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At least 19 recordsLinked to original sources
PubMed1979

Soluble factors in tolerance and contact sensitivity to 2,4-dinitrofluorobenzene in mice. III. Histocompatibility antigens associated with the hapten dinitrophenol serve as target molecules on 2,4-dinitrofluorobenzene-immune T cells for soluble suppressor factor.

Previous studies have shown that suppression of 2,4-dinitrofluorobenzene (DNFB) contact sensitivity by soluble suppressor factor (SSF) requires that the donor of immune lymph node (LN) cells and of SSF share either the H-2K and/or H-2D region of the major histocompatibility complex. Thus, target or acceptor molecules for SSF appear to be coded for by genes within the H-2K and H-2D loci. Experiments were done to investigate the nature of these target molecules and to determine what cell types expressed them. It was found that purified lymph node T cells are suppressed by SSF indicating that T cells express the acceptor molecules. Adsorption experiments showed that the only cells capable of adsorbing the suppressor factor are DNFB-immune T cells from donors which share with the factor-producing strain either the H-2K or H-2D locus. This adsorption can be specifically blocked by pretreating the immune LN cells with antibodies directed against H-2K and/or H-2D determinants or against the hapten DNP but not by antibodies against Ia or theta-antigens. Collectively, these results indicate that the target molecules are expressed only by DNFB-immune T cells and are comprised of histocompatibility antigens associated with DNP.

Animals↗
PubMed1981

Quantitative cytochemistry of nuclear and cytoplasmic proteins using the Naphthol Yellow S and dinitrofluorobenzene staining methods.

The 'total protein staining' of biological specimens with the electrostatically binding Naphthol Yellow S or the covalently binding dinitrofluorobenzene must be interpreted as methods which yield data on the specific amino acid pool of the proteins concerned. Both dyes bind to certain free amino-acid side-chains, giving different dye--protein ratios for various proteins. In the presence of DNA, dinitrofluorobenzene stains all proteins present in cell nuclei, whereas Naphthol Yellow S only stains the majority of the non-histone proteins. When protein staining methods are combined with the Feulgen--Pararosanile (SO2) procedure for DNA, decreased Feulgen--DNA contents were measured in dinitrofluorobenzene-stained isolated nuclei and lymphocytes.

Animals↗
PubMed1988

Metabolic myopathy produced by dinitrofluorobenzene inhibition of creatine phosphokinase.

A previously developed animal model of exercise-induced muscle contractures, which utilized intra-aortic injection of iodoacetate (IOA) to inhibit the second stage glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, showed histological evidence of selective type II muscle fiber involvement with sparing of the type I muscle fibers. A new model has been developed using dinitrofluorobenzene (DNFB) as a selective inhibitor of creatine phosphokinase in a similar, but slightly modified distal aortic injection protocol. Two hours after the injection of a dinitrofluorobenzene solution of 2.22 mg/kg body weight, spontaneous electrically-silent contracture developed in the injected lower extremity, involving principally the soleus muscle. Histologically, selective damage was apparent in the type I muscle fibers, with sparing of the type II muscle fibers. The contrast in findings associated with iodoacetate inhibition of glycolysis or with DNFB inhibition of the phosphocreatine shuttle suggests that type I and type II fibers have markedly different usable pools of readily available ATP: type II fibers must rely on the minute-by-minute replenishment of the usable pool of ATP from glycolysis, while type I fibers must regenerate the usable pool of ATP from phosphocreatine through a creatine phosphokinase-mediated process.

Animals↗
PubMed1999

Isotope edited product ion assignment by alpha-N labeling of peptides with [2H3(50%)]2,4-dinitrofluorobenzene.

An isotopic modification of Sanger's method for identifying peptide N-termini has been developed to assist peptide sequencing by tandem mass spectrometry. Tryptic peptides, such as Val-His-Leu-Thr-Pro-Val-Glu-Lys, are derivatized with an equimolar mixture of 2,4-dinitrofluorobenzene and [2H3]2,4-dinitrofluorobenzene. Under optimized derivatization conditions, the alpha-amino group could be derivatized while the epsilon-amine of the lysine side chain and the imidazole of histidine remained underivatized. The alpha-dinitrophenyl modified peptides were characterized by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) and liquid chromatography (LC)-ESI-MS. The [M + H]+ ions showed a doublet pattern with a delta m/z of 3 and the [M + 2H]2+ ions were recognized as doublets with a delta m/z of 1.5. MS/MS was employed where both isotopic [M + 2H]2+ ions were alternately subjected to collision-induced dissociation in the second quadrupole. Fragmentation in the ionization source generated identical product ion patterns that were observed during fragmentation in the second quadrupole. In the product ion mass spectra, the N-terminal a and b ions (no c ion observed) are doublets with a delta m/z of 3 or 1.5, while the C-terminal y and z ions (no x ion observed) are singlets appearing at identical masses. Thus, the product ions containing the N-terminus derivatized with a dinitrophenyl group are unequivocally distinguished from the product ions containing the C-terminus. The dinitrophenyl modification generally enhanced the production of a and b ions without diminishing y and z ion yields.

Amino Acid Sequence↗
PubMed1985

[The micelle catalysis of cetyltrimethyl ammonium bromide on phenylalanine arylation by 2,4-dinitrofluorobenzene].

The reaction of phenylalanine with 2,4-dinitrofluorobenzene is increased by substances forming micells. As against the basic test, the increase is not dependent on conditions using solutions of 2.42 X 10(-3) mol phenylalanine and 1.62 X 10(-4) mol dinitrofluorobenzene the rate constant of the reaction proportionally increases by addition of 6.2 X 10(-4) to 4.4 X 10(-3) mol cetyltrimethyl ammonium bromide solution, then remains constant and finally decreases with further increase of the concentration. These curves could continuously be registered with varying pH values, although the basic and the catalytic reaction rate of both the reactions will increase according to the pH value with any buffer. A further increase of the basic and the catalyzed reactions can be enabled by increasing the temperature. The validity of the Arrhenius equation could be proved.

Catalysis↗
PubMed1980

[Mechanism of action of 2,4-dinitrofluorobenzene on bacterial luminescence in vitro].

2,4-Dinitrofluorobenzene (DNFB) changes the parameters of the bioluminescent reaction involving two enzymes, i.e. NADH: FMN-oxidoreductase and luciferase, by decreasing the maximal intensity of luminescence and increasing the time of maximal intensity. Modification of the proteins does not affect the changes of the reaction parameters. The effects of DNFB on each one of the reactions, i.e. reduction of FNM and light emission, were studied. DNFB does not inhibit the reaction of FMN reduction. It was shown that the mechanism of DNFB effect of bioluminescence consists in uncoupling of FMN reduction and light emission due to competitive inhibition of the FMNH2 active center by 2,4-dinitrofluorobenzene.

Binding, Competitive↗
PubMed1997

Histopathologic features of T-cell mediated colonic injury induced with 2,4-dinitrofluorobenzene in BALB/c mice.

The aim of the study was to reveal the histopathologic features of intestinal inflammation as demonstrated in BALB/c mice, using the challenge of 2,4-dinitrofluorobenzene (DNFB) with or without previous sensitization. Forty mice were randomized into 5 groups. Two groups of animals were treated with rectal enema of 0.2% or 1.0% of 2,4-dinitrofluorobenzene solution. Third group was pretreated with 2 sensitizing doses of DNFB. Two control groups were treated with PBS or acetone and vehicle enema only (acetone and olive oil). In order to assess the extent of colonic inflammation and damage, a histopathologic score scale was developed. In contrast to scanty superficial ulcerations and mild edema observed in the control group of animals, edema, ulcerations, hemorrhage, necrosis and infiltration of inflammatory cells were observed in experiment groups treated with enema of DNFB. Total score of lesion as well almost all inflammatory parameters of injury observed were highest in previously sensitized animals. The results of this study clearly demonstrated the pattern of colonic inflammation induced with DNFB using the histopathologic scoring scale system.

Animals↗
PubMed1982

Soluble factors in tolerance and contact sensitivity to 2,4-dinitrofluorobenzene in mice. IV. Characterization of migration inhibition factor-producing lymphocytes and genetic requirements for activation.

The production of migration inhibition factor (MIF) in vitro by lymph node cells from mice with contact sensitivity to 2,4-dinitrofluorobenzene (DNFB) was investigated. MIF activity of cell-free culture supernatants was measured using a micro, indirect "hanging-drop" assay system. We found that DNFB-sensitized lymph node cells are stimulated to produce MIF by co-culture with DNP-labeled spleen cells or splenic adherent cells. The stimulation was quantitatively antigen-specific, as co-culture with TNP-spleen cells or TNP-splenic adherent cells induced only low levels of MIF activity. Pretreating the immune lymph node cells with different antisera plus complement, before addition of DNP-spleen cells, showed that MIF production is dependent on Ia- T cells. Additional experiments showed that in order for the T cells to be stimulated, homology at the I-A subregion of the major histocompatibility complex between the T cells and DNP-spleen cells is required. Collectively, these results correlate with our previous finding that transfer of contact sensitivity is mediated by Ia- T cells and indicate that both tests, i.e., transfer in vivo and MIF production in vitro, are measuring effector functions of the same T cell subset.

Animals↗
PubMed1995

Major histocompatibility complex class I-restricted CD8+ T cells and class II-restricted CD4+ T cells, respectively, mediate and regulate contact sensitivity to dinitrofluorobenzene.

Contact sensitivity (CS) is a form of delayed-type hypersensitivity to haptens applied epicutaneously and is thought to be mediated, like classical delayed-type hypersensitivity responses, by CD4+ T helper-1 cells. The aim of this study was to identify the effector T cells involved in CS. We studied CS to the strongly sensitizing hapten dinitrofluorobenzene (DNFB) in mice rendered deficient by homologous recombination in either major histocompatibility complex (MHC) class I, MHC class II, or both, and which exhibited deficiencies in, respectively, CD8+, CD4+, or both, T cells. MHC class I single-deficient and MHC class I/class II double-deficient mice, both of which have a drastic reduction in the number of CD8+ T cells, were unable to mount a CS response to DNFB. In contrast, both MHC class II-deficient mice and normal mice treated with an anti-CD4 monoclonal antibody (mAb) developed exaggerated and persistent responses relative to heterozygous control littermates. Furthermore, anti-CD8 mAb depletion of class II-deficient mice totally abolished their ability to mount an inflammatory response to DNFB. Removal of residual CD4+ T cells in class II-deficient mice by anti-CD4 mAb treatment did not diminish the intensity of CS. These data clearly demonstrate that class I-restricted CD8+ T cells are sufficient for the induction of CS to DNFB, and further support the idea that MHC class II-restricted CD4+ T cells down-regulate this inflammatory response.

Animals↗
PubMed1996

Lack of oral tolerance but oral priming for contact sensitivity to dinitrofluorobenzene in major histocompatibility complex class II-deficient mice and in CD4+ T cell-depleted mice.

Oral tolerance is defined by immune unresponsiveness after oral administration of soluble antigens and by antigen-specific inhibition of peripheral immune responses induced by prior antigen feeding. The aim of this study was to investigate the implication of the major histocompatibility complex (MHC) class II presentation pathway to CD4+ T cells in oral tolerance of contact sensitivity (CS) to the hapten dinitrofluorobenzene (DNFB). We used MHC class II knockout (AB0/0) and invariant chain knockout (Ii0/0) mice, which have, respectively, a total or partial defect in class II-restricted activation of CD4+ T cells, as well as normal C57BL/6 mice depleted of CD4+ T cells by injection of a specific antibody. Intragastric administration of DNFB prior to skin sensitization induced specific inhibition of contact sensitivity to DNFB in A beta +/0 and Ii+/0 heterozygotes comparable to that observed in C57BL/6 mice. In contrast, no oral tolerance was observed in either MHC class II-deficient A beta 0/0 and Ii0/0 homozygote mutants or in syngeneic anti-CD4-depleted C57Bl/6 mice. Moreover, a single oral administration of DNFB, without skin sensitization, could prime A beta 0/0, Ii0/0 as well as anti-CD4-depleted C57BL/6 mice for DNFB-specific CS. These findings demonstrate that the class II/CD4 pathway is involved in oral tolerance manifested both as the inhibition of CS by hapten feeding prior to skin sensitization, and as immune unresponsiveness of normal mice to oral administration of hapten. Furthermore, our data provide evidence that a single oral feeding with DNFB is able to prime mice for hapten-specific CS, provided that the class II/ CD4 pathway is bypassed.

Administration, Oral↗
PubMed1995

Tumor-promoting activity of 2,4-dinitrofluorobenzene.

The aim of this study was to determine whether the skin-sensitizing agent 2,4-dinitrofluorobenzene (DNFB) would elicit the same morphological and biochemical events that are characteristic of 12-O-tetradecanoylphorbol-13-acetate (TPA). While single applications of 0.1% or 0.2% DNFB produced only mild epidermal hyperplasia, multiple applications produced pronounced hyperplasia. Compared with TPA, a single application of DNFB produced small increases in ODC activity, although a second DNFB treatment produced a greater response. Both DNFB and TPA caused marked induction of ODC, c-fos and c-jun mRNA. Vascular permeability increased significantly in response to DNFB, such that after 15 hr the response was quantitatively the same as for TPA. Repeated TPA produced the same response as a single application, but repeated DNFB resulted in a response that was half that of TPA. In contrast to TPA, DNFB failed to activate partially purified protein kinase C (PKC), although it did cause transient down-regulation of activity 15 hr after treatment. The ability of DNFB to induce ODC activity, however, was unaffected by prior down-regulation of PKC. DNFB was also shown to promote tumors in initiated SSIN mice. Twice-weekly applications of 0.1% or 0.2% DNFB resulted in approximately 65% and 85% of the mice developing an average of 2.0 or 3.2 tumors each, respectively. These results demonstrate that DNFB elicits many of the same changes as TPA and that it does so in a PKC-independent manner.

Animals↗
PubMed1984

Colorimetric determination of gentamicin, kanamycin, tobramycin, and amikacin aminoglycosides with 2,4-dinitrofluorobenzene.

The reaction of 2,4-dinitrofluorobenzene (Sanger's reagent) is used to form colored products with aminoglycoside antibiotics. Stopping the progress of the reaction with acid after a fixed time allows aqueous solubility to be maintained while discharging any color due to excess reactant. The choice of an appropriate analytical wavelength results in adherence to Beer's law. Although this colorimetric method is not expected to be stability-indicating, it is convenient and should be useful in content uniformity determinations for pharmaceutical dosage forms (e.g., ointments).

Amikacin↗
PubMed2001

The determination of draining lymph node cell cytokine mRNA levels in BALB/c mice following dermal sodium lauryl sulfate, dinitrofluorobenzene, and toluene diisocyanate exposure.

Differential modulation has been demonstrated in interleukin-4 (IL-4), IL-10, and interferon gamma (IFN-gamma) mRNA and protein secretion patterns of cells isolated from the draining lymph nodes of mice following exposure to T cell and respiratory sensitizers. Using a multiprobe ribonuclease protection assay, the following investigation examined the mRNA expression patterns of multiple cytokines associated with respiratory sensitization for modulation following exposure to chemicals known primarily to induce irritation (sodium lauryl sulfate), respiratory sensitization (toluene diisocyanate), or T cell-mediated hypersensitivity (dinitrofluorobenzene) responses. On days 0 and +5 female BALB/c mice were exposed to either test article or vehicle on the shaven dorsal lumbar region; on days +10 through +12 the mice received test article on the dorsal aspect of each ear. On day +13 animals were euthanized, draining lymph nodes were excised, and mRNA was isolated immediately or following 24 or 48 h of culture in the presence or absence of concanavalin (Con) A. Differential expression of cytokine mRNA was most notable following 24 h incubation with Con A. Modulation of IL-4, -10, and IFN-gamma following chemical exposure was consistent with previous studies. In addition, IL-9, -13, and -15 were significantly elevated only following toluene diisocyanate exposure. Further investigations of these cytokines may provide additional insight into the mechanisms of chemically induced respiratory sensitization and provide endpoints for the detection of a chemical's ability to elicit IgE-mediated hypersensitivity responses.

Administration, Topical↗
PubMed1993

Lymphokine profiles in contact sensitivity induced by dinitrofluorobenzene and tolerance induced by dinitrothiocyanobenzene.

We determined the lymphokines involved in the immune response to epicutaneously applied dinitrofluorobenzene (DNFB), a sensitizer, and dinitrothiocyanobenzene (DNTB), a tolerogen. Hapten-dependent T-cell proliferation and production of interleukin-2, interleukin-3 and interleukin-4 by lymph node cells (LNC) in mice painted with these cross-reactive haptens were measured by specific lymphokine assays. Proliferation of LNC in tolerized animals was lower than in sensitized mice. LNC from DNTB-treated mice produced lower amounts of interleukin-2, interleukin-3 and interleukin-4 than cells from DNFB-painted mice. These results may explain hapten-specific tolerance induced by DNTB which results in deficient production of both type 1 T-helper cell (Th1) and type 2 T-helper cell (Th2) lymphokines in response to hapten re-exposure. Deficient interleukin-4 production by cells from tolerized mice was corrected by the addition of exogenous interleukin-2. The suppressor function of adoptively transferred T cells from animals tolerized with dinitrothiocyanobenzene may be related to a shift in the balance of Th1 and Th2 lymphokines in favour of the latter, since recipient T cells might provide the source of interleukin-2 that induces interleukin-4 production by donor T cells.

Animals↗
PubMed1981

Combined staining procedures for cytophotometry of protein and DNA Feulgen-Naphthol Yellow S and dinitrofluorobenzene-Feulgen.

A comparison has been made between dinitrofluorobenzene (DNFB) and Naphthol Yellow S (NYS) as protein stains in combination with the pararosaniline-SO2 Feulgen procedure. Chicken erythrocytes were used as test cells. Cytophotometric measurements were made using a Zeiss scanning stage cytophotometer coupled to a PDP 11/10 minicomputer using the BICOSCAN program to obtain values for protein per cell, protein per "nuclear area' and DNA per nucleus. With 5N HCl as the Feulgen hydrolysis agent, DNFB staining, applied before the Feulgen procedure, was found to be unaffected by hydrolysis conditions required to give optimum Feulgen staining and showed only small losses after longer hydrolysis times. On the other hand measurements of NYS staining, of necessity applied after the Feulgen procedure, seem to be susceptible to the duration of Feulgen hydrolysis. This susceptibility is probably due to the interaction of the DNA phosphates with the basic amino acid residues, potential binding sites for NYS. Since the degree of this interaction may be variable, it is argued that NYS binding will measure the available basicity of proteins at the time of staining but no specific protein fraction. DNFB binding is unaffected by DNA-protein interactions and therefore can give a more reliable measure of "nuclear' protein, particularly in conjunction with Feulgen-DNA measurements.

Animals↗
PubMed1991

Cellular and subcellular distribution of 2,4-dinitrophenyl groups in mouse epidermis and regional lymph nodes after epicutaneous application of 2,4-dinitrofluorobenzene.

The cellular and subcellular distribution of 2,4-dinitrophenyl (DNP) groups in the epidermis and regional lymph nodes of the mouse was investigated after epicutaneous application of 2,4-dinitrofluorobenzene (DNFB) to sensitized and non-sensitized mice. The peroxidase-antiperoxidase method and the immunogold technique were used to visualize the DNP groups at both light and electron microscopic levels. The highest intensity of immunolabelling was found on tonofilaments of keratinocytes present in the upper layers of the epidermis. On the other hand, in vitro experiments showed that DNFB has the capacity to bind keratin which, together with immunocytochemistry, suggests that this molecule may be one of the skin protein carriers for DNFB. In addition, intense immunostaining for DNP was observed in the Golgi area of some epidermal Langerhans cells. Cells immunoreactive to DNP were also observed in the marginal sinus of cervical lymph nodes 6, 12 and 24 h after challenge. Immunoelectron microscopy revealed immunoreactive DNP groups in phagosomes of Langerhans cells at this site. The present findings support the hypothesis that the hapten DNFB penetrates passively into the cytoplasm of Langerhans cells, concentrates in the Golgi area and, during the migration of Langerhans cells to the lymph nodes, it is probably processed in the lysosomes before its presentation to T lymphocytes.

Animals↗
PubMed2002

Differential activation of nuclear factor kappa B subunits in a skin dendritic cell line in response to the strong sensitizer 2,4-dinitrofluorobenzene.

Dendritic cell (DC) maturation is essential for the initiation of T-dependent immune responses. Nuclear factor kappa B (NF-kappaB) transcription factors are ubiquitously expressed signalling molecules, known to regulate the transcription of a large number of genes involved in immune responses, including cytokines and cell surface molecules. In this work, we studied the time-dependent activation of five members of the NF-kappaB family, p50, p52, p65, RelB and cRel, in a mouse skin DC line in response to stimulation with the strong sensitizer, 2,4-dinitrofluorobenzene (DNFB). Western blot assay revealed that exposure of fetal skin DC (FSDC) to DNFB induced the degradation of the inhibitor of NF-kappaB (IkappaB). Three out of its five members, i.e. p50, p52, and RelB, were similarly activated upon DNFB stimulation, with subsequent translocation of these subunits from the cytosol to the nucleus, but with different kinetics. In contrast, p65 expression was diminished in both the nucleus and the cytosol. The electrophoretic mobility shift assay (EMSA) showed that exposure of FSDC to DNFB induced DNA binding to NF-kappaB. Together, these results show that DNFB differentially activates the various members of the NF-kappaB family in skin DC.

Animals↗
PubMed1983

Inhibition of protein and lipid synthesis in muscle by 2,4-dinitrofluorobenzene, an inhibitor of creatine phosphokinase.

The incorporation of [3H]-valine into protein and [14C]-acetate into lipid was measured in rat diaphragm and hepatocytes after pretreatment of the tissue with 2,4-dinitrofluorobenzene (FDNB), an inhibitor of creatine phosphokinase (CPK) (EC2.7.3.2). The activity of CPK was also measured. Increasing concentrations of FDNB inhibited protein and lipid synthesis in muscle in parallel with the inhibition of CPK activity. In hepatocytes, which have little CPK activity, similar concentrations of FDNB had no effect on protein synthesis and little effect on lipid synthesis. The possible role of CPK and the creatine phosphate shuttle in muscle metabolism is discussed.

Animals↗