Microbiota and Dose Response: Evolving Paradigm of Health Triangle.

SRA Dose-Response and Microbial Risk Analysis Specialty Groups jointly sponsored symposia that addressed the intersections between the "microbiome revolution" and dose response. Invited speakers presented on innovations and advances in gut and nasal microbiota (normal microbial communities) in the first decade after the Human Microbiome Project began. The microbiota and their metabolites are now known to influence health and disease directly and indirectly, through modulation of innate and adaptive immune systems and barrier function. Disruption of healthy microbiota is often associated with changes in abundance and diversity of core microbial species (dysbiosis), caused by stressors including antibiotics, chemotherapy, and disease. Nucleic-acid-based metagenomic methods demonstrated that the dysbiotic host microbiota no longer provide normal colonization resistance to pathogens, a critical component of innate immunity of the superorganism. Diverse pathogens, probiotics, and prebiotics were considered in human and animal models (in vivo and in vitro). Discussion included approaches for design of future microbial dose-response studies to account for the presence of the indigenous microbiota that provide normal colonization resistance, and the absence of the protective microbiota in dysbiosis. As NextGen risk analysis methodology advances with the "microbiome revolution," a proposed new framework, the Health Triangle, may replace the old paradigm based on the Disease Triangle (focused on host, pathogen, and environment) and germophobia. Collaborative experimental designs are needed for testing hypotheses about causality in dose-response relationships for pathogens present in our environments that clearly compete in complex ecosystems with thousands of bacterial species dominating the healthy superorganism.

Nanoaerosols reduce required effective dose of liposomal levofloxacin against pulmonary murine Francisella tularensis subsp. novicida infection.

BACKGROUND: The Institute of Theoretical and Experimental Biophysics in Moscow recently developed a new nanoaerosol generator. This study evaluated this novel technology, which has the potential to enhance therapeutic delivery, with the goal of using the generator to treat pulmonary Francisella tularensis subsp. novicida (F. novicida) infections in BALB/c mice. RESULTS: First, the analysis of quantum dots distribution in cryosections of murine lungs demonstrated that nanoaerosols penetrate the alveoli and spread more homogenously in the lungs than upon intranasal delivery. Second, the generator was used to aerosolize the antibiotic levofloxacin to determine the effectiveness of nanoaerosolized levofloxacin as treatment against F. novicida. The generator was capable of delivering a sufficient dose of nanoaerosolized liposome-encapsulated levofloxacin to rescue mice against 100LD50 of F. novicida. CONCLUSIONS: The nanoaerosol-delivered dosage of liposome-encapsulated levofloxacin required to rescue mice is approximately 94× lower than the oral required dose and approximately 8× lower than the intraperitoneal dose required for rescue. In addition, treatment with nanoaerosols consumes less total volume of therapeutic solutions and is gentler on sprayed material than the aerosolization by a conventional three-jet Collison nebulizer as seen by the preservation of liposomes. This could represent a significant advance for the use of expensive therapeutics and lung directed therapies.

Dose-Response Modeling for Inhalational Anthrax in Rabbits Following Single or Multiple Exposures.

There is a need to advance our ability to characterize the risk of inhalational anthrax following a low-dose exposure. The exposure scenario most often considered is a single exposure that occurs during an attack. However, long-term daily low-dose exposures also represent a realistic exposure scenario, such as what may be encountered by people occupying areas for longer periods. Given this, the objective of the current work was to model two rabbit inhalational anthrax dose-response data sets. One data set was from single exposures to aerosolized Bacillus anthracis Ames spores. The second data set exposed rabbits repeatedly to aerosols of B. anthracis Ames spores. For the multiple exposure data the cumulative dose (i.e., the sum of the individual daily doses) was used for the model. Lethality was the response for both. Modeling was performed using Benchmark Dose Software evaluating six models: logprobit, loglogistic, Weibull, exponential, gamma, and dichotomous-Hill. All models produced acceptable fits to either data set. The exponential model was identified as the best fitting model for both data sets. Statistical tests suggested there was no significant difference between the single exposure exponential model results and the multiple exposure exponential model results, which suggests the risk of disease is similar between the two data sets. The dose expected to cause 10% lethality was 15,600 inhaled spores and 18,200 inhaled spores for the single exposure and multiple exposure exponential dose-response model, respectively, and the 95% lower confidence intervals were 9,800 inhaled spores and 9,200 inhaled spores, respectively.

Evaluation of Inhaled Versus Deposited Dose Using the Exponential Dose-Response Model for Inhalational Anthrax in Nonhuman Primate, Rabbit, and Guinea Pig.

The application of the exponential model is extended by the inclusion of new nonhuman primate (NHP), rabbit, and guinea pig dose-lethality data for inhalation anthrax. Because deposition is a critical step in the initiation of inhalation anthrax, inhaled doses may not provide the most accurate cross-species comparison. For this reason, species-specific deposition factors were derived to translate inhaled dose to deposited dose. Four NHP, three rabbit, and two guinea pig data sets were utilized. Results from species-specific pooling analysis suggested all four NHP data sets could be pooled into a single NHP data set, which was also true for the rabbit and guinea pig data sets. The three species-specific pooled data sets could not be combined into a single generic mammalian data set. For inhaled dose, NHPs were the most sensitive (relative lowest LD50) species and rabbits the least. Improved inhaled LD50 s proposed for use in risk assessment are 50,600, 102,600, and 70,800 inhaled spores for NHP, rabbit, and guinea pig, respectively. Lung deposition factors were estimated for each species using published deposition data from Bacillus spore exposures, particle deposition studies, and computer modeling. Deposition was estimated at 22%, 9%, and 30% of the inhaled dose for NHP, rabbit, and guinea pig, respectively. When the inhaled dose was adjusted to reflect deposited dose, the rabbit animal model appears the most sensitive with the guinea pig the least sensitive species.

Modeling low-dose mortality and disease incubation period of inhalational anthrax in the rabbit.

There is a need to advance our ability to conduct credible human risk assessments for inhalational anthrax associated with exposure to a low number of bacteria. Combining animal data with computational models of disease will be central in the low-dose and cross-species extrapolations required in achieving this goal. The objective of the current work was to apply and advance the competing risks (CR) computational model of inhalational anthrax where data was collected from NZW rabbits exposed to aerosols of Ames strain Bacillus anthracis. An initial aim was to parameterize the CR model using high-dose rabbit data and then conduct a low-dose extrapolation. The CR low-dose attack rate was then compared against known low-dose rabbit data as well as the low-dose curve obtained when the entire rabbit dose-response data set was fitted to an exponential dose-response (EDR) model. The CR model predictions demonstrated excellent agreement with actual low-dose rabbit data. We next used a modified CR model (MCR) to examine disease incubation period (the time to reach a fever >40 °C). The MCR model predicted a germination period of 14.5h following exposure to a low spore dose, which was confirmed by monitoring spore germination in the rabbit lung using PCR, and predicted a low-dose disease incubation period in the rabbit between 14.7 and 16.8 days. Overall, the CR and MCR model appeared to describe rabbit inhalational anthrax well. These results are discussed in the context of conducting laboratory studies in other relevant animal models, combining the CR/MCR model with other computation models of inhalational anthrax, and using the resulting information towards extrapolating a low-dose response prediction for man.

Ultraviolet dosage and decontamination efficacy were widely variable across 14 UV devices after testing a dried enveloped ribonucleic acid virus surrogate for SARS-CoV-2.

Aims: The dosages and efficacy of 14 ultraviolet (UV) decontamination technologies were measured against a SARS-CoV-2 surrogate virus that was dried onto different materials for laboratory and field testing. Methods and results: A live enveloped, ribonucleic acid (RNA) virus surrogate for SARS-CoV-2 was dried on stainless steel 304 (SS304), Navy Top Coat-painted SS304 (NTC), cardboard, polyurethane, polymethyl methacrylate (PMMA), and acrylonitrile butadiene styrene (ABS) materials at > 8.0 log10 plaque-forming units (PFU) per test coupon. The coupons were then exposed to UV radiation during both laboratory and field testing. Commercial and prototype UV-emitting devices were measured for efficacy: four handheld devices, three room/surface-disinfecting machines, five air disinfection devices, and two larger custom-made machines. UV device dosages ranged from 0.01 to 729 mJ cm-2. The antiviral efficacy among the different UV devices ranged from no decontamination up to nearly achieving sterilization. Importantly, cardboard required far greater dosage than SS304. Conclusion: Enormous variability in dosage and efficacy was measured among the different UV devices. Porous materials limit the utility of UV decontamination. Significance and impact of the study: UV devices have wide variability in dosages, efficacy, hazards, and UV output over time, indicating that each UV device needs independent technical measurement and assessment for product development prior to and during use.

Hot, Humid Air Decontamination of Aircraft Confirmed That High Temperature and High Humidity Are Critical for Inactivation of Infectious, Enveloped Ribonucleic Acid (RNA) Virus.

Aims: To develop infectious (live/dead) enveloped virus test indicators and response surface methodology (RSM) models that evaluate survival of an enveloped ribonucleic acid (RNA) virus on contaminated aircraft materials after exposure to hot, humid air (HHA). Methods and Results: Enveloped RNA bacteriophage Phi6 (Φ6) was dried on wiring insulation, aircraft performance coating (APC), polypropylene, and nylon at ≥ 8 log10 plaque-forming units (PFU) test coupon-1. Only 2.4 log10 inactivation was measured on APC at 70°Celsius (°C), 5% relative humidity (RH) after 24 h. In contrast, HHA RSM models showed a 90% probability of a 7 log10 inactivation at ≥63°C, 90% RH after 1 h, and decontamination kinetics were similar across different materials. HHA decontamination of C-130 and C-17 aircraft showed >7 log10 and ≥5.9 log10 inactivation of enveloped virus on 100 and 110 test indicators, respectively, with a 1-h treatment, excluding ramp-up and ramp-down times. Conclusions: Enveloped RNA virus test indicators were successfully developed, lab tested for HHA decontamination, analyzed for RSM, and field-tested in aircraft demonstrations. Significance and Impact of the Study: The utility of HHA decontamination was demonstrated after inactivating enveloped RNA virus on aircraft with a 1-h HHA treatment within aircraft temperature and RH limits.

Repurposing FDA-approved drugs as therapeutics to treat Rift Valley fever virus infection.

There are currently no FDA-approved therapeutics available to treat Rift Valley fever virus (RVFV) infection. In an effort to repurpose drugs for RVFV treatment, a library of FDA-approved drugs was screened to determine their ability to inhibit RVFV. Several drugs from varying compound classes, including inhibitors of growth factor receptors, microtubule assembly/disassembly, and DNA synthesis, were found to reduce RVFV replication. The hepatocellular and renal cell carcinoma drug, sorafenib, was the most effective inhibitor, being non-toxic and demonstrating inhibition of RVFV in a cell-type and virus strain independent manner. Mechanism of action studies indicated that sorafenib targets at least two stages in the virus infectious cycle, RNA synthesis and viral egress. Computational modeling studies also support this conclusion. siRNA knockdown of Raf proteins indicated that non-classical targets of sorafenib are likely important for the replication of RVFV.

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

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

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

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

Deterministic models of inhalational anthrax in New Zealand white rabbits.

Computational models describing bacterial kinetics were developed for inhalational anthrax in New Zealand white (NZW) rabbits following inhalation of Ames strain B. anthracis. The data used to parameterize the models included bacterial numbers in the airways, lung tissue, draining lymph nodes, and blood. Initial bacterial numbers were deposited spore dose. The first model was a single exponential ordinary differential equation (ODE) with 3 rate parameters that described mucociliated (physical) clearance, immune clearance (bacterial killing), and bacterial growth. At 36 hours postexposure, the ODE model predicted 1.7×107 bacteria in the rabbit, which agreed well with data from actual experiments (4.0×107 bacteria at 36 hours). Next, building on the single ODE model, a physiological-based biokinetic (PBBK) compartmentalized model was developed in which 1 physiological compartment was the lumen of the airways and the other was the rabbit body (lung tissue, lymph nodes, blood). The 2 compartments were connected with a parameter describing transport of bacteria from the airways into the body. The PBBK model predicted 4.9×107 bacteria in the body at 36 hours, and by 45 hours the model showed all clearance mechanisms were saturated, suggesting the rabbit would quickly succumb to the infection. As with the ODE model, the PBBK model results agreed well with laboratory observations. These data are discussed along with the need for and potential application of the models in risk assessment, drug development, and as a general aid to the experimentalist studying inhalational anthrax.

Achieving consistent multiple daily low-dose Bacillus anthracis spore inhalation exposures in the rabbit model.

Repeated low-level exposures to biological agents could occur before or after the remediation of an environmental release. This is especially true for persistent agents such as B. anthracis spores, the causative agent of anthrax. Studies were conducted to examine aerosol methods needed for consistent daily low aerosol concentrations to deliver a low-dose (less than 10(6) colony forming units (CFU) of B. anthracis spores) and included a pilot feasibility characterization study, acute exposure study, and a multiple 15 day exposure study. This manuscript focuses on the state-of-the-science aerosol methodologies used to generate and aerosolize consistent daily low aerosol concentrations and resultant low inhalation doses to rabbits. The pilot feasibility characterization study determined that the aerosol system was consistent and capable of producing very low aerosol concentrations. In the acute, single day exposure experiment, targeted inhaled doses of 1 × 10(2), 1 × 10(3), 1 × 10(4), and 1 × 10(5) CFU were used. In the multiple daily exposure experiment, rabbits were exposed multiple days to targeted inhaled doses of 1 × 10(2), 1 × 10(3), and 1 × 10(4) CFU. In all studies, targeted inhaled doses remained consistent from rabbit-to-rabbit and day-to-day. The aerosol system produced aerosolized spores within the optimal mass median aerodynamic diameter particle size range to reach deep lung alveoli. Consistency of the inhaled dose was aided by monitoring and recording respiratory parameters during the exposure with real-time plethysmography. Overall, the presented results show that the animal aerosol system was stable and highly reproducible between different studies and over multiple exposure days.

Inhalational anthrax (Ames aerosol) in naïve and vaccinated New Zealand rabbits: characterizing the spread of bacteria from lung deposition to bacteremia.

There is a need to better understand inhalational anthrax in relevant animal models. This understanding could aid risk assessment, help define therapeutic windows, and provide a better understanding of disease. The aim here was to characterize and quantify bacterial deposition and dissemination in rabbits following exposure to single high aerosol dose (> 100 LD(50)) of Bacillus anthracis (Ames) spores immediately following exposure through 36 h. The primary goal of collecting the data was to support investigators in developing computational models of inhalational anthrax disease. Rabbits were vaccinated prior to exposure with the human vaccine (Anthrax Vaccine Adsorbed, AVA) or were sham-vaccinated, and were then exposed in pairs (one sham and one AVA) so disease kinetics could be characterized in equally-dosed hosts where one group is fully protected and is able to clear the infection (AVA-vaccinated), while the other is susceptible to disease, in which case the bacteria are able to escape containment and replicate uncontrolled (sham-vaccinated rabbits). Between 4-5% of the presented aerosol dose was retained in the lung of sham- and AVA-vaccinated rabbits as measured by dilution plate analysis of homogenized lung tissue or bronchoalveolar lavage (BAL) fluid. After 6 and 36 h, >80% and >96%, respectively, of the deposited spores were no longer detected in BAL, with no detectable difference between sham- or AVA-vaccinated rabbits. Thereafter, differences between the two groups became noticeable. In sham-vaccinated rabbits the bacteria were detected in the tracheobronchial lymph nodes (TBLN) 12 h post-exposure and in the circulation at 24 h, a time point which was also associated with dramatic increases in vegetative CFU in the lung tissue of some animals. In all sham-vaccinated rabbits, bacteria increased in both TBLN and blood through 36 h at which point in time some rabbits succumbed to disease. In contrast, AVA-vaccinated rabbits showed small numbers of CFU in TBLN between 24 and 36 h post-exposure with small numbers of bacteria in the circulation only at 24 h post-exposure. These results characterize and quantify disease progression in naïve rabbits following aerosol administration of Ames spores which may be useful in a number of different research applications, including developing quantitative models of infection for use in human inhalational anthrax risk assessment.

Bacillus anthracis interacts with plasmin(ogen) to evade C3b-dependent innate immunity.

The causative agent of anthrax, Bacillus anthracis, is capable of circumventing the humoral and innate immune defense of the host and modulating the blood chemistry in circulation to initiate a productive infection. It has been shown that the pathogen employs a number of strategies against immune cells using secreted pathogenic factors such as toxins. However, interference of B. anthracis with the innate immune system through specific interaction of the spore surface with host proteins such as the complement system has heretofore attracted little attention. In order to assess the mechanisms by which B. anthracis evades the defense system, we employed a proteomic analysis to identify human serum proteins interacting with B. anthracis spores, and found that plasminogen (PLG) is a major surface-bound protein. PLG efficiently bound to spores in a lysine- and exosporium-dependent manner. We identified α-enolase and elongation factor tu as PLG receptors. PLG-bound spores were capable of exhibiting anti-opsonic properties by cleaving C3b molecules in vitro and in rabbit bronchoalveolar lavage fluid, resulting in a decrease in macrophage phagocytosis. Our findings represent a step forward in understanding the mechanisms involved in the evasion of innate immunity by B. anthracis through recruitment of PLG resulting in the enhancement of anti-complement and anti-opsonization properties of the pathogen.

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

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

Oxazolone and diclofenac-induced popliteal lymph node assay reactions are attenuated in mice orally pretreated with the respective compound: potential role for the induction of regulatory mechanisms following enteric administration.

The murine popliteal lymph node assay (PLNA) was examined as a preclinical assay with the potential to identify low-molecular-weight compounds (LMWCs) that are likely to be associated with immune-mediated drug hypersensitivity reactions (IDHRs) in humans. We hypothesized that the contact sensitizer oxazolone (OX) would cause a strong PLN reaction in naive mice and that the PLN reaction would be attenuated in mice orally pretreated with OX due to the induction of oral tolerance. In naive mice, OX induced a strong PLN reaction and caused dose-dependent increases in PLN size, weight, cellularity, percentage of CD4(+) PLN T cells, and percentage of PLN B cells, with a concomitant decrease in the percentage of CD8(+) PLN T cells. Next, the PLNA was conducted in mice gavaged three times with either OX or vehicle alone (olive oil). Mice pretreated with OX had suppressed PLN reactions following the footpad injection of OX (decrease in PLN size, weight, and cellularity), which was associated with an increase in the percentage of PLN CD8(+)T cells. In contrast, oral pretreatment with OX had no observable effect on the PLN reaction induced following footpad injection of the irrelevant hapten dinitrochlorobenzene (DNCB). Adoptive transfer studies were conducted to examine the mechanism of PLN hyporesponsiveness. It was found that either (1) unfractionated splenocytes or (2) purified CD8(+) splenocytes, but not (3) purified CD4(+) splenocytes isolated from mice gavaged with OX adoptively transferred PLN suppression to naive BALB/c mice. Because OX is not a pharmaceutical, we also examined the NSAID diclofenac (DF) (Voltaren). Like OX, DF caused dose-dependent increases in PLN size, weight, and cellularity in naive mice. Furthermore, like OX, the diclofenac-induced PLN reaction was attenuated in mice that had been orally pretreated three times with DF. However, splenocytes from mice orally treated with DF were not able to adoptively transfer PLN hyporesponsiveness. Collectively, these observations demonstrate that both OX and DF are potent immunostimulators in the PLNA. As importantly, these results demonstrate that the immunostimulating potential of OX and DF in the PLNA is significantly decreased in mice orally exposed to the respective drug, possibly due to the presence of a cellular mechanism of oral tolerance. For OX, the mechanism appears to involve, in part, CD8(+) T cells, whereas the mechanism(s) associated with PLN hyporesponsiveness using DF remain to be defined.