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Increased expectations from a number of regulatory agencies, e.g., Environmental Protection Agency (EPA), Food and Drug Administration (FDA), European Medicines Agency (EMEA), and the Ministry of Health, Labour and Welfare (MHLW) of Japan, call for the evaluation of potential adverse effects on the immune system. As recently summarized in the ICH S8 guideline, the T-cell-dependent antibody response (TDAR) has been identified in a regulatory context as a main functional test of immunotoxicity. While the characterization of immunotoxic potential is pertinent to both the chemical and pharmaceutical industries, the use of immunotoxicology data for hazard identification and/or risk assessment in each case is different. Therefore, multiple approaches to immunotoxicity testing have evolved. The assays that evaluate TDAR function include both well-established tests, e.g., anti-sheep red blood cell plaque-forming cell (PFC) assay, and newer models, e.g., anti-keyhole limpet hemocyanin (KLH) antibody ELISA. These tests vary in the study design, antigen application and analytical methods. However, they all evaluate the same endpoint - a competent immune (e.g., antibody) response to an antigen. Numerous issues have been identified in the application of TDAR tests, including high animal to animal variability; differences in antigen source and potency; a lack of established "normal range" of the immune response and uncertainty about the degree of inhibition of the TDAR to be considered toxicologically important. As such, the need for a forum to discuss these issues was recognized by the immunotoxicology community, and was addressed at the 2006 Society of Toxicology (SOT) Workshop. A series of papers will summarize that forum with the ultimate objectives being to build a consensus among immunotoxicologists on the implications of these factors on using TDAR results in hazard identification and/or risk assessment, and to establish a criteria to classify compounds as immunotoxicants. [ABSTRACT FROM AUTHOR]

The immunotoxic potential of drug candidates is assessed through the examination of results from a variety of studies and endpoints. While the functional assessment of CD8+ cytotoxic T-lymphocytes (CTL) is well-characterized in the clinic, the lack of a robust macaque CTL functional assay has been an important hurdle in evaluating and accurately quantifying cell-mediated CD8+ T-cell effector responses in the nonclinical setting. This paper describes the development of an assay to measure CTL activity in peripheral blood mononuclear cells (PBMC) isolated from Cynomolgus macaques. A human EGFR/CD3 Bispecific T-cell Engager (BiTE®) was used to mount a robust CD8+ T-cell response in the presence of target-expressing cells. Upon target engagement, degranulation of CD107a and production of interferon (IFN)-γ both reliably indicated a robust functional response in CD8+ T-cells. The BiTE®-mediated stimulation method proved to be favorable when compared to other methods of stimulation in the absence of target cells. These studies demonstrated acceptable longitudinal variability of the functional assay and sensitivity to dexamethasone-mediated immunosuppression. Taken together, the results indicated an assay leveraging CD3-bispecific antibodies and target-expressing cells can provide a robust approach to the in vitro or ex vivo assessment of CTL function in Cynomolgus macaques. Because the impairment of CTL activity by immunomodulators is recognized to be an important contributor to decreased antiviral defense and increased carcinogenicity risk, we believe that this novel assay to be a valuable addition to the immunotoxicology assessment of therapeutic drug candidates. [ABSTRACT FROM AUTHOR]

The number of anti-inflammatory and immunomodulatory drugs being developed in the pharmaceutical industry has increased considerably in the past decade. This increase in research and development has been paralleled by questions from both regulatory agencies and industry on how best to assess decreased host resistance to infections or adverse immunostimulation caused by immunomodulatory agents such as anti-cytokine antibodies (e.g., the tumor necrosis factor-α inhibitors), anti-adhesion molecule antibodies (e.g., anti-α-4 integrin inhibitors) and immunostimulatory molecules (e.g., anti-CD28 antibodies). Although several methods have been developed for nonclinical assessment of immunotoxicity, highly publicized adverse events have brought to light significant gaps in the application of nonclinical immunotoxicity testing in assessing potential risk in humans. Confounding this problem is inconsistent application of immunotoxicology methods for risk assessment within the scientific community, limited understanding of appropriate immunotoxicity testing strategy for immunomodulators and inconsistent testing requests by regulatory agencies. To address these concerns, The Immunotoxicology Technical Committee (ITC) of the International Life Science Institute (ILSI) Health and Environmental Sciences Institute (HESI) organized a workshop on Immunomodulators and Clinical Immunotoxicology in May 2007. The Workshop was convened to identify key gaps in nonclinical and clinical immunotoxicity testing of anti-inflammatory and immunomodulatory agents and to begin to develop consistent approaches for immunotoxicity testing and risk assessment. This paper summarizes the outcome of the HESI ITC Immunomodulators and Clinical Immunotoxicology Workshop. Topics not discussed at the Workshop were outside the scope of this report. Although more work is needed to develop consistent approaches for immunotoxicity assessment of immunomodulators, this Workshop provided the foundation for future discussion. [ABSTRACT FROM AUTHOR]

It has long been known that the developing immune system is more sensitive and susceptible than the adult immune system to some drugs and environmental contaminants. However, notable advances have been made in the database of studies supporting developmental immunotoxicity (DIT) over the past 5 years. There is considerable evidence that responses of the immune system can be quantitatively or qualitatively different from normal adult responses when xenobiotic exposure occurs during critical periods of immune system development. Qualitative differences of DIT relative to adult exposures include examples of more persistent effects, a latency of effects, and immune dysfunction that is fundamentally different than effects observed when adults are exposed. A symposium was presented at the Society of Toxicology annual meeting to provide an update on advances in the maturing field of developmental immunotoxicology and to facilitate discussion on the range of DIT and later life effects following developmental exposure. In particular, presentations focused on implications of neuroendocrine cross-talk for DIT, the association between developmental air pollutant exposure and asthma, and recent evidence that developmental exposure to 2,3,7,8-tetrachlorodibenzo- p-dioxin may increase the risk of autoimmune responses. Several important concepts relative to DIT assessment were illustrated, i.e., (1) Screening for immunosuppression alone is not sufficient to identify all potential immunotoxic effects; (2) DIT cannot be reliably predicted from studies that only utilize adult exposures; (3) Functional testing protocols are preferred in the assessment of DIT; (4) Gender-related differences should be routinely assessed; (5) Latency (i.e., later-life adverse outcomes resulting from developmental exposures) is an important consideration that cannot be detected in adult exposure studies; and, (6) There is increasing support for DIT testing protocols with continuous exposure throughout development until the immune assay is performed. [ABSTRACT FROM AUTHOR]

Micro- and nanoplastics (MNP) are ubiquitously present in the environment due to their high persistence and bioaccumulative properties. Humans get exposed to MNP via various routes and consequently, they will encounter dendritic cells (DC) which are antigen-presenting cells involved in regulating immune responses. The consequences of DC exposure to MNP are an important, yet understudied, cause of concern. Therefore, this study aimed to assess the uptake and effect of MNP in vitro by exposing human monocyte-derived dendritic cells (MoDC) to virgin and environmentally weathered polystyrene (PS) particles of different sizes (0.2, 1, and 10 µm), at different concentrations ranging from 1 to 100 µg/ml. The effects of these particles were examined by measuring co-stimulatory surface marker (i.e. CD83 and CD86) expression. In addition, T-cell proliferation was measured via a mixed-leukocyte reaction (MLR) assay. The results showed that MoDC were capable of absorbing PS particles, and this was facilitated by pre-incubation in heat-inactivated (HI) plasma. Furthermore, depending on their size, weathered PS particles in particular caused increased expression of CD83 and CD86 on MoDC. Lastly, weathered 0.2 µm PS particles were able to functionally activate MoDC, leading to an increase in T-cell activation. These in vitro data suggest that, depending on their size, weathered PS particles might act as an immunostimulating adjuvant, possibly leading to T-cell sensitization. [ABSTRACT FROM AUTHOR]

In development of peptide therapeutics, rodents are commonly-used preclinical models when screening compounds for efficacy endpoints in the early stages of discovery projects. During the screening process, some peptides administered subcutaneously to rodents caused injection site reactions manifesting as localized swelling. Screening by postmortem evaluations of injection site swelling as a marker for local subcutaneous histamine release, were conducted in rats to select drug candidates without this adverse effect. Histological analysis of skin samples revealed that the injection site reactions were concurrent with mast cell degranulation, resulting in histamine release. Mast cell activation can be mediated by MRGPRX2, a GPCR that induces a pseudo-allergenic immune response. The present study demonstrates that a commercially-available cell-based MRGPRX2 assay reliably identifies compounds that induce histamine release or localized edema in ex vivo human and rodent skin samples. In vitro screening was subsequently implemented using the MRGPRX2 assay as a substitute for postmortem injection site evaluation, thus achieving a significant reduction in animal use. Thus, in cases where injection site reactions are encountered during in vivo screening, to enable faster screening during the early drug discovery process, an MRGPRX2 in vitro assay can be used as an efficient, more ethical tool with human translational value for the development of safer pharmacotherapies for patients. [ABSTRACT FROM AUTHOR]

Resveratrol is a naturally occurring polyphenol that is being investigated to treat and prevent various diseases, both experimentally and in the clinic. Despite increased use and interest in resveratrol due to its immunomodulatory properties, there is a lack of studies evaluating potential toxicities, particularly immunotoxicity, associated with resveratrol use. A previous 2-week study found decreasing thymus weight in male B6C3F1/N mice with increasing exposure to trans-resveratrol. This study is a follow-up on those findings by evaluating immune function. Male adult B6C3F1/N mice were given trans-resveratrol (0, 156, 312, 625, 1250, 2500 mg/kg/day) via oral gavage for 28 days and functional immune tests and histopathology were evaluated. There were no treatment-related effects on body weight during the study. Humoral, cell-mediated, and innate immune function were not altered after 28 days of trans-resveratrol treatment. There were also no changes in organ weight or microscopic alterations in immune organs. Overall, under the conditions of this study, there was no evidence of immunotoxicity or improvements in immune function associated with oral exposure to trans-resveratrol in male mice. Importantly, the immunomodulatory benefits of resveratrol may require a prerequisite level of inflammatory activity and may not be observable in healthy individuals. [ABSTRACT FROM AUTHOR]

The immunotoxicant bisphenol A (BPA) may produce toxic effects on organs and systems, in part, by altering the secretion of cytokines and chemokines. However, systematic studies of the effects of BPA, let alone of its analogs and in cases when there are interactions with other chemicals, on innate immunity and cytokine modulation are limited. The objectives of this study were to investigate the immunomodulatory effects of: (1) BPA and its analogs, BPS and BPAF; and (2) the interaction between BPA and genistein (GEN), a partial estrogen agonist or antagonist. BPA, BPS, and BPAF were incubated with PMA-differentiated-U937 cells (a widely used cell line for primary human macrophages) at concentrations of 0, 0.1, 1, 10, 100 µM for up to 96 h. BPA (0, 0.1, 1, 10 µM) and GEN (0, 1, 10 µM) were also applied at various combinations. Cell viability and 30 cytokines/chemokines were measured. The results showed that the cell viability-inhibiting effect of these three bisphenols was BPAF > BPA > BPS. At 0.1 µM, BPA and BPAF generally increased the secretion of cytokines/chemokines, while BPS had minimal effects. All three bisphenols generally suppressed the secretion of cytokines/chemokines at 1 µM, while increased their secretion at 10 µM. The most increased cytokines/chemokines were interferon (IFN)-γ, interleukin (IL)-1RA, IL-8 and MIP-1β, and the most decreased was IL-10. GEN increased cell viability at low BPA concentrations but had no effect when BPA levels were high. In general, GEN attenuated the BPA-induced secretion of cytokines/chemokines but enhanced it at low BPA concentrations. In conclusion, this study showed that BPA, BPS, and BPAF were immunotoxic to macrophages: BPS was the least toxic, while BPAF was the most toxic. Further, GEN reversed suppressive effects on macrophages that resulted from exposure to high concentrations of BPA and produced synergetic effects with BPA at low concentrations. [ABSTRACT FROM AUTHOR]

The aim of the present study was to explore the potential hematotoxic and immunotoxic effects of melamine (MA) in the absence and presence of formaldehyde (FA) in mice. Forty adult Swiss mice were equally allocated into four groups and daily treated with water, MA (50 mg/kg), FA (25 mg/kg), and MA + FA respectively via feeding needle for 60 consecutive days. Hematological status was evaluated using erythrogram and leukogram profiling. Innate immune functions were assessed by measuring white blood cells lysozyme and phagocytic activities. Serum immunoglobulin levels were evaluated as indicators of humoral immunity. In addition, histologic and immunohistochemical evaluations of splenic tissues were performed. The results indicated that either MA or FA treatment resulted in significant decreases in RBCs, Hb, MCHC, total WBC, lymphocyte, and basophile levels as well as in WBCs phagocytosis and lysozyme activity. In contrast, MCV, PCV%, and reticulocyte levels were significantly increased in these hosts. The total IgM level was significantly reduced in the MA-only-exposed mice but markedly increased in the FA-only-treated ones. A significant decrease in serum IgG levels was detected following either MA or FA treatment. The combined exposure to MA and FA, compared to levels of either toxicant alone, was revealed to evoke a significant improvement in Hb, PCV%, MCV, MCHC, neutrophil, eosinophil, total IgM level, and lysozyme activity; however these values did not reach that of the controls. Furthermore, compared to control mice, both MA-only- and FA-only-treated mice showed a strong distribution of CD4+and CD8+cells in their spleens, while a moderate presence of the former cells was obvious at their co-exposure. Taken together, these findings revealed that exposure to MA or FA resulted in significant alterations in hemato-immune parameters at variable degrees while a co-exposure resulted in the mitigation of most effects of either toxicant alone. [ABSTRACT FROM AUTHOR]

The potential to engineer extracellular vesicles (EV) that target specific cells and deliver a therapeutic payload has propelled a growing interest in their development as promising therapeutics. These EV are often produced from cultured cells. Very little is known about the interaction of cell culture-derived EV with cells of the immune system and their potential immunomodulatory effects. The present study evaluated potential immunotoxic effects of HEK293T-derived EV on the human monocytic cell lines THP-1 and U937. Incubation of cells with different doses of EV for 16–24 h was followed by assessment of cytotoxicity and cell function by flow cytometry. Changes in cell functionality were evaluated by the capacity of cells to phagocytize fluorescent microspheres. In addition, the internalization of labeled EV in THP-1 and U937 cells was evaluated. Exposure to EV did not affect the viability of THP-1 or U937 cells. Although lower doses of the EV increased phagocytic capacity in both cell lines, phagocytic efficiency of individual cells was not affected by EV exposure at any of the doses evaluated. This study also demonstrated that THP-1 and U937 monocytic cells are highly permissive to EV entry in a dose-response manner. These results suggest that, although HEK293T-derived EV are efficiently internalized by human monocytic cells, they do not exert a cytotoxic effect or alter phagocytic efficiency on the cell lines evaluated. [ABSTRACT FROM AUTHOR]

The development of immune-dependent drug hypersensitivity reactions (IDHR) is likely to involve activation of the innate immune system to stimulate neo-antigen specific T-cells. Previously it has been shown that, upon oral exposure to several drugs with immune-adjuvant capacity, mice developed T-cell-dependent responses to TNP-OVA. These results were indicative of the adjuvant potential of these drugs. The present study set out to evaluate the nature of this adjuvant potential by focusing on early immune changes in the spleen, by testing several drugs in the same experimental model. Mice were exposed to one or multiple oral doses of previously-tested drugs: the non-steroidal-anti-inflammatory drug (NSAID) diclofenac (DF), the analgesic acetaminophen (APAP), the anti-epileptic drug carbamazepine (CMZ) or the antibiotic ofloxacin (OFLX). Within 24 h after the final dosing, early innate and also adaptive immune parameters in the spleen were examined. In addition, liver tissue was also evaluated for damage. Exposure to APAP resulted in severe liver damage, increased levels of serum alanine aminotransferase (ALT) and local MIP-2 expression. DF exposure did not cause visible liver damage, but did increase liver weight. DF also elicited clear effects on splenic innate and adaptive immune cells, i.e. increased levels of NK cells and memory T-cells. Furthermore, an increase in plasma MIP-2 levels combined with an influx of neutrophils into the spleen was observed. OFLX and CMZ exposure resulted in increased liver weights, MIP-2 expression and up-regulation of co-stimulatory molecules on antigen-presenting cells (APC). The data suggested that multiple immune parameters were altered upon exposure to drugs known to elicit immunosensitization and that broad evaluation of immune changes in straightforward short-term animal models is needed to determine whether a drug may harbor the hazard to induce IDHR. The oral exposure approach as used here may be applied in the future as an immunotoxicological research tool in this type of evaluation. [ABSTRACT FROM AUTHOR]

Vanadium (V) is a transition metal often adhered to particulate matter and released into the atmosphere as vanadium pentoxide (V2O5) by the burning of fossil fuels. This air pollutant causes adverse effects in the immune system. Lymphocytosis and splenomegaly have been reported with increased white pulp in mice after V inhalation. The effect of V on the immune system as related to sex has been poorly investigated. This study sought to determine if V inhalation (a) produced lymphoproliferation that could explain the changes previously observed in the spleen and in peripheral blood lymphocyte counts and (b) whether any observed effects differed due to gender. Immunohistochemical analyses of Ki-67, a specific proliferation marker, was made in the spleens of CD-1 male and female mice exposed for 1 h, twice a week, over a 12-week period to V2O5(at 1.4 mg V2O5/m3) by whole-body inhalation; similar analyses were performed on spleens of control mice exposed to vehicle (filtered air). The results showed that in male mice there was a significant increase in percentage of Ki-67 immunopositive lymphocytes starting from the second week and until the end of the exposure. The Ki-67 signal was cytoplasmic and nuclear in the exposed males, while in controls the signal was only nuclear. In female mice, V inhalation singificantly increased the percentage of proliferating lymphocytes only after 1 week of exposure. Ki-67 signal was observed only in the nucleus of lymphocytes from the control and exposed females. The results here help to explain the splenomegaly and lymphocytosis observed previously in male mice and support the lymphoproliferative effect induced by V. Lastly, the finding that there was a sex difference in the effect of vanadium on lymphocyte proliferation suggests a role for sex hormones in potential protection against V immunotoxicity; however, further studies are needed to support this hypothesis. [ABSTRACT FROM AUTHOR]

The objective of this study was to characterize the variability of rat lymphoid organ weights and morphology following treatment with a known immunotoxicant, with a focus on the usefulness of evaluating popliteal lymph node weight and histology. Cyclophosphamide was administered to male Sprague-Dawley rats by oral gavage at doses of 2, 7 or 12 mg/kg/day for 10 consecutive days. Left and right popliteal lymph nodes (PLN), spleen and thymus were collected at necropsy, weighed, fixed and processed for histopathology. Femoral bone marrow was also collected, fixed and processed for histology. Organ weight variability was greater for PLN than for either spleen or thymus in control animals. There was a significant but weak correlation between paired left and right PLN weights (p < 0.005;r2 = 0.2774). Significant treatment-related decreases in lymphoid organ weights were observed in spleen and thymus at ≥ 7 mg/kg/day (p < 0.01), whereas in PLN a significant decrease (p < 0.05) was noted only at 12 mg/kg/day. The inclusion of PLN did not enhance the sensitivity of detection of systemic treatment-related changes in lymphoid organs in a rat cyclophosphamide model. [ABSTRACT FROM AUTHOR]

The immunotoxic effects of the isoquinoline alkaloid berberine (BBR) were investigated in Balb/c mice. Here, BBR was administered daily by intraperitoneal injection at doses of 5 and 10 mg/kg for 14 days. Following the exposure, host spleen weight, cellularity and histopathology, as well as delayed-type hypersensitivity (DTH) responses, hemagglutination titers (HA), spleen cell subtype profiles, splenocyte cytokine production and lymphocyte proliferation were studied in all of the test groups of animals. The results showed that the high dose of BBR (10 mg/kg) could suppress both cellular and humoral immune functions in the treated hosts. BBR at 5 mg/kg only appeared to impact on DTH responses and lymphoproliferation. Based on the finding here, it would seem that BBR has effective immunosuppressive properties. Mechanistic studies are required to determine exactly how this material is acting to impart many of the immunotoxic effects demonstrated here. At the same time, further research should also be performed on BBR to further develop its potential use as an effective immunosuppressant or co-adjuvant for the treatment of diseases caused by an exaggerated or unwanted immune response. [ABSTRACT FROM AUTHOR]

An important component of safety assessment of new pharmaceuticals is evaluation of their potential to increase the risk of developing cancer in humans. The traditional 2-year rodent bioassay often is not feasible or scientifically applicable for evaluation of biotherapeutics. Additionally, it has poor predictive value for non-genotoxic immunosuppressive compounds. Thus, there is a need for alternative testing strategies. A novel 3-stage tumor model in syngeneic C3H/HeN mice was evaluated here to study the effects of immunosuppressive drugs on tumor promotion and progressionin vivo. The model employed a skin squamous cell carcinoma cell line (SCC VII) due to the increased prevalence of squamous cell carcinoma (SCC) in humans associated with immunosuppression after transplants. Local invasion, colonization and tumor progression were evaluated. The validation set of immunosuppressive drugs included: Cyclosporin (CSA), cyclophosphamide (CTX), azathioprine, etanercept, abatacept and prednisone. Local invasion was evaluated by histological assessment as well as fluorescence trafficking from Qdot®-labeled tumor cells from the site of inoculation to the draining lymph node. Colonization was evaluated by lung colony counts following intravenous inoculation. Tumor progression was assessed by morphometric analysis of lesion area, angiogenesis and growth fraction of established metastatic neoplasia. Immunosuppressive drugs in the validation set yielded mixed results, including decreased progression. The methods and results described herein using anin vivosyngeneic mouse tumor model can provide insight about the assessment of immunosuppressive drugs in carcinogenicity risk assessment. [ABSTRACT FROM AUTHOR]

Triclosan is an antimicrobial chemical commonly used occupationally and by the general public. Using select immune function assays, the purpose of these studies was to evaluate the immunotoxicity of triclosan following dermal exposure using a murine model. Triclosan was not identified to be a sensitizer in the murine local lymph node assay (LLNA) when tested at concentrations ranging from 0.75–3.0%. Following a 28-day exposure, triclosan produced a significant increase in liver weight at concentrations of ≥ 1.5%. Exposure to the high dose (3.0%) also produced a significant increase in spleen weights and number of platelets. The absolute number of B-cells, T-cells, dendritic cells and NK cells were significantly increased in the skin draining lymph node, but not the spleen. An increase in the frequency of dendritic cells was also observed in the lymph node following exposure to 3.0% triclosan. The IgM antibody response to sheep red blood cells (SRBC) was significantly increased at 0.75% – but not at the higher concentrations – in the spleen and serum. These results demonstrate that dermal exposure to triclosan induces stimulation of the immune system in a murine model and raise concerns about potential human exposure. [ABSTRACT FROM AUTHOR]

Signaling networks are essential elements that are involved in diverse cellular processes. One group of fundamental components in various signaling pathways concerns protein tyrosine kinases (PTK). Various toxicants have been demonstrated to exert their toxicity via modulation of tyrosine kinase activity. The present study aimed to identify common cellular signaling pathways that are involved in chemical-induced direct immunotoxicity. To this end, an antibody array-based profiling approach was applied to assess effects of five immunotoxicants, two immunosuppressive drugs and two non-immunotoxic control chemicals on the phosphorylation of 28 receptor tyrosine kinases and 11 crucial signaling nodes in Jurkat T-cells. The phosphorylation of ribosomal protein S6 (RPS6) and of kinases Akt, Src and p44/42 were found to be commonly regulated by immunotoxicants and/or immunosuppressive drugs (at least three compounds), with the largest effect observed upon RPS6. Flow cytometry and Western blotting were used to further examine the effect of the model immunotoxicant TBTO on the components of the mTOR-p70S6K-RPS6 pathway. These analyses revealed that both TBTO and the mTOR inhibitor rapamycin inactivate RPS6, but via different mechanisms. Finally, a comparison of the protein phosphorylation data to previously obtained transcriptome data of TBTO-treated Jurkat cells resulted in a good correlation at the pathway level and indicated that TBTO affects ribosome biogenesis and leukocyte migration. The effect of TBTO on the latter process was confirmed using a CXCL12 chemotaxis assay. [ABSTRACT FROM PUBLISHER]

Aflatoxins (AF) are important foodborne mycotoxins implicated in human health and have immunocytotoxic effects. The aims of this study were to evaluate a new aflatoxin B1(AFB1) and fumonisin B1(FB1)-binding/degrading micro-organism for biological detoxification, to examine its ability to degrade AFB1and FB1in liquid medium, and to evaluate its potentialin vivoprotective role against any combined effects from AFB1and FB1on host splenocyte caspase-3 activity (reflecting DNA damage/cell death) and mRNA levels of select inflammation-regulating cytokines. Balb/c mice were divided into groups (10/group) and treated daily for 2 weeks by oral gavage with AFB1(80 µg/kg BW), FB1(100 µg/kg), AFB1 + FB1, or lactic acid bacteria (Lactobacillus paracaseiBEJ01, 2 × 109 CFU/L, ∼2 mg/kg) – alone or in combination with the AFB1and/or FB1. After the exposures, spleens were collected for measures of caspase-3 activity, lipid peroxidation (LP), and glutathione (GSH) content, expression of anti-oxidation protective enzymes (GPx and SOD), and mRNA levels of inflammation-regulating cytokines (e.g.IL-10, IL-4, IFNγ, TNFα). Thymii were also removed for analysis of apoptosis. The results indicated that, in the spleen, exposure to the mycotoxins led to increased caspase-3 activity, LP, andIL-10andIL-4mRNA levels, but decreased GSH content and down-regulated expression of GPx and SOD, and ofIFNγandTNFαmRNA. Co-treatment using Lactic Acid Bacteria (LAB) with AFB1or FB1suppressed levels of DNA fragmentation, normalized splenic LP and increased GSH levels, up-regulated expression of GPx and SOD, and normalized mRNA levels of the analyzed cytokines. It is concluded that AFB1and FB1might have combinational (synergistic moreso than additive) toxic effectsin situ. Further, it can be seen that use of LAB induced protective effects against the oxidative stress and (immuno)toxicity of these agents in part through adhesion (and so likely diminished bioavalability). [ABSTRACT FROM PUBLISHER]

The potential immunotoxicity of tabalumab was assessed as a component of standard pre-clinical toxicology studies in cynomolgus monkeys. To evaluate potential tabalumab-associated immunosuppression after antigen challenge, cynomolgus monkeys were administered placebo control or tabalumab in three immunotoxicological safety studies. Study 1, a 4-week pilot study, evaluated biweekly intravenous (IV) control, and 0.3, 1.0, 5.0, and 15.0 mg/kg tabalumab doses. Study 2 evaluated IV control, and 0.1, 1.0, and 30.0 mg/kg tabalumab doses biweekly for 6 weeks. Study 3 evaluated IV control and 0.1, 1.0, 30.0 mg/kg, and subcutaneous (SC) 30.0 mg/kg tabalumab biweekly for 6 months, with recovery (16 weeks) to monitor standard immunotoxicity endpoints. T-cell dependent primary and secondary antibody responses to tetanus toxoid antigen challenge (4-week and 6-week studies) or keyhole limpet hemocyanin (KLH; 6-week and 6-month studies) were evaluated as a measure of immunocompetence, together with quantitation of T- and B-cell subsets. In addition, anti-tabalumab antibody formation (6-week and 6-month studies) was assessed. The results indicated that, despite expected decreases in circulating B-cell populations, no changes in follicle histopathology or organ weights, except decreases in spleen weight (after 6-months of 30 mg/kg IV/SC treatment only), were attributed to tabalumab. Non-adverse microscopic decreases in size or number of germinal centers in spleen, mesenteric, and mandibular lymph nodes occurred, but without an effect on antibody responses to KLH or tetanus. At 16-weeks recovery, microscopic compound-related changes observed after 6 months of treatment were completely reversed (0.1 mg/kg group) and partially reversed (1.0 and 30.0 mg/kg groups), while peripheral blood B cells remained 66–72% reduced from baseline. Despite reduced germinal centres in lymphoid organs, and reductions in circulating B cells, T-cell-dependent humoral immunity was maintained following tabalumab administration in three safety studies in cynomolgus monkeys. [ABSTRACT FROM PUBLISHER]

In vitrogene profiling studies have associated the molecular pathways of Nrf2-Keap1 and Toll-like receptor (TLR) signaling with skin sensitization. In this study, the role of these pathways in the regulation of protein biomarkers for skin sensitization was further elucidated using transient gene knock-down of key components of the signaling cascades in HaCaT cells after exposure to dinitrochlorobenzene (DNCB). The effect of targeting these pathways was established through evaluation of heme oxygenase1 (HMOX1) and interleukin (IL)-8 production. These experiments showed that Nrf2 is not involved in regulating HMOX1 after exposure to DNCB, but that activation of TLR signaling moderates the expression of HMOX1. The regulation of IL-8 depended on Nrf2, but also on the Toll/interleukin-1 receptor (TIR)-domain-containing adapter-inducing interferon-β(TRIF) adaptor protein in TLR signaling. This study provides new insights into the regulation of HMOX1 and IL-8, but the exact regulating mechanisms remain to be further elucidated. [ABSTRACT FROM PUBLISHER]

Delayed-type hypersensitivity (DTH) is a T-cell-mediated immune response that may be used for immunotoxicity testing in non-clinical species. However, in some cases DTH assays using T-dependent antigens may be confounded by the production of antibodies to the antigen. The authors have previously modified a DTH assay, initially validated in the mouse, for use in juvenile rats to assess the effect of immunosuppressive drugs on the developing rat immune system. The assay measures footpad swelling induced by subcutaneous footpad injection ofCandida albicans(C. albicans) derived-chitosan in rats previously sensitized withC. albicans. Antibodies to chitosan are not produced in this model. However, considerable inter-animal variability inherent in the footpad swelling assay can make it difficult to precisely quantify the magnitude of the immune response and inhibition by immunosuppressants, particularly if complete suppression is not observed. This report describes the development of anex vivoassay to assess DTH in rats using interferon (IFN)-γ production by splenocytes, obtained from rats sensitized withC. albicans, as the quantifiable measure of the DTH response. Adult and neonatal rats administered dexamethasone (DEX), a known immunosuppressant, exhibited immunosuppression as evidenced by a reduction inex vivoIFNγ production from splenocytes challenged withC. albicans-derived chitosan. Current data indicate that theex vivobased DTH assay is more sensitive than the conventional footpad swelling assay due to a lower background response and the ability to detect a response as early as post-natal day (PND) 12. Theex vivobased rat DTH assay offers a highly sensitive and quantitative alternative to the footpad swelling assay for the assessment of the immunotoxic potential of drugs. The increased sensitivity of theex vivoDTH assay may be useful for identifying smaller changes in response to immunotoxic drugs, as well as detecting responses earlier in animal development. [ABSTRACT FROM PUBLISHER]

Paraquat (PQ), one of the most widely used herbicides, has been used for several decades in agriculture. Some studies suggest that PQ has effects on the immune system. Moreover, previous studies have shown that PQ imparted some immunosuppressive effects. In the present study, cytotoxicity assays using splenic NK cells from mice treated for 28 days with PQ (at 0.2, 1, and 5 mg/kg) were performed to determine whether PQ altered the function of NK cells. Given that PQ was expected to induce an immunosuppressive effect, it was hypothesized that a gene involved in cellular metal ion homeostasis, metallothionein-1 (MT-1), could play an important role in this outcome. This belief was based on the fact thatMT1encodes a protein responsible for zinc ion homeostasis, and that a reduction in free zinc ion levels impairs NK cell function. The results showed that PQ treatments led to increased MT expression in several organs (liver, kidneys, testes) and in splenocytes, caused a reduction of both free zinc ions in sera and in free intracellular zinc, and reduced the expression of GATA-3, a zinc-finger transcription factor important for maturation and activity of T-cells and NK cells. These results provide a basis for a new molecular mechanism to describe potential immunosuppressive effects of PQin vivo. [ABSTRACT FROM PUBLISHER]

Deoxynivalenol (DON) is one of the most abundant mycotoxins worldwide and mostly detected in cereals and grains. As such, DON poses a risk for many adverse health effects to human and animals. In particular, immune cells are very sensitive to DON, with the initiating step leading to toxicity being a binding to the eukaryotic 60S ribosomal subunit and induction of ribotoxic stress. The present study aimed to: (1) extend insight into the mechanism of action (MOA) of DON in immune cells; and (2) understand why immune cells are more sensitive to DON than most other cell types. Previously published microarray studies have described the effects of DON on immune cells. To build upon these findings, here, immunocytological and biochemical studies were performed using human T-lymphocyte Jurkat cells that were exposed for 3 h to 0.5 µM DON. Induction of ER stress by DON was confirmed by immunocytology demonstrating increased protein expression of two major ER stress markers ATF3 and DDIT3. T-cell activation was confirmed by induction of phosphorylation of protein kinases JNK and AKT, activation of NF- κB (p65), and increased expression of NFAT target gene NUR77; each of these are known inducers of the T-cell activation response. Induction of an oxidative stress response was also confirmed by monitoring the nuclear translocation of major oxidative stress markers NRF2 and KEAP1, as well as by changes (i.e. decreases) in cell levels of reduced glutathione. Lastly, this study showed that DON induced cleavage of caspase-3, an event known to mediate apoptosis. Taken together, these results allowed us to formulate a potential mechanism of action of DON in immune cells, i.e. binding to eukaryotic 60S ribosomal subunit → ribotoxic stress → ER stress → calcium release from the ER into cytoplasm → T-cell activation and oxidative stress → apoptosis. It is proposed that immune cells are more sensitive to DON than other cell types due to the induction of a T-cell activation response by increased intracellular calcium levels. [ABSTRACT FROM AUTHOR]

Aflatoxin B1 (AFB1) and M1 (AFM1) are mycotoxins produced by numerous Aspergillus species in pre- or post-harvest cereals and milk. AFB1 and AFM1 display a potent economic loss in livestock and also cause severe immunological problems. The aims of this study were to: evaluate a new AFB1 and AFM1-binding/degrading micro-organism for biological detoxification; examine its ability to degrade AFB1 and AFM1 in liquid medium; and evaluate its potential for in vivo preventative effects against AFB1- and AFM1-induced immunomodulation in mice. Lactobacillus plantarum MON03 (LP) isolated from Tunisian artisanal butter was found to display significant binding ability to AFB1 and AFM1 in PBS (i.e. 82% and 89%, respectively) within 24 h of incubation and able to tolerate gastric acidity, have strongly hydrophilic cells surface properties, and adhere efficacy to Caco-3 cells in vitro. The in vivo study was conducted using Balb/c mice that received by oral gavage vehicle (control), LP only (2 × 109 CFU/L, ∼2 g/kg BW), AFB1 or AFM1 alone (0.25 and 0.27 mg/kg, respectively), or AFB1 + LP or AFM1 + LP daily for 15 days. Compared to in control mice, treatments with AFB1 and AFM1 led to significantly decreased body weight gains, histopathological changes, and decrements in all hematologic and immune parameters assessed. Co-treatment with LP strongly reduced the adverse effects of each mycotoxin. In fact, the mice receiving AFB1 + LP or AFM1 + LP co-treatment displayed no significant differences in the assayed parameters as compared to the control mice. By itself, the bacteria alone had no adverse effects in the mice. From these data, it is concluded that the tested bacteria could be beneficial in biotechnology detoxification of contaminated food and feed for humans and animals. [ABSTRACT FROM AUTHOR]

The toxic effects of highly carcinogenic mycotoxins, especially aflatoxins (AF), on key antigen-presenting cells, such as dendritic cells (DC), are largely unknown. To elucidate the effect of AF on DC function, porcine monocyte-derived DC (MoDC) were treated with a mixture of several AF (i.e., AFB1, AFB2, AFG1, and AFG2) and the phagocytic capacity, the membrane expression level of several DC activation markers, the T-cell proliferation-inducing capacity, and the cytokine secretion pattern were assessed. As compared to untreated MoDC, AF significantly up-regulated the expression of the co-stimulatory molecules CD25 and CD80/86. However, the phagocytic activity of MoDC was not affected by AF treatment. While the cytokine secretion pattern of AF-treated MoDC was similar to control MoDC, the T-cell proliferation-inducing capacity of MoDC was increased upon aflatoxin treatment. The results indicate that a mixture of naturally occurring AF enhances the antigen-presenting capacity of DC, which could explain the observed immunotoxicity of AF by breaking down tolerance and further emphasizes the need to reduce the admissible level of AF in agricultural commodities. [ABSTRACT FROM AUTHOR]

Aflatoxin M1 (AFM1) is a mycotoxin produced by numerous Aspergillus species in pre- or post-harvest cereals and milk. Exposure to AFM1 imparts potent economic losses in the livestock industry. Toxicologically, it also causes severe immune system problems. The aims of this study were to evaluate a new AFM1-binding/degrading microorganism for biologic detoxification, to examine its ability to degrade AFM1 in liquid medium, and to evaluate its potential for in vivo preventative effects against AFM1-induced immunotoxicity and genotoxicity in mice. Lactobacillus plantarum MON03 (LP) isolated from Tunisian artisanal butter was found to display significant binding ability to AFM1 in PBS (93%) within 24 h of incubation. Further, the LP was able to tolerate gastric acidity, bile salts, and adhere efficiently to Caco-3 cells in vitro. The in vivo study used Balb/c mice that received either vehicle (control), LP only (at 1 × 109 CFU/L, ∼1 mg/kg bw), AFM1 (100 mg/kg bw), or AFM1 + LP daily for 15 days (by gavage); two other groups received a single dose of colchicine (4 mg/kg) or mitomycin C (1 mg/kg) as positive controls for induction of micronuclei and chromosomal aberrations, respectively. The results showed that, compared to in control mice, AFM1 treatment led to significantly decreased body weight gains, and caused cytotoxic/genotoxic effects as indicated by increases in frequencies of polychromatic erythrocytes, as well as those with micronucleation (PCEMN) and chromosomal aberrations, among bone marrow cells. The concurrent administration of LP with AFM1 strongly reduced the adverse effects of AFM1 on each parameter. Mice receiving AFM1 + LP co-treatment displayed no significant differences in the assayed parameters as compared to the control mice. By itself, the bacteria caused no adverse effects. Based on the data, it is concluded that the test bacteria could potentially be beneficial in the detoxification of AFM1-contaminated foods and feeds for humans and animals. [ABSTRACT FROM AUTHOR]

5-Fluorouracil (5-FU) is a widely used anti-cancer agent; however, it induces immunosuppression in patients undergoing a chemotherapy regime. The mode of action by which 5-FU induces immunosuppression is primarily via inhibition of hematopoietic growth factors. In the present study, immunoprotective effects of a semiquinone glucoside derivative (SQGD), a bacterial metabolite isolated from Bacillus sp. INM-1, were evaluated in a model of 5-FU-induced immunotoxicity in C57Bl/6 male mice. The evaluation was done by analyzing G-CSF, GM-CSF, and M-CSF expression in the serum, spleen, and bone marrow cells of the mice at different timepoints after 5-FU treatment. Mice received a single intraperitoneal injection of either 5-FU (75 mg/kg) alone, SQGD (50 mg/kg) alone, or SQGD 2 h prior to the 5-FU treatment. Control mice received saline vehicle only. The results demonstrated that 5-FU treatment significantly inhibited G-CSF, GM-CSF, and M-CSF expression in all three sites at all timepoints from 6-72 h post 5-FU. In SQGD treated mice, up-regulation of factor expression was observed in each compartment, and significantly so most often after 12 h. SQGD treatment prior to 5-FU administration to the mice significantly increased in all sites evaluated - relative to values in both control mice and 5-FU only-treated mice - G-CSF, M-CSF, and GM-CSF expression at almost every timepoint. The present findings suggest that SQGD provides protection against 5-FU-induced immunotoxicity in mice and could protect bone marrow progenitor cells against the effects of cytotoxic drugs used for treatment of cancer. The findings also suggested to us that SQGD is a potential immunomodulator and could protect hematopoiesis against toxic assault caused by anti-cancer drugs in the clinical setting. [ABSTRACT FROM AUTHOR]

Cadmium (Cd), a known carcinogen and potent immunotoxicant in humans and animals, is dispersed throughout the environment as a result of pollution from a variety of sources. Tunisian radish ( Raphanus sativus) extract (TRE) is a known anti-oxidant and free radical scavenger that has been shown to help alleviate immune system disorders, including some induced by environmental toxicants. The present study was undertaken to investigate potential protective effects of TRE against Cd-induced immunotoxicities (and general toxicities) in situ. Cadmium chloride (at 2.5 mg CdCl2/kg BW) and TRE (5, 10, or 15 mg/kg BW) were given (alone or in combination [actually, in sequence of Cd and then TRE]) to rats daily by oral gavage for 2 weeks. Results indicated that treatment with CdCl2 alone resulted in significant decreases in plasma levels of total protein, triglycerides, creatine kinase, creatinine, IgG and IgA, T-lymphocyte sub-types (CD4+, CD3+, CD56+, and CD8+), and in thymic and hepatic indices (relative weights). In contrast, CdCl2 treatment caused significant increases in serum LDH, AST, and ALT, in the formation/release of pro-inflammatory cytokines (IL-1 and TNF α), and in the relative weights of host spleen and kidneys. Rats treated with TRE alone had no discernable changes compared to the controls with regard to all test parameters. Combined treatment of CdCl2 and TRE-at any dose-resulted in a significant improvement of all test parameters compared to those seen with Cd alone. These results illustrated (and provided further support for a continuing belief in) the beneficial effects of TRE in reducing the harmful outcomes of commonly encountered toxicants (like Cd) on the immune system and on overall host health status. [ABSTRACT FROM AUTHOR]

Despite the transient immunosuppressive properties of local radiotherapy (RT), this classical treatment modality of solid tumors is capable of inducing immunostimulatory forms of tumor-cell death. The resulting 'immunotoxicity' in the tumor, but not in healthy tissues, may finally lead to immune-mediated destruction of the tumor. However, little is known about the best irradiation scheme in this setting. This study examines the immunological effects of differently irradiated human colorectal tumor cells on human monocyte-derived dendritic cells (DC). Human SW480 tumor cells were irradiated with a norm-fractionation scheme (5 × 2 Gy), a hypo-fractionated protocol (3 × 5 Gy), and with a high single irradiation dose (radiosurgery; 1 × 15 Gy). Subsequently, human immature DC (iDC) were co-incubated with supernatants (SN) of these differently treated tumor cells. Afterwards, DC were analyzed regarding the expression of maturation markers, the release of cytokines, and the potential to stimulate CD4+ T-cells. The co-incubation of iDC with SN of tumor cells exposed to norm- or hypo-fractionated RT resulted in a significantly increased secretion of the immune activating cytokines IL-12p70, IL-8, IL-6, and TNF α, compared to iDC co-incubated with SN of tumor cells that received a high single irradiation dose or were not irradiated. In addition, DC-maturation markers CD80, CD83, and CD25 were also exclusively elevated after co-incubation with the SN of fractionated irradiated tumor cells. Furthermore, the SN of tumor cells that were irradiated with norm- or hypo-fractionated RT triggered iDC to stimulate CD4+ T-cells not only in an allogenic, but also in an antigen-specific manner like mature DC. Collectively, these results demonstrate that norm- and hypo-fractionated RT induces a fast human colorectal tumor-cell death with immunogenic potential that can trigger DC maturation and activation in vitro. Such findings may contribute to the improvement of irradiation protocols for the most beneficial induction of anti-tumor immunity. [ABSTRACT FROM AUTHOR]

People are constantly exposed to environmental chemicals through contact with the atmosphere or by ingestion of food. Therefore, when conducting safety assessments, the immunotoxic effects of combinations of chemicals in addition to toxicities produced by each chemical alone should be considered. The objective of the studies reported here were to demonstrate the combined effects of three well-known environmental immunotoxic chemicals -- methoxychlor (MXC), an organochlorine compound; parathion (PARA), an organophosphate compound; and piperonyl butoxide (PBO), an agricultural insecticide synergist -- by using a short-term oral exposure method. Seven-week-old Balb/cAnN mice received daily oral exposure to either one or two of the environmental immunotoxic chemicals for 5 consecutive days. On Day 2, all mice in each group were immunized with sheep red blood cells (SRBC), and their SRBC-specific IgM responses were analyzed by using an enzyme-linked immunosorbent assay and plaque-forming cell assay. T- and B-cell counts in the mouse spleens were also assessed via surface antigen expression. Mice that received MXC + PARA and PBO + MXC treatment showed marked decreases in SRBC-specific IgM production and T- and B-cell counts compared with those in mice that received vehicle control or the corresponding individual test substance. This suggests that simultaneous exposure to multiple environmental chemicals increases the immunotoxic effects of the chemicals compared to individual exposure. [ABSTRACT FROM AUTHOR]

An historical perspective of immunotoxicology is presented beginning from early observations in which exposure to workplace environments led to unexpected immune-mediated lung diseases to its eventual evolution as a sub-discipline in toxicology. As with most toxicology disciplines, immunotoxicology originated from concerns by scientists within industry and government as well as medical professionals to limit human exposure to agents that can potentially effect human health. The basis for these concerns originated from laboratory studies in experimental models and clinical observations that suggested certain industrial and agrochemicals, pharmaceuticals and consumer products were capable of inadvertently interacting with the immune system and cause adverse health effects. The types of immunopathologies observed and mechanisms responsible were found to be broad, being dependent upon the physiochemical properties of an agent, exposure route, and target organ/tissue, and included allergic/hypersensitivity responses, immune dysfunction, manifested by suppression or, in rare instances, stimulation, autoimmune or autoimmune-like diseases, and chronic inflammatory disorders. [ABSTRACT FROM AUTHOR]

This study was designed to evaluate the protective activity of Vitamin E (Vit E) on the immunotoxic effects induced by aflatoxin B1 (AFB1) in the progeny of breeder hens. For this purpose, 192 White Leghorn (WL) layer breeder hens were divided into 12 groups (A-L) and then fed test diets for either 1, 2 or 3 weeks. Group A was kept on basal feed (2900 Kcal/kg metabolizable energy) and served as control, while group B was offered a feed supplemented with Vit E at 100 mg/Kg. Groups C-G were offered feed containing 0.1, 0.5, 2.5, 5.0, and 10.0 mg/Kg AFB1, respectively, whereas groups H-L were offered the same dietary levels of AFB1 along with 100 mg/Kg Vit E supplementation. Hatching eggs were shifted to an incubator on a weekly basis to get progeny chicks. Hatched chicks in each group were maintained on basal ration and then subjected to different immunological assays. Lymphoproliferative responses (against PHA-P), antibody titers (against SRBC), oxidative damage to RBC, as well as phagocytic and nitrite production potential of the peritoneal macrophages from the chicks, were all adversely impacted by hen exposure to the higher doses of AFB1 or by increased intake (time) by the hens at a given dose of the toxin. No consistent ameliorative effects from Vit E were noted in these studies, i.e. effects seen against lower AFB1 doses were no longer apparent with the highest doses of AFB1. As such, for now it can be concluded that, with this particular single dose level of Vit E, AFB1-associated immunotoxic effects in progeny chicks can potentially be mitigated by dietary intake of Vit E by their hen dams. However, this is clearly an outcome that is driven by the level of the mycotoxin present in the feed. Future studies need to examine what impact higher Vit E doses than those employed herein might have in these ameliorative outcomes. [ABSTRACT FROM AUTHOR]

Non-human primates may be the only relevant species for pharmacology or toxicology studies of certain biologics, due to lack of activity in other species. Flow cytometry immunophenotyping is often included as a minimally invasive adjunct to standard toxicity testing. A retrospective inter-laboratory analysis was conducted to assess counts and variability of the main cell types monitored in toxicity studies, and to provide guidance for conduct and interpretation of immunophenotyping assessments in cynomolgus monkeys. Univariate and multivariate models were developed. Study design factors influencing cell counts and variability were identified and a power analysis was performed. Pre-study and on-study counts were generally similar; longitudinal analysis showed little drift in mean counts or within-animal variability over time. Within-animal variability was lower than inter-animal variability. Gender was associated with small but significant differences in mean counts and variability. Age was associated with significant differences in variability. Immunophenotype definitions were associated with significant differences in mean counts and within-animal variability for most cell types. Power analysis for groups of 6-8 animals showed that differences of ≈50% in counts of T-cells, T-cell subsets, and B-cells compared to pre-treatment values may be detected; for NK cells and monocytes, differences of ≈60-90% may be detected. This review yields some general points to consider for immunophenotyping studies, i.e. (a) analysis of log-transformed cell count data and comparisons using each animal as its own reference will improve ability to detect changes, (b) the magnitude of change detectable given study group size should be considered, (c) multiplication of sampling timepoints during a study seems unnecessary, (d) consideration should be given to using only one gender, when applicable, to increase power while minimizing animal usage, and (e) the choice of immunophenotype has impacts on cell counts and variability. [ABSTRACT FROM AUTHOR]

Aflatoxin M1 (AFM1) has been detected in many parts of the world both in raw milk and many dairy products, causing great economic losses and human disease. Unfortunately, there are few studies dealing with AFM1 immunotoxicity/interactions with lactic acid bacteria for potential application as a natural preventive agent. The aim of this study was to isolate (from dairy products) food-grade probiotic bacteria able to degrade/bind AFM1 in vitro and evaluate whether the same organism(s) could impart a protective role against AFM1-induced immunotoxicity in exposed Balb/c mice. Bacteria ( Lactobacillus plantarum MON03 and L. rhamnosus GAF01) were isolated from Tunisian artisanal butter and then tested for abilities to eliminate AFM1 from phosphate-buffered saline (PBS) and reconstituted milk (containing 0.05, 0.10, and 0.20 µg AFM1/ml) after 0, 6, and 24 h at 37°C. Results showed that the selected bacteria could 'remove' AFM1 both in PBS and skimmed milk. The binding abilities of AFM1 by L. plantarum MON03 and L. rhamnosus GAF01 strains (at 108 CFU/ml) in PBS and reconstituted milk ranged, respectively, from 16.1-78.6% and 15.3-95.1%; overall, L. rhamnosus showed a better potential for removal than L. plantarum. 'Removal' appeared to be by simple binding; the bacteria/AFM1 complex was stable and only a very small proportion of mycotoxin was released back into the solution. L. rhamnosus GAF01 had the highest binding capacity and was selected for use in the in vivo study. Those results indicated that use of the organism prevented AFM1-induced effects on total white and red blood cells, and lymphocyte subtypes, after 15 days of host treatment. These studies clearly indicated that L. rhamnosus GAF01 was able to bind AFM1 in vitro and-by mechanisms that might also be related to a binding effect-counteract AFM1-induced immunotoxicity. Moreover, by itself, this bacterium was not toxic and could potentially be used as an additive in dairy products and in biotechnology for mycotoxin detoxification. [ABSTRACT FROM AUTHOR]

Associations between painting, sensitization, and respiratory disease have received little attention, despite the extensive use of paint and paint removal products. The objectives of this study were to investigate the possible immunotoxicity and pulmonary toxicity induced by paints in Egyptian painter workers. This study was carried out on 60 adult males. Subjects were designated as controls ( n = 30 healthy persons) or paint-exposed workers ( n = 30). The controls and workers were then divided into four equal groups (15 individuals/group): Group I, Control group-never smoked; Group II, Smoker controls; Groups III, paint-exposed non-smoking workers; and Group IV, paint-exposed smoker workers. A complete physical examination, chest radiograph, and pulmonary function test (PFT) were performed with each subject. Serum levels of immunoglobulin (Ig) E and interleukin (IL)-4, -6, and -10, WBC sub-set counts, total numbers of WBC, and leukocyte differentials were also assessed. The pulmonary toxicity due to the paint exposures appeared in the form of allergic manifestations in the respiratory tract, significant reductions in FVC, FEV1, FEV1/FVC ratio and PEF parameters, and a reticular pattern in both lung fields. Immunotoxicity was evidenced by increases in total leukocyte levels, total lymphocytes, CD8+ T-lymphocytes, B (CD19+)-lymphocytes, NK (CD3+CD16+CD56+) cells, and eosinophils, as well as a significant decrease in CD4+ T-lymphocyte; there were also significant elevations in serum IgE, IL-4, and IL-6, and a significant reduction in IL-10, levels in these hosts. Based on these results, we assert that repeated paint exposure is associated with pulmonary and immune system toxicities that may lead to an augmentation of allergic diseases. [ABSTRACT FROM AUTHOR]

Chlorpyrifos (CPF) is a widely used agricultural organophosphorus insecticide (OP). Epidemiological studies have reported that children living in an OP contaminated environment, showed altered immune function. However, there is a paucity of experimental evidence for CPF-induced toxicity in the developmental phases of immune system. The current studies sought to ascertain the effect of CPF on the developing immune system of mice. Swiss albino dams were exposed orally with 0, 0.3, and 3.0 mg CPF/kg/day from GD12 to PND7, and then pups were directly dosed with CPF (by gavage) through PND42. One week after the final dose of CPF (i.e. on PND49), immunotoxicities in these offspring were assessed. These analyses revealed that there were increases in Foxp3+CD25+CD4+ T-regulatory ( Treg) cell frequency in the spleens of 3.0 mg CPF/kg/day-exposed female mice, but not in males. In contrast, the anti-sheep red blood cells IgM response was reduced in both genders. Moreover, splenic lymphoproliferative response to phytohemagglutinin (PHA), concanavalin A (ConA), and lipopolysaccharide (LPS), as well as interferon (IFN)- γ, tumor necrosis factor (TNF)- α, and interleukin (IL)-6 production following LPS stimulation decreased in mice of both sexes at higher CPF doses. Together, these findings suggest that developmental CPF exposure might cause immunosuppression in mice. Some of these outcomes might arise, in part, through a suppression of pro-inflammatory cytokine production and/or changes in the responsivity (specifically to mitogens) of lymphocytes in these hosts. [ABSTRACT FROM AUTHOR]

Several types of pesticides, including organochlorines, are known to suppress or modulate immune responses. The present study evaluated the immunotoxicity of the organochlorine pesticide methoxychlor (MXC) in female BALB/c, C3H/He, and ICR mice. Mice were given oral MXC doses of 0, 30, 100, and 300 mg/kg each day for 7 consecutive days. On day 4, the mice also received an intravenous injection of sheep red blood cells (SRBC). The splenic plaque-forming cell (PFC) IgM response and the serum anti-SRBC IgM antibody titer were evaluated while splenic lymphocytes were counted by flow cytometry and the spleen underwent histopathological analysis. Significant decreases in IgM PFC responses were seen in BALB/c, C3H/He, and ICR mice that received MXC doses of 100 and 300 mg/kg. Similar changes in serum anti-SRBC IgM antibody titers occurred in three strain mice. Flow cytometric analysis revealed significantly decreased splenic T-cell (CD3+) populations in a dose dependent manner in BALB/c mice, and in the 300 mg/kg of MXC-treated group of C3H/He mice. Germinal center (GC) B-cell (CD19+PNA+) populations were significantly decreased in the 300 mg/kg of MXC-treated groups of all three mouse strains and in the 30 and 100 mg/kg of MXC-treated groups of BALB/c and C3H/He strain mice. Histopathological analysis revealed decreased cellularity of the periarteriolar lymphoid sheath (PALS; T-cell area) and decreased GC development in all three strains of mice treated with 300 mg/kg MXC. These results suggest that MXC has an immune-suppressive effect in mice, and that our protocol may be useful for rapidly detecting immunosuppression induced by environmental chemicals. [ABSTRACT FROM AUTHOR]

Exposure to environmental agents can compromise numerous immunological functions. Immunotoxicology focuses on the evaluation of the potential adverse effects of xenobiotics on immune mechanisms that can lead to harmful changes in host responses such as: increased susceptibility to infectious diseases and tumorigenesis; the induction of hypersensitivity reactions; or an increased incidence of autoimmune disease. In order to assess the immunosuppressive response to short-term exposure to some commonly used pesticides, the studies here focused on the response of mice after exposures to the organochlorine pesticide methoxychlor, the organophosphorus pesticide parathion, or the agricultural insecticide synergist piperonyl butoxide. In these studies, 7-week-old mice were orally administered (by gavage) methoxychlor, parathion, or piperonyl butoxide daily for five consecutive days. On Day 2, all mice in each group were immunized with sheep red blood cells (SRBC), and their SRBC-specific IgM responses were subsequently assessed. In addition, levels of B-cells in the spleen of each mouse were also analyzed via surface antigen expression. The results of these studies indicated that treatments with these various pesticides induced marked decreases in the production of SRBC-specific IgM antibodies as well as in the expression of surface antigens in IgM- and germinal center-positive B-cells. Based on these outcomes, it is concluded that the short-term exposure protocol was able to detect potential immunosuppressive responses to methoxychlor, parathion, and piperonyl butoxide in situ, and, as a result, may be useful for detecting other environmental chemical-related immunotoxicities. [ABSTRACT FROM AUTHOR]

The use of recombinant human proteins for the treatment of several diseases has increased considerably during the last decades. A major safety and efficacy issue of biopharmaceuticals is their potential immunogenicity. To prevent immunogenicity, biotechnology-derived proteins are engineered to be as human-like as possible. Immunogenicity is mainly determined in non-human primates (NHP), as they are considered to be the best predictive animal species for human safety, based on their close relatedness to man. As minipigs are increasingly used in the safety evaluation of (bio)pharmaceuticals, the predictive value of the minipig in immunogenicity testing was evaluated in this study, using anakinra as a model compound. Animals were treated subcutaneously with either placebo, low- (0.5 mg/kg), or high-dose (5 mg/kg) anakinra daily on 29 consecutive days. After the first and last dose, the pharmacokinetic (PK) profile of anakinra was evaluated. Antibodies directed to anakinra were measured on several time points during the treatment period. Furthermore, hematology, clinical chemistry, body weight, clinical signs, and histopathology of several organs were evaluated. No signs of toxicity were observed upon treatment with anakinra. PK parameters were comparable with those found in human and NHP studies performed with anakinra. All animals developed anti-anakinra antibodies. The results obtained in minipigs were comparable to those observed in monkeys. For anakinra, the predictive value of the minipig for immunogenicity testing was found to be comparable to that seen in NHP. However, more studies evaluating additional biopharmaceutical products are needed to support the use of the minipig as an alternative model for (immuno)toxicity testing, including immunogenicity. [ABSTRACT FROM AUTHOR]

Limited non-clinical immunotoxicity data are available in the dog, although this is a major non-rodent species in regulatory safety studies. The present study aimed to test whether widely accepted immunotoxicity endpoints including lymphocyte subset immunophenotyping, the anti-KLH TDAR assay, and histological examination of the main lymphoid organs were reliable to detect immunosuppression induced by cyclosporine and cyclophosphamide in dogs and could, therefore, be used for non-clinical immunotoxicity evaluation in this species. Male and female Beagle dogs were treated orally from Day 1 for 4 weeks with 25 mg/kg cyclosporine daily, or with 2 mg/kg cyclophosphamide on 4 consecutive days each week, or the same volume of drinking water daily. Blood samples were withdrawn pre-test and on Days 11, 18, and 23 to measure standard hematology parameters and analyze lymphocyte subsets. All animals received an intramuscular injection of 5 mg KLH on Day 11. Sandwich ELISA assays were used to quantify anti-KLH IgM and anti-KLH IgG levels in blood samples taken pre-test, on Days 18 and 23, and pre-test, on Days 23 and 28, respectively. At the end of the treatment period, all animals were submitted to histological examination of lymphoid organs, liver, and kidneys. No signs of marked toxicity were observed. No changes in lymphocyte subsets, but markedly decreased primary anti-KLH IgM and IgG responses, and a slightly-to-markedly increased cortex/medulla ratio in the thymus were observed in cyclosporine-treated dogs. Lower total WBC counts correlating with lower total and B-lymphocyte subset and decreased germinal center development in mesenteric lymph nodes, but no changes in primary anti-KLH IgM and IgG responses were observed in cyclophosphamide-treated dogs. These results demonstrate that widely accepted immunotoxicity endpoints can adequately detect the effects of known immunosuppressive drugs in the dog and support the conclusion that it is a relevant animal species for immunotoxicity evaluation. [ABSTRACT FROM AUTHOR]

The failure of regulatory science to keep pace with and support the development of new biological medicines was very publically highlighted in March 2006 when the first-in-man Phase I clinical trial of the immunomodulatory CD28-specific monoclonal antibody (mAb) TGN1412 ended in disaster when all six volunteers suffered a life-threatening adverse reaction termed a 'Cytokine Storm'. The poor predictive value of standard pre-clinical safety tests and animal models applied to TGN1412 demonstrated the need for a new generation of immunotoxicity assays and animal models that are both sensitive and predictive of clinical outcome in man. The non-predictive result obtained from pre-clinical safety testing in cynomolgus macaques has now been attributed to a lack of CD28 expression on CD4+ effector memory T-cells that therefore cannot be stimulated by TGN1412. In contrast, high levels of CD28 are expressed on human CD4+ effector memory T-cells, the source of most TGN1412-stimulated pro-inflammatory cytokines. Standard in vitro safety tests with human cells were also non-predictive as they did not replicate in vivo presentation of TGN1412. It was subsequently shown that, if an immobilized therapeutic mAb-based assay or endothelial cell co-culture assay was used to evaluate TGN1412, then these would have predicted a pro-inflammatory response in man. New in vitro assays based on these approaches are now being applied to emerging therapeutics to hopefully prevent a repeat of the TGN1412 incident. It has emerged that the mechanism of pro-inflammatory cytokine release stimulated by TGN1412 is different to that of other therapeutic mAbs, such that standard pro-inflammatory markers such as TNF α and IL-8 are not discriminatory. Rather, IL-2 release and lymphoproliferation are optimal readouts of a TGN1412-like pro-inflammatory response. [ABSTRACT FROM AUTHOR]

Silymarin (SL) is the bioactive extract of the plant Silybum marianum and Vitamin E (VE) is an important anti-oxidant. The present study was designed to evaluate potential ameliorative effects of SL and VE against Ochratoxin A (OTA)-induced immunotoxic effects in White Leghorn cockerels. One day-old birds were divided into 12 groups (20 birds/group) and fed basal diets amended with OTA (1.0 or 2.0 mg/kg) alone or in combination with SL (10 g/kg) and/or VE (200 mg/kg) for 42 days. Immunological in situ responses, including antibody formation against sheep red blood cells (7 and 14 days after both primary and booster injections), lymphoproliferative responses to avian tuberculin (30 days of age), and mononuclear phagocytic system function (i.e. by clearance of injected carbon particles) assay (42 days of age), were assessed. Results suggested that silymarin and Vitamin E alone or in combination ameliorated the immunotoxic effects induced by 1.0 mg OTA/kg but could not significantly impact on the effect from ingestion of 2.0 mg OTA/kg. The results of the present study suggested that both SL and VE possess an ability to ameliorate OTA-induced immunotoxicity in chicks. However, it remains to be determined whether/what SL:OTA or VE:OTA ratios are required to assure such mitigation of OTA-induced immunotoxicities. [ABSTRACT FROM AUTHOR]

Functional innate immune assessments, including phagocytosis and respiratory burst, are at the forefront of immunotoxicology evaluation in pre-clinical animal species. Although in the clinic and in academic science, phagocytosis, and respiratory burst assessments have been reported for over two decades, the implementation of phagocytosis and respiratory burst analyses in toxicology safety programs is just recently gaining publicity. Discussed herein are general methods, both microtiter plate-based and flow cytometric-based, for assessing phagocytosis and respiratory burst in pre-clinical species including mouse, rat, dog, and monkey. This methods-centric discussion includes a review of technologies and descriptions of method applications, with examples of results from analyses testing reported inhibitors (rottlerin, wortmannin, and SB203580) of phagocytosis and respiratory burst. Justification of implementation, strategic experimental design planning, and feasibility aspects of evaluating test article effects on phagocytosis and respiratory burst function are described within the context of a case study. The case study involves investigation of the effects of a small molecule p38 kinase inhibitor, BMS-582949, on phagocytosis and respiratory burst functions in rat and monkey neutrophils and monocytes in vitro, as well as ex vivo in these innate immune cells from monkeys administered BMS-582949 during a 1-week repeat dose investigative study. The results of the in vitro and ex vivo assessments demonstrated that BMS-582949 inhibited phagocytosis and respiratory burst. These findings correlated with incidences of opportunistic infections observed in rat and monkey toxicity studies. [ABSTRACT FROM AUTHOR]

Dimethyl carbonate (DMC) is an industrial chemical, used as a paint and adhesive solvent, with the potential for significant increases in production. Using select immune function assays, the purpose of these studies was to evaluate the immunotoxicity of DMC following dermal exposure using a murine model. Following a 28-day exposure, DMC produced a significant decrease in thymus weight at concentrations of 75% and greater. No effects on body weight, hematological parameters (erythrocytes, leukocytes, and their differentials), or immune cell phenotyping (B-cells, T-cells, and T-cell sub-sets) were identified. The IgM antibody response to sheep red blood cell (SRBC) was significantly reduced in the spleen but not the serum. DMC was not identified to be an irritant and evaluation of the sensitization potential, conducted using the local lymph node assay (LLNA) at concentrations ranging from 50-100%, did not identify increases in lymphocyte proliferation. These results demonstrate that dermal exposure to DMC induces immune suppression in a murine model and raise concern about potential human exposure and the need for occupational exposure regulations. [ABSTRACT FROM AUTHOR]

Although translational research is a rapidly evolving area of biomedical sciences, translational immunologic safety evaluation has so far attracted only very limited attention. Assays and animal models have been developed to identify immunotoxic hazards related to immunosuppression, but less attention has been paid to immunostimulation, hypersensitivity, and autoimmunity. Some of these assays and models are recommended by regulatory bodies, even though it is as yet unsure to what extent they can predict the potential of, or lack of, new chemical entities and drug candidates for inducing significant immunotoxic effects. A translational approach should attempt to standardize and validate those models, assays, and biomarkers that could be used in regulatory non-clinical safety studies as well as clinical studies. Beyond translational immunologic safety, immune monitoring during clinical studies is intended to identify and evaluate potential immune safety issues not seen in non-clinical studies. Based on this overview of the current knowledge, it can be concluded that much remains to be done to conduct translational studies helpful to enhance the immunologic safety of drugs and chemicals. [ABSTRACT FROM AUTHOR]

Arc welding processes generate complex aerosols composed of potentially hazardous metal fumes and gases. Millions of workers worldwide are exposed to welding aerosols daily. A health effect of welding that is of concern to the occupational health community is the development of immune system dysfunction. Increased severity, frequency, and duration of upper and lower respiratory tract infections have been reported among welders. Specifically, multiple studies have observed an excess mortality from pneumonia in welders and workers exposed to metal fumes. Although several welder cohort and experimental animal studies investigating the adverse effects of welding fume exposure on immune function have been performed, the potential mechanisms responsible for these effects are limited. The objective of this report was to review both human and animal studies that have examined the effect of welding fume pulmonary exposure on local and systemic immune responses. [ABSTRACT FROM AUTHOR]