BALB/c mice orally pretreated with diclofenac have augmented and accelerated PLNA responses to diclofenac

The nonsteroidal anti-inflammatory drug (NSAID) diclofenac (DF) is associated with idiosyncratic hepatotoxicity and several other distinct hypersensitivity reactions. The mechanism(s) are unknown but evidence suggests both cell-mediated and antibody-mediated immune effector systems may be involved. In the present studies, the immunostimulating potential of DF was evaluated using the direct and TNP-Ficoll (trinitrophenyl (TNP)-Ficoll) popliteal lymph node assays (PLNA). These assays were conducted in naive mice, T-cell-deficient mice, or in mice that had been pretreated with a single oral dose of DF. In naive mice, DF induced a dose-, and time-dependent reaction in the direct PLNA. A significant increase in popliteal lymph node (PLN) weight and PLN cellularity was detected 7 days after the injection of 0.50 and 0.75 mg DF, whereas 0.25 mg DF produced no observable effect. With 0.75 mg, there was a rapid accumulation of cells in the PLN between days 5 and 6, with maximum PLN cellularity observed between days 7 and 10. The immunostimulating effects of DF were significantly attenuated in T-cell-deficient mice. In the TNP-Ficoll PLNA conducted in naive mice, DF caused a dose-dependent increase in PLN cellularity on day 7 with a time-dependent increase in anti-TNP antibody forming cells (AFCs) in the PLN; the reaction was dominated by IgM anti-TNP AFCs from day 4 through day 7, but IgG1 anti-TNP AFCs and IgG3 anti-TNP AFCs were detected beginning on day 5 and day 6, respectively. Relative to mice pretreated with vehicle (ddH2O), mice orally pretreated with DF had a significantly greater increase in PLN weight 5 days following the injection of 0.25 mg DF and a significantly greater increase in PLN weight and cellularity 4 days following the injection of 0.50 mg DF. Oral pretreatment with DF had no observable effect on the direct PLN reaction induced following the footpad injection of the irrelevant drugs, D-penicillamine (D-PEN) or streptozotocin. When 0.50 mg DF was co-injected with TNP-Ficoll, mice orally pretreated with DF, compared to vehicle-pretreated mice, and had a significantly greater increase in IgM anti-TNP AFCs on day 4, and a significant increase in both IgG1 and IgG3 anti-TNP AFCs on day 7. Additionally, IgG1 anti-TNP AFCs were detected in the PLN of DF-pretreated mice as early as day 4. No differences in anti-TNP AFCs were detected when orally pretreated mice were injected with 0.50 mg D-PEN. Collectively, these results demonstrated that DF (i) is an immunostimulating drug that induced a dose-, time- and T-cell-dependent PLN reaction in naive mice, (ii) provided non-cognate help that produced antibody against co-injected TNP-Ficoll, and (iii) mice orally pretreated with DF had DF-specific increased responsiveness in the direct PLNA, which (iv) resulted in accelerated and augmented AFC production against co-injected TNP-Ficoll. These novel findings suggest that oral administration of DF may result in primed T cells that respond with footpad injection. Thus, the oral pretreatment modification of the PLNA should be further explored as a possible alternative to hypersensitivity testing with drugs administered via the oral route. Additional studies with other compounds known to produce hypersensitivity reactions are needed.     

Diclofenac activates T cells in the direct popliteal lymph node assay and selectively induces IgG(1) and IgE against co-injected TNP-OVA

Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently associated with immune-mediated hypersensitivity reactions. The NSAID diclofenac is associated with several distinct allergic and autoimmune-like reactions including anaphylaxis, idiosyncratic hepatotoxicity and autoimmune hemolytic anemia. The aim of this study was to examine the immunostimulating potential of diclofenac in the direct popliteal lymph node assay (PLNA) and reporter antigen PLNA. In BALB/c mice, diclofenac caused dose-dependent increases in PLN weight and PLN cellularity in the direct PLNA; 0.25 mg was non-immunostimulating whereas 0.50-1.00 mg caused a significant PLN reaction. In the direct PLNA, diclofenac also increased the percent of T cells in the PLN with activated phenotypes (CD44(high)CD62L(low) and CD44(high)CD62L(high)). Finally, the magnitude of the diclofenac-induced direct PLN reaction was significantly reduced when the assay was conducted in T-cell-deficient mice. When co-injected with the reporter antigen TNP-Ficoll (trinitrophenyl Ficoll), 0.50 mg diclofenac caused significant increases in PLN weight, PLN cellularity, and induced IgM and IgG(1) anti-TNP antibody forming cells (AFCs) in the PLN. In a final set of studies, a TNP-OVA PLNA was conducted using diclofenac, phenobarbital (negative control) and streptozotocin (positive control). As expected, phenobarbital (1.00 mg) failed to cause an increase in PLN cellularity or induce AFCs in the PLN. Streptozotocin (1.00 mg) caused significant increases in PLN cellularity, IgM AFCs, and selectively induced IgG(2a) and IgG(2b) AFCs against TNP-OVA. Likewise, diclofenac caused dose-dependent increases (0.25-1.00 mg) in PLN cellularity and IgM AFCs. However, in contrast to streptozotocin, diclofenac caused a selective dose-dependent increase in both IgG(1) and IgE AFCs. Finally, an increase in the intracellular level of IL-4, but not INFgamma, was detected in CD4(+) PLN cells following the injection of diclofenac mixed with TNP-OVA. Collectively, these data suggest that diclofenac: (i) induces a T-cell-dependent direct PLN reaction that; (ii) provides non-cognate help for IgG AFC production when co-injected with TNP-Ficoll, possibly through the formation of neo-antigens; and (iii) possesses intrinsic adjuvant activity that selectively induces IL-4 mediated production of IgG(1) and IgE against co-injected TNP-OVA.     

Popliteal lymph node assay: facts and perspectives

The popliteal lymph node assay (PLNA) derives from the hypothesis that some supposedly immune-mediated adverse effects induced by certain pharmaceuticals involve a mechanism resembling a graft-versus-host reaction. The injection of many but not all of these compounds into the footpad of mice or rats produces an increase in the weight and/or cellularity of the popliteal lymph node in the treated limb (direct PLNA). Some of the compounds known to cause these adverse effects in humans, however, failed to induce a positive PLNA response, leading to refinements of the technique to include pretreatment with enzyme inducers, depletion of CD4(+) T cells or additional endpoints such as histological examination, lymphocyte subset analysis and cytokine fingerprinting. Alternative approaches have been used to improve further the predictability of the assay. In the secondary PLNA, the test compound is injected twice in order to illicit a greater secondary response, thus suggesting a memory-specific T cell response. In the adoptive PLNA, popliteal lymph node cells from treated mice are injected into the footpad of naive mice; a marked response to a subsequent footpad challenge demonstrates the involvement of T cells. Finally, the reporter antigens TNP-Ficoll and TNP-ovalbumin are used to differentiate compounds that induce responses involving neo-antigen help or co-stimulatory signals (modified PLNA). The PLNA is increasingly considered as a tool for detection of the potential to induce both sensitization and autoimmune reactions. A major current limitation is validation. A small inter-laboratory validation study of the direct PLNA found consistent results. No such study has been performed using an alternative protocol. Other issues include selection of the optimal protocol for an improved prediction of sensitization vs autoimmunity, and the elimination of false-positive responses due to primary irritation. Finally, a better understanding of underlying mechanisms is essential to determine the most relevant endpoints. The confusion resulting from use of the PLNA to predict autoimmune-like reactions as well as sensitization should be clarified. Interestingly, most drugs that were positive in the direct PLNA are also known to cause drug hypersensitivity syndrome in treated patients. This observation is expected to open new avenues of research.     

Evaluation of the use of reporter antigens in an auricular lymph node assay to assess the immunosensitizing potential of drugs.

Immune-mediated idiosyncratic drug reactions are a major problem for susceptible patients, physicians, and the pharmaceutical industry. Validated screening tools to assess the immunosensitizing capacity of orally or intravenously administered pharmaceuticals are currently not available. To date, the popliteal lymph node assay (PLNA) seems the most promising tool for this purpose. The PLNA has recently been extended with the use of reporter antigens (RA) that are coinjected together with the drug of interest. The measurement of isotypes of RA-specific antibody-secreting cells (ASC) enables the distinction of sensitizing chemicals and (nonsensitizing) irritants without radio-isotopic end points. However, the use of footpad injections raises ethical concerns. Therefore, we examined the use of RA after intradermal injection into the ear of BALB/c mice and measured RA-specific ASC in the draining auricular lymph node (ALN). We show that RA-specific IgG isotype ASC numbers are very useful and sensitive parameters to identify drug-induced hypersensitivity in both PLN and ALN. However, the type 1-associated parameters (CD8(+) cells, macrophages, IFN-gamma, TNF-alpha, and IL-1 beta) that are induced in the PLN by streptozotocin were less pronounced in the ALN. Thus, the PLNA may provide more immunologically relevant information on the mechanisms of certain chemical-induced hypersensitivity reactions. The RA-ALN assay may provide an alternative for the RA-PLNA; both assays can be used to distinguish sensitizing compounds from nonsensitizing ones.     

Differentiation of responses to allergenic and irritant compounds in mouse popliteal lymph node assay

The mouse popliteal lymph node assay (PLNA) has been proposed as an immunotoxicological test to predict allergenic chemicals. However, PLN response is also observed in association with non-specific activation induced by some irritants. We, therefore, examined the kinetics of the PLN cellularity indices of primary and secondary responses. Flow cytometric analysis was used to measure the proportions of T and B cells in PLN. Male ICR mice were subcutaneously injected with TNBS (an allergenic compound) or SDS (an irritant compound) in the left hind footpad, and with vehicle in the right one. On day 28 after first injection, mice were injected with 1/10 or 1/100 dose of the initial injection. On day 7 after first injection of TNBS, primary response reached maximal PLN cellularity index (16.5). On day 2 after second injection, secondary response reached maximal PLN cellularity index (13.1). A marked increase in proportion of B cells was observed in the PLN. On the other hand, after first injection of SDS, primary response reached maximal PLN cellularity index (2.8) on day 10, but neither secondary response nor increase in the B cell proportion were observed. These results suggest that the detection of secondary response and the flow cytometric analysis are useful in differentiating responses to allergenic and irritant compounds in the PLNA.     

Investigating the TNP-OVA and direct popliteal lymph node assays for the detection of immunostimulation by drugs associated with anaphylaxis in humans

Using current animal models, it is not possible to identify low-molecular-weight compounds (LMWCs) that are likely to be associated with anaphylaxis. It is generally accepted that the ultimate effector mechanism involves drug-induced IgE antibody. The objective of the present study was to determine if diclofenac, zomepirac and glafenine, which are associated with anaphylaxis in humans, have immunostimulating potential in the murine TNP-OVA (trinitrophenyl-ovalbumin) popliteal lymph node assay (PLNA), and more specifically to determine if the immunostimulation caused by these LMWCs results in IgE antibody production. These LMWCs were chosen because both zomepirac and glafenine were removed from the market due to high association with anaphylaxis, and diclofenac, which remains on the market, is frequently associated with anaphylaxis. In addition to conducting a TNP-OVA PLNA, the immunostimulating potential of these compounds was examined in the direct PLNA. When co-administered with TNP-OVA, all three LMWCs caused dose-dependent (0.25, 0.50, 1.00 and 1.25 mg) increases in popliteal lymph node (PLN) weight and cellularity that were observed beginning with the 0.25-mg dose. In addition, beginning with the 0.25-mg dose, all three compounds caused dose-dependent increases in TNP-OVA specific IgM and IgG(1) antibody-forming cells (AFCs). Diclofenac induced an isotype switch and caused a dose-dependent increase in the number of IgE AFCs with no detectable IgG(2a) AFCs and minimal high-dose-only IgG(2b) AFCs. Zomepirac induced IgE, IgG(2a) and IgG(2b) AFCs following the injection of 0.50 mg only, and glafenine induced IgE, IgG(2a) and IgG(2b) AFCs following the injection of 0.50-1.00 mg. In the direct PLNA, diclofenac caused dose-dependent increases in PLN weight and cellularity that were observed beginning with dose of 0.50 mg, whereas zomepirac failed to increase any PLN parameter and glafenine only increased the PLN weight. These results suggest that diclofenac, zomepirac and glafenine are immunostimulating LMWCs in the TNP-OVA PLNA with the potential to induce IgE antibody against a co-administered hapten-conjugate. Furthermore, these results suggest that the TNP-OVA PLNA offered significant advantages over the direct PLNA. Although it is not realistic to suggest that a single assay, based on a low number of test compounds, can identify all LMWCs with the potential to cause anaphylaxis in humans, these observations do demonstrate the potential utility of the PLNAs in examining LMWC-induced immunomodulation and support further development and investigation of the assays.     

The effects of TCDD on the activation of ovalbumin (OVA)-specific DO11.10 transgenic CD4(+) T cells in adoptively transferred mice.

Exposure to the environmental contaminant 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) suppresses the generation of T cell-dependent immunity, both humoral and cell-mediated. However, the mechanism of TCDD-induced immune suppression remains to be defined. We hypothesized that exposure to TCDD suppresses the activation of naive CD4(+) T cells and prevents their expansion and differentiation into effector T-helper cells capable of driving T cell-dependent immune responses. To test this hypothesis, we adoptively-transferred DO11.10 OVA-specific T-cell receptor (TCR) transgenic T cells into syngeneic recipients and used a TCR-specific monoclonal antibody to track the in vivo activation of naive CD4(+) T lymphocytes following exposure to OVA. The production of OVA-specific antibodies was suppressed in a dose-dependent manner in adoptively transferred mice that had been exposed to TCDD. Although TCDD exposure had little effect on the expansion or activation of the adoptively transferred, OVA-specific CD4(+) T cells, these cells disappeared from the spleen more rapidly in TCDD-treated mice and produced significantly decreased levels of the T cell-derived cytokines IL-2 and IL-10. There was also a trend towards reduced IFN-gamma and IL-4 production following in vitro re-stimulation. These data suggest that TCDD may interfere with the survival and/or differentiation of OVA-specific T-helper cells. These results demonstrate for the first time the potential of the DO11.10 adoptive transfer system to directly assess immunotoxic effects of xenobiotics on antigen-specific CD4(+) T cells in vivo.     

硫普罗宁注射液腘窝淋巴结试验研究

目的用小鼠腘窝淋巴结试验（Popliteal lymph node assay,PLNA）以及检测淋巴细胞表面分子（CD4＋/CD8＋）来评价硫普罗宁注射液的免疫毒性。方法将昆明种小鼠随机分为5组,每组12只,设阳性对照组（盐酸氯丙嗪20.83mg.kg-1BW）、阴性对照组（氯化钠注射液）和硫普罗宁注射液（3.33、13.33、53.33mg.kg-1BW）3个剂量组,采用小鼠腘窝淋巴结试验方法测定腘窝淋巴结重量及细胞指数,淋巴细胞亚群测定采用流式细胞技术法,观察不同剂量组与对照组的差别。结果小鼠分别给予不同剂量的硫普罗宁注射液后,PLN（腘窝淋巴结）重量指数、细胞指数未见统计学意义的改变,但随用药剂量的增大而表现为增加的趋势。CD4＋/CD8＋淋巴细胞的比例无明显变化。结论硫普罗宁注射液有引起小鼠淋巴结肿大、增殖细胞的趋势。     

Immunomodulatory effects of tetrachlorobenzoquinone,a reactive metabolite of hexachlorobenzene

Hexachlorobenzene (HCB) is an environmental pollutant that causes autoimmune-like effects in humans and rats. It is not completely clear whether T cells are involved and, if so, how they are stimulated after oral exposure to HCB. HCB as a rather inert chemical is not likely to bind covalently to macromolecules. The oxidative metabolite of HCB, tetrachlorobenzoquinone (TCBQ), which is in a redox equilibrium with tetrachlorohydroquinone (TCHQ), can bind to macromolecules, hence may form hapten-carrier complexes in vivo. We have assessed in the reporter antigen-popliteal lymph node assay whether HCB or TCHQ and TCBQ are able to induce a 2,4,6-trinitrophenyl (TNP) specific IgG1 response to the T cell-independent antigen TNP-Ficoll, which is indicative of neoantigen specific T cell help. To this end, these compounds and silica were injected into the footpad of Balb/c mice. Silica was included as an inert model compound, which causes autoimmune-like effects by activating macrophages. Seven days later, cell number and TNP specific antibody-secreting cells (ASC) in the popliteal lymph node (PLN) were determined. Furthermore, a secondary PLNA was performed to find out if TCHQ was capable of eliciting a memory response. Silica, TCHQ, and TCBQ, but not HCB, increased PLN cellularity and the number of IgM-producing ASC by ELISPOT. Both oxidative metabolites were able to induce the formation of germinal centers as assessed by immunohistochemistry and an IgG1 response to TNP-Ficoll. In the secondary PLNA, only mice primed with TCHQ and challenged with TCHQ together with TNP-Ficoll showed a significant increase in TNP specific IgG1 ASC. Present data show that TCHQ and TCBQ are capable of inducing neoantigen specific T cell help and that TCHQ can induce a compound specific memory response.     

腘窝淋巴结试验在药物超敏反应研究中的应用

免疫介导药物超敏反应(IDHR)是一种最常见、也最为复杂的药物不良反应。迄今为止,尚无可靠的动物模型可以有效预测IDHR。已有研究发现,腘窝淋巴结试验(PLNA)操作简单,省时,灵敏,特异性高,可靠性和重复性好,实验动物用量少。尽管其反应机制不清,给药途径为非常规途径,缺乏公认的评价标准,剂量水平、溶媒种类及实验周期的选择等各不一致,但PLNA是一种最有希望可成功预测IDHR的动物模型方法。本文综述了PLNA的原理、分类和应用,并分析了PLNA在成分复杂的中药超敏反应研究方面的应用前景。     

Increased production of interferon-gamma, but not IL-4 mRNA, by streptozotocin in the popliteal lymph node assay

The popliteal lymph node (PLN) assay has been proposed as a tool to predict systemic autoimmune reactions induced by medicinal products and chemicals, the mechanisms of which are poorly understood. To determine whether PLN responses involved Th1 or Th2 cell control, or both, the effects of streptozotocin (STZ), a prototypic immunotoxic compound, were analysed on the production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) mRNA by lymph node cells after injection into the hind footpad of C57 BL/6 mice. Streptozotocin induced a dramatic increase in IFN-gamma mRNA production, which correlated with PLN responses as evidenced by augmented weight and cellularity indices. No effect on IL-4 mRNA synthesis was noted. These results suggest that a Th1 response is involved in the PLN response to STZ.     

Assessment of sensitization potential of monoterpenes using the rat popliteal lymph node assay.

The popliteal lymph node assay (PLNA) has been proposed as a screening test for detecting chemicals with potential of inducing allergic and auto-immune-like reactions in humans. In the present study, we used the rat PLNA to evaluate the immuno-sensitizing potential of 10 monoterpenes found in the essential oils of a variety of aromatic, edible and medicinal plants. The primary or direct PLNA was performed with the monoterpenes, and chlorpromazine (CPZ) and barbital were used as positive and negative controls, respectively. Female, 7-8 week-old Wistar rats were injected subcutaneously (50 microL) with the test substance (0.5, 2.5 or 5mg) into the right hind footpad while the contralateral footpad was injected with the vehicle (DMSO) alone. Weight (WI) and cellularity (CI) indices for draining PLNs were determined 7 days after treatment. PLNA was positive (WI >or= 2 and CI >or= 5) for CPZ, citral, alpha-terpinene, beta-myrcene and (-)-alpha-pinene, and negative for barbital, DMSO, (-)-menthol, 1,8-cineole, (+/-) citronellal, (+)-limonene, (+/-) camphor and terpineol. A secondary PLNA, a T-cell priming test, was carried out with the four substances that had been positive in the primary assay. Six weeks after being locally primed with 5 mg/paw, rats were sc injected into the same footpad with a dose (0.5 mg/paw) of the substance that had been previously found to be insufficient to cause a positive response. WI and CI were then calculated 4 and 7 days after the second injection. CPZ was also positive in the secondary assay thereby confirming that it is a sensitizing agent. Citral, alpha-terpinene, beta-myrcene and (-)-alpha-pinene, however, were negative in the secondary assay. In summary, citral, alpha-terpinene, beta-myrcene and (-)-alpha-pinene induced a clear immuno-stimulatory response due to their irritant properties but no monoterpene proved to be a sensitizing agent in the PLNA.     

应用腘窝淋巴结实验评价干扰素的免疫毒性

目的用小鼠腘窝淋巴结实验(Popliteallymphnodassay,PLNA)以及检测淋巴细胞表面分子来预测干扰素的免疫毒性,探讨该方法的预测价值。方法采用小鼠腘窝淋巴结实验方法测定腘窝淋巴结重量及细胞指数,淋巴细胞亚群测定采用流式细胞技术法。结果干扰素可引起小鼠腘窝淋巴结肿大,并造成腘窝淋巴结CD4+/CD8+T细胞比例改变。结论本实验中干扰素的测试结果呈阳性,提示用腘窝淋巴结实验对干扰素(Interferon,IFN)的免疫毒性进行初步评估。     

The effect of the food matrix on in vivo immune responses to purified peanut allergens.

There is little knowledge about the factors that determine the allergenicity of food proteins. One aspect that remains to be elucidated is the effect of the food matrix on immune responses to food proteins. To study the intrinsic immunogenicity of allergens and the influence of the food matrix, purified peanut allergens (Ara h 1, Ara h 2, Ara h 3, or Ara h 6) and a whole peanut extract (PE) were tested in the popliteal lymph node assay (PLNA) and in an oral model of peanut hypersensitivity. In the PLNA, peanut proteins were injected into the hind footpad of BALB/c mice; in the oral exposure experiments C3H/HeOuJ mice were gavaged weekly with PE or allergens in the presence of cholera toxin (CT). Upon footpad injection, none of the allergens induced significant immune activation. In contrast, PE induced an increase in cell number, cytokine production, and activation of antigen-presenting cells. Furthermore, the presence of a food matrix enhanced the immune response to the individual allergens. Oral exposure to the purified allergens in the presence of CT induced specific IgE responses, irrespective of the presence of a food matrix. These results suggest that purified peanut allergens possess little intrinsic immune-stimulating capacity in contrast to a whole PE. Moreover, the data indicate that the food matrix can influence responses to individual proteins and, therefore, the food matrix must be taken into account when developing models for allergenic potential assessment.     

In-vitro generation and characterisation of murine CD4+CD25+ regulatory T cells with indirect allospecificity

Naturally arising CD4(+)CD25(+) regulatory T cells play a pivotal role in the prevention of autoimmunity and in the induction of donor-specific transplantation tolerance. Harnessing regulatory cells for potential adoptive cell therapy is hampered by their lack of antigen-specificity and their limited numbers. Here we describe the generation and expansion of murine CD4(+)CD25(+) T cells with antigen-specificity for an K(d) peptide as potential reagents for adoptive cell therapy in promoting donor-specific transplantation tolerance. Using bone marrow-derived autologous dendritic cells pulsed with the K(d) peptide, we generated T cell lines from purified CD4(+)CD25(+) T cells from C56BL/6 mice. The T cell lines expressed high level of CD25 and low level of CD45RB and CD69. They maintained the expression of CD62L, GITR, CTLA-4 and more importantly FoxP3. The CD4(+)CD25(+) T cell lines were anergic after TCR stimulation and produced little cytokine such as IL-2 and IFN-gamma. Importantly, they were more potent than freshly isolated CD4(+)CD25(+) T cells in suppressing proliferation and cytokine secretion by effector CD4(+) T cells. Furthermore, the CD4(+)CD25(+) T cell lines could be expanded to large cell numbers and maintained in culture up to 1 year. The K(d)-specific CD4(+)CD25(+) T cell lines will be invaluable in devising a strategy for the induction of cardiac transplantation tolerance in wild-type B6 mice carrying a full mismatch BALB/c heart.     

Immunosuppressory activity of an isoxazolo[5,4-e]triazepine-compound RM-33 II. Effects on the carrageenan-induced inflammation

The aim of this investigation was to evaluate effectiveness of RM-33, a new isoxazolotriazepine, in the model of carrageenan-induced inflammation in rats. Wistar rats were pretreated with intraperitoneal (ip) or oral (po) doses of RM-33, at daily doses ranging from 250 to 1000 microg, administered 1-3 days before elicitation of the carrageenan reaction. We showed that both routes of RM-33 administration were effective in significantly diminishing the footpad edema. The effects were dose-dependent and better pronounced at the ip administration of the compound. We found a lower production of tumor necrosis factor alpha (TNF-alpha) by mitogen-stimulated splenocytes isolated from rats pretreated with RM-33 and injected with carrageenan, as well as lower serum TNF-alpha levels in these rats, as compared to the respective control. Histological analysis of the skin reaction site revealed that in the rats pretreated with RM-33, the carrageenan-induced inflammation was reduced, as reflected by a lesser damage of mast cells, smaller infiltration by macrophages and a diminished edema of the connective tissue. Together with our previous data, indicating the antagonistic action of RM-33 in the adjuvant-induced footpad inflammation in mice, the present results confirm the anti-inflammatory activity of RM-33 compound.     

Local popliteal lymph node reactions to hexachlorobenzene and pentachlorobenzene: Comparison with systemic effects

The effects of the presumed autoimmunogenic chemical hexachlorobenzene (HCB), and the closely related non-autoimmunogenic pentachlorobenzene (PCB) in the local popliteal lymph node assay (PLNA) were investigated. To that end 1–5 mg of HCB, equimolar amounts of PCB or the vehicle only, were injected into the hind footpads of rats or mice, and the reaction in the draining lymph node was evaluated on days 7 and 21 after injection. PLN were isolated, weighed, and cell suspensions were prepared to determine PLN cell numbers, and antibody production of PLN cells with an ELISPOT assay or a line immunoassay. The extent of the lymphoproliferative effect was examined by detection of proliferating cells with the BrdU method, and by measurement of paracortex and follicle areas, by combined immunohistochemistry and morphometry of PLN cryosections. We demonstrate here that HCB elevated PLN weights and cell numbers of the rat PLN, by day 7 after injection, but no elevation of antibody production in the PLN. Moreover, HCB caused an enlargement of both the PLN paracortical and follicular areas, and an elevation of proliferating paracortical T cells. None of the HCB-induced effects were found on day 21. HCB caused the same effects in the mouse PLNA, but they tended to sustain at least until day 21. Hardly any of the HCB-induced changes were found when PCB was injected. Previously, we have shown that oral exposure of Wistar rats to HCB elevated the number of splenic T cells and B cells, but also the serum levels of (auto-)antibodies and the production of these antibodies in the spleen, which is thus only partly in accordance with the results of the local reaction to HCB described in this study. This seeming contradiction is discussed.     

Evaluation of the immunosensitizing potential of chlorogenic acid using a popliteal lymph node assay in BALB/c mice

It has yet to be established whether chlorogenic acid (CGA), a common xenobiotic with potential exposure risk to humans, is associated with immune-mediated hypersensitivity reactions (HRs). The primary limitation in evaluating this potential relationship is the lack of an effective animal model for use in predicting the immunosensitizing potential of low molecular weight compounds (LMWCs). Currently, the popliteal lymph node assay (PLNA) is considered a very promising tool for assessing immunosensitizing potential of LMWCs. To determine whether CGA may possess an intrinsic capacity to stimulate or dysregulate immune responses, and if so, what mechanisms may be involved, we characterized the popliteal lymph node reaction induced by CGA in naive female BALB/c mice using both a direct PLNA (d-PLNA) and a reporter antigen PLNA (RA–PLNA) method. Our results show that CGA failed to induce immunoreactivity following a single subcutaneous injection either alone or when combined with TNP–OVA or TNP–Ficoll. These results indicated that CGA lacks the intrinsic capacity to sensitize or stimulate immune responses in BALB/c mice. Moreover, these results suggest that exposure to CGA may not represent a safety concern for humans and that removal of CGA from Traditional Chinese Medicine Injections may not significantly decrease the prevalence of HRs.     

Neurotoxicity and immunotoxicity assessment in CBA/J mice with chronic Toxoplasma gondii infection and single-dose exposure to methylmercury

Toxoplasma gondii is a protozoan parasite that localizes in the brain where it can cause life-threatening disease. Methylmercury (MeHg) is a well-documented neurotoxicant that accumulates in the brain. We investigated end points associated with immunotoxicity and neurotoxicity in mice exposed to MeHg during a chronic T. gondii infection. Two groups of 6-week-old, female CBA/J mice were either fed 25 T. gondii tissue cysts of the ME-49 strain or given vehicle. Six weeks later, half of the mice in each group were orally gavaged with a single dose of 20 mg/kg body weight of MeHg, creating four groups of mice (vehicle control, T. gondii, MeHg, and T. gondii/MeHg). Mice were sacrificed 7 days post MeHg exposure. MeHg exposure caused a significant decrease in mouse body weight. MeHg administration resulted in an increase of splenic cellularity and spleen-to-body weight ratios. MeHg had no significant effect on the percentages of CD4(+), CD8(+), or non-T-cell subpopulations in the spleen. MeHg dosed mice demonstrated an increase in absolute numbers of splenic CD4(+), CD8(+), or non-T cells when compared to mice in control and T. gondii-infected groups. Thymic CD4(+)CD8(+) T-cell subpopulations were decreased (p <.05) by MeHg with or without a concurrent T. gondii infection. There was a significant (p <.05) increase in brain tissue cyst counts within the group exposed to both MeHg and T. gondii (16 +/- 4, mean +/- SE, n = 7) versus T. gondii alone (4 +/- 1, n = 8). Histopathological examination demonstrated encephalitis, gliosis, and meningitis in brains from mice infected with T. gondii. These data indicate that exposure to both MeHg and T. gondii has synergistic effects, with effects of MeHg especially on the immune system.     

The popliteal lymph node assay: a tool for predicting drug allergies

A considerable number of drugs is able to induce systemic hypersensitivity in man. Systemic hypersensitivity can be drug- or autoantigen-specific, but in either case a complex of immunological processes and predisposing factors are involved and it is rarely if ever noticed in standard toxicity testing. The popliteal lymph node assay (PLNA) is regarded a suitable test to screen for the immunostimulating ability of a chemical, which may indicate its immunosensitizing potential. The most simple, primary PLNA measures popliteal lymph node hyperplasia after subcutaneous injection of a chemical into the footpad of the hindpaw of a mouse or rat. In order to assess the involvement of T cells, and hence immunosensitizating potential of a chemical, anamnestic immune reactions to a chemical or its metabolite can be measured in previously exposed (and sensitized) animals or in naive animals that received an adoptive transfer of syngeneic T cells from previously exposed animals. In the recently introduced modified PLNA, defined reporter antigens TNP-OVA (T cell-dependent antigen) and TNP-Ficoll (T cell-independent antigen) are used to distinguish between sensitizing and non-sensitizing (IgG1-response or not to TNP-Ficoll, respectively) and between mere inflammatory and complete innocent (no IgG1-response to TNP-Ficoll and an IgG1-response or not to TNP-OVA, respectively) drugs. Results with about 130 compounds (drugs and environmental pollutants) with the various types of the PLNA show a good correlation with documented immunostimulating (both autoimmunogenic and allergic) potential and no false negative chemicals were detected if metabolism was considered. The PLNA awaits further validation before this test can be recommended as a tool for prediction of drug allergy.