Mouse Ear Swelling Test Research Articles

Ketoconazole-p aminobenzoic cocrystal, an improved antimycotic drug formulation, does not induce skin sensitization on the skin of BALBc mice.

Fungal infections are a growing global health problem. Therefore, our group has synthetized and characterized an improved antimycotic by co-crystallization of ketoconazole and para-amino benzoic acid, named KET-PABA. The aim was to increase bioavailability, biocompatibility, and efficiency of the parent drug-ketoconazole. Based on our previous results showing the cocrystal improved physical properties, such as stability in suspension, solubility, as well as antimycotic efficiency compared to ketoconazole, the current study investigated the local possible side effects induced on the skin of BALBc mice by the application of KET-PABA cocrystal, in view of a further use as a topically applied antimycotic drug. A specific test (mouse ear-swelling test) was used, combined with the histopathological examination and the measurement of pro and anti-inflammatory cytokines and inflammation mediators. KET-PABA application was safe, without signs of skin sensitization shown by the mouse ear sensitization test, or histopathology. KET-PABA strongly inhibited proinflammatory cytokines such as IL1 α, IL1 β, IL6 and TNF α, and other proinflammatory inducers such as NRF2, compared to vehicle. KET-PABA had no effect on the levels of the anti-inflammatory cytokine IL10, or proinflammatory enzyme COX2 and had minimal effects on the activation of the NF-κB pathway. Overall, KET-PABA application induced no sensitization, moreover, it decreased the skin levels of proinflammatory molecules. The lack of skin sensitization effects on BALBc mice skin along with the inhibition of the proinflammatory markers show a good safety profile for topical applications of KET-PABA and show promise for a further clinical use in the treatment of cutaneous mycosis.

Evaluation of 4-methylcyclohexanemethanol (MCHM) in a combined irritancy and Local Lymph Node Assay (LLNA) in mice

4-Methylcyclohexanemethanol (MCHM) is a flotation reagent used in fine coal beneficiation. On January 9, 2014, crude MCHM, a mixture containing predominantly MCHM, was inadvertently released into the Elk River, a municipal water source that serves about 300,000 people in the Charleston, WV area, resulting in temporary contamination of 15 percent of the state's tap water and causing significant dermal exposure. The current studies were undertaken to determine whether crude MCHM or MCHM has the potential to produce dermal irritancy and/or sensitization. BALB/c female mice were treated daily for 3 consecutive days by direct epicutaneous application of 25 μL of various concentrations of crude MCHM or MCHM to the dorsum of each ear. A mouse ear-swelling test was used to determine irritancy potential and was undertaken in combination with the standardized Local Lymph Node Assay (LLNA) to determine skin sensitizing potential. MCHM was found to produce skin irritation at concentrations above 20% and did not produce sensitization. Crude MCHM also produced irritation, although weaker, and in addition was found to be a weak to moderate skin sensitizer. The results are discussed in terms of potential human health hazard.

Biomimetic synthesis of proline-derivative templated mesoporous silica for increasing the brain distribution of diazepam and improving the pharmacodynamics of nimesulide

Herein a new kind of proline-derivative templated mesoporous silica with curved channels (CMS) was biomimetically synthesized and applied as carrier to improve the drug dissolution and bioavailability of hydrophobic diazepam (DZP) and nimesulide (NMS). Drugs can be incorporated into CMS with high efficiency; during this process, they successfully transformed to amorphous phase. As a result, the dissolution rate of DZP and NMS was significantly improved. Biodistribution study confirmed that CMS converted DZP distribution in mice with the tendency of lung targeting and brain targeting. At 45 min postadministration, the concentrations of DZP in plasma, lung and brain were 8.57-fold, 124.94-fold and 19.55-fold higher from 1:3 DZP/CMS sample than that of pure DZP sample, respectively. At 90 min postadministration, the content of DZP in brain was 62.31-fold higher for 1:3 DZP/CMS sample than that of pure DZP. Besides, the anti-inflammatory and analgesic effects of 1:3 NMS/CMS were systematic evaluated using mouse ankle swelling test (MAST), mouse ear swelling test (MEST) and mouse writhing test (MWT). The results indicated that after incorporating into CMS, the therapeutic effects of NMS were obviously improved, and the inhibition rates of 1:3 NMS/CMS in all pharmacodynamics tests varied from 102.2% to 904.3%.

Oxysophocarpine Ameliorates Carrageenan-induced Inflammatory Pain via Inhibiting Expressions of Prostaglandin E2 and Cytokines in Mice.

Oxysophocarpine is an alkaloid extracted from Sophora alopecuroides. We investigated the analgesic effect of oxysophocarpine on carrageenan-induced inflammatory pain in mice, in order to explore its possible mechanisms. Mouse ear swelling tests and carrageenan-induced paw edema tests were used to investigate the effects of oxysophocarpine on inflammatory pain in mice. Morphological changes on inflamed paw sections were measured by hematoxylin-eosin staining. The mRNA and protein expression of extracellular signal-regulated kinase, phosphorylation of extracellular signal-regulated kinase 1/2, cyclooxygenase-2, tumor necrosis factor α, interleukin-1 beta, interleukin-6 and prostaglandin E2 were investigated by real-time quantitative polymerase chain reaction, immunohistochemistry, western-blot and enzyme-linked immunosorbent assay. In our results, oxysophocarpine shows a significant anti-inflammatory effect in the mouse ear swelling test. Oxysophocarpine also significantly reduced the paw edema volume and improved mechanical allodynia threshold value on carrageenan-induced inflammatory pain, as well as relieved paw tissues inflammatory damage and reduced the numbers of neutrophils in mice. Oxysophocarpine significantly suppressed over-expression of cyclooxygenase-2, tumor necrosis factor α, interleukin-1 beta, interleukin-6 and prostaglandin E2, and inhibited the over-phosphorylation of extracellular signal-regulated kinase 1/2. Based on these findings we propose that oxysophocarpine attenuates inflammatory pain by suppressing the levels of phosphorylation of extracellular signal-regulated kinase 1/2, cyclooxygenase-2, prostaglandin E2, tumor necrosis factor α, interleukin-1 beta and interleukin-6.

Reduced Inflammatory Threshold Indicates Skin Barrier Defect in Transglutaminase 3 Knockout Mice

Recently, a transglutaminase 3 knockout (TGM3/KO) mouse was generated that showed impaired hair development, but no gross defects in the epidermal barrier, although increased fragility of isolated corneocytes was demonstrated. Here we investigated the functionality of skin barrier in vivo by percutaneous sensitization to FITC in TGM3/KO (n=64) and C57BL/6 wild-type (WT) mice (n=36). Cutaneous inflammation was evaluated by mouse ear swelling test (MEST), histology, serum IgE levels, and by flow cytometry from draining lymph nodes. Inflammation-induced significant MEST difference (P<0.0001) was detected between KO and WT mice and was supported also by histopathology. A significant increase of CD4+ CD25+-activated T cells (P<0.01) and elevated serum IgE levels (P<0.05) in KO mice indicated more the development of FITC sensitization than an irritative reaction. Propionibacter acnes-induced intracutaneous inflammation showed no difference (P=0.2254) between the reactivity of WT and KO immune system. As in vivo tracer, FITC penetration from skin surface followed by two-photon microscopy demonstrated a more invasive percutaneous penetration in KO mice. The clinically uninvolved skin in TGM3/KO mice showed impaired barrier function and higher susceptibility to FITC sensitization indicating that TGM3 has a significant contribution to the functionally intact cutaneous barrier.

Contact sensitizing potential of pyrogallol and 5-amino-o-cresol in female BALB/c mice

Hair dye components such as pyrogallol and cresol have been shown previously to promote allergic reactions such as rashes, dermal inflammation, irritation and dermatitis. The objective of this study was to determine the contact sensitization potential of pyrogallol (PYR) and 5-amino-o-cresol (AOC) when applied dermally to female BALB/c mice. Measurement of the contact hypersensitivity response was initially accomplished using the local lymph node assay. For PYR, significant increases in the proliferation of lymph node cells were observed at concentrations of 0.5% (w/v) and higher. For AOC, borderline increases, albeit significant, in auricular lymph node cell proliferation were observed at 5% and 10%. Results from the irritancy assay suggested that PYR, but not AOC, was an irritant. To further delineate whether PYR was primarily an irritant or a contact sensitizer, the mouse ear swelling test (MEST) was conducted. A significant increase in mouse ear thickness was observed at 72h following challenge with 0.5% PYR in mice that had been sensitized with 5% PYR. In contrast, no effects were observed in the MEST in mice sensitized and challenged with the highest achievable concentration of AOC (10%). Additional studies examining lymph node subpopulations and CD86 (B7.2) expression by B cells further support the indication that PYR was a sensitizer in BALB/c mice. The results demonstrate that PYR is both a sensitizer and an irritant in female BALB/c mice. However, the contact sensitization potential of AOC is minimal in this strain of mouse.

Route-dependent systemic and local immune effects following exposure to solutions prepared from titanium dioxide nanoparticles

Nanoparticle titanium dioxide (nano-TiO2) is a white pigment widely used in foods, sunscreens, and other cosmetic products. However, it remains unclear whether exposure to nano-TiO2 results in immunosuppressive effects or induces a contact hypersensitivity response. To address these data gaps, studies were conducted with the hypothesis that nano-TiO2 exposure could alter immune responses. After 28 days of oral gavage, nano-TiO2 (1.25–250 mg/kg in 0.5% methylcellulose) produced no significant effects on innate, humoral, or cell-mediated immune functions in female B6C3F1 mice. Furthermore, there were no effects on the weights of selected organs (spleen, thymus, liver, lung, and kidneys with adrenals). Following dermal exposure on the ears for 3 days, nano-TiO2 (2.5–10% w/v in 4:1 acetone:olive oil) did not affect auricular lymph node cell proliferation, although an irritancy response was observed following treatment with 5% and 10% nano-TiO2. Dermal sensitization (2.5–10%) on the back and subsequent challenge (10%) on the right ear with nano-TiO2 produced no significant effects on percentage ear swelling in the Mouse Ear Swelling Test (MEST). However, when nano-TiO2 was injected subcutaneously along the mid-line on top of the head at 125–250 mg/kg (in 0.5% methylcellulose), significant increases in auricular lymph node cell proliferation resulted. These results demonstrate that immune effects of nano-TiO2 exposure are route-of-exposure dependent, and they suggest that irritancy and/or potential hypersensitivity responses may occur following parenteral exposure or dermal administration of nano-TiO2 to compromised skin.

Allergic Skin Inflammation Induced by Chemical Sensitizers Is Controlled by the Transcription Factor Nrf2

Allergic contact dermatitis (ACD) is induced by low-molecular weight electrophilic chemicals and metal ions. Chemical contact sensitizers trigger reactive oxygen species production and provoke electrophilic stress, leading to the accumulation of the transcription factor nuclear-related factor 2 (Nrf2) in innate immune cell types. The objective of this work was to identify the role of Nrf2 in the regulation of ACD. We used the local lymph node assay (LLNA) and the mouse ear swelling test (MEST) to study the role of Nrf2 in both the sensitization and elicitation phase in nrf2 knockout (nrf2(-/-)) and wild-type (nrf2(+/+)) mice. Five chemicals were used: two compounds known to react with cysteine residues, 2,4-dinitrochlorobenzene (DNCB) and cinnamaldehyde (CinA); one sensitizer known to exhibit mixed reactivity to cysteine and lysine residues, isophorone diisocyanate; and one reacting specifically with lysine residues, trimellitic anhydride and croton oil, a well-known irritant. In the MEST assay, DNCB (1 and 2%) induced a significant increase in ear thickness in nrf2(-/-) compared with nrf2(+/+) mice, suggesting a role for Nrf2 in the control of the inflammatory process. When DNCB was used at 0.25 and 0.5% or when mice were treated with CinA, inflammation was found only in nrf2(-/-) mice. In the LLNA, all chemical sensitizers induced an increase of lymphocyte proliferation in nrf2(-/-) compared with nrf2(+/+) mice for the same chemical concentration. These results reveal an important role for Nrf2 in controlling ACD and lymphocyte proliferation in response to sensitizers.

Potential Immunotoxicological Health Effects Following Exposure to COREXIT 9500A during Cleanup of the Deepwater Horizon Oil Spill

Workers involved in the Deepwater Horizon oil spill cleanup efforts reported acute pulmonary and dermatological adverse health effects. These studies were undertaken to assess the immunotoxicity of COREXIT 9500A, the primary dispersant used in cleanup efforts, as a potential causative agent. COREXIT 9500A and one of its active ingredients, dioctyl sodium sulfosuccinate (DSS), were evaluated using murine models for hypersensitivity and immune suppression, including the local lymph node assay (LLNA), phenotypic analysis of draining lymph node cells (DLN), mouse ear swelling test (MEST), total serum immunoglobulin E (IgE), and the plaque-forming cell (PFC) assay. Dermal exposure to COREXIT 9500A and DSS induced dose-responsive increases in dermal irritation and lymphocyte proliferation. The EC3 values for COREXIT 9500A and DSS were 0.4% and 3.9%, respectively, resulting in a classification of COREXIT 9500A as a potent sensitizer and DSS as a moderate sensitizer. A T-cell-mediated mechanism underlying the LLNA was supported by positive responses in the MEST assay for COREXIT and DSS, indicated by a significant increase in ear swelling 48 h post challenge. There were no marked alterations in total serum IgE or B220+/IgE+ lymph-node cell populations following exposure to COREXIT 9500A. Significant elevations in interferon (IFN)-γ but not interleukin (IL)-4 protein were also observed in stimulated lymph node cells. The absence of increases in IgE and IL-4 in the presence of enhanced lymphocyte proliferation, positive MEST responses, and elevations in IFN-γ suggest a T-cell-mediated mechanism. COREXIT 9500A did not induce immunosuppression in the murine model.

Nickel sensitisation in mice: A critical appraisal

The market release of new domestic and industrial chemical and metal products requires certain safety certification, including testing for skin sensitisation. Although various official guidelines have described how such testing is to be done, the validity of the available test models are in part dubious, for which reason regulatory agencies and research aim to further improve and generalise the models for testing of skin sensitisation. We applied a recently published murine model of nickel allergy as to test its applicability in a regulatory setting and to study and better understand the events leading to type-IV hypersensitivity. Nickel was chosen as model hapten since it induces allergic contact dermatitis with high incidence in the general population. Typically, C57BL/6 mice were sensitised and challenged by intradermal applications of nickel, and cutaneous inflammation was analysed by the mouse ear-swelling test, by histology, and by lymphocyte reactivity in vitro. Surprisingly, the study suggested that the skin reactions observed were results of irritant reactions rather than of adaptive immune responses. Non-sensitised mice responded with cutaneous inflammation and in vitro lymphocyte reactivity which were comparable with nickel-sensitised mice. Furthermore, histological examinations as well as experiments in T-cell deficient mice demonstrated that lymphocytes were not involved and that nickel caused an irritant contact dermatitis rather a true allergic type-IV contact dermatitis. The authors question the validity of the described murine model of nickel allergy.

Genetic determinants of sensitivity to beryllium in mice

Chronic beryllium disease (CBD), an irreversible, debilitating granulomatous lung disease is caused by exposure to beryllium. This occupational hazard occurs in primary production and machining of Be-metal, BeO, beryllium - containing alloys, and other beryllium products. CBD begins as an MHC Class II-restricted, TH1 hypersensitivity, and the Human Leukocyte Antigen, HLA-DPB1E69, is associated with risk of developing CBD. Because inbred strains of mice have not provided good models of CBD to date, three strains of HLA-DPB1 transgenic mice in an FVB/N background were developed; each contains a single allele of HLA-DPB1 that confers a different magnitude of risk for chronic beryllium disease: HLA-DPB1*0401 (OR ≈ 0.2), HLA-DPB1*0201 (OR ≈ 3), and HLA-DPB1*1701 (OR ≈ 46). The mouse ear swelling test (MEST) was employed to determine if these different alleles would support a hypersensitivity response to beryllium. Mice were first sensitized on the back and subsequently challenged on the ear. In separate experiments, mice were placed into one of three groups (sensitization/challenge): C/C, C/Be, and Be/Be. In the HLA-DPB1*1701 mice, the strain with the highest risk transgene, the Be/Be group was the only group that displayed significant maximum increased ear thickness of 19.6% ± 3.0% over the baseline measurement (p < 0.05). No significant changes were observed in the other transgenic strains for any treatment condition. In addition, inter-strain differences in response to beryllium in seven inbred strains were investigated through use of the MEST, these included: FVB/N, AKR, Balb/c, C3H/HeJ, C57/BL6, DBA/2, and SJL/J. The FVB/N strain was least responsive, while the SJL/J and C57/BL6 strains were the highest responders. Our results suggest that the HLA-DPB1*1701 transgene product is an important risk factor for induction of the beryllium-sensitive phenotype. This model should be a useful tool for investigating beryllium sensitization.

Toxicity studies on dermal application of plant extract of Plumbago zeylanica used in Ethiopian traditional medicine

Plant-based therapeutic preparations are cyclically returning to complement dermatologic therapy, however, data on the toxicity profile of such plants are lacking. In the present study, Plumbago zeylanica, a medicinal plant commonly used in Ethiopia for skin diseases was subjected to a systematic dermatotoxicity study. To this effect, the dermatotoxicity of 80% methanol extract of the root part of Plumbago zeylanica was investigated in animals following standard procedures for irritation, sensitization, acute toxicity and repeated toxicity tests. Extraction of plant material with 80% methanol resulted in 9.45% of crude extract of Plumbago zeylanica. The skin irritation test on rabbits showed Plumbago zeylanica extract to be a moderate irritant, with a primary irritation index of 2.00. Sensitization test on mice by the Mouse Ear Swelling Test method revealed the extract to be non-sensitizer in a dose range of 4–10 mg/ml and the percent responder was zero. Acute dermal toxicity test on rats did not produce any overt signs of toxicity, except that there was a weight gain difference between the test and control groups of female rats. This was not, however, supported by other parameters, like the absolute and relative organ weights. Repeated dose toxicity test was associated with increased relative testis weight ( P < 0.05) as well as higher values for Blood urea nitrogen and K + ( P < 0.05) in both sexes with the highest dose (1000 mg/kg) group, although histopathological analyses failed to lend support to these observations. Taken together, the dermatotoxicity test results from this study suggest that Plumbago zeylanica toxic effects might be limited to effects like moderate irritation.

Combined dermal exposure to permethrin and cis-urocanic acid suppresses the contact hypersensitivity response in C57BL/6N mice in an additive manner

Cutaneous exposure to the pyrethroid insecticide permethrin significantly suppresses contact hypersensitivity (CH) response to oxazolone in C57BL/6N mice. Additionally, cis-urocanic acid ( cUCA), an endogenous cutaneous chromophore isomerized to its active form following exposure to ultraviolet radiation, modulates cell-mediated cutaneous immune responses. This study describes cutaneous immune alterations following combined topical permethrin and intradermal cUCA exposure. Female C57BL/6N mice were administered 5, 50 or 100 μg cUCA daily for 5 consecutive days. CH was then evaluated by the mouse ear swelling test (MEST) response to oxazolone. Decreased responses of 52.3%, 76.3% and 76.3%, respectively, as compared to controls were observed. Then, mice were co-exposed to 5 μg cUCA daily for 5 days and 1.5, 5, 15, or 25 μL permethrin, on either day 1, 3 or 5 of the cUCA treatment to evaluate combined immunomodulatory effects of the two chemicals, or cUCA daily for 5 days followed by permethrin on day 3, 5, or 7 after the last cUCA injection to demonstrate prolonged immunosuppressive effects. Two days after final treatment, mice were sensitized with oxazolone and MEST was performed. Mice receiving five cUCA injections and permethrin topically on cUCA injection day 1 showed up to 93.3% suppression of MEST compared to vehicle control. CH was suppressed by 87.5%, 86.6% and 74.2% in mice treated with 25 μL permethrin on days 3, 5 and 7 after cUCA, respectively, compared to vehicle control. Taken together, these data indicate co-exposure to cUCA and permethrin profoundly suppresses cell-mediated cutaneous immunity.

Divergent immunological responses following glutaraldehyde exposure

Although Glutaraldehyde (Glut) has been demonstrated to be a moderate contact sensitizer, numerous cases of occupational asthma related to Glut exposure have been reported. The purpose of these studies was to examine the dose–response relationship between Glut exposure and the development of T cell-mediated vs. IgE- mediated responses. Initial evaluation of the sensitization potential was conducted using the local lymph node assay (LLNA) at concentrations ranging from 0.75% to 2.5%. A concentration-dependent increase in lymphocyte proliferation was observed with EC3 values of 0.072% and 0.089% in CBA and BALB/c mice, respectively. The mouse ear swelling test (MEST) was used to evaluate the potential for Glut to elicit IgE (1/2 h post challenge) and contact hypersensitivity (24 and 48 h post challenge) responses. An immediate response was observed in animals induced and challenged with 2.5% Glut, whereas animals induced with 0.1% or 0.75% and challenged with 2.5% exhibited a delayed response 48 h post challenge. IgE-inducing potential was evaluated by phenotypic analysis of draining lymph node cells and measurement of total serum IgE levels. Only the 2.5% exposed group demonstrated a significant increase ( P < 0.01) in the percentage of IgE +B220 + cells and serum IgE. Following 3 days of dermal exposure, a significant increase in IL-4 mRNA in the draining lymph nodes was observed only in the 2.5% exposed group. These results indicate that the development of an immediate vs. a delayed hypersensitivity response following dermal exposure to Glut is at least in part mediated by the exposure concentration.

Langerhans' cells in the murine oral mucosa in the inductive phase of delayed type hypersensitivity with 1-chloro-2, 4-dinitrobenzene.

We created a murine model of delayed-type hypersensitivity (DTH) to 1-chloro-2, 4-dinitrobenzene (DNCB). Using this murine model, we compared oral mucosal sensitization and skin sensitization for the difference in reaction during the elicitation phase. Evaluation of sensitizability, using the mouse ear swelling test (MEST) after oral mucosal or skin sensitization, showed that the ear swelling response peaked 24 h after challenge. The optimal induction concentration was 1.0% in both oral mucosal and skin sensitization, resulting in a positive reaction rate of 100%. However, the ear swelling response 24 h after challenge with the optimal concentration of DNCB (1.0%) was significantly lower in oral mucosal than in skin sensitization. We compared the oral mucosal and skin sensitization sites for the number of Langerhans' cells (LC) and the antigen-presenting capability in the induction phase. The numbers of F4/80+ major histocompatibility complex (MHC) class II+ LC before induction did not differ significantly between the oral mucosa and the skin. After induction, F4/80+ MHC class II+ LC increased in number, but the increase was significantly smaller in the oral mucosa than in the skin. MEST on anti-CD86 antibody-administered mice showed that ear swelling was similarly suppressed after oral mucosal or skin sensitization. In murine models of DTH after oral mucosal sensitization, the number of F4/80+CD86+ LC increased after induction, but the increase was significantly smaller than that in murine models of DTH after skin sensitization. This study showed that, in murine models of DTH, oral mucosal sensitization elicited a weaker reaction than skin sensitization. This was presumably because oral mucosal sensitization induced fewer LC, resulting in lower antigen-presenting capability.

Evaluation of cell proliferation in ear and lymph node using BrdU immunohistochemistry for mouse ear swelling test

The mouse ear swelling test (MEST) was developed as an alternative to guinea pig models for measuring the contact sensitization potential. However, the MEST relies on the quantitative measurement of ear swelling by micrometer as the means of determining the endpoint. The purpose of this study was to investigate the possibility of using cell proliferation in the ear and lymph node by bromodeoxyuridine (BrdU) immunohistochemistry as a reliable marker for MEST. Female Balb/c mice were treated by the topical application of various sensitizers, 2,4-dinitrochlorobenzene (DNCB), toluene diisocyanate (TDI) and α-hexylcinnamaldehyde (HCA) and an irritant, sodium lauryl sulfate (SLS) following the protocol of MEST. The proliferation of cells in the ear and auricular lymph node was analyzed by BrdU incorporations into cells. There were significant increases in the cell proliferations of the ear and auricular lymph node in mice treated with DNCB and TDI compared to the vehicle control. All allergens and the irritant were correctly identified by the MEST using BrdU immunohistochemistry of lymph node responses. The standard MEST assay showed positive results in the case of the strong sensitizers, DNCB and TDI. However, HCA and SLS were not correctly identified in the ear swelling assay. These results suggest that the measurement of cell proliferation in the auricular lymph node using BrdU immunohistochemistry could provide a reliable marker for MEST.

Sodium Metasilicate Hypersensitivity in BALB/c Mice

Background: Sodium metasilicate (SMS) is a key ingredient for a number of industrial and consumer products. Although little is known about potential for this chemical to cause allergic reactions, a similar silicate compound, sodium silicate, was reported to elicit IgE-mediated contact urticaria. Objective: The aim of this study was to evaluate the potential for sodium metasilicate to elicit an allergic response in female BALB/c mice after dermal exposure. Methods: The primary irritancy assay (IA), local lymph node assay (LLNA), and a mouse ear swelling test (MEST) were used to evaluate the hypersensitivity response elicited by SMS exposure. An evaluation of lymph node subpopulations, cytokine mRNA expression, and serum IgE levels was also conducted. Results: SMS caused significant dermal irritation at concentrations ≥6% and an allergic response after mice were sensitized with 4% SMS then challenged with 6% SMS in the MEST. Lymph node cell proliferation was not observed in the LLNA after treatment with SMS (2% to 6% SMS). Increases in lymph node cellularity, the percentage of B cells, and the expression of certain cytokine mRNAs were observed in mice treated with SMS. Changes in the concentration of serum IgE after SMS treatment, however, were not observed. Conclusions: SMS appears to elicit a chemical hypersensitivity response in mice, as indicated by the MEST, but not by the LLNA. Increases in auricular lymph node cellularity, the percentage of B cells, and certain cytokine mRNAs support classifying SMS as a weak chemical allergen.

The effect of a vitamin A acetate diet on ultraviolet radiation-induced immune suppression as measured by contact hypersensitivity in mice.

The adverse health effects caused by increased exposure to ultraviolet radiation (UVR) due to deterioration of stratospheric ozone are of major concern. These health effects include sunburn, skin cancer, cataracts and immune suppression. Immune suppression has been associated with the release of cytokines, a defect in antigen presentation, induction of suppressor T cells and suppression of contact hypersensitivity (CH). CH is typically assessed by the mouse ear swelling test (MEST). Previous studies have demonstrated enhanced CH responses with vitamin A acetate (VAA) dietary supplementation assessed by MEST and the local lymph node assay (LLNA). To determine the effect that VAA has on UVR-induced immune suppression, we examined both the induction and elicitation phases of CH using murine models. The MEST was used to evaluate the interaction of UVR and VAA on CH elicitation. However, a positive MEST response requires that the induction phase as well as the elicitation phase of CH be functional. The LLNA was used to evaluate the interaction of UVR and VAA only on CH induction. We tested the hypothesis that mice maintained on a VAA-enriched diet are more resistant to UVR-induced immune suppression (CH) than those maintained on a control diet. Mice were maintained on a VAA-enriched or the control diet for 3 weeks and then exposed to UVR 3 days prior to sensitization with 2,4-dinitrofluorobenzene (DNFB). VAA enhanced the MEST response in both UVR-exposed and non-UVR-exposed mice. The VAA-enriched diet did not significantly alter the LLNA response in either UVR- or non-UVR-exposed mice. However, there was significant suppression in CH by UVR as measured by the LLNA. These results indicate that (1) the VAA-enriched diet does not restore the number of proliferating cells in the CH induction phase of UVR-induced immunosuppression; (2) the immunosuppressive effects of UVR affect the induction phase of CH; and (3) the LLNA should be examined as an alternative to the MEST for measurement of UVR-induced immunosuppression. The data indicate that the VAA-enriched diet enhanced the elicitation response (MEST) but not the earlier induction phase (LLNA). Further studies are necessary to define mechanisms of action, but modulation of cytokines and effects of specific lymphocyte subsets, as well as systemic effects and local modulation at the site of elicitation are possible. Additionally, future studies to evaluate the effect of the VAA-enriched diet when multiple doses of both UVR and DNFB are used would be of interest for both the LLNA and MEST end-points.

COMPARISON OF MURINE ASSAYS FOR THE IDENTIFICATION OF CHEMICAL SENSITIZERS

Efforts are under way to develop a phenotypic analysis assay capable of identifying and differentiating chemicals with the potential to induce irritation and IgE-mediated and T-cell-mediated sensitization. These studies compare results obtained with the irritancy/phenotypic analysis assay to those obtained in the mouse ear-swelling test (MEST) and the local lymph node assay (LLNA). Female BALB/c mice (5 animals per group) were exposed topically to the human sensitizers 2,4-dinitrochlorobenzene (DNCB) and potassium dichromate (PDC) as well as the irritant methyl salicylate (MSC), following the protocol for each assay. DNCB was identified as a sensitizer in the LLNA and phenotypic analysis assay at concentrations as low 0.25%. In the MEST, DNCB was considered positive in concentrations as low as 0.01 and 0.05% at the 24- and 48-h measurements of the MEST, respectively. PDC tested positive as a sensitizer in the LLNA and the phenotypic analysis assay at concentrations as low as 0.25 and 0.1%, respectively. I...

&lt;I&gt;N&lt;/I&gt;-Pyridinyl(alkyl)polyhalogenobenzamides Acting as TNF-α Production Inhibitors

A series of N-pyridinyl(methyl)fluorobenzamides issued from 2,4-dimethyl-6-aminopyridine and 3-aminomethylpyridine were synthesized and evaluated as inhibitors of TNF-α production. Although less active than the corresponding phthalimides, several pentafluorobenzamides exhibited significant activity at 10 μM. N-(4,6-dimethylpyridin-2-yl)pentafluorobenzamide was selected for a preliminary in-vivo assay. Although its inhibitory activity against carrageenan-induced rat paw oedema was moderate, it induced a significant reduction in ear thickness in the PMA-induced mouse ear-swelling test (49± 6% inhibition after a dose of 0.1 mM kg−1, p.o.).