Proliferation Of Lymph Node Cells Research Articles

Evaluating the Effects of Chronic Oral Exposure to the Food Additive Silicon Dioxide on Oral Tolerance Induction and Food Sensitivities in Mice.

The increasing prevalence of food sensitivities has been attributed to changes in gut microenvironment; however, ubiquitous environmental triggers such as inorganic nanoparticles (NPs) used as food additives have not been thoroughly investigated. We explored the impact of the NP-structured food-grade silicon dioxide () on intestinal immune response involved in oral tolerance (OT) induction and evaluated the consequences of oral chronic exposure to this food-additive using a mouse model of OT to ovalbumin (OVA) and on gluten immunopathology in mice expressing the celiac disease risk gene, HLA-DQ8. Viability, proliferation, and cytokine production of mesenteric lymph node (MLN) cells were evaluated after exposure to . C57BL/6J mice and a mouse model of OT to OVA were orally exposed to or vehicle for 60 d. Fecal lipocalin-2 (Lcn-2), anti-OVA IgG, cytokine production, and immune cell populations were analyzed. Nonobese diabetic (NOD) mice expressing HLA-DQ8 (NOD/DQ8), exposed to or vehicle, were immunized with gluten and immunopathology was investigated. MLN cells exposed to presented less proliferative T cells and lower secretion of interleukin 10 (IL-10) and transforming growth factor beta () by T regulatory and cells compared to control, two factors mediating OT. Mice given exhibited intestinal Lcn-2 level and interferon gamma () secretion, showing inflammation and less production of IL-10 and . These effects were also observed in OVA-tolerized mice exposed to , in addition to a breakdown of OT and a lower intestinal frequency of T cells. In NOD/DQ8 mice immunized with gluten, the villus-to-crypt ratio was decreased while the intraepithelial lymphocyte counts and the Th1 inflammatory response were aggravated after treatment. Our results suggest that chronic oral exposure to blocked oral tolerance induction to OVA, and worsened gluten-induced immunopathology in NOD/DQ8 mice. The results should prompt investigation on the link between exposure and food sensitivities in humans. https://doi.org/10.1289/EHP12758.

Chronic oral exposure to low-concentration fumonisin B2 significantly exacerbates the inflammatory responses of allergies in mice via inhibition of IL-10 release by regulatory T cells in gut-associated lymphoid tissue.

Contamination with fumonisins produced by Fusarium spp. is rapidly growing in both developing and developed countries. The purpose of this study was to determine whether oral exposure to fumonisin contributed to the development of allergic diseases. We initially examined the immunotoxic potential of short-term, oral administration of fumonisin B1 (FB1, 1mg/kg) and fumonisin B2 (FB2, 1mg/kg), both naturally occurring fumonisins, using a BALB/c mouse model of allergic contact dermatitis and Dermatophagoides farina-induced asthma. Using an NC/nga mouse model of atopic dermatitis (AD), we evaluated the adverse effects of subchronic oral exposure to low concentrations of FB2 (2 or 200μg/kg). Finally, we explored the influence of FB2 on regulatory T cell proliferation and function in mesenteric lymph nodes after 1-week oral exposure to FB2 in BALB/c mice. Oral exposure to FB2 markedly exacerbated the symptoms of allergy, including skin thickness, histological evaluation, immunocyte proliferation, and proinflammatory cytokine production, although no change was observed following exposure to FB1. Furthermore, oral exposure to low concentrations of FB2 considerably exacerbated the AD scores, skin thickness, transepidermal water loss, histological features, and proinflammatory cytokine production. The aggravated allergic symptoms induced by oral exposure to FB2 could be attributed to the direct inhibition of IL-10 production by regulatory T cells in mesenteric lymph nodes. Our findings indicate that the recommended maximum fumonisin level should be reconsidered based on the potential for allergy development.

Ionizing Radiation Reduces Head and Neck Squamous Cell Carcinoma Cell Viability and Is Associated with Predictive Tumor-Specific T Cell Responses

Head and neck squamous cell carcinoma (HNSCC) is common and deadly, and there is a need for improved strategies to predict treatment responses. Ionizing radiation (IR) has been demonstrated to improve HNSCC outcomes, but its effects on immune responses are not well characterized. We determined the impact of IR on T cell immune responses ex vivo. Human and mouse HNSCC cells were exposed to IR ranging from 20 to 200 Gy to determine cell viability and the ability to stimulate T-cell-specific responses. Lymph node cells of LY2 and MOC2 tumor-bearing or non-tumor-bearing mice were re-stimulated with a tumor antigen derived from LY2 or MOC2 cells treated with 200 Gy IR, ultraviolet (UV) exposure, or freeze/thaw cycle treatments. T cell proliferation and cytokine production were compared to T cells restimulated with plate-bound CD3 and CD28 antibodies. Human and mouse HNSCC cells showed reduced viability in response to ionizing radiation in a dose-dependent manner, and induced expression of T cell chemotactic cytokines. Tumor antigens derived from IR-treated LY2 and MOC2 cells induced greater proliferation of lymph node cells from tumor-bearing mice and induced unique T cell cytokine expression profiles. Our results demonstrate that IR induces potent tumoral immune responses, and IR-generated tumor antigens can potentially serve as an indicator of antitumor immune responses to HNSCC in ex vivo T cell restimulation assays.

Abstract 3276: Tumor cell-intrinsic PGE2-EP4 signaling is a major immunosuppressive pathway in pancreatic cancer

Abstract Pancreatic adenocarcinoma (PDA) is the third leading cause of cancer-related deaths in the US partly because of its resistance to conventional chemotherapies. Immunotherapies that improve survival in many cancers, almost universally fail in PDA. The resistance to immune checkpoint blockade is at least partially explained by the lack of T cells in the tumor microenvironment (TME) in PDA. We have previously demonstrated that tumor cell-derived prostaglandin E2 (PGE2) is one of the tumor intrinsic factors that creates/supports immunosuppressive TME in PDA presumably through its paracrine effect on the stromal cells. In the current study, we investigate the role of autocrine PGE2 signaling through its cognate EP4 receptor in shaping the T cell low, myeloid cell rich PDA TME. The knockdown (EP4 KD) or knockout (EP4 KO) of the receptors on KrasG12D/p53R172H/YFP/PdxCre (KPCY) tumor cells was sufficient to suppress or abolish the growth of implanted tumors, respectively (control vs EP4 KD, p=0.01, and control vs EP4 KO, p<0.0001). The rejection of the EP4 KO PDA cell lines was T cell dependent as it did not happen in hosts depleted of CD4+ and CD8+ T cells. Upon rechallenge, 80-90% of mice that rejected the primary EP4 KO tumors were able to reject the control, EP4-sufficient tumors in a T cell-dependent manner. While EP4 KO tumors were universally rejected by wild-type (WT) or EP4 global KO (KOgl) hosts, control cell lines formed tumors and grew in both hosts albeit with significantly slower growth in EP4 KOgl hosts (p<0.0001). The TME analysis revealed changes in the T cell presence, subtypes, and functional state in EP4 KO tumors demonstrating more T cell inflamed TME with increased proportions of central memory CD4+ and CD8+ cells (p=0.02 both) along with downregulation of exhaustion markers PD-1 (p=0.02) and CD39 (p=0.009) on CD4+ and CD8+ cells, respectively. Deletion of EP4 on tumor cells changed the systemic anti-tumor immunity by inducing T cell proliferation in lymph nodes (EP4 KO vs control, Ki67+CD8+ p<0.0001 and Ki67+CD4+ p=0.003) and depleting gMDSC population in the spleens of EP4 KO tumor bearing mice (p<0.0001). RNAseq analysis of tumor cells (cultured and in vivo) as well as bulk tumors identified TNFa, IFNa, and IFNg pathways as top upregulated ones in EP4 KO tumors while TGFb and Myc pathways were among the significantly downregulated pathways. The study demonstrates a primary role of tumor cell PGE2-EP4 signaling in creating immunosuppressive TME in PDA suggesting EP4 blockade as a novel approach in treatment of pancreatic cancer. Citation Format: Nune Markosyan, Charu Arora, Liz Quinones, Il-Kyu Kim, Nikhil Joshi, Noah Cheng, John Tobias, Ben Stanger, Robert Vonderheide. Tumor cell-intrinsic PGE2-EP4 signaling is a major immunosuppressive pathway in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3276.

Assessment of skin sensitization properties of few-layer graphene and graphene oxide through the Local Lymph Node Assay (OECD TG 442B)

Skin contact is one of the most common exposure routes to graphene-based materials (GBMs) during their small-scale and industrial production or their use in technological applications. Nevertheless, toxic effects in humans by cutaneous exposure to GBMs remain largely unexplored, despite skin contact to other related materials has been associated with adverse effects. Hence, this in vivo study was carried out to evaluate the cutaneous effects of two GBMs, focusing on skin sensitization as a possible adverse outcome.Skin sensitization by few-layer graphene (FLG) and graphene oxide (GO) was evaluated following the Organization for Economic Cooperation and Development (OECD) guideline 442B (Local Lymph Node Assay; LLNA) measuring the proliferation of auricular lymph node cells during the induction phase of skin sensitization. Groups of four female CBA/JN mice (8–12 weeks) were daily exposed to FLG or GO through the dorsal skin of each ear (0.4–40 mg/mL, equal to 0.01–1.00 mg/ear) for 3 consecutive days, and proliferation of auricular lymph node cells was evaluated 3 days after the last treatment. During this period, no clinical signs of toxicity and no alterations in body weight and food or water consumptions were observed. In addition, no ear erythema or edema were recorded as signs of irritation or inflammation. Bromo-deoxyuridine (BrdU) incorporation in proliferating lymphocytes from ear lymph nodes (stimulation indexes <1.6) and the histological analysis of ear tissues excluded sensitizing or irritant properties of these materials, while myeloperoxidase activity in ear biopsies confirmed no inflammatory cells infiltrate. On the whole, this study indicates the absence of sensitization and irritant potential of FLG and GO.

Observation of hapten-induced sensitization responses for the development of a mouse skin sensitization test, including the elicitation phase

The only official method that can detect the skin sensitizing potential of chemicals, including the elicitation response, is the OECD test guideline (TG) 406. However, this guideline uses guinea pigs, which requires complex procedures. Since a simple and complete test method for evaluating skin sensitization is needed, especially for mechanistic studies of skin sensitization, this study confirmed the reactivity of mice to skin sensitizing substances. We set up a protocol involving one induction exposure of the test substance to the back skin, followed by three challenge exposures to the auricle (Protocol 2), and compared their skin sensitization responses with the results of two exposures to the auricle and back skin every 2 weeks (Protocol 1) and a local lymph node assay (TG442B). A hapten 2,4-dinitrofluorobenzene caused significant auricular thickening, skin inflammation, and enlarged auricular lymph nodes in Protocols 1 and 2. These changes were more pronounced in Protocol 2. Plasma IgE and IgG1 and gene expression of IL4, IFNγ, and perforin were significantly increased in Protocol 2. Cell proliferation in the auricular lymph nodes was observed in both protocols as in TG442B. These results indicate that Protocol 2 can be a good candidate for a relatively simple skin sensitization test.

Prevention of Atopic Dermatitis in Mice by Lactobacillus Reuteri Fn041 Through Induction of Regulatory T Cells and Modulation of the Gut Microbiota.

The development of atopic dermatitis (AD) in infants is closely related to the lagging development of intestinal microbiota, including that inoculated by breast milk bacteria, and immune development. Lactobacillus reuteri Fn041 is a secretory immunoglobulin A (sIgA) -coated bacterium derived from human milk. We intervene with L. reuteri Fn041 in maternal and offspring BALB/C mice during late gestation and lactation and after weaning of the pups, respectively. AD is then induced with MC903. L. reuteri Fn041 significantly suppresses AD symptoms such as skin swelling, mast cell, and eosinophil infiltration. This effect is attributed to the regulation of the systemic Th1 and Th2 cytokine ratios and the promotion of CD4+ CD25+ Foxp3+ regulatory T cell proliferation in mesenteric lymph nodes. It is also associated with the regulation of intestinal microbiota, particularly promoting Lactobacillus and Akkermansia. Our study strengthens the understanding that breast milk-derived sIgA coated potential probiotics are involved in the development of infant intestinal microbiota, thus promoting immune development and preventing allergic diseases, and expanding the knowledge of breast milk sIgA and bacterial interactions on infant immune development.

Evaluation of skin sensitization induced by four ionic liquids.

Ionic liquids (ILs) are synthetic solvents used as replacements for volatile organic solvents. Human exposure occurs through dermal or oral routes. In rodents, several ILs were reported to induce dermal toxicity, irritation, and sensitization. Due to the potential for occupational exposure, and industrial use as nonvolatile solvents, 1-ethyl-3-methylimidazolium chloride (EMIM, 6.25% to 50% v/v), 1-butyl-3-methylimidazolium chloride (BMIM, 3.12% to 12.5% v/v), 1-butyl-1-methylpyrrolidinium chloride (BMPY, 0.825% to 6.25% v/v), and N-butylpyridinium chloride (NBuPY, 0.825% to 12.5% v/v) were nominated to the National Toxicology Program and evaluated for skin sensitization. The test compound was applied to the ears of female BALB/c mice daily for 3 days in a primary irritancy (IRR)/local lymph node assay (LLNA). Sensitization was assessed in vitro in the direct peptide reactivity assay (DPRA), KeratinoSens™ assay, and human cell line activation test (h-CLAT). In the LLNA, the butylated ILs, BMIM, and BMPY were more potent than NBuPY (butylated) or EMIM (ethylated), which was neither an irritant nor a sensitizer. NBuPY induced skin irritation in vivo at ≥3.12% (p ≤ 0.01), and sensitization in vitro in the KeratinoSens™ assay and h-CLAT, but was negative for sensitization in vivo and in the DPRA. Although SI3 was not achieved, dermal treatment with 12.5% BMIM or 6.25% BMPY increased (p ≤ 0.01) lymph node cell proliferation in the LLNA. In vitro, BMIM was positive for sensitization in the h-CLAT, and BMPY was positive in the h-CLAT and KeratinoSens™ assay; both were negative in the DPRA. Integrated data analyses, weighted toward in vivo data, suggested that BMIM and BMPY may induce weak to mild sensitization.

The evaluation of lymph node cell proliferation response by liposomes loaded with major histocompatibility complex class II binding aquaporin 4 antigen peptide.

Autoimmune responses to aquaporin 4 (AQP4) cause neuromyelitis optica (NMO); thus, specific immunotolerance to this self-antigen could represent a new NMO treatment. We generated the liposome-encapsulated AQP4 peptide 201-220 (p201-220) to induce immunotolerance. Liposomes were generated using phosphatidylserine and the polyglycidol species PG8MG. The in vivo tissue distribution of the liposomes was tested using an ex vivo imaging system. To confirm the antigen presentation capacity of PG8MG liposomes, dendritic cells were treated with PG8MG liposome-encapsulated AQP4 p201-220 (AQP4-PG8MG liposomes). Immunotolerance induction by AQP4-PG8MG liposomes was evaluated using the ex vivo cell proliferation of lymph node cells isolated from AQP4 p201-220-immunized AQP4-deficient mice. Fluorescent dye-labeled PG8MG liposomes were distributed to the lymph nodes. AQP4 p201-220 was presented on dendritic cells. AQP4-PG8MG liposomes were tended to suppress immune responses to AQP4 p201-220. Thus, the encapsulation of AQP4 peptides in PG8MG liposomes represents a new strategy for suppressing autoimmune responses to AQP4.

Evaluation of 2-methoxy-4-nitroaniline (MNA) in hypersensitivity, 14-day subacute, reproductive, and genotoxicity studies

2-Methoxy-4-nitroaniline (MNA), an intermediate in the synthesis of azo dyes used in textiles and paints, is structurally similar to carcinogenic anilines. Human exposure occurs primarily in the occupational setting through handling of dye dust, and through use and disposal of MNA-containing products. MNA has been reported to induce contact hypersensitivity in a human, myocardial necrosis in rats, and bacterial mutagenicity. This study assessed the subacute toxicity, genotoxicity, contact hypersensitivity, and reproductive toxicity of MNA in rodents in an effort to more fully characterize its toxicological profile. B6C3F1/N mice were exposed to 0, 650, 1250, 2500, 5000, or 10,000 ppm MNA by dosed feed for 14-days to evaluate subacute toxicity and histopathological endpoints. In female mice, decreased body weight (13.5 %) and absolute kidney weight (14.8 %), compared to control, were observed at 10,000 ppm MNA; increased relative liver weight (10–12 %), compared to control, occurred at 5,000–10,000 ppm MNA. In male mice, absolute (15 %) and relative liver weights (9–13 %) were increased at 2,500−5,000 ppm and 1250–10,000 ppm MNA, compared to control, respectively. In both sexes of mice, minimal elevations of hemosiderin pigmentation (a breakdown product of erythrocytes), relative to control, were observed in the liver (10,000 ppm); minimal to moderate elevations of hemosiderin pigmentation (5,000–10,000 ppm) and minimal increases in hematopoietic cell proliferation occurred in the spleen (≥ 1250 ppm). In a reproductive toxicity study, timed-mated female Harlan Sprague Dawley rats were exposed to 0–10,000 ppm MNA by dosed feed from gestation day 6 through postnatal day (PND) 21. Decreases in mean litter weights were observed at 5000 ppm MNA, compared to control, beginning at PND1. To evaluate potential contact hypersensitivity, MNA (2.5–50 %, in dimethylformamide) was applied to the dorsa of both ears of female Balb/c mice once daily for three days. The increase observed in lymph node cell proliferation (10–50 % increase in thymidine uptake compared to control) did not reproducibly achieve the Sensitization Index (SI) 3 level, and there was no ear swelling evident following sensitization with 10–50 % MNA and challenge with 25 % MNA in the mouse ear swelling test. In bacterial mutagenicity assays, MNA (250−1000 μg/plate) induced significant increases, compared to control, in mutant colonies with and without metabolic activation enzymes in Salmonella typhimurium strains TA100 and TA98. These data indicate that MNA is genotoxic, and may induce erythrocyte damage and reactive phagocytosis by macrophages in the liver and spleen.

Evaluation of skin sensitization potential of chemicals by local lymph node assay using 5-bromo-2-deoxyuridine with flow cytometry

The local lymph node assay using 5-bromo-2-deoxyuridine with flow cytometry (LLNA: BrdU-FCM) is a modified LLNA used to identify skin sensitizers. This assay measures the proliferation of auricular lymph node cells (LNCs) during the induction phase of skin sensitization and the number of BrdU-positive LNCs using flow cytometry. We determined if LLNA: BrdU-FCM can evaluate the skin sensitization potential of 20 substances, including 16 sensitizers and 4 non-sensitizers, that were tested using LLNA: DA and LLNA: BrdU-ELISA but not listed in OECD TG 429. After selecting appropriate vehicles and conducting pre-screen tests in 2 phases, solvents and test concentrations for the main test were determined. In the main study, we measured changes in LN weight, the number of LNCs, and the proportion of BrdU incorporated into LNCs to calculate stimulation indexes (SI). SI was calculated based on the total number of LNCs and BrdU incorporation in LNCs. We found that all substances were correctly classified as sensitizers or non-sensitizers. Overall, we confirmed that the LLNA: BrdU-FCM can evaluate skin sensitization potential of the 20 substances. Additionally, our results of combining 22 reference substances listed in OECD TG 429 and 20 additional substances showed that concordance of LLNA: BrdU-FCM with the LLNA was higher than before.

Systemic administration of orexin A ameliorates established experimental autoimmune encephalomyelitis by diminishing neuroinflammation

BackgroundOrexins (hypocretins, Hcrt) A and B are GPCR-binding hypothalamic neuropeptides known to regulate sleep/wake states and feeding behavior. A few studies have shown that orexin A exhibits anti-inflammatory and neuroprotective properties, suggesting that it might provide therapeutic effects in inflammatory and neurodegenerative diseases like multiple sclerosis (MS). In MS, encephalitogenic Th1 and Th17 cells trigger an inflammatory response in the CNS destroying the myelin sheath. Here, we investigated the effects of peripheral orexin A administration to mice undergoing experimental autoimmune encephalomyelitis (EAE), a widely used model of MS.MethodsMice were subcutaneously immunized with myelin oligodendrocyte glycoprotein peptide (MOG)35–55 in CFA. Mice were treated intraperitoneally for five consecutive days with either PBS or 300 μg of orexin A starting at a moderate EAE score. Molecular, cellular, and histological analysis were performed by real-time PCR, ELISA, flow cytometry, and immunofluorescence.ResultsOrexin A strongly ameliorated ongoing EAE, limiting the infiltration of pathogenic CD4+ T lymphocytes, and diminishing chemokine (MCP-1/CCL2 and IP-10/CXCL10) and cytokine (IFN-γ (Th1), IL-17 (Th17), TNF-α, IL-10, and TGF-β) expressions in the CNS. Moreover, orexin A treatment was neuroprotective, decreasing demyelination, astrogliosis, and microglial activation. Despite its strong local therapeutic effects, orexin A did not impair peripheral draining lymph node cell proliferation and Th1/Th17 cytokine production in response to MOG35–55 in vitro.ConclusionsPeripherally-administered orexin A ameliorated EAE by reducing CNS neuroinflammation. These results suggest that orexins may represent new therapeutic candidates that should be further investigated for MS treatment.

New approach to predict photoallergic potentials of chemicals based on murine local lymph node assay.

Photoallergic dermatitis, caused by pharmaceuticals and other consumer products, is a very important issue in human health. However, S10 guidelines of the International Conference on Harmonization do not recommend the existing prediction methods for photoallergy because of their low predictability in human cases. We applied local lymph node assay (LLNA), a reliable, quantitative skin sensitization prediction test, to develop a new photoallergy prediction method. This method involves a three-step approach: (1) ultraviolet (UV) absorption analysis; (2) determination of no observed adverse effect level for skin phototoxicity based on LLNA; and (3) photoallergy evaluation based on LLNA. Photoallergic potential of chemicals was evaluated by comparing lymph node cell proliferation among groups treated with chemicals with minimal effect levels of skin sensitization and skin phototoxicity under UV irradiation (UV+) or non-UV irradiation (UV-). A case showing significant difference (P<.05) in lymph node cell proliferation rates between UV- and UV+ groups was considered positive for photoallergic reaction. After testing 13 chemicals, seven human photoallergens tested positive and the other six, with no evidence of causing photoallergic dermatitis or UV absorption, tested negative. Among these chemicals, both doxycycline hydrochloride and minocycline hydrochloride were tetracycline antibiotics with different photoallergic properties, and the new method clearly distinguished between the photoallergic properties of these chemicals. These findings suggested high predictability of our method; therefore, it is promising and effective in predicting human photoallergens.

Vaccine potential of antigen cocktails composed of recombinant Toxoplasma gondii TgPI-1, ROP2 and GRA4 proteins against chronic toxoplasmosis in C3H mice

The development of an effective and safe vaccine to prevent Toxoplasma gondii infection is an important aim due to the great clinical and economic impact of this parasitosis. We have previously demonstrated that immunization with the serine protease inhibitor-1 (TgPI-1) confers partial protection to C3H/HeN and C57BL/6 mice. In order to improve the level of protection, in this work, we combined this novel antigen with ROP2 and/or GRA4 recombinant proteins (rTgPI-1+rROP2, rTgPI-1+rGRA4, rTgPI-1+rROP2+rGRA4) to explore the best combination against chronic toxoplasmosis in C3H/HeN mice. All tested vaccine formulations, administered following a homologous prime-boost protocol that combines intradermal and intranasal routes, conferred partial protection as measured by the reduction of brain cyst burden following oral challenge with tissue cysts of Me49 T. gondii strain. The highest level of protection was achieved by the mixture of rTgPI-1 and rROP2 proteins with an average parasite burden reduction of 50% compared to the unvaccinated control group. The vaccine-induced protective effect was related to the elicitation of systemic cellular and humoral immune responses that included antigen-specific spleen cell proliferation, the release of Th1/Th2 cytokines, and the generation of antigen-specific antibodies in serum. Additionally, mucosal immune responses were also induced, characterized by secretion of antigen-specific IgA antibodies in intestinal lavages and specific mesenteric lymph node cell proliferation. Our results demonstrate that rTgPI-1+rROP2 antigens seem a promising mixture to be combined with other immunogenic proteins in a multiantigenic vaccine formulation against toxoplasmosis.

Gene expression profiles in auricle skin as a possible additional endpoint for determination of sensitizers: A multi-endpoint evaluation of the local lymph node assay

The murine local lymph node assay (LLNA) is widely used to test chemicals to induce skin sensitization. Exposure of mouse auricle skin to a sensitizer results in proliferation of local lymph node T cells, which has been measured by in vivo incorporation of H3-methyl thymidine or 5-bromo-2′-deoxyuridine (BrdU). The stimulation index (SI), the ratio of the mean proliferation in each treated group to that in the concurrent vehicle control group, is frequently used as a regulatory-authorized endpoint for LLNA. However, some non-sensitizing irritants, such as sodium dodecyl sulfate (SDS) or methyl salicylate (MS), have been reported as false-positives by this endpoint. In search of a potential endpoint to enhance the specificity of existing endpoints, we evaluated 3 contact sensitizers; (hexyl cinnamic aldehyde [HCA], oxazolone [OXA], and 2,4-dinitrochlorobenzene [DNCB]), 1 respiratory sensitizer (toluene 2,4-diisocyanate [TDI]), and 2 non-sensitizing irritants (MS and SDS) by several endpoints in LLNA. Each test substance was applied to both ears of female CBA/Ca mice daily for 3 consecutive days. The ears and auricle lymph node cells were analyzed on day 5 for endpoints including the SI value, lymph node cell count, cytokine release from lymph node cells, and histopathological changes and gene expression profiles in auricle skin. The SI values indicated that all the test substances induced significant proliferation of lymph node cells. The lymph node cell counts showed no significant changes by the non-sensitizers assessed. The inflammatory findings of histopathology were similar among the auricle skins treated by sensitizers and irritants. Gene expression profiles of cytokines IFN-γ, IL-4, and IL-17 in auricle skin were similar to the cytokine release profiles in draining lymph node cells. In addition, the gene expression of the chemokine CXCL1 and/or CXCL2 showed that it has the potential to discriminate sensitizers and non-sensitizing irritants. Our results suggest that multi-endpoint analysis in the LLNA leads to a better determination of the sensitizing potential of test substances. We also show that the gene expression of CXCL1 and/or CXCL2, which is involved in elicitation of contact hypersensitivity (CHS), can be a possible additional endpoint for discrimination of sensitizing compounds in LLNA.

The effects of increased heme oxygenase-1 on the lymphoproliferative response in dogs with visceral leishmaniasis

Canine visceral leishmaniasis (CVL) is known to affect the cellular immunity of infected dogs, through impairing lymphoproliferation and microbicidal mechanisms. This study examined heme oxygenase-1 (HO-1) and its metabolites, oxidative stress and IL-10 levels in CVL and investigated correlations between these parameters. Additionally, the effects of HO-1 inhibition on the lymphoproliferative response and cytokine production in lymph node cells (LNCs) from infected dogs were evaluated. Forty-four dogs, 24 controls and 20 dogs with CVL were selected. Plasma and splenic levels of HO-1, haptoglobin, soluble CD163 receptor, ferritin and IL-10 were determined using capture ELISA. The HO-1 levels and relative gene expression in peripheral blood and bone marrow mononuclear cells were also determined. LNCs proliferation was evaluated with an HO-1 activator and with an HO-1 inhibitor, in the presence of the Leishmania infantum soluble antigen (SAgL), using flow cytometry. HO-1, IL-2, IFN-gamma and IL-10 were also determined in these cultures using capture ELISA. Infected dogs presented oxidative stress and increased HO-1 levels and relative gene expression, with correlation between oxidative stress and HO-1. The substances from heme metabolism and IL-10 were also elevated in the plasma and spleens of infected dogs. IL-10 and HO-1 levels were positively correlated with one another. Inhibition of HO-1 increased LNCs proliferation and decreased IL-10 and IL-2 production in the presence of SAgL. The increased HO-1 metabolism observed in CVL is probably associated with oxidative stress and increased IL-10, which could be one of the mechanisms responsible for inhibition of the lymphoproliferative response in sick dogs.

Strain differences in intestinal toxicity of warfarin in rats.

Intestinal hemorrhage characterizes effectiveness of warfarin (WF) as rodenticide and is among adverse effects of therapy in humans. Having in mind genetic variations in the effectiveness of WF in wild rats and in the doses required for therapeutic effect, strain differences in the intestinal toxicity of oral warfarin in rats were examined in this study. High WF dose (3.5mg/l) led to mortality in Albino Oxford (AO) rats, with no lethality in Dark Agouti (DA) rats. Higher values of prothrombin time were noted at low WF dose (0.35mg/l) in the former strain. Leukocyte infiltration in intestine noted at this dose in both strains was associated with oxidative injury and more pronounced anti-oxidative response in AO rats. Suppression of mesenteric lymph node cell proliferation and IFN-γ and IL-10 production in AO rats and lack of these effects in DA rats, represent different strategies to protect vulnerable intestine from harmful immune responses.

JAK inhibitor JTE-052 regulates contact hypersensitivity by downmodulating T cell activation and differentiation

BackgroundUsing JAK inhibitors to inhibit cytokine signaling is presumed to be a possible means of treating skin inflammatory disorders such as contact dermatitis. ObjectiveTo clarify the action site of JAK inhibitors in skin inflammatory disorders. MethodsWe analyzed the mechanism of action of the JAK inhibitor JTE-052 using murine skin inflammation models, including contact hypersensitivity (CHS) and irritant contact dermatitis. Cells isolated from ear tissue or lymph node (LN) were analyzed by flow cytometry. The amounts of cytokines in the culture medium were measured by ELISA or bead array system. Proliferation of LN cells was evaluated by measurement of tritiated thymidine incorporation. ResultsOral administration of JTE-052 during both sensitization and elicitation phase attenuated CHS, but did not affect croton oil-induced irritant contact dermatitis. JTE-052 potently inhibited T cell proliferation and activation by antigen presentation in vitro, and attenuated skin inflammation in a sensitized-lymphocyte transfer model without suppressing T cell migration. JTE-052 did not affect hapten-induced cutaneous dendritic cell migration into draining lymph nodes or their costimulatory molecule expressions. ConclusionThe JAK inhibitor JTE-052 exerts an inhibitory effect on antigen-specific T cell activation and subsequent inflammation in acquired skin immunity, such as CHS.

Dietary litchi pulp polysaccharides could enhance immunomodulatory and antioxidant effects in mice

The purpose of this study was to investigate the immunomodulatory and antioxidant activities of litchi pulp polysaccharides (LP) in cyclophosphamide (Cy)-induced mice. The administration of LP (50, 100 and 200mg/kg/d) remarkably enhanced mesenteric lymph node cells proliferation and serum IgA. Moreover, IL-6, TNF-α, IgG and IgM levels in serum were significantly improved in a dose-dependent manner with LP treatment. Dietary LP induced systemic immune responses including stimulating the proliferation of splenocytes, balancing the ratio of spleen lymphocyte subsets, up-regulating the thymus and spleen indices. Thus, we speculate litchi polysaccharides would express their systemic immunomodulatory effects by triggering the intestinal mucosal immunity. Likewise, LP also significantly increased total antioxidant capacity, as well as superoxidase dismutase and glutathione peroxidase activity, while decreasing malondialdehyde levels in the serum and liver. The immunomodulatory activity of LP was accompanied with its antioxidant activity, which might be related with its structure and prebiotic effect. This is a novel study on the relationship between the immunomodulatory and antioxidant activities of litchi polysaccharides.

Characterization and mesenteric lymph node cells-mediated immunomodulatory activity of litchi pulp polysaccharide fractions

Three water-soluble hetero-polysaccharides, designated LP1–3, were isolated from litchi pulp. Their structures, solution properties and immunomodulatory activities were evaluated. LP1 contained (1→4,6)-β-d-Glc and (1→4)-α-l-Gal, while LP2 contained (1→3)-α-l-Ara and (l→2)-β-d-Gal, and LP3 contained α-l-Ara and (l→4)-β-Rha. Their molecular weights ranged from 105,880 to 986,470g/mol. LP1 had a spherical conformation with hyper-branched structure and LP2 was semi-flexible chain, while the polysaccharide chains of LP3 were cross linked to form network-like conformation in solution. In addition, all fractions strongly stimulated mesenteric lymph node cell proliferation, IFN-γ and IL-6 secretion in the dose range of 25–100μg/mL compared with untreated control group (p<0.05). LP1 exhibited the strongest stimulation of mesenteric lymph node cell proliferation and cytokine secretion, which may be attributed to its unique chemical structure and chain conformation. This is the first report on the solution properties and intestinal immunity activities of polysaccharides from litchi pulp.