Adverse hepatic drug reactions: inflammatory episodes as consequence and contributor

Susceptibility to drug toxicity is influenced by a variety of factors, both genetic and environmental. The focus of this article is the evidence addressing the hypothesis that inflammation is both a result of and a susceptibility factor for drug toxicity, with an emphasis on liver as a target organ. Results of studies suggesting a role for inflammatory mediators in the hepatotoxicity caused by acetaminophen or ethanol are discussed. For several drugs, the evidence from animal models that concurrent inflammation increases injury is presented. In addition, the occurrence of adverse drug reactions in people with preexisting inflammatory diseases is considered. The special case of idiosyncratic drug reactions is discussed and the potential raised for development of animal models for this type of drug toxicity. The conclusion is that inflammatory factors should be considered as determinants of sensitivity to adverse drug reactions.     

Mucosal modulation of immune responses to heat shock proteins in autoimmune arthritis

Induction of oral tolerance to antigens that are targets of self-reactive immune responses is an attractive approach to antigen-specific immune therapy of autoimmune diseases. Oral tolerization has indeed proven to be safe and effective in amelioration of autoimmune diseases in animal models. In humans, results have been somewhat controversial. The emphasis given to clinical outcome rather than to immunomodulation, and the difficulty in identifying appropriate candidate antigens contribute to the controversy. Heat shock proteins are promising targets for immune intervention. Immune reactivity to heat shock proteins has indeed been correlated with autoimmune arthritis in animal models, and abnormal immune responses to heat shock proteins have been described in human arthritis as well. Despite significant recent progress, little is known at a molecular level regarding the mechanisms which are responsible for a switch from autoimmunity to tolerance in humans. This is particularly true with respect to sequential analysis of several molecular and immunologic markers during both the course and treatment of disease. Novel approaches are currently under way to fill the gaps. We will briefly detail here the experience gained to date, and identify some of the avenues which future research will explore.     

Plant cell-made protein antigens for induction of Oral tolerance

The gut associated lymphoid tissue has effective mechanisms in place to maintain tolerance to food antigens. These can be exploited to induce antigen-specific tolerance for the prevention and treatment of autoimmune diseases and severe allergies and to prevent serious immune responses in protein replacement therapies for genetic diseases. An oral tolerance approach for the prevention of peanut allergy in infants proved highly efficacious and advances in treatment of peanut allergy have brought forth an oral immunotherapy drug that is currently awaiting FDA approval. Several other protein antigens made in plant cells are in clinical development. Plant cell-made proteins are protected in the stomach from acids and enzymes after their oral delivery because of bioencapsulation within plant cell wall, but are released to the immune system upon digestion by gut microbes. Utilization of fusion protein technologies facilitates their delivery to the immune system, oral tolerance induction at low antigen doses, resulting in efficient induction of FoxP3⁺ and latency-associated peptide (LAP)⁺ regulatory T cells that express immune suppressive cytokines such as IL-10. LAP and IL-10 expression represent potential biomarkers for plant-based oral tolerance. Efficacy studies in hemophilia dogs support clinical development of oral delivery of bioencapsulated antigens to prevent anti-drug antibody formation. Production of clinical grade materials in cGMP facilities, stability of antigens in lyophilized plant cells for several years when stored at ambient temperature, efficacy of oral delivery of human doses in large animal models and lack of toxicity augur well for clinical advancement of this novel drug delivery concept.     

The effect of diabetes mellitus on pharmacokinetics,pharmacodynamics and adverse drug reactions of anticancer drugs

Diabetes mellitus (DM) and cancer are global problems carrying huge human, social, and economic impact. Type 2 diabetes (T2DM) is associated with an increased risk for a number of cancers, including breast, pancreatic, and liver cancer. Moreover, adverse drug reactions are higher in paitents with cancer with T2DM compared to cancer patients without T2DM. Cellular mechanisms of hyperglycemia and chemotherapy efficacy may be different depending upon the particular cancer type and the condition of the patient. This review evaluates the effect of DM on the pharmacokinetic, pharmacodynamic, and adverse drug reactions of commonly used anticancer drugs such as cisplatin, methotrexate, paclitaxel, doxorubicin, and adriamycin in both clinical and animal models. A literature search was conducted in scientific databases including Web of Science, PubMed, Scopus, and Google Scholar including the relevant keywords. The results of the effectiveness of anticancer therapies in patients with DM are, however, inconsistent because DM can negatively impact multiple diverse entities including nerves and vascular structures, insulin-like growth factor 1, the function of the innate immune system, drug pharmacokinetics, the expression levels of hepatic CYP₄₅₀, Mdr _1b and enzymes that then lead to drug toxicity. However, in a few circumstances, DM led to attenuation of the toxicity of anticancer drugs secondary to attenuation of the energy-dependent renal uptake process. Overall, the impact of DM on patients with cancer is variable because of the diverse types of cancers and the spectrum of anticancer drugs. With respect to the evidence for cancer involvement in DM pathophysiology and the response to anticancer treatment in patients with DM, many questions still remain and further clinical trials are needed.     

Spontaneous adenocarcinoma mouse models for immunotherapy

Recent discoveries regarding the identification of tumor-associated antigens and antigen presentation have made successful immunotherapy strategies possible with little, if any, toxicity. Here, we describe transgenic mammary, pancreas, prostate, stomach and lung adenocarcinoma animal models that can be used to study various immunotherapeutic strategies. The challenge in developing a tumor vaccine is effective antigen presentation that elicits anti-tumor immune responses without precipitating autoimmunity. Clinical trials must be preceded by appropriate animal studies to demonstrate that the concepts can be translated into efficacious therapy for cancer. Although many xenograph or transplantable tumor models have been used, the most effective studies are in spontaneous tumor models. These models are clinically relevant, as tumors arise in an appropriate tissue background and in a host conditioned by the physiological events of neoplastic progression and tumorigenesis and in the context of a viable immune system.     

Animal models of human-derived cancer vaccines

Preclinical cancer vaccine studies must address vaccine safety, immunogenicity, and efficacy, as well as mechanism of vaccine action. Animal models of vaccines employing human tumor-associated antigen or epitopes (TAA, TAE) differ fundamentally from those employing tumor-specific antigens or epitopes (TSA, TSE). TSA and TSE vaccines will most likely demonstrate similar toxicity, immunogenicity, and efficacy in both tumor-bearing animals and patients. In contrast, TAA/TAE immunizations may have to overcome a host’s immunological tolerance to TAA/TAE expressed not only on tumor, but also on normal tissues; immunity to TAA/TAE will potentially target normal tissues and thus may induce autoimmunity. Various experimental models for human-derived TAA/TAE vaccines have been developed. These models include transgenic mice, mice with severe combined immunodeficiency (SCID), and non-human primates. Recently, unique animal models of TAA/TAE cancer vaccines have been developed, taking advantage of the discovery of animal tissue antigens with significant sequence homologies to human TAA/TAE. These models mimic perhaps most closely the situation in cancer patients.     

The role of cytokines in immunological tolerance: potential for therapy

Current immunosuppression protocols, although often effective, are nonspecific and therefore hazardous. Consequently, immunological tolerance that is antigen specific and does not globally depress the patient's immune system has become one of the Holy Grails of immunology. Since the discovery that cytokines have immunomodulatory effects, extensive research has investigated the potential of these molecules to induce and maintain specific immunological tolerance in the context of transplantation, allergy and autoimmunity. In this article, we review the possible mechanisms by which cytokines can modulate the immune response and the animal models that frequently confound the theory that a single cytokine, or group of cytokines, can induce tolerance in a predictable manner. Finally, we discuss the role of cytokines at a paracrine level, particularly in the context of inducing and maintaining antigen-specific, regulatory T cells with the clinical potential to suppress specific immune responses.     

Anthrax lethal toxin activates the inflammasome in sensitive rat macrophages

Anthrax lethal toxin (LT) is an important virulence factor for Bacillus anthracis. In mice, LT lyses macrophages from certain inbred strains in less than 2 h by activating the Nlrp1b inflammasome and caspase-1, while macrophages from other strains remain resistant to the toxin’s effects. We analyzed LT effects in toxin-sensitive and resistant rat macrophages to test if a similar pathway was involved in rat macrophage death. LT activates caspase-1 in rat macrophages from strains harboring LT-sensitive macrophages in a manner similar to that in toxin-sensitive murine macrophages. This activation of caspase-1 is dependent on proteasome activity, and sensitive macrophages are protected from LT’s lytic effects by lactacystin. Proteasome inhibition also delayed the death of rats in response to LT, confirming our previous data implicating the rat Nlrp1 inflammasome in animal death. Quinidine, caspase-1 inhibitors, the cathepsin B inhibitor CA-074Me, and heat shock also protected rat macrophages from LT toxicity. These data support the existence of an active functioning LT-responsive Nlrp1 inflammasome in rat macrophages. The activation of the rat Nlrp1 inflammasome is required for LT-mediated rat macrophage lysis and contributes to animal death.     

Ex vivo activated OVA specific and non-specific CD4+CD25+ regulatory T cells exhibit comparable suppression to OVA mediated T cell responses

CD4+CD25+ regulatory T cells (Tr) are important in maintaining immune tolerance to self-antigen (Ag) and preventing autoimmunity. Reduced number and inadequate function of Tr are observed in chronic autoimmune diseases. Adoptively transferred Tr effectively suppress ongoing autoimmune disease in multiple animal models. Therefore, strategies to modulate Tr have become an attractive approach to control autoimmunity. Activation of Tr is necessary for their optimal immune regulatory function. However, due to the low ratio of Tr to any given antigen (Ag) and the unknown nature of Ag in many autoimmune diseases, specific activation is not practical for potential therapeutic intervention. It has been shown in animal models that once activated, Tr can exhibit immune suppression in a bystander Ag-non-specific fashion, suggesting the effector phase of Tr is Ag independent. To investigate whether the immune suppression by activated bystander Tr is as potent as that of the Ag specific Tr, Tr cells were isolated from BALB/c or ovalbumin (OVA) specific T cell receptor (TCR) transgenic mice (DO11.10) and their immune suppression of an OVA specific T cell response was compared. We found that once activated ex vivo, Tr from BALB/c and DO11.10 mice exhibited comparable inhibition on OVA specific T cell responses as determined by T cell proliferation and cytokine production. Furthermore, their immune suppression function was compared in a delayed type hypersensitivity (DTH) model induced by OVA specific T cells. Again, OVA specific and non-specific Tr exhibited similar inhibition of the DTH response. Taken together, the results indicate that ex vivo activated Ag-non-specific Tr are as efficient as Ag specific Tr in immune suppression, therefore our study provides additional evidence suggesting the possibility of applying ex vivo activated Tr therapy for the control of autoimmunity.     

Vitamin D and autoimmunity: is vitamin D status an environmental factor affecting autoimmune disease prevalence?

The environment in which the encounter of antigen with the immune system occurs determines whether tolerance, infectious immunity, or autoimmunity results. Geographical areas with low supplies of vitamin D (for example Scandinavia) correlate with regions with high incidences of multiple sclerosis, arthritis, and diabetes. The active form of vitamin D has been shown to suppress the development of autoimmunity in experimental animal models. Furthermore, vitamin D deficiency increases the severity of at least experimental autoimmune encephalomyelitis (mouse multiple sclerosis). Targets for vitamin D in the immune system have been identified, and the mechanisms of vitamin D-mediated immunoregulation are beginning to be understood. This review discusses the possibility that vitamin D status is an environmental factor, which by shaping the immune system affects the prevalence rate for autoimmune diseases such as multiple sclerosis, arthritis, and juvenile diabetes.     

D-penicillamine-induced granulomatous hepatitis in brown Norway rats

The mechanism of idiosyncratic drug reactions (IDRs) remains poorly understood. D-penicillamine treatment is associated with a wide range of autoimmune reactions including liver injury. An animal model which utilizes brown Norway (BN) rats has been used to investigate the mechanism of D-penicillamine-induced IDRs because it mimics the autoimmune reactions that occur in humans. The purpose of this study was to investigate the type of liver injury that results from D-penicillamine treatment in BN rats. We had previously noted that D-penicillamine caused histological changes in the liver, but there was no increase in alanine transaminase (ALT), and we assumed that there was no significant injury. However, we subsequently discovered that D-penicillamine inhibits the ALT assay. In the present study, we found that treatment of BN rats with a low doses of D-penicillamine (10 or 15 mg/day) resulted in a mild increases in glutamate dehydrogenase (GLDH) and sorbitol dehydrogenase (SDH) activities; however, this was not associated with histological changes. A higher dose of D-penicillamine (20 mg/day) resulted in 63 % of the rats developing a skin rash, and these rats had elevated serum GLDH and SDH levels with histopathological changes characteristic of granulomatous hepatitis. This included large clusters of leukocytes in the form of granulomas that contained neutrophils, macrophages, and CD8 T cells. These changes did not occur in the rats that did not get sick. This model may be a good model to investigate the characteristics of drug-induced granulomatous hepatitis.     

TNFR1 and the TNFα axis as a targetable mediator of liver injury from stereotactic body radiation therapy

IntroductionRadiation therapy for the management of intrahepatic malignancies can adversely affect liver function. Liver damage has been associated with increased levels of inflammatory cytokines, including tumor necrosis factor alpha (TNFα). We hypothesized that an inflammatory state, characterized by increased soluble TNFα receptor (sTNFR1), mediates sensitivity of the liver to radiation.Materials/MethodsPlasma samples collected during 3 trials of liver radiation for liver malignancies were assayed for sTNFR1 level via enzyme-linked immunosorbent assay (ELISA). Univariate and multivariate logistic regression and longitudinal models were used to characterize associations between liver toxicity (defined as a ≥2-point increase in Child-Pugh [CP] score within 6 months of radiation treatment) and sTNFR1 levels, ALBI score, biocorrected mean liver dose (MLD), age, and baseline laboratory values.ResultsSamples from 78 patients given liver stereotactic body radiation therapy [SBRT] (92%) or hypofractionated radiation were examined. There was a significant association between liver toxicity and sTNFR1 levels, and higher values were associated with increased toxicity over a range of mean liver doses. When ALBI score and biocorrected dose were included in the model with sTNFR1, baseline ALBI score and change in ALBI (ΔALBI) were significantly associated with toxicity, but sTNFR1 was not. Baseline aminotransferase levels also predicted toxicity but not independently of ALBI score.ConclusionsElevated plasma sTNFR1 levels are associated with liver injury after liver radiation, suggesting that elevated inflammatory cytokine activity is a predictor of radiation-induced liver dysfunction. Future studies should determine whether administration of agents that decrease inflammation prior to treatment is warranted.     

Sercarzian immunology--In memoriam. Eli E. Sercarz, 1934-2009

During his long career as a principal investigator and educator, Eli Sercarz trained over 100 scientists. He is best known for developing hen egg white lysozyme (HEL) as a model antigen for immunologic studies. Working in his model system Eli furthered our understanding of antigen processing and immunologic tolerance. His work established important concepts of how the immune system recognizes antigenic determinants processed from whole protein antigens; specifically he developed the concepts of immunodominance and crypticity. Later in his career he focused more on autoimmunity using a variety of established animal models to develop theories on how T cells can circumvent tolerance induction and how an autoreactive immune response can evolve over time. His theory of "determinant spreading" is one of the cornerstones of our modern understanding of autoimmunity. This review covers Eli's entire scientific career outlining his many seminal discoveries.     

Animal models of autoimmune hepatitis

Many animal models for autoimmune hepatitis (AIH) have been described in the past. Most models had to deal with the relative immunosuppressive environment of the liver. Therefore, some models used a combination of several triggering factors often on a susceptible background to generate an aggressive immune response that targets the liver. In addition, in order to be able to track the immune response the models used specific model autoantigens as targets that are either not present or have not been identified as a natural autoantigen in AIH patients. Thereby the feasibility of such models is somewhat questionable. Although many historic approaches included challenges of experimental animals with liver homogenates it was only in the last decade that natural occurring liver autoantigens have been used in animal models. This article reflects on the requirements for breaking liver tolerance and on how an ideal experimental model for AIH would look like. In addition, it discusses historic as well as recent animal models in the context of feasibility of induction, similarity of the clinical outcome to human AIH, and gain of knowledge for possible future therapies.     

Animal models of autoimmune liver disease

Autoimmune liver diseases in humans are characterized by chronic active hepatitis with serum autoantibodies, hypergammaglobulinemia and liver pathology showing necroinflammatory disease and fibrosis. There are an increasing number of autoantigens believed to be associated with various autoimmune liver diseases. This review will briefly outline human autoimmune hepatitis and the immunology of the liver. Various murine models of liver inflammation will be discussed, including transgenic and non-transgenic models, with emphasis on how these models aid in our knowledge of the mechanisms of disease development and chronicity. There are limitations with all of the models, including a preponderance of T-cell-focused responses. Murine models do not easily develop fibrosis, a hallmark of autoimmune hepatitis in humans. Different experimental models may not reach the same conclusions with differences between immune responses. However, this multiplicity of responses does not necessarily imply that these models are inappropriate for the study of liver immunology and autoimmune liver diseases, as different autoantigens may induce different liver responses. Knowledge of how the liver differs from other immune organs is essential to further our understanding of liver-specific autoimmunity. The plethora of antigens implicated in autoimmune hepatitis in humans predicts that multiple mechanisms may play a role in precipitating disease in the susceptible individual.     

Autoimmunity, immunodeficiency and mucosal infections: Chronic intestinal inflammation as a sensitive indicator of immunoregulatory defects in response to normal luminal microflora

Despite the fact that target antigens and the genetic basis of several autoimmune diseases are now better understood, the initial events leading to a loss of tolerance towards self-components remain unknown. One of the most attractive explanations for autoimmune phenomena involves various infections as possible natural events capable of initiating the process in genetically predisposed individuals. The most accepted explanation of how infection causes autoimmunity is based on the concept of “molecular mimicry” (similarity between the epitopes of an autoantigen and the epitopes in the environmental antigen). Infectious stimuli may also participate in the development of autoimmunity by inducing an increased expression of stress proteins (hsp), chaperones and transplantation antigens, which leads to abnormal processing and presentation of self antigens. Superantigens are considered to be one of the most effective bacterial components to induce inflammatory reactions and to take part in the development and course of autoimmune mechanisms. It has long been known that defects in the host defense mechanism render the individual susceptible to infections caused by certain microorganisms. Impaired exclusion of microbial antigens can lead to chronic immunological activation which can affect the tolerance to self components. Defects in certain components of the immune system are associated with a higher risk of a development of autoimmune disease. The use of animal models for the studies of human diseases with immunological pathogenesis has provided new insights into the influence of immunoregulatory factors and the lymphocyte subsets involved in the development of disease. One of the most striking conclusion arising from work with, genetically engineered immunodeficient mouse models is the existence of a high level of redundancy of the components of the immune system. However, when genes encoding molecules involved in T cell immunoregulatory functions are deleted, spontaneous chronic inflammation of the gut mucosa (similar to human inflammatory bowel disease) develops. Surprisingly, when such immunocompromised animals were placed into germfree environment, intestinal inflammation did not develop. Impairment of the mucosal immune response to the normal bacterial flora has been proposed to play a crucial role in the pathogenesis of chronic intestinal inflammation. The use of immunodeficient models colonized with defined microflora for the analysis of immune reactivity will shed light on the mode of action of different immunologically important molecules responsible for the delicate balance between luminal commensals, nonspecific and specific components of the mucosal immune system.     

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.     

The future of organ transplantation: from the laboratory to the clinic

This is a short review of tolerance from the point of view of the clinician. Various examples of tolerance occurring in patients and animal models that relate to the clinical experience are described. It is suggested that there may be different mechanisms by which tolerance is achieved, but from the patient's point of view operational tolerance is the goal, whereby, after a short induction procedure, the patient will maintain good function in the grafted organ indefinitely without maintenance immunosuppression. It is pointed out that such a goal may be difficult to achieve with any given protocol due to the enormous variation between donors and recipients of organ grafts of tissue matching, innate immune reactivity and susceptibility to disturbance of a tolerant state by infections or allergic reactions. Thus the case is made for prope or almost tolerance in which graft acceptance is maintained by a low, non-toxic dosage of maintenance immunosuppression that may not be required indefinitely.     

Risk assessment and mitigation strategies for reactive metabolites in drug discovery and development

Drug toxicity is a leading cause of attrition of candidate drugs during drug development as well as of withdrawal of drugs post-licensing due to adverse drug reactions in man. These adverse drug reactions cause a broad range of clinically severe conditions including both highly reproducible and dose dependent toxicities as well as relatively infrequent and idiosyncratic adverse events. The underlying risk factors can be split into two groups: (1) drug-related and (2) patient-related. The drug-related risk factors include metabolic factors that determine the propensity of a molecule to form toxic reactive metabolites (RMs), and the RM and non-RM mediated mechanisms which cause cell and tissue injury. Patient related risk factors may vary markedly between individuals, and encompass genetic and non-genetic processes, e.g. environmental, that influence the disposition of drugs and their metabolites, the nature of the adverse responses elicited and the resulting biological consequences. We describe a new strategy, which builds upon the strategies used currently within numerous pharmaceutical companies to avoid and minimize RM formation during drug discovery, and that is intended to reduce the likelihood that candidate drugs will cause toxicity in the human population. The new strategy addresses drug-related safety hazards, but not patient-related risk factors. A common target organ of toxicity is the liver and to decrease the likelihood that candidate drugs will cause liver toxicity (both non-idiosyncratic and idiosyncratic), we propose use of an in vitro Hepatic Liability Panel alongside in vitro methods for the detection of RMs. This will enable design and selection of compounds in discovery that have reduced propensity to cause liver toxicity. In vitro Hepatic Liability is assessed using toxicity assays that quantify: CYP 450 dependent and CYP 450 independent cell toxicity; mitochondrial impairment; and inhibition of the Bile Salt Export Pump. Prior to progression into development, a Hepatotoxicity Hazard Matrix combines data from the Hepatic Liability Panel with the Estimated RM Body Burden. The latter is defined as the level of covalent binding of radiolabelled drug to human hepatocyte proteins in vitro adjusted for the predicted human dose. We exemplify the potential value of this approach by consideration of the thiazolidinedione class of drugs.