Influenza virus host resistance model

Methods 41 (2007) 31–37 www.elsevier.com/locate/ymeth

InXuenza virus host resistance model Gary R. Burleson ¤, Florence G. Burleson BRT-Burleson Research Technologies, Inc., 120 First Flight Lane, Morrisville, NC 27560, USA Accepted 22 September 2006

Abstract Host resistance (HR) models are used to evaluate the eVect of a test article on clearance of an infectious microorganism in order to assess total immunocompetence. HR models serve as biomarkers of net immunological health or immunological well-being. Immunotoxicity can result either in an impaired clearance of an infectious agent, increased susceptibility to an opportunistic microorganism, prevention of immunization, or exacerbation of latent viral infections. The purpose of immunotoxicity testing is to obtain data that is meaningful for safety assessment, and for immunosuppression the major objective is to determine the signiWcance with respect to increased susceptibility to infectious disease. Host resistance models provide the only sure method of examining the inXuence of test articles on the functional integrity of the immune system and its ability to eliminate pathogenic microorganisms and tumor cells. They provide the means to directly assess the functional reserve of the immune system. Clearance of inXuenza virus requires an intact and functional immune system that incorporates a cascade of immune responses. Mechanistic studies can be included in the inXuenza virus host resistance model by measuring the eVect of a test article on innate immunity (cytokine and interferon production, macrophage function, and natural killer (NK) cell function) and acquired or adaptive immunity (cytotoxic T lymphocyte (CTL) activity as well as inXuenza-speciWc IgM and/or IgG antibody). © 2006 Elsevier Inc. All rights reserved. Keywords: Host resistance (HR) models; InXuenza virus; Natural killer (NK) cell activity; Cytotoxic T lymphocyte (CTL) activity; T-Dependent antibody response (TDAR)

1. Introduction The purpose of immunotoxicity testing is to obtain data that is meaningful for safety assessment, and for immunosuppression the major objective is to predict increased susceptibility to infectious disease [1]. Resistance to infectious and neoplastic disease is the raison d’être of the immunological armamentarium [2]. Immunotoxicity caused by a test compound may be reXected in an impaired clearance of an infectious microorganism, increased susceptibility to an opportunistic microorganism, prevention of immunization, or exacerbation of latent viral infections. Panels of immune function assays have been used as surrogates for measuring actual resistance to disease; however, host resistance mod-

*

Corresponding author. Fax: +1 919 719 2505. E-mail address: [email protected] (G.R. Burleson).

1046-2023/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2006.09.007

els are considered to be the ultimate predictor of adverse eVects [3]. HR models provide the only sure method of examining the inXuence of xenobiotics on the functional integrity of the immune system and its ability to eliminate pathogenic microorganisms and tumor cells and they provide the only means to directly assess the functional reserve of the immune system [1]. An aggregate, integral, intact, and functional immune system consisting of both local and systemic, innate and acquired, as well as humoral and cell-mediated immunity is required for resistance to infectious disease. Several small (by themselves not statistically signiWcant) decreases in one or more immunological functions, may result in additive or synergistic eVects that together result in eVects on disease resistance. A small statistically signiWcant decrease in several immunological functions may not result in increased disease susceptibility due to immunological reserve [2]. The biological signiWcance of “x” percent

32

G.R. Burleson, F.G. Burleson / Methods 41 (2007) 31–37

change in any immunological parameter is not known. It is not known what percent change correlates with an increased susceptibility to disease and this may vary depending on the virulence of the infectious agent and the immunological reserve of the individual or test animal [4]. Clearance of an infectious agent is the culmination of a variety of immunological functions working together to protect from infection or to clear an infectious agent. A fully functional, healthy immune system will eliminate the infectious challenge through a complex but interrelated series or cascade of immunological functions involving: (a) cytokines plus the neuroendocrine and biochemical immunological mediators that are nonspeciWc and are important in signaling the presence and activation of immunological eVector cells, (b) nonspeciWc innate immunity mediated by neutrophils, macrophages, and natural killer (NK) cells, and (c) adaptive or speciWc immunity mediated by cytotoxic T lymphocytes (CTL) and humoral immunity through B lymphocytes [2,5,6]. In summary, HR models provide a means to determine if a test agent results in an adverse eVect (decreased clearance) as well as providing the mechanism(s) of the adverse eVect (cytokines, innate immune function or adaptive immunity).

survival in mice infected intranasally with S. pneumoniae [10]. The streptococcal pulmonary host resistance model is thus an important means to assess the functional immunological capacity of macrophages and neutrophils as well as certain macrophage cytokines and has been used in both mice and rats to assess immunotoxicity of pharmacological agents [Burleson, personal communication] and to rank order anti-inXammatory agents [Komocsar, Wierda and Burleson, personal communication]. Streptococcal-speciWc IgG and cytokines may also be quantiWed. L. monocytogenes, a gram positive bacillus, is a widely used HR assay measuring mortality [11] or measuring bacterial clearance in the liver and spleen [Burleson, personal communication].

2. HR models

Parasite HR models have also been used for immunotoxicity testing. These include malaria [13] and Trichinella spiralis [14].

HR models exist for viral, bacterial, fungal, parasitic, and neoplastic diseases. Models for each of these infectious or neoplastic diseases have been used by immunologists to study immunological functions associated with clearance, by immunopharmacologists to evaluate anti-viral, anti-parasitic, anti-bacterial agents, anti-fungal, anti-tumor agents and biological response modiWers, and by immunotoxicologists to assess the immunotoxicity of a test article by quantifying the adverse eVect on disease susceptibility. 2.1. Bacterial HR models Numerous bacterial HR models have been used for immunotoxicity testing. The most commonly used models are Streptococcus pneumoniae and Listeria monocytogenes. S. pneumoniae is a gram positive coccus that has been used in mice as a systemic infection following intravenous infection and mortality recorded [7] or bacterial clearance quantiWed in the liver, spleen and blood [Burleson, personal communication]. S. pneumoniae has also been used in mice as a pulmonary infection following intranasal infection [Burleson, personal communication]. Macrophages were demonstrated to be important in the clearance of streptococci from the lungs of mice [8] and in mice and rats [8,9]. Further studies [9] demonstrated the importance of neutrophils in pulmonary streptococcal disease in rats by pretreatment with antibody to neutrophils. This model is also valuable for evaluating the eVect of macrophage cytokines on bacterial host resistance. Treatment with antibody to TNF increased TNF in the lungs and serum, decreased neutrophils and increased bacterial load with decreased

2.2. Fungal HR model Candida albicans is a well-characterized fungal host resistance model [12]; [Burleson, personal communication]. Candida is administered intravenously and mortality or clearance monitored. Candida-speciWc IgG and cytokines may also be quantiWed. 2.3. Parasite HR models

2.4. Tumor HR models Tumor HR models have been used for immunotoxicity testing using the syngeneic tumor cell models B16F10 and PYB6 [15]. 2.5. Viral HR models Several viral host resistance models have been used for immunotoxicity testing including reovirus, murine cytomegalovirus (MCMV), rat cytomegalovirus (RCMV), encephalomyocarditis (EMC) virus, vesicular stomatitis virus (VSV), and inXuenza virus. The reovirus model in mice has been used for gastrointestinal immunotoxicity testing [16]. Deoxynivalenol, also known as DON or vomitoxin, has been shown to target gut lymphoid tissue and IgA production and in the murine reovirus host resistance model increased both the severity of the infection and shedding in feces with an elevated reovirus IgA response and a cytokine pattern corresponding to a suppressed TH1 and enhanced TH2 cytokine pattern [17]. Murine cytomegalovirus (MCMV) and rat cytomegalovirus (RCMV) are well-characterized models for human cytomegalovirus (CMV) disease in humans and have been used for immunotoxicity testing [18–24]. These viruses cause a primary infection with infectious virus detectable in a variety of organs (lung, liver, spleen, and salivary gland). After primary infection, virus is cleared and viral replication is terminated by immune control mechanisms. The

G.R. Burleson, F.G. Burleson / Methods 41 (2007) 31–37

virus can remain in a nonproductive latent state for a lifetime [25] unless viral reactivation or recrudescence in patients immunocompromised via treatment, e.g., transplant patients, or individuals immunosuppressed by infection with HIV, or idiopathic immunosuppression. The MCMV latent viral model is therefore an excellent model to study reactivation of latent viral disease as a result of immunosuppression. Lymphocyte depletion studies revealed a hierarchy of immune control functions of CD4+, CD8+, and NK cells. Reactivation was rare if only one cell type was deleted, but was evident after deletion of another cell type [26] demonstrating immunological reserve. 3. InXuenza virus HR models InXuenza virus host resistance models in mice and rats have been used extensively and are thoroughly characterized. Early studies with inXuenza host resistance models used mortality as an endpoint. Successive passage of inXuenza A/Hong Kong/8/68 (H3N2) virus in mice results in a highly virulent virus between passages 3 and 9 [27]. The molecular mechanism is not known by which a virus becomes a killer virus. Fewer numbers of infectious particles are required for death in mice along with a greater variability in mortality occurring with the more virulent virus. Mortality studies with passage 14 of this highly virulent virus resulted in unacceptable variability and excessive use of animals, as demonstrated by the study design for evaluating immunotoxicity to TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) [28]. Thirty mice were used per dose group (6 groups). Because of the inherent variability of the virulent virus, three diVerent challenge levels were used. The three challenge levels were not large (150 plaque forming units (PFU) per mouse, 95 PFU per mouse, or 60 PFU per mouse). This was done to assure that at least one control group would have 30% or less deaths. The use of diVerent dilutions of the stock virus was required because of the inherent variability of the highly virulent challenge virus. The study also reported that, at a dose of TCDD that would have resulted in signiWcant mortality, there was no eVect on viral clearance [29]. Clearance of infectious virus is a more sensitive and meaningful measure of immunological function [5,18,29] than mortality although much more labor intensive. Mortality, viral replication, and immune function assessment are diVerent endpoints that can be used depending on the question addressed [29]. Challenging the immune system with an extremely virulent or with an extremely high titer of virus overwhelms the immune system with death often occurring before development of the cascade of immunological responses required for viral clearance. Challenge with a highly virulent or with a high titer of virus may more accurately reXect a model of sepsis or result in the ill-deWned “cytokine storm”. Furthermore, viral titer does not necessarily correlate with mortality. Similar titers of virus were observed in the lungs of mice infected with the mouse-adapted lethal inXuenza A/Hong

33

Kong/8/68 or the mouse-adapted nonlethal inXuenza A/ Port Chalmers/1/73 [29]. Viral titers also did not correlate with mortality in studies with TCDD [28]. 3.1. Non-lethal inXuenza viruses Numerous nonlethal inXuenza viruses have been used to study pathogenesis of the viral disease, in immunology studies of the host immune response to infection, in immunopharmacology studies to evaluate the eVectiveness of antiviral or immunomodulatory agents, and for immunotoxicological evaluations to evaluate adverse eVects occurring as a result of exposure to test articles. Lawrence’s laboratory has published elegant studies to assess the immunotoxicity of TCDD and to investigate immunological mechanisms responsible for TCDD immunotoxicity [30–35]. The inXuenza virus used for these studies is the inXuenza A/HKx31 (H3N2). A/HKx31 is a murine-adapted strain containing the internal components of A/PR8/34 (H1N1) and the external components of A/Aichi (H3N2). Mouse-adapted inXuenza A/Port Chalmers/1/73 and ratadapted inXuenza A/Port Chalmers/1/73 viruses have been characterized with regard to viral pathogenesis, histopathology, and the cascade of immunological functions occurring in response to infection and used to evaluate adverse consequences of exposure to test articles. Clearance of inXuenza virus requires an intact and functional immune system that incorporates a cascade of immune responses. An example of viral clearance and the eVect of immunosuppression is presented in Fig. 1. Mechanistic studies can be included in the inXuenza virus HR model by measuring the eVect of a test article on innate immunity (cytokine and interferon production, macrophage function, and natural killer (NK) cell function) and acquired or adaptive immunity (cytotoxic T lymphocyte (CTL) activity as well as inXuenza-speciWc IgM and/or IgG antibody). InXuenza virus replicates in the rat and causes sequential histopathological lesions in the upper respiratory tract. The hallmarks of damage include epithelial necrosis, descending inXammation, regenerative epithelium, epithelial hyperplasia, mucus pooling, and atelectasis. These lesions resolve 14 days after infection. The rat inXuenza host resistance model resembles human inXuenza infection, both with respect to time course and morphogenesis of the respiratory tract lesions [36–39]. 4. Immunology of a non-lethal inXuenza virus HR model InXuenza virus replicates to high titer in the lungs of mice or rats following intranasal infection. Intranasal infection of mice or rats with inXuenza A/Port Chalmers/1/73 (H3N2) virus results in replication of the virus to high titer in the lungs of both species. HR models provide the opportunity to measure an increased or decreased replication of microorganisms in order to assess the eVect of a test article on disease resistance. Moreover, host resistance models also provide the opportunity to measure antigen-driven or

34

G.R. Burleson, F.G. Burleson / Methods 41 (2007) 31–37

Mean Log Virus Titer/g Lung Tissue

Virus Clearance 9

Dexamethasone 2 mg/kg/day

8

Vehicle Control

7 6 5 4 3 2 1 0 0

2

4

6

8

10 12 Day post-infection

14

16

18

20

Fig. 1. Kinetics of viral clearance in Fischer (CDF) rats infected with rat-adapted inXuenza A/Port Chalmers/1/73 (H3N2) virus treated with either dexamethasone (2 mg/kg/day) or vehicle.

dy An tib o

Cy tot Ly oxic mp T ho cy te

Int e Cy rfero tok ns Ne ine u s M trop ac hil r NK oph Ce age ll

Generalized Immune Response to Infectious Disease

Infection

Days Post-Infection

Fig. 2. Generalized immune response to virus infection. ModiWed from Burleson [2].

microorganism (infectious agent)—augmented immune functions associated with the particular host resistance model to assess the eVect of a xenobiotic on host resistance in order to mechanistically investigate the immunological defect. Viral infection stimulates a cascade of immunological functions that are important in immunological defense against viral disease [5]. The following immune functions have been used to evaluate local immunocompetence: (a) interferon response, (b) cytokine response, (c) macrophage function, (d) natural killer (NK) activity, and (e) cytotoxic T lymphocyte (CTL) activity. Humoral immunity is assessed by measuring the production of IgM, IgG, and IgA antibody. This cascade of immunological functions is demonstrated in Fig. 2 and each immunological function is discussed below. 4.1. Interferon activity Interferon is known to have potent antiviral eVects whether administered exogenously or produced endogenously as a result of virus infection [40]. The exogenous

eVectiveness of interferon as an antiviral agent was demonstrated after intramuscular and intravenous injections [41,42]. Endogenous interferon has been demonstrated to be important in the pathogenesis of viral infections [43,44]. Antibody to interferon administered intranasally prior to aerosol infection of mice with inXuenza virus resulted in enhanced mortality thus demonstrating the importance of endogenous interferon on local virus infections [45] as well as the systemic viral infections [43,44]. Mice infected with inXuenza A/Hong Kong/68 virus have titers of interferon in bronchoalveolar lavage Xuid that directly correlates with lung virus titers [46]. Rats infected with inXuenza A/Port Chalmers/1/73 (H3N2) virus had titers in the whole-lung homogenate as well as in the nasal lavage Xuid that correlated with viral titers [47]. Treatment with a dual toll-like receptor 7 and toll-like receptor 8 agonist protected against inXuenza in rats [47]. This dual toll-like receptor 7 and tolllike receptor 8 agonist induced interferon, as well as other cytokines, in both whole lung homogenate and nasal lavage Xuid and resulted in a large suppression of inXuenza viral titers. 4.2. Cytokine activity Cytokines (CK’s) are important immunological mediators and messengers that serve to aid in cell–cell communication among cells of the immune system. Interferon (discussed above) is classiWed as a cytokine to reXect its immunological mediator and messenger activity as opposed to its antiviral activity. Cytokines were measured in the bronchoalveolar lavage Xuid of mice following infection with 10 LD50 of inXuenza A/PR8/34 virus [48]. Mice died on day 6 of infection. Interleukin-1 (IL-1), IL-1B, IL-6, tumor necrosis factor (TNF), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon- (IFN) and leukotriene B4 (LTB4), peaked between 36 h and 3 days postinfection with the exception of IL-6 which remained elevated throughout all 6 days of infection. G-CSF and M-

G.R. Burleson, F.G. Burleson / Methods 41 (2007) 31–37

CSF peaked on day 5 after infection. IL-2, IL-3, and IL-4 were not detected. Platelet-activating factor (PAF) remained at the same level throughout the infection. IFN is produced primarily by NK cells and T cells and is an important antiviral TH1 cytokine that is central to many important immunological functions. IFN exerts the following eVects: (a) antiviral eVect; (b) aVects several T cell functions including enhancing cytotoxicity; (c) aVects macrophage functions including increased antimicrobial and antitumor eVects; (d) B cells including counteracting IL-4 and decreased CD23 and decreased proliferation; and (e) increased activity of NK cells. Symptoms and fever in humans with acute inXuenza as well as in experimental inXuenza correlate with the release of IL-6 [49]. 4.3. Macrophage activity Alveolar macrophages are present in large numbers in the airways of the lung and are the predominant cell obtained by bronchoalveolar lavage. By virtue of their location and specialized functions, these cells facilitate a critical early interaction with airborne infectious agents and pollutants. Macrophages are important contributors to early nonspeciWc immunity and they also participate in speciWc immunological responses. Macrophages may initiate and modulate both speciWc and nonspeciWc immune responses due to the following properties: processing and presentation of antigen, phagocytosis of microorganisms and particles, antiviral and antibacterial killing, cytostasis and cytotoxicity of tumor cells, secretion of a variety of soluble mediators including interferons, interleukins, tumor necrosis factor, eicosanoids, chemotactic factor for neutrophils, superoxide anion, and numerous enzymes. Alveolar macrophages play an important role in the clearance of inXuenza virus from infected mice [50]. However, macrophages have acted in concert with speciWc antiviral antibody resulting in increased viral titers. Low levels of antiviral antibody were shown in vivo to increase virus infectivity [51] and to be important in antibody-mediated growth of inXuenza virus in a macrophage-like cell line [52]. 4.4. Natural killer (NK) cell activity NK cell activity has an important role in the interaction of diVerent immunological cell types and cell functions that are important in immunological defense against viral, bacterial, and neoplastic disease. Measurement of NK cell activity is an important functional assay of innate immunity following exposure to both pharmaceutical and environmental test articles [53–56]. NK cell activity is enhanced following viral infection and reaches peak activity 2 days following infection in both mice and rats infected with mouse-adapted or rat-adapted inXuenza A/Port Chalmers/ 1/73 (H3N2) virus. Evaluation of NK activity in mice or rats infected with inXuenza virus provides an important mechanistic probe if an increase or decrease in viral clearance (immunopharmacology or immunotoxicology) is

35

observed. Measurement of an antigen-driven cytokine enhanced immunological function is more meaningful than the measurement of endogenous or unstimulated NK cell activity. NK activity can be measured in collagenasetreated whole lung homogenate or spleen cells [5] from inXuenza virus-infected mice or rats demonstrating the systemic immunological response following intranasal infection. 4.5. Cytotoxic T lymphocyte (CTL) activity The cytotoxic T lymphocyte (CTL) response to inXuenza virus is a component of the speciWc or acquired immune response and has been used to evaluate cell-mediated immunity following exposure to test articles [57]. The CTL response entails the polymorphic HLA Class I molecule presentation of viral antigen to CD8+ T lymphocytes that are the functional CTL. The Class I molecules of the major histocompatibility complex (MHC) are important for recognition of viral antigen by CTL [58]. CTL are generated against several of the inXuenza viral antigens. CTL activity was measured in the lungs and the bronchoalveolar lavage Xuid after intravenous or intranasal infection of mice [59]. CTL activity was detected in the spleen on days 4–12 and in the lymph node peaking on day 4 and gone by day 6 [60]. The importance of CTL in recovery from viral infection was demonstrated by the adoptive transfer of spleen cells enriched for inXuenza virus-speciWc eVector CTL into mice resulting in a reduction in both mortality and pulmonary virus titers [61,62]. Nude mice do not have a functional cellmediated immunity and following intranasal infection with inXuenza virus these mice have a prolonged and chronic shedding of virus [63] and adoptive transfer of spleen cells to inXuenza-infected nude mice results in recovery [62]. CTL activity is enhanced following viral infection and reaches peak activity 8 days following infection in both mice and rats infected with mouse-adapted or rat-adapted inXuenza A/Port Chalmers/1/73 (H3N2) virus. Evaluation of CTL activity in mice or rats infected with inXuenza virus provides another important mechanistic probe if an increase or decrease in viral clearance (immunopharmacology or immunotoxicology) is observed. Measurement of CTL provides an important measure of cell-mediated immunity. CTL activity can be measured in collagenasetreated whole lung homogenate or spleen cells [5] from inXuenza virus-infected mice or rats demonstrating the systemic immunological response following intranasal infection. 4.6. Humoral immunity (TDAR response) InXuenza virus is a T-dependent antigen [63–67] and measurement of inXuenza-speciWc IgG therefore serves as a Tdependent antibody response (TDAR) thus requiring and assessing functionality of T cells, B cells and macrophage antigen processing and presentation activity. Measurement of inXuenza-speciWc IgG evaluates humoral immunity. Mea-

36

G.R. Burleson, F.G. Burleson / Methods 41 (2007) 31–37

surement of inXuenza-speciWc IgG allows an evaluation of T cells, B cells, and the antigen processing and presentation activity of macrophages. The role of antibody in protection from reinfection with inXuenza virus is well established and is the basis for the use of vaccines in an attempt to control this important viral infection. The importance of physiological amounts of speciWc antibody is shown by their protection against lethal inXuenza infection, even in mice immunosuppressed with cyclophosphamide [68]. Antibody directed against the hemagglutinin is more important than antibody directed against the neuraminidase in protection against lethal inXuenza virus infection; however, antibody to neuraminidase has protective value [69]. Antibody in response to a primary inXuenza viral infection in mice showed hemagglutination-inhibiting (HI) antibody on day 8 systemically and on day 12 in the bronchoalveolar lavage Xuid while antibody detected by neutralization was detected systemically and in the lung on day 12 [61]. InXuenza virus-speciWc antibody secreting cells following a primary inXuenza virus infection was measured using an enzyme linked immunosorbent plaque assay in mice [70]. Antibody secreting cells were more numerous in the lung than in the spleen. Lung antibody secreting cells producing IgM appeared on day 5 and peaked on day 10 while IgG and IgA appeared at the end of week 2 and peaked during week 3. Secretory IgA is thought to play an important role as a mediator of local mucosal immunity. Following infection of mice with inXuenza virus the number of lymphocytes increased greatly and lymphocytes with surface immunoglobulin A demonstrated the largest relative increase [71]. These cells increased on day 8 and peaked on day 14 following infection. Passive immunization of polymeric IgA administered intravenously protected against inXuenza virus infection [72]. Polymeric IgA was transported across mucosal epithelium into nasal secretions via a secretory component-mediated mechanism. 5. Conclusions InXuenza virus HR assays are used to evaluate the eVect of a test article on clearance of an infectious microorganism in order to assess total immunocompetence and serves as biomarkers of net immunological health or immunological well-being. While immunotoxicity can result either in an impaired clearance of an infectious agent, increased susceptibility to an opportunistic microorganism, prevention of immunization, or exacerbation of latent viral infections, the inXuenza virus HR assay evaluates potential immunosuppression following exposure to a test article by quantifying viral clearance. An adverse outcome in the inXuenza virus HR assay may be followed, or performed concurrently, by mechanistic evaluation of a cascade of immunological function assays including: innate immunity (cytokine and interferon production, macrophage function, and natural killer (NK) cell function) and acquired or adaptive immunity (cytotoxic T lymphocyte (CTL) activity as well as inXuenza-speciWc TDAR IgM and/or IgG antibody).

References [1] E. Gleichmann, I. Kimber, I.F.H. Purchase, Arch. Toxicol. 63 (1989) 257–273. [2] G.R. Burleson, Pulmonary immunocompetence and pulmonary immunotoxicology, in: R. Smialowicz, M.P. Holsapple (Eds.), Experimental Immunotoxicology, CRC Press, Inc., Boca Raton, Florida, 1996, pp. 113–135, Chapter 7. [3] D.R. Germolec, Toxicol. Lett. 149 (2004) 109–114. [4] G.R. Burleson, J.H. Dean, Immunotoxicology: Past, present, and future., in: G.R. Burleson, J.H. Dean, A.E. Munson (Eds.), Methods in Immunotoxicology, Wiley-Liss Inc., New York, N.Y., 1995, pp. 3–10, Volume 1, Chapter 1. [5] G.R. Burleson, InXuenza virus host resistance model for assessment of immunotoxicity, immunostimulation, and antiviral compounds, in: G.R. Burleson, J.H. Dean, A.E. Munson (Eds.), Methods in Immunotoxicology, Wiley-Liss Inc., New York, NY, 1995, pp. 181–202, Volume 2, Chapter 14. [6] G.R. Burleson, Immunopharmacology 48 (2000) 315–318. [7] S.G. Bradley, Streptococcus host resistance model, in: G.R. Burleson, J.H. Dean, A.E. Munson (Eds.), Methods in Immunotoxicology, WileyLiss Inc., New York, N.Y., 1995, pp. 159–168, Chapter 12. [8] M.I. Gilmour, P. Park, M.K. Selgrade, Am. Rev. Respir. Dis. 147 (1993) 753–760. [9] M.I. Gilmour, M.K. Selgrade, Toxicol. Appl. Pharmacol. 123 (1993) 211–218. [10] K. Takashima, K. Tateda, T. Matsumoto, Y. Iizawa, M. Nakao, K. Yamaguchi, Infect. Immun. 65 (1997) 257–260. [11] S.G. Bradley, Listeria host resistance model, in: G.R. Burleson, J.H. Dean, A.E. Munson (Eds.), Methods in Immunotoxicology, Wiley Liss Inc., New York, N.Y., 1995, pp. 169–179, Volume 2, Chapter 13. [12] D.J. Herzyk, E.R. Gore, R. Polsky, K.L. Nadwodny, C.C. Maier, S. Liu, T.K. Hart, A.G. Harmsen, P.J. Bugelski, Infect. Immun. 69 (2) (2001) 1032–1043. [13] R.W. Luebke, Assessment of host resistance to infection with rodent malaria, in: G.R. Burleson, J.H. Dean, A.E. Munson (Eds.), Methods in Immunotoxicology, WileyLiss, Inc., New York, N.Y., 1995, pp. 221–242, Volume 2, Chapter 16. [14] H. Van Loveren, R.W. Luebke, J.G. Vos, Assessment of immunotoxicity with the parasitic infection model Trichinella spiralis, in: G.R. Burleson, J.H. Dean, A.E. Munson (Eds.), Methods in Immunotoxicology, Wiley-Liss, Inc., New York, N.Y., 1995, pp. 243–271, Volume 2, Chapter 17. [15] J.A. McCay, Syngeneic tumor cell models B16F10 and PYB6, in: G.R. Burleson, J.H. Dean, A.E. Munson (Eds.), Methods in Immunotoxicology, Wiley-Liss Inc., New York, N.Y., 1995, pp. 143–157, Volume 2, Chapter 11. [16] C.F. CuV, J.R. Fulton, J.B. Barnett, C.S. Boyce, Toxicol. Sci. 42 (1998) 99–108. [17] M. Li, C.F. CuV, J. Pestka, Toxicol. Sci. 87 (1) (2005) 134–145. [18] M.J.K. Selgrade, M.J. Daniels, G.R. Burleson, L.D. Lauer, J.H. Dean, Int. J. Immunopharmacol. 10 (1998) 811–818. [19] M.J.K. Selgrade, M.J. Daniels, Host resistance models: Murine cytomegalovirus, in: G.R. Burleson, J.H. Dean, A.E. Munson (Eds.), Methods in Immunotoxicology, Wiley-Liss Inc., New York, N.Y., 1995, pp. 203–219, Volume 2, Chapter 15. [20] P.S. Ross, R.L. de Swart, H. van der Vliet, L. Willemsen, A. De Klerk, G. Van Amerongen, J. Groen, A. Brouwer, I. Schipholt, D.C. Morse, H. van Loveren, Arch. Toxicol. 71 (9) (1997) 563–574. [21] P.S. Ross, H. van Loveren, R.L. de Swart, H. van der Vliet, A. de Klerk, H.H. Timmerman, R. Van Binnendijk, A. Brouwer, J.G. Vos, A.d. Osterhaus, Arch. Toxicol. 70 (10) (1996) 661–671. [22] J. Garssen, H. van der Vliet, A. De Klerk, W. Goettsch, J.A. Dormans, C.A. Bruggeman, A.D. Osterhaus, H. Van Loveren, Eur. J. Pharmacol. 292 (3-4) (1995) 223–231. [23] H. Van Loveren, Hum. Exp. Toxicol. 14 (1) (1995) 137–140. [24] W. Goettsch, J. Garssen, F.R. de Gruijl, H. Van Loveren, Toxicolology 72 (1-3) (1994) 359–363.

G.R. Burleson, F.G. Burleson / Methods 41 (2007) 31–37 [25] M.C. Jordan, J.D. Shanley, and J.G. Stevens, J. Gen. Virol. 37, (2):419423. [26] B. PoliT, H. Hengel, A. KrmpotiT, J. Trgovcich, I. PaviT, P. Lubin, S. JonjiT, U.H. Koszinowski, J. Exp. Med. 188 (1998) 1047–1054. [27] P.R. Wyde, R.B. Couch, B.F. Mackler, T.R. Cate, B.M. Levy, Infect. Immun. 15 (1) (1977) 221–229. [28] G.R. Burleson, H. Lebrec, Y.G. Yang, J.D. Ibanes, K.N. Pennington, L.S. Birnbaum, Fund. Appl. Toxicol. 29 (1996) 40–47. [29] H. Lebrec, G.R. Burleson, Toxicology 91 (2) (1994) 179–188. [30] T.K. Warren, K.A. Mitchell, B.P. Lawrence, Toxicol. Sci. 56 (2000) 114–123. [31] K.A. Mitchell, B.P. Lawrence, Toxicol. Sci. 74 (2003) 74–84. [32] B.P. Lawrence, B.A. Vonderstrasse, Toxicol. Sci. 79 (2004) 304–314. [33] S. Teske, A.A. Bonn, J.F. Regal, J.J. Neumiller, B.P. Lawrence, Am. J. Physiol. Lung Cell. Mol. Physiol. 289 (1:L) (2005) 111–124. [34] B.A. Vonderstrasse, J.A. CundiV, B.P. Lawrence, J. Toxicol. Environ. Health A 69 (6) (2006) 445–463. [35] A.A. Bonn, K.S. Harrod, S. Teske, B.P. Lawrence, Chem. Biol. Interact. 155 (3) (2005) 181–190. [36] T.T. Kawabata, G.R. Burleson, P.B. Ernst, S.E. Ullrich, Fund. Appl. Toxicol. 26 (1) (1995) 8–19. [37] G.R. Burleson, J.D. Ibanes, K.T. Morgan, Fund. Appl. Toxicol. 26 (1) (1995) 11–19. [38] J.D. Ibanes, K.T. Morgan, G.R. Burleson, Vet. Pathol. 33 (1996) 412–418. [39] J.A. Dye, K.T. Morgan, D.L. Neldon, J.S. Tepper, G.R. Burleson, D.L. Costa, Vet. Pathol. 33 (1996) 43–54. [40] A. Isaacs, J. Lindenmann, Proc. R. Soc. London Ser. B 147 (1957) 258–267. [41] N.B. Finter, J. Exp. Pathol. 47 (1996) 361–371. [42] N.B. Finter, J. Gen. Virol. 1 (1967) 395–397. [43] I. Gresser, M.G. Tovey, M.-T. Bandu, C. Maury, D. Brouty-Boye, J. Exp. Med. 144 (1976) 1305–1315. [44] I. Gresser, M.G. Tovey, C. Maury, M.-T. Bandu, J. Exp. Med. 144 (1976) 1316–1323. [45] A. Hoshino, H. Takenaka, O. Mizukoshi, J. Imanishi, T. Kishida, M.G. Tovey, Antiviral. Res. 3 (1983) 59–65. [46] P.R. Wyde, M.R. Wilson, T.R. Cate, Infect. Immun. 38 (1982) 1249– 1255. [47] D.M. Hammerbeck, G.R. Burleson, C.J. Schuller, J.P. Vasilakos, M. Tomai, E. Egging, F.R. Cochran, S. Woulfe, R.L. Miller, Antiviral Research 2006, (in press).

37

[48] T. Hennet, H.J. Ziltener, K. Frei, E. Peterhans, J. Immunol. 149 (1992) 932–939. [49] L. Kaiser, R.L.S. Fritz, S.E. Straus, L. Gubareve, F.G. Hayden, J. Med. Virol. 64 (2001) 262–288. [50] G. Lemercier, S. Mavet, Ann. Microbiol. (Instit. Pasteur.) 130B (1979) 235–244. [51] R.G. Webster, B.A. Askonas, Eur. J. Immunol. 10 (1980) 396–401. [52] H. Ochiai, M. Kurokawa, K. Hayashi, S. Niwayama, J. Virol. 62 (1988) 20–26. [53] G.R. Burleson, Alteration of cellular interactions in the immune system: Natural killer activity and N lymphocytes, in: H.A. Milman, E. Elmore (Eds.), Biochemical Mechanisms and Regulation of Intercellular Communication, in: M.A. Mehlman (Ed.), Advances in Modern Environmental Toxicology, vol. XIV, Princeton ScientiWc Publishing Co. Inc., Princeton, New Jersey, 1987, pp. 51–96. [54] H. Van Loveren, E.I. Krajnc, P.J.A. Rombout, F.A. Blommaert, J.G. Vos, Toxicol. Appl. Pharmacol. 102 (1990) 21–33. [55] G.R. Burleson, L.L. Keyes, Immunopharmacol. Immunotoxicol. 11 (1989) 421–443. [56] G.R. Burleson, L.L. Keyes, J.D. Stutzman, Immunopharmacol. Immunotoxicol. 11 (1989) 715–735. [57] J.P. Ehrlich, A.F. Gunnison, G.R. Burleson, Inhal. Toxicol. 1 (1989) 129–138. [58] R.M. Zinkernagel, P.C. Doherty, Nature (London) 251 (1974) 547–548. [59] K.L. Yap, G.L. Ada, Scand. J. Immunol. 7 (1978) 73–80. [60] K.L. Yap, G.L. Ada, I.F.C. McKenzie, Nature 273 (1978) 238–240. [61] M.A. Wells, F.A. Ennis, S. Daniel, J. Gen. Virol. 43 (1979) 685–690. [62] M.A. Wells, P. Albrecht, F.A. Ennis, J. Immunol. 126 (1981) 1036–1041. [63] R.M. Kris, R.A. Yetter, R. Cogliano, et al., Immunology 63 (1988) 349–353. [64] M.Y. Sangster, J.M. Riberdy, M. Gonzalez, D.J. Topham, N. Baumgarth, P.C. Doherty, J. Exp. Med. 198 (2003) 1011–1021. [65] W. Burns, L.C. Billups, A.L. Notkins, Nature 256 (1975) 654–656. [66] T. Iwasaki, T. Nozima, J. Immunol. 118 (1977) 256–263. [67] P.A. Scherle, W. Gerhard, J. Exp. Med. 164 (1986) 1114–1128. [68] J-L. Virelizier, J. Immunol. 115 (1975) 434–439. [69] J.L. Schulman, M. Khakpour, E.D. Kilbourne, J. Virol. 2 (1968) 778–786. [70] P.D. Jones, G.L. Ada, J. Virol. 60 (1986) 614–619. [71] G.H. Scott, J.S. Walker, Infect. Immun. 13 (1976) 1525–1527. [72] K.B. Renegar, P.A. Small Jr, J. Immunol. 146 (1991) 1972–1978.