Topical Application Of Dinitrochlorobenzene Research Articles

Saikosaponin A Recovers Impaired Filaggrin Levels in Inflamed Skin by Downregulating the Expression of FRA1 and c-Jun.

Filaggrin (FLG) is an essential structural protein expressed in differentiated keratinocytes. Insufficient FLG expression contributes to the pathogenesis of chronic inflammatory skin diseases. Saikosaponin A (SSA), a bioactive oleanane-type triterpenoid, exerts anti-inflammatory activity. However, the effects of topically applied SSA on FLG expression in inflamed skin remain unclear. This study aimed to evaluate the biological activity of SSA in restoring reduced FLG expression. The effect of SSA on FLG expression in HaCaT cells was assessed through various biological methods, including reverse transcription PCR, quantitative real-time PCR, immunoblotting, and immunofluorescence staining. TNFα and IFNγ decreased FLG mRNA, cytoplasmic FLG protein levels, and FLG gene promoter-reporter activity compared to the control groups. However, the presence of SSA restored these effects. A series of FLG promoter-reporter constructs were generated to investigate the underlying mechanism of the effect of SSA on FLG expression. Mutation of the AP1-binding site (mtAP1) in the -343/+25 FLG promoter-reporter abrogated the decrease in reporter activities caused by TNFα + IFNγ, suggesting the importance of the AP1-binding site in reducing FLG expression. The SSA treatment restored FLG expression by inhibiting the expression and nuclear localization of FRA1 and c-Jun, components of AP1, triggered by TNFα + IFNγ stimulation. The ERK1/2 mitogen-activated protein kinase signaling pathway upregulates FRA1 and c-Jun expression, thereby reducing FLG levels. The SSA treatment inhibited ERK1/2 activation caused by TNFα + IFNγ stimulation and reduced the levels of FRA1 and c-Jun proteins in the nucleus, leading to a decrease in the binding of FRA1, c-Jun, p-STAT1, and HDAC1 to the AP1-binding site in the FLG promoter. The effect of SSA was evaluated in an animal study using a BALB/c mouse model, which induces human atopic-dermatitis-like skin lesions via the topical application of dinitrochlorobenzene. Topically applied SSA significantly reduced skin thickening, immune cell infiltration, and the expression of FRA1, c-Jun, and p-ERK1/2 compared to the vehicle-treated group. These results suggest that SSA can effectively recover impaired FLG levels in inflamed skin by preventing the formation of the repressor complex consisting of FRA1, c-Jun, HDAC1, and STAT1.

Prebiotics supplementation modulates pre-weaning stress in male cattle calves by improving growth performance, health scores and serum biomarkers

Neonatal calves are prone to gastrointestinal infections and microbial dysbiosis that lead to high morbidity and mortality. Prebiotics can be used to mitigate the adverse effects of gut diseases and microbial dysbiosis. Forty male Holstein-Friesian calves (2 ± 1 day old) were divided into four dietary treatments: control (milk without prebiotics), YCW-2, YCW-4 (milk containing 2 or 4 g/day/calf of yeast cell wall, respectively), and cMOS (milk containing commercial mannan-oligosaccharides 4 g/day/calf). Milk intake, feed intake, and health scores were recorded daily, whereas body weight, dry matter intake (DMI), and body measurements were recorded weekly. Feed efficiency (FE) was determined at the end of the trial (eight weeks). Cell-mediated immunity (CMI) was assessed by the topical application of dinitrochlorobenzene. Blood samples were collected fortnightly to determine glucose, non-esterified fatty acid (NEFA), blood urea nitrogen (BUN), and beta-hydroxybutyric acid (βHBA). Cell wall supplemented calves had significantly higher (P < 0.05) final body weights, DMI, and body measurements along with improved (P < 0.05) faecal scores. Feed intake was higher (P < 0.05) in both the YCW-supplemented calves. Glucose was lower (P < 0.001), whereas BUN and βHBA were significantly higher in the YCW-2 animals. No differences were observed in FE, CMI, and NEFA between all the experimental animals. The yeast cell wall (2 g) may have the potential to improve the growth performance and health status of neonatal calves.

Effect of 6'-acetylpaeoniflorin on dinitrochlorobenzene-induced allergic contact dermatitis in BALB/c mice.

Allergic contact dermatitis (ACD) is a classical experimental model of allergic inflammatory skin disease, which is a delayed-type hypersensitivity reaction that is mediated by hapten-specific T lymphocytes. The goal of this study was to investigate the pharmacokinetic profiles of 6'-acetylpaeoniflorin (6-AP) and the effect of 6-AP on the ACD model. 6-AP was synthesized from paeoniflorin (Pae) via acetylation, and the structure was confirmed. There were statistically significant differences in the pharmacokinetic parameters including t 1/2α , t 1/2β , AUC, MRT and C max among the animals that were orally administered Pae and 6-AP. An ACD model was induced using immunization with dinitrochlorobenzene (DNCB) in BALB/c mice. The mice were orally administered 6-AP (35, 70 and 140mg/kg/d), Pae (70mg/kg/d) and prednisone (Pre, 5mg/kg/d) from day 1 to day 7 after immunization. The results indicated that the topical application of DNCB to the skin provoked obvious inflammatory responses. 6-AP significantly inhibited ear swelling and decreased inflammatory cell infiltration and epidermal keratinization. Additionally, 6-AP observably alleviated the hyperplasia of red pulp and germinal center appearance, decreased the spleen index and inhibited splenocyte proliferation in the ACD model compared to that of Pae. Furthermore, the study indicated that 6-AP could increase the IL-10 level, while simultaneously reducing the IL-17 level in splenocytes. In summary, these results suggest that 6-AP has a significantly higher anti-inflammatory effect than Pae and that 6-AP might be a candidate for the treatment of allergic skin diseases.

Paeoniflorin inhibits inflammatory responses in mice with allergic contact dermatitis by regulating the balance between inflammatory and anti-inflammatory cytokines

Paeoniflorin (Pae) was previously reported to inhibit inflammation in the skin of mice with allergic contact dermatitis (ACD); however, the mechanism remains unclear. The primary purpose of this study was to investigate the effect of Pae on the regulation of cytokine production in a murine model of ACD. ACD was induced in the mice by repeated application of dinitrochlorobenzene (DNCB) to their skin. Cutaneous inflammation was evaluated by measuring ear swelling and by histological examination. The cytokine levels were measured by enzyme-linked immunosorbent assays. The results showed that topical application of DNCB caused obvious swelling and inflammatory cell infiltration. Treatment with Pae (70 or 140mg/kg/d) significantly inhibited the cutaneous inflammation and decreased thymocyte proliferation in the mice with ACD. Additional data indicated that Pae increased interleukin-4 (IL-4) and IL-10 production but reduced IL-2 and IL-17 levels in the serum as well as in thymocyte and splenocyte culture supernatants. As expected, IL-2 and IL-17 levels in the serum displayed a significant positive correlation with the severity of skin inflammation. In contrast, IL-4 and IL-10 levels were negatively correlated with the inflammation. The anti-inflammatory action of Pae in the murine model of ACD may be related to its regulation of an imbalanced cytokine production.

Systemic immunomodulatory effects of topical dinitrochlorobenzene (DNCB) in rats. Activity of peripheral blood polymorphonuclear cells

Topical application of dinitrochlorobenzene (DNCB) is employed in the immunotherapy of skin diseases. Activation of T-cell mediated immune responses (Th1/type1) is the supposed mechanism of the clinical effect of DNCB, but there are no data concerning innate/inflammatory mechanisms. In this study, the effect of repeated topical DNCB application on peripheral blood polymorphonuclear (PMN) leukocytes has been examined in two rat strains which differ in the propensity to mount Th1/type1 or Th2/type2 responses. The dynamics of changes in PMN numbers and effector activities (respiratory burst, nitric oxide production and myeloperoxidase content), as well as in adhesion and TNF-α production following the rat skin sensitization with low (0.4%) and high (4%) DNCB doses were measured. Both priming and activation of PMNs were observed following skin sensitization with DNCB, with dose-dependent as well as time-dependent differences in some PMN activities. Obtained data might be relevant for understanding the immune mechanisms of topical DNCB therapy.

NCB-02 (standardized Curcumin preparation) protects dinitrochlorobenzene-induced colitis through down-regulation of NFκ-B and iNOS

To evaluate the efficacy and mechanism of action of NCB 02, a standardized Curcumin preparation, against 2, 4 dinitrochlorobenzene (DNCB) induced ulcerative colitis in rats. Ulcerative colitis was induced in male rats by sensitizing with topical application of DNCB in acetone for 14 d and intra-colonol challenge with DNCB on day 15. A separate group of animals with vehicle treatment in similar fashion served as control group. Colitis rats were divided into different groups and treated with NCB-02 at doses of 25, 50 and 100 mg/kg b.wt p.o. for 10 d. Sulfasalazine at a dose of 100 mg/kg b.wt for 10 d served as a reference group. On day 10 after respective assigned treatment, all the animals were euthanized and the length of the colon, weight of entire colon and distal 8 cm of the colon were recorded. The distal part of the colon was immediately observed under a stereomicroscope and the degree of damage was scored. Further distal 8 cm of the colon was subject to the determination of colonic myeloperoxidase (MPO), lipid peroxidation (LPO) and alkaline phosphatase (ALP) activities. A small piece of the sample from distal colon of each animal was fixed in 10% neutral buffered formalin and embedded in paraffin wax and sectioned for immunohistochemical examination of NFkappa-B and iNOS expression. NCB-02 showed a dose dependent protection against DNCB-induced alteration in colon length and weight. NCB-02 treatment also showed a dose dependent protection against the elevated levels of MPO, LPO and ALP, induced by DNCB. NCB-02 demonstrated a significant effect at a dose of 100 mg/kg b.wt., which was almost equipotent to 100 mg/kg b.wt. of sulfasalazine. Treatment with sulfasalazine and curcumin at a dose of 100 mg/kg b.wt. inhibited the DNCB-induced overexpression of NFkappa-B and iNOS in the colon. Curcumin treatment ameliorates colonic damage in DNCB induced colitic rats, an effect associated with an improvement in intestinal oxidative stress and downregulation of colonic NFkappa-B and iNOS expression.

Assessment of Preferential T-Helper 1 or T-Helper 2 Induction by Low Molecular Weight Compounds Using the Local Lymph Node Assay in Conjunction with RT-PCR and ELISA for Interferon-γ and Interleukin-4

The local lymph node assay (LLNA) is a new and promising test in mice used to identify contact allergens by means of dermal exposure. Experimentally this assay, which comprises a sensitizing phase only, is also used to identify respiratory allergens. Another, experimentally used test in mice to identify allergens is also based on dermal exposure, but comprises both a sensitizing and effector phase. In this latter test, it has been shown that contact allergens preferentially induce a T-helper 1 (TH1) response, whereas respiratory allergens preferentially induce a T-helper 2 (TH2) response. These responses can be discriminated on the basis of cytokine production, such as IFN-γ, which is produced by TH1 cells, and IL-4, which is produced by TH2 cells. The aim of the study was to establish whether the LLNA was sufficient to not only identify allergens but also mark them as either a contact or a respiratory allergen. To this end, LLNA responses to the contact allergen dinitrochlorobenzene (DNCB) and the respiratory allergen trimellitic anhydride (TMA) were determined using IFN-γ and IL-4 mRNA expression and production as parameters. Topical application of TMA resulted in a threefold higher lymphocyte proliferation compared to DNCB 3 and 5 days after the first application, while a similar proliferation was found from Day 7 and onward. RT-PCR showed a similar induction of IFN-γ and IL-4 mRNA expression. While both DNCB and TMA induced IFN-γ production, TMA but not DNCB induced IL-4 production. Thus, only IL-4 production seemed a suitable parameter to discriminate between the two compounds. In a second study, the respiratory allergens toluene-2,4-diisocyanate (TDI) and phthalic anhydride (PA) were also assayed 7 days after the first application. Topical application of DNCB and PA resulted in a similar lymphocyte proliferation, while application of TMA and TDI resulted in a 1.8-fold higher proliferation. IFN-γ production was similar for DNCB, TMA, and TDI, and fourfold lower for PA, while IL-4 production was similar for TMA, TDI, and PA, and 24-fold lower for DNCB. In summary, both studies showed induction of IL-4 production by respiratory allergens, with little or no induction by the contact allergen, holding promise for the possibility of identifying respiratory allergens within the LLNA by measuring IL-4 production 7 days after the first application.

Improvement of lichen nitidus after topical dinitrochlorobenzene application

Recently, a relationship between immunologic alterations and the development of lichen nitidus (LN) lesions has been described. We treated LN lesions with topical dinitrochlorobenzene (DNCB) application in a patient with peripheral CD4 + T lymphocytopenia. After 4 months, the eruption cleared. We investigated histologically and immunohistochemically biopsy specimens obtained before and after treatment. Our result suggests that local alterations of the pattern of cells and cytokines by topical DNCB application could have contributed to the resolution of the LN lesions.(J Am Acad Dermatol 1998;39:305-8.)

Improved results of delayed-type hypersensitivity skin testing in HIV-infected patients treated with topical dinitrochlorobenzene

Background: Infection with HIV results in progressive deterioration of cell-mediated immunity, with consequent impairment of delayed-type hypersensitivity (DTH) skin reactivity. Topical dinitrochlorobenzene (DNCB) is thought to increase cellular immune function in HIV infection. Objective: Our goal was to evaluate changes in DTH skin reactivity of HIV-infected patients who were given DNCB therapy. Methods: DTH skin testing and lymphocyte subset analysis were performed in five HIV-infected patients before the start of DNCB treatment and after 3 months of therapy. Topical DNCB application was carried out by the patients according to a standardized protocol. Results: DNCB therapy enhanced DTH skin test responses in four of five patients. Three patients who were anergic before the start of treatment reacted to skin test antigens while using DNCB. The skin test results did not correlate with lymphocyte subset changes. Conclusion: DNCB treatment improves DTH skin test responses in HIV-infected patients, indicating a positive effect on cellular immune function.

Pilot study of topical dinitrochlorobenzene (DNCB) in human immunodeficiency virus infection

Dendritic cells, the primary antigen presenting cells of the human immune system, are heavily infected with human immunodeficiency virus (HIV) in patients with the acquired immunodeficiency syndrome (AIDS). Dinitrochlorobenzene (DNCB) is a contact sensitizing agent that acts as a potent immune modulator of dendritic cells. In this pilot study, we examined the safety and efficacy of topical DNCB application in patients with early HIV disease. Topical DNCB was well tolerated by these patients, with an adverse reaction rate of 10%. CD4 + T-cell counts remained stable with repeated DNCB use. In contrast, CD8 + T-cell counts and natural killer cells increased significantly following DNCB sensitization. This increase in CD8 + T-cell and natural killer cell subsets was accompanied by a decrease in HIV replication, as measured by serum HIV RNA levels. Based on this pilot study, we conclude that topical DNCB is safe in early HIV disease and may decrease viral load via a systemic effect on dendritic cells, CD8 + T-cells and natural killer cells. These results require confirmation in larger controlled trials.

Activation of the Human Immunodeficiency Virus Type 1 Long Terminal Repeat by Skin-Sensitizing Chemicals in Transgenic Mice

Topical dinitrochlorobenzene (DNCB) is often used for evaluating contact skin hypersensitivity in immunocompromised patients. We have determined, in this study, that topical application of DNCB alone, even without induction of contact skin hypersensitivity, was sufficient to observe activation of the human immunodeficiency virus promoter (long terminal repeat) in the skin of an HIV-1 long terminal repeat-luciferase transgenic mouse model. Such treatment might be contra-indicative in patients infected with the human immunodeficiency virus, because in earlier studies DNCB-exposed skin dendritic cells might migrate into draining lymph nodes which play an important role in AIDS pathogenesis.

A simple and effective method of monitoring free skin flaps

A simple and effective method of monitoring a free skin flap transfer when the flap skin color is white in spite of having a good blood circulation is described. It is easy to confirm that the anastomosed vessels are patent or not by visualizing the flap skin color, because erythema will appear after topical application of dinitrochlorobenzene even if skin color is white.

Eosinophil leukocytic response in dinitrochlorobenzene skin hypersensitivity reaction in the chicken

The cutaneous inflammation evoked by topical application of dinitrochlorobenzene in sensitised chickens was studied at 24- and 48-hours post-challenge. A slight increase in vascular permeability was present at 24 hours but absent at 48 hours. Histologically, the reaction comprised oedema and a diffuse infiltration of polymorphonuclear granulocytes in the superficial dermis, and intense perivascular mononuclear infiltrations in the deep dermis. The apparent participation of eosinophils was a noteworthy feature of the reaction.