Development of non-radio isotopic endpoint of murine local lymph node assay based on 5-bromo-2′-deoxyuridine (BrdU) incorporation

Takeyoshi, Masahiro; Yamasaki, Kanji; Yakabe, Yoshikuni; Takatsuki, Mineo; Kimber, Ian

doi:10.1016/s0378-4274(00)00315-5

Cited by 67 publications

(52 citation statements)

References 11 publications

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“…This chemical is also known as an irritant on the skin [10,17]. Dex is reported to be a non-sensitizer on the skin [7,39].…”

Section: Methodsmentioning

confidence: 99%

“…Recently, the local lymph node assay (LLNA) was developed in mice as an alternative approach based on the characterization of initial proliferative responses in draining lymph nodes caused by chemicals exposure [16,21,34,39], and this method is now widely used for estimation of the allergenic potency of chemical substances. Although these approaches are sensitive and reliable, more advantageous ways in terms of cost performance, safety, readiness and animal welfare are expected.…”

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confidence: 99%

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Gene Expression Changes Induced by Type IV Allergy-Inducible Chemicals in Dendritic Cells

Tarama

Kato

Ishikawa

et al. 2008

The Journal of Veterinary Medical Science

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ABSTRACT. In the present study, the changes of gene expression profile in dendritic cell (DC)-derived DC2.4 cells sensitized with two allergenic chemicals were analyzed by microarray analysis to develop a basis for an in vitro assessment system of type IV allergenic chemicals. Consequently, 26 genes were significantly up-regulated, and 53 were down-regulated in both groups. Interestingly, some of upregulated genes were associated with the maturation process of DCs. A set of genes was further evaluated by real-time reverse transcription-polymerase chain reaction to identify the gene expression changes specifically induced by type IV allergy-inducible chemicals in DC2.4 cells, and 2 possible candidates, syndecan-1 (Sdc1) and smoothened (SMO) genes were identified. Thus, up-regulation of Sdc1 gene and down-regulation of SMO gene in DC2.4 cells may be diagnostic markers for the screening of type IV-allergenic chemicals. KEY WORDS: chemical, dendritic cell, microarray analysis, type IV allergy.J. Vet. Med. Sci. 70 (7): [673][674][675][676][677][678][679][680] 2008 Allergic contact dermatitis (ACD), a type IV allergy, is one of the most common inflammatory diseases of the skin with unknown genetic basis and is often an occupationally related disorder in industrialized countries with an important socio-medical impact [14,28]. At present, many chemicals are considered to have allergenic potency and thus risk assessment of such chemical substances is important. ACD has been intensively studied, and the development of an allergic hypersensitivity reaction in the skin is considered to be processes depending on the induction of specific T-lymphocyte responses [23]. At the initial step, chemical allergens exposed on the skin are recognized by Langerhans cells (LCs), the principal DC residing in the epidermis and known to play a key role in the development of ACD. Following an encounter with a chemical allergen, LCs are activated and subsequently migrate from the skin to the draining lymph nodes, undergoing a maturation process during the journey [20,22].For many years, guinea pigs have been applied for the hazard analysis of skin-sensitizing chemicals [9,26]. Recently, the local lymph node assay (LLNA) was developed in mice as an alternative approach based on the characterization of initial proliferative responses in draining lymph nodes caused by chemicals exposure [16,21,34,39], and this method is now widely used for estimation of the allergenic potency of chemical substances. Although these approaches are sensitive and reliable, more advantageous ways in terms of cost performance, safety, readiness and animal welfare are expected. More recently, in vitro assay for chemical substances with allergenic potency is extensively explored by using cultured cells, especially macrophages [5,41]. In these experiments, up-regulation of several molecules including cell surface markers were reported to be induced by chemical exposure, suggesting that these markers may be candidates for evaluating chemicals with allergenic potency [5...

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“…This chemical is also known as an irritant on the skin [10,17]. Dex is reported to be a non-sensitizer on the skin [7,39].…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Gene Expression Changes Induced by Type IV Allergy-Inducible Chemicals in Dendritic Cells

Tarama

Kato

Ishikawa

et al. 2008

The Journal of Veterinary Medical Science

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“…These newly developed alternative methods are expected to be implemented for practical use in the future. However, a considerable time will be required for these in vitro test methods to become reliable and accepted international standard test methods.In 2001, Takeyoshi et al developed the non-radioisotope (non-RI) LLNA [12,13] in Japan. The novel test method was expected to become an alternative to the tra--Note-…”

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confidence: 99%

“…In 2001, Takeyoshi et al developed the non-radioisotope (non-RI) LLNA [12,13] in Japan. The novel test method was expected to become an alternative to the tra--Note-ditional LLNA [14,16].…”

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confidence: 99%

Effects of Strain Differences and Vehicles on Results of Local Lymph Node Assays

Anzai

Ullmann²,

Hayashi

et al. 2010

Exp. Anim.

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Abstract:The Local Lymph Node Assay (LLNA) is now regarded as the worldwide standard. The analysis of accumulated LLNA data reveals that the animal strains and vehicles employed are likely to affect LLNA results. Here we show that an obvious strain difference in the local lymph node response was observed between DMSO-treated CBA/CaOlaHsd and CBA/ CaHsdRcc mice. We also show that a vehicle difference in the response was observed when CBA/CaHsdRcc mice were exposed to 6 vehicles; 4:1 v/v acetone/olive oil (AOO), ethanol/ water (70% EtOH), N,N-dimethylformamide (DMF), 2-butanone (BN), propylene glycol (PG), and dimethylsulfoxide (DMSO). The dpm/LN level was lowest in the 70% EtOH group and highest in the DMSO group. When alpha-hexylcinnamaldehyde (HCA) was used as a sensitizer for the LLNA, HCA was a weak sensitizer when AOO or DMSO was used as a vehicle, but a moderate sensitizer when the other 4 vehicles were used. This study showed that there are vehicle differences in the local lymph node response (dpm/LN level) in the LLNA and that the sensitization potency of HCA may be classified in different categories when using different vehicles. This suggests that careful consideration should be exercised in selecting a vehicle for the LLNA. A further comprehensive study will be needed to investigate why vehicle differences are observed in the LLNA. Key words: animal strain, local lymph node assay, vehicle-effect The local lymph node assay (LLNA) is the most commonly used among several alternatives of skin sensitization tests [1,8,10], including the human cell line activation test (h-CLAT), the peptide-binding test [7], and the quantitative structure-activity relationship (QSAR) system. Furthermore, a three-dimensional human skin model is now being actively developed as an in vitro model for the assessment of primary skin irritation [15], and put into trials for use as an in vitro model for the evaluation of skin sensitization. These newly developed alternative methods are expected to be implemented for practical use in the future. However, a considerable time will be required for these in vitro test methods to become reliable and accepted international standard test methods.In 2001, Takeyoshi et al. developed the non-radioisotope (non-RI) LLNA [12,13] in Japan. The novel test method was expected to become an alternative to the tra--Note-

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“…In the standard LLNA, cell proliferation is measured using the incorporation of radiolabeled thymidine or uridine into draining lymph node cells, and this requires specific facilities and handling conditions. We previously developed a non-radioisotopic alternative method for the LLNA which uses 5-bromo-2'-deoxyuridine (BrdU) incorporation in place of radioisotopes [9,10]. The responsiveness of mouse strains against antigen is known to vary with their H-2 haplotypes.…”

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confidence: 99%

Differences in Responsiveness of Mouse Strain against p-Benzoquinone as Assessed by Non-Radioisotopic Murine Local Lymph Node Assay.

2004

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Abstract:The non-radioisotopic modification of murine local lymph node assay (LLNA) by using 5-bromo-2'-deoxyuridine (BrdU) was conducted to investigate the strain-related difference of the responsiveness of mice to p-benzoquinone (PBQ) with BALB/cAnN, CBA/ JN and CD-1 mouse strains. Strain and dose related differences were analyzed by two-way analysis of variance (two-way ANOVA). CBA/JN was considered to be the highest responsive strain to PBQ, and interaction was detected between CD-1 and each of the other inbred strains. These results support the recommendation in the OECD test guideline 429 and the skin sensitization test guideline of US-EPA with regard to the selection of mouse strain for LLNA.

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Development of non-radio isotopic endpoint of murine local lymph node assay based on 5-bromo-2′-deoxyuridine (BrdU) incorporation

Cited by 67 publications

References 11 publications

Gene Expression Changes Induced by Type IV Allergy-Inducible Chemicals in Dendritic Cells

Gene Expression Changes Induced by Type IV Allergy-Inducible Chemicals in Dendritic Cells

Effects of Strain Differences and Vehicles on Results of Local Lymph Node Assays

Differences in Responsiveness of Mouse Strain against p-Benzoquinone as Assessed by Non-Radioisotopic Murine Local Lymph Node Assay.

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