque-forming cells by a chemically reactive hapten.

The ability of the chemically reactive hapten 2-4-6-trinitrobenzene sulphonic acid (TNBSA) to inhibit anti-TNP IgM antibody plaque-forming cells (APFC) was investigated. TNBSA at a concentration of 1 mg (3 X 10(-3)M) to 0.3 mg (10(-3] completely inhibited IgM anti-TNP APFC after a 10-min incubation period at 37 degrees C. This inhibition was specific and did not alter the antifluorescein IgM antibody response. After a 24-h culture period in tissue culture medium without antigen, the non-dividing APFC remained inhibited, whereas the dividing APFC escaped this inhibition.

Animals↗

Detection of nitroaromatic and cyclic nitramine compounds by cyclodextrin assisted capillary electrophoresis quadrupole ion trap mass spectrometry.

An Agilent 3DCE capillary electrophoresis system using sulfobutylether-beta-cyclodextrin (SB-beta-CD)-ammonium acetate separation buffer pH 6.9 was coupled to a Bruker Esquire 3000+ quadrupole ion trap mass detector via a commercially available electrospray ionization interface with acetonitrile sheath flow. The CE-MS system was applied in negative ionization mode for the resolution and detection of nitroaromatic and polar cyclic or caged nitramine energetic materials including TNT [2,4,6-trinitrotoluene, formula mass (FW) 227.13], TNB (1,3,5-trinitrobenzene, FW 213.12), RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine, FW 222.26) HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine, FW 296.16), and CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane, FW 438.19). The CE-MS system conformed to the high-performance liquid chromatography with ultraviolet absorbance detection (HPLC-UV) and HPLC-MS reference methods for the identification of energetic contaminants and their degradation products in soil and marine sediment samples.

Aza Compounds↗

Method for preparation of fine TATB (2-5 microm) and its evaluation in plastic bonded explosive (PBX) formulations.

There is a need of fine 1,3,5-triamino-2,4,6-trinitrobenzene (TATB) (2-5 microm) for various high explosive formulations to achieve desired mechanical strength, ease in processing and finally, provide better performance of end product. The reprecipitation method for TATB has been developed using concentrated sulfuric acid as a solvent. The reprecipitation parameters of TATB were optimized to achieve required fine TATB of particle size approximately 2-5 microm. The characteristic properties of fine TATB thus obtained have been confirmed by FTIR, DSC and TG-FTIR. The spectroscopic and thermal data obtained for fine TATB were compared with standard coarse TATB and found chemically unchanged during particle size reduction. In the present study, the preparation of fine TATB was also attempted using ultrasonication method. The fine (2-5 microm) TATB has been introduced to study in the bimodal high explosive formulations. High explosive formulations based on coarse (55 microm) and fine TATB ( approximately 2-5 microm) with 10% polyurethane were studied. It was observed that properties like bulk density (1.70 g/cm(3)), mechanical strength/compressed strength (115.9 mg/cm(2)), %elongation (6.36) were improved for fine TATB in comparison with coarse TATB ( approximately 55 microm) alone in high explosive formulations.

Calorimetry, Differential Scanning↗

Influence of solvents and gas chromatographic injector conditions on the detectability of nitroaromatic compounds.

We investigated the influence of four common solvents and of several liner packings of a split/splitless injector on the gas chromatographic behavior of trinitrotoluenes and related nitroaromatic compounds. The highest peaks are observed using toluene in combination with an empty liner or with a prepacked CarboFrit liner. In particular, the peaks of trinitrotoluene isomers and 1,3,5-trinitrobenzene significantly decreased or even totally disappeared when using quartz wool or glass wool, even when treated with dimethylchlorosilane. Similiar peak reductions are obtained with methanol or acetonitrile. Effects of decreasing peak are accompanied by the formation of two additional products when using methanol.

Chromatography, Gas↗