The development and validation of methods for evaluating the immune system in preweaning piglets

Food and Chemical Toxicology 84 (2015) 197e207

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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox

The development and validation of methods for evaluating the immune system in preweaning piglets Brandon M. Zeigler a, *, 1, Mark Cameron b, 1, Keith Nelson a, 1, Kristi Bailey a, Myra L. Weiner c, Brinda Mahadevan d, Bjorn Thorsrud e a

MPI Research, Mattawan, MI, 49009, USA Beckman Coulter, Southfield, MI, 48033, USA TOXpertise, Princeton, NJ, 08540, USA d Abbott Laboratories, Columbus, OH, 43219, USA e Experimur, Chicago, IL, 60609, USA b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 June 2015 Received in revised form 26 August 2015 Accepted 28 August 2015 Available online 2 September 2015

The preweaning piglet has been found to be a valuable research model for testing ingredients used in infant formula. As part of the safety assessment, the neonates' immune system is an important component that has to be evaluated. In this study three concurrent strategies were developed to assess immune system status. The methods included (1) immunophenotying to assess circulating innate immune cell populations, (2) monitoring of circulating cytokines, particularly in response to a positive control agent, and (3) monitoring of localized gastrointestinal tissue cytokines using immunohistochemistry (IHC), particularly in response to a positive control agent. All assays were validated using white papers and regulatory guidance within a GLP environment. To validate the assays precision, accuracy and sample stability were evaluated as needed using a fit for purpose approach. In addition animals were treated with proinflammtory substances to detect a positive versus negative signal. In conclusion, these three methods were confirmed to be robust assays to evaluate the immune system and GIT-specific immune responses of preweaning piglets. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Immunotoxicology Flow cytometry ELISA Immunohistochemistry (ICH) Immunophenotyping (IP) Porcine

1. Introduction When evaluating the safety of new ingredients in infant formula it is important to select an appropriate research model that will replicate the conditions in infants during the nursing period. Some compounds in infant formula may have potential effects on the immune system; therefore testing ingredients within a formula for an inflammatory response is crucial to affirm safety (Nicklin and Miller, 1984; Tsuji et al., 2003). A comprehensive review of neonatal testing paradigms and their suitability for infant formula testing can be found in the literature (Flamm, 2013). Of the different

Abbreviations: CCE, circulating cytokine evaluation; CV, coefficient of variance; DDS, Dextran Sodium Sulfate; H&E, Hematoxylin & Eosin; IHC, Immunohistochemistry; IP, Immunophenotyping; GLP, Good Laboratory Practices; LLOQ, lower limit of quantification; LPS, lipopolysaccharide; NBF, Neutral Buffered Formalin; RT, Room Temperature; PBS, Phosphate Buffered Saline; QC, Quality Control. * Corresponding author. E-mail address: [email protected] (B.M. Zeigler). 1 These authors contributed equally to this manuscript. http://dx.doi.org/10.1016/j.fct.2015.08.027 0278-6915/© 2015 Elsevier Ltd. All rights reserved.

animal models available, the neonatal piglet is considered to be the most appropriate for this type of an assessment (Guilloteau et al., 2010; Helm et al., 2007). In addition, swine are favored for toxicology testing due to the extensive historical data sets for toxicology endpoints and their anatomic, physiological and immunological similarity to humans (Barrow, 2012; Guilloteau et al., 2010; Helm et al., 2007; NIH, 1993; Odle et al., 2014; Penninks et al., 2012). For this study, an artificially reared preweaning Yorkshire cross-bred farm piglet was chosen because it is readily available; has a relatively short nursing period (3e4 weeks) and is similar in size to a human neonate. The Yorkshire piglet is a robust and relevant research model that has been used to study pediatric nutrition and safety, as related to growth and the development (Fedorova-Dahms et al., 2014; Hanlon and Thorsrud, 2014; Mahadevan et al., 2014; Weiner et al., 2015). Passive immunity is imparted by the sow through the colostrum during the first few hours of life and is necessary for the normal growth and development of the suckling pig and for its role in the maturation of the gastrointestinal tract (Dividich et al., 2005; Xu et al., 2002).

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Using the piglet animal model, the development, assessment and validation of three different assays to monitor immune system function are described for use with infant formula ingredients. This work includes assessment of the innate immunologic subpopulations via immunophenotyping, and evaluation of systemic and local gastrointestinal competency after a pro-inflammatory challenge. The assays were chosen because they are recommended for use by current regulatory guidance for immunotoxic responses in adult laboratory animals (ICH, 1997). The FDA recommends that Good Laboratory Practice guidelines (GLP) be followed for all data to be submitted to the Agency (FDA, 2014). GLPs provides guidance regarding the organization, process and conditions under which laboratory studies are planned, performed, recorded, monitored and reported. Data collected following GLP guidelines are intended to promote the quality and validity of the test data. The GLPs mandate that all assays must be validated: proven reproducible and accurate for the specific purpose for which they will be generating data. Published GLP regulations do not specifically state validation implementation details, however, regulatory agencies, such as the FDA, have published guidance for analytical assay development and validation (FDA, 2015). There is still ongoing discussion and varying practices in the design of preclinical biomarker validations (Green et al., 2011; O'Hara et al., 2011; Owens et al., 2000). This ambiguity is due to a combination of challenges, such as heterogeneous cell populations, lack of reference material for accurate evaluation, and instrumentation complexity (Cunliffe et al., 2009; Green et al., 2011; Owens et al., 2000). Furthermore, due to the diverse nature of cytokine analysis via ELISA and Immunohistochemistry (IHC), neither FDA bioanalytical drug guidance, nor Clinical and Laboratory Science Institute (CLSI) guidance fully define a validation strategy ((CLSI), 2011). Regulatory white papers suggest constructing a validation plan based upon assay type and the endpoints of the assay (NCCLS, 1997). A “fit-for-purpose” approach has been recommended for biomarker validation due to the complexity of assay design and the necessity for biomarker assay customization (Lee et al., 2006). In a fit for purpose model, the validation plan should meet the predefined needs of the study, reflect the nature of the assay methodology, and take into consideration biomarker variability within and between study populations. In the assays validated here each parameter had a different validation plans and acceptance criteria based upon the objects of each assay. ELISA assays are considered in the relative quantitative category of methods by the biomarker community, in other words, a calibration standard can be utilized to estimate absolute quantitative concentration for an unknown analyte sample (Lee et al., 2006). Since reference standard (calibrators) can be spiked into the sample or matrix, a calibration curve is established and utilized for accuracy estimation. Thus relative quantitative assays can be assessed for precision and accuracy values. In fit for purpose analytical assay, accuracy describe the closeness of the mean interpolated (actual) test results to the theoretical (expected) value of the sample (Lee et al., 2006). For the ELISA validations performed on this study inter- and intra-accuracy and precision were assessed. The matrix was spiked with a known concentration of the cytokines and these values observed in the assay were compared with expected value in terms of relative error (RE) between the values. Accuracy within ±20% RE of nominal concentration was used as a component of the acceptance criteria. Precision in the case of this assay describes the closeness or variance of individual measurements performed in triplicate on the same sample with the same conditions (Davis et al., 2013). Precision is expressed as coefficient of variance (% CV) which is the standard deviation of the replicates divided by the mean of the replicates. The establishment of CVs parameters for the

acceptance criteria in ELISA-based assays was determined by a number of factors, such as how the quality of reagents is utilized in the assay and method of signal detection (Lee et al., 2006). Given the dynamic nature of cytokines in neonatal development, these analytes were designated to have a CV less than 30% in order to meet the validation acceptance criteria. In order to confirm the assay's ability to detect a positive inflammatory signal, a portion of the animals were LPS treated. LPS is routinely used as an inducer systemic inflammation for animal models making it an appropriate proinflammatory agent for testing assay development (Carroll et al., 2005). Immunophenotyping (IP) assays generally fall into the quasiquantitative assay category (Lee et al., 2006). This category is defined by the assay not having reference standards to establish a quantitative reference curve, as in the ELISA assay. In these types of assays, there is a numerical effect of the response and the measurement, however without a biological reference standard, the accuracy cannot be assessed. This study validated the IP panel by the assessment of numerous precision variables, in addition to whole and fixed cell stability assessment. Data sets for each precision parameter must have a CV less than 30% in order to meet acceptance criteria. Precision values describes the closeness or variance of individual measurements performed in triplicate on the same sample in identical conditions (Davis et al., 2013). The establishment of CV parameters for acceptance criteria in cytometry-based assays are determined by a number of variables such as the rarity of a cell population, the quality of reagents utilized in the assay, fluorochrome conjugate and the instrumentation used (Wood et al., 2013). Given the dynamic nature of these cell populations in neonatal development, these subpopulations were designated to have a CV less than 30% in order to meet the validation acceptance criteria. Immunohistochemistry (IHC) assays commonly fall into the qualitative assay category (Lee et al., 2006). Qualitative assays do not generate results with direct numerical values. Assessment of histological and immunohistochemical targeted tissues with assessment by a board certified pathologist is an accepted method for evaluation of overt tissue changes in toxicology studies (Haschek and Rousseaux, 2013). Histological observation of microscopic cellular changes is a standard endpoint for toxicology and safety studies and is well characterized and cited within the literature (Crissman et al., 2004). The four step grading system used to define lesions in this study consisted of minimal, mild, moderate and severe gradations of severity. The severity gradings were determined based on the amount of tissue affected, the degree of tissue perturbation, and the pathologist's knowledge of potential systemic deleterious effects on the animal (Gibson-Corley et al., 2013). For the IHC staining, the acceptance criterion was a positive signal confirmation versus negative control assessed by a clinical pathologist. In order to confirm the assay's ability to detect a positive inflammatory signal, a portion of the animals were DSS treated. DSS is routinely used as an inducer of intestinal inflammation for animal models of inflammatory bowel disease and other enteric inflammatory disease, making it an appropriate proinflammatory agent for testing assay efficacy (Bassaganya-Riera and Hontecillas, 2006; Kim et al., 2012). Multiple methods of evaluation focusing on a particular assessment are often necessary to reduce the overall uncertainty of scientific findings. By utilizing a weight of evidence approach using multiple assays the authors of this manuscript confirmed assay robustness and created a panel of tests which monitored immunotoxic effects of a potential proinflammatory compound with greater certainty (Weed, 2005). For instance, the physiological assessment of a positive control sample showed gastrointestinal damage in parallel with increased cytokine up regulation, thus

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suggesting assay efficacy. The subsequent validation of all the methods strengthened their predictive ability to detect immune response further. Based upon the results of the validation the assays were deemed fit for use to detect positive inflammation based on responses to substance exposure. 2. Materials and methods

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ten minutes per animal ensuring their safety. Necropsy on group 4 and 5 animals was performed on study day 29. For the circulating cytokine evaluation (CCE) assay animals in groups 2 and 3 were intramuscular injected with PBS or LPS on day 14 and day 29 (Table 1). The dosage of LPS used has been previously observed to illicit a cytokine immune response (Carroll et al., 2005). 2.2. Matrix collection and storage for validations

2.1. Animals Domestic Yorkshire Crossbred Swine (farm pigs) were received on Lactation Day (LD) 2 from Bailey Terra Nova Farms, Schoolcraft, Michigan. The day on which all piglets of a litter were delivered by the sow was designated as LD 0. The piglets nursed from the sow at the supplier for 48 h to allow for colostrum ingestion, which is necessary for the proper growth and development of the animal. The piglets were also injected with an iron supplement and a broad spectrum antibiotic prophylactically to insure good health (Farmer and Quesnel, 2009; Guilloteau et al., 2010). MPI Research is registered with the USDA and has an approved Animal Welfare Assurance from the National Institute of Health office. In addition, all components of MPI Research are accredited by Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Percutaneous endoscopic gastrostomy (PEG) tube surgery was performed on Group 4 and 5 animals (Table 1). All animals were anesthetized according to standard operating procedure. During the procedure animals were intubated and ventilated, as needed. Animals were implanted with a 20 French Bard PEG tube kit and a flexible scope was threaded through the esophagus into the stomach for internal visualization of the procedure. A small incision was made in the skin and a catheter was inserted percutaneously into the stomach. A looped wire was threaded through the catheter, grasped with endoscopic graspers and exteriorized through the oral cavity. The looped wire was wound around the PEG tube and pulled out of the abdomen, exteriorizing the PEG tube. Medical tape and an external bumper were placed to further secure the device, and a clamp was placed on the distal end of the PEG tube. Triple antibiotic ointment was placed at the exteriorization site, the site was bandaged, and the animals were jacketed for the remainder of the study. The stomach and implant site were grossly examined by trained veterinary staff. Dextran sodium sulfate (DSS) dosing was conducted under surgeon supervision to ensure tube patency and dosed at levels previously observed to elicit an immune response (BassaganyaRiera and Hontecillas, 2006; Kim et al., 2010). Group 4 and 5 animals were dosed through the PEG tube with approximately 80 mL of PBS or test material using a 60 cc syringe. DSS was administered to all group 5 animals twice a day on study days 26, 27 and 28 at a dose of 1250 mg/kg, as a positive control. The PEG tube was closed with an injection cap, the jacket was secured, and animals were returned to their home cage. This procedure took approximately

The animals were placed into five groups for the three different immunologic assessments (Table 1). Group 1 animals were used for immunophenotyping (IP), Groups 2 and 3 animals were used for the circulating cytokine evaluation (CCE), and Group 4 and 5 animals were used for the IHC evaluation. For IP assays venous blood was collected from Group 1 piglets in Vacutainer Tubes (BD Bioscience) containing the anti-coagulant, sodium heparin. Samples were maintained at room temperature (RT), 18e22
C, until processing occurred. For CCE assay blood was drawn and processed to serum and plasma from groups 2 and 3 animals 24 h after LPS injections. For serum collection whole blood was collected into tubes without anticoagulant and maintained at room temperature for 60 min until clot formed, after centrifugation serum phase was removed from the tube. For plasma whole blood was collected in EDTA tubes and centrifugation occurred within 1 h of collection, plasma phase was then pulled from the tube. The serum and plasma were frozen and stored at 70
C. These samples served as positive controls for use in pre-validation studies and for dilution linearity assessments. For IHC analysis, upon dissection tissue samples were placed in 10% Neutral Buffered Formalin (NBF) fixative for 48e72 h, and then held in 70% ethanol prior to tissue processing for up to 48 h prior to paraffin block stage. For histology assessment at terminal necropsy (Study Day 29), histopathology evaluations were conducted on gastrointestinal tract tissues, including stomach, duodenum, jejunum, ileum, cecum, proximal colon, distal colon, and rectum. Tissues were fixed with 10% NBF for Hematoxylin & Eosin (H&E) stain. 2.3. Immunohistochemistry (IHC) assay development and evaluation of validation samples Mouse monoclonal anti-Interleukin-8 antibody (Abcam), Polyclonal rabbit anti-interleukin-6 antibody (Abcam), Polyclonal rabbit and anti-Tumor Necrosis Factor Alpha antibody (Abcam) were optimized for the detection of porcine cells (Table 2). Staining parameters such as pretreatment, dilution, and incubation time were varied to achieve optimal conditions, resulting in the final method. Staining protocols were run on the automated staining platform Ventana Discovery XT (Roche). For the validation, staining was performed on the negative control and DSS-dosed study animal gastrointestinal tissues over a series of dilutions to visualize antigen presence. IHC staining for all markers was performed on sections of proximal and distal colon for animals from each group at varying

Table 1 Animal study design. Group

Description

Piglets

Dose level mg/kg/dose

Dose volume mL/kg/dose

Dose conc. mg/mL

1 2 3 4 5

Control PBS Controla LPS Challengeb PBS Controlc DSS Challenged

10 3 3 3 3

NA 0 0.05 0 1250

NA 0.25 0.25 31.25 31.25

NA 0 0.20 0 40

a b c d

PBS e Phosphate buffered saline (negative control) intramuscular (IM) injection on Study Days 14 and 29. LPS e Lipopolysaccharide (positive control) IM injection on Study Days 14 and 29. PBS control solution (negative control) provided through percutaneous endoscopic gastrostomy (PEG) tube, twice a day from Study Days 26e27. DSS e Dextran sodium sulfate solution (positive control) provided through a PEG tube, twice a day from Study Day 26e27.

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Table 2 Antibodies used in porcine immunohistochemistry assays. Phenotype

Specificity

Host

Dilutions

Clone

Vendor

IL6 IL8 TNFa Recombinant Porcine TNF-alpha

Cytokine Cytokine Cytokine Protein

Rabbit Mouse Rabbit E. coli derived

1:400, 1:800, and 1:1200 1:100, 1:200, 1:400, 1:800 1:160, 1:200, 1:400, 1:800, 1:1600 25 ug/mL

Polyclonal 807 Polyclonal NA

Abcam Abcam Abcam R&D Systems

In the dilution column bold and underline text indicate the concentration of reagent used for tissue optimal staining.

Microscopic examination of fixed H&E-stained paraffin sections was performed on three animals from each treatment group, with slides examined by a board-certified veterinary pathologist. A fourstep grading system, or Score Method, was utilized to define gradable lesions for comparison between dose groups (Holland, 2001). IHC samples were also examined by a board-certified veterinary pathologist. Staining intensity level was semi-quantitated on a scale of increase, marked increase, and no increase compared to controls, using visual light microscopic assessment of individual cell staining within multiple microscopic fields compared to appropriate controls for each tissue examined.

sample in a 96 round bottom deep well plate (BD Falcon). The mixture was incubated for 30 min and the red blood cells lysed using 500 mL VersaLyse (Beckman Coulter) for 15 min. Next, 10 mL of IO Test Fixative (Beckman Coulter) was added to the solution and incubated for 15 min. All incubations were performed at room temperature and protected from light. After fixation, the samples were stored at 2e8
C and protected from light until analysis. At least 5.0  103 gated lymphocyte events were collected for each sample. Raw data were saved into an FCS file and analyzed using FACSDiva software to obtain relative percentage data of the subpopulations. A Becton Dickinson LSRFortessa (LSR) was optimized to measure eight parameters with three lasers; red, blue and violet. Cytometer System & Tracking reports were used daily to adjust system application settings for optimal positive fluorescent detection and laser delay. For compensation eBioscience Ultra Comp Beads capture beads were incubated with cross-reactive single antibodies (Table 3) used within the panel. Data were collected for each single stain fluorochrome-bead conjugate and FACSDiva software automatically calculated the compensation matrix for the panel. Samples were run on a 96 well high throughput system linked with the LSR. For IP, lymphocyte and monocyte absolute cell counts were directly analyzed using the Advia 120 system (Seimens), according to the manufacturer's instructions. Absolute cell count statistics (cell number/ml) of B cell, monocyte, NK cell and T cell subset populations were calculated based upon the Advia lymphocyte absolute cell count and LSR percent gated data. All post-fixation stability data obtained from the LSR used the 0e4 h post-collection whole blood sample leukocyte count to ascertain absolute cell counts of the corresponding cell subpopulations. Thirty hour post-collection Advia lymphocyte absolute cell count readings were used to extrapolate cellular subsets' absolute cell count.

2.5. IP assay development and evaluation of validation samples

2.6. Method development and validation for cytokine ELISA assay

Table 3 summarizes the antibodies used in the porcine IP evaluation. A single, six-color panel was developed to evaluate leukocyte populations (CD45þ), B cells (CD45þCD21þ), mature T cells (CD45þCD3þ), CD8þ T cells (CD45þCD3þCD8þ), CD4þ T cells (CD45þCD3þCD4þ), NK cells (CD45þCD8þCD3CD4), and monocytes (CD45þCD14þ) (Yang and Parkhouse, 1996). After optimal antibody concentrations were established via titration to obtain optimal signal to background levels (Table 3), the antibodies were pooled together then added to 50 mL of each porcine whole blood

Quantikine ELISA kits for IL-1b, IL-6, IL-8 and TNF-a (catalog numbers PBL00B, P6000B, P8000 and PTA00 respectively R&D systems) were validated in a fit-for-purpose (Lee et al., 2006) approach in a GLP compliant manner for inter- and intra-accuracy and precision (using recombinant calibrator spiked into porcine serum or porcine plasma), dilution linearity, and stability (short term, freeze and thaw, bench top, and long term). Porcine serum was used for the validation of the IL-6 and IL-8 ELISA kits, and plasma was used for the validation of the IL-1b and TNF-a kits.

dilutions. Antigen positive controls for TNF-a were created by injection of 100 mL of 25 mg/mL recombinant swine TNF-a (rTNF-a) (R&D Systems) in Histogel (Thermo Fisher) into fixed naïve colon tissue. This method was utilized to provide a semi-solid, sectionable suspension of a known positive antigen that would be retained within the colonic tissue (Joiner and Spangler, 2012). This allowed a clear assessment of both the background tissue staining and the stabilized cytokine positive control. Antigen positive controls for IL-6 and IL-8 were unavailable. Slides stained as reagent-negative controls were subjected to the same staining conditions with the absence of the primary antibody. Detection and counterstaining for all staining methods was performed using a ChromoMap DAB kit (Ventana). Counterstain reagents used were Hematoxylin (Ventana) and Bluing Reagent (Ventana). Finally, the slides were dehydrated, cleared, and cover-slipped with synthetic mounting medium. 2.4. Histopathology evaluation of validation samples

Table 3 Antibodies used in porcine immunophenotyping assay. Phenotype

Specificity

Host

Fluorochrome

Dilution

Clone

Vendor

CD4a CD3e CD8a CD14 CD21 CD45

Helper T cell Pan T cell Cytotoxic T cell Monocyte Pan B cell Pan Leukocyte

Mouse Mouse Mouse Mouse Mouse Mouse

PerCP-Cy5.5 FITC PE Pacific Blue PE-Cy7 Alexa Fluor 647

1:40 1:40 1:40 1:10 1:20 1:10

74-12-4 BB23-8E6-8C8 MIL-12 TUK4 B-ly4 K252-1E4

BD Bioscience BD Bioscience Serotec BD Bioscience BD Bioscience Serotec

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Serum and plasma matrix (Bioreclamation) was obtained from piglets 6e10 weeks of age. The quality control samples (QC) were made by spiking calibrator into serum or plasma. The QC samples were frozen and maintained at 70
C degrees until evaluated. Data was analyzed by SOFTmax Pro GxP V5.3 software. 2.7. Statistical analysis For each cellular subpopulation within the IP data set two measurements were analyzed; absolute cell count and percentage gated. Absolute cell counts refers to the cell number/mL of whole blood. Percent gated is a cytometry statistic indicating the frequency of the number of cells within the gated subpopulation compared with all the cells within the dot plot (Herzenberg et al., 2006). In order to determine the amount of variability present and the contributions of various design factors to variability, the data from the LSR instrument were used to estimate the variance components for between animal, within animal, between period and between instruments. The MIXED procedure in SAS software was used in estimating these components (Little et al., 2006). In addition, coefficients of variations (CV) were computed for the standard deviation with regards to the average and comparison of variables. The within animal (CVwithin) and between age of piglet Day 13, 14, 28 and 29 (CVperiod) components of the assay variation were estimated using standard statistical techniques (Snedecor and Cochran, 1980). For ELISA based assays, measurements of precision and accuracy were calculated. Precision, the closeness of individual measures of an analyst, was calculated as a CV. The formula for CV was CV ¼ (Standard deviation of replicates/Mean of replicates)  100. Accuracy was calculated as a product of relative error. Relative error was defined as ((Interpolated Result  Nominal concentration)/ Nominal concentration)  100. These values were calculated by Microsoft excel spreadsheet. 3. Results 3.1. Histopathology observations DSS-dosed animals had microscopic evidence of inflammation, edema, erosion and ulceration of the proximal and distal colon (Fig. 1). The consistent findings (seen to some degree in all animals) consisted of minimal to moderate acute inflammation, minimal chronic inflammation, minimal to mild edema, minimal to mild erosion/ulceration and minimal to mild dilation of the colonic crypts. These colonic findings were not seen in the other two groups, negative control animals (Fig. 1A). Acute inflammation was characterized by infiltrates of numbers of neutrophils expanding and occasionally effacing the lamina propria and submucosa of the colon, as well as migrating into the crypts and forming crypt abscesses with accompanying cellular debris (Fig. 1B). Chronic inflammation consisted of small numbers of lymphocytes, plasma cells, and histocytes and was generally seen accompanying acute inflammatory infiltrates. Edema was associated with inflammatory infiltrates and formed part of the inflammatory response in the colon, expanding the submucosa and, to a lesser extent, the lamina propria. The mucosa was multifocally eroded to ulcerated, with varying degrees of loss of mucosal epithelial cells, including extension into the lamina propria, with accompanying inflammation. Colonic crypts were generally prominent and dilated, with increased cellular debris and neutrophilic infiltrates within a subset of affected crypts. Those groups with implanted PEG tubes had findings of minimal peritoneal inflammation of the stomach (cardia or pylorus), which was considered to be associated with the implantation and

Fig. 1. Hematoxylin and eosin stained photomicrographs of colon from animals at study termination (100). A) Image of colon from PBS-dosed negative control animal. B) Image of colon from DSS-treated positive control animal. Representative areas of ulceration are marked by black arrows, with edema marked with an asterisk (*) and submucosal and mucosal inflammation marked by arrowheads. Chronic and acute inflammation is intermingled in this image and cannot be readily separated.

presence of the PEG tube in these groups and was not considered to be related to the DSS-treatment, as it was seen equally in PBStreated and DSS-treated groups. One DSS-dosed animal had minimal acute inflammation in the lamina propria of the duodenum. This was not seen in any other male or female animals in this treatment group or in other sections of small intestine and is not considered to be clearly related to DSS-induced toxicity. All other microscopic findings were typical of those seen in swine of this strain, sex, and age or were considered incidental. 3.2. Immunohistochemistry observations Figs. 2e4 show sections of the proximal colon of PBS-dosed and DSS-dosed animals stained for IL-6, IL-8 and TNF-a, respectively. There was increased positive staining for IL-8 and TNF-a in the proximal colon of DSS-treated animals, generally localized to the areas of increased inflammation and edema. There were no effects of DSS on the immunohistochemical staining of any gastrointestinal tract tissues for IL-6 (Fig. 2). Microscopic findings in the colon on H&E-stained tissue were broadly co-localized with increased IHC staining for IL-8 and TNF-a in the DSS-dosed animals (Figs. 3 and 4, respectively). Both IL-8 and TNF-a staining was generally increased in areas of inflammation, edema, and in gut-associated lymphoid tissue (GALT). Erosion and ulceration did not seem to be associated with increased cytokine production. There was some increase in mucosal epithelial cytokine staining, but this was not as prominent as the staining seen in the submucosal layers, associated with the

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Fig. 2. IL-6 immunohistochemical staining of colon from animals at study termination (100). A) Photomicrograph of colon from PBS-dosed negative control animal stained at 1:800 dilution with IL-6 antibody. B) Photomicrograph of colon from DSS-dosed positive control animal stained at 1:800 dilution with IL-6 antibody.

edema and inflammation found there. A marked increase in the level of IL-8 positive staining compared to controls was seen at 1:400 and 1:800 dilutions in DSS-dosed animals (Fig. 3). An increased level of TNF-a positive staining compared to controls was seen at a 1:800 dilution in DSS-dosed animals (Fig. 4B). The increased level of TNF-a staining in DSS-dosed animals was similar to the diffusely increased staining found in the rTNF-a injected positive controls that did not have DSS (Fig. 4C). IL-6 staining was assessed at 1:400, 1:800, and 1:1200 dilutions, but no clear differences were seen in staining between the negative controls (both PEG þ PBS and formula-fed controls with no PEG tube implanted) and the positive DSS-treated group. 3.3. Immunophenotyping panel strategy and development Panel development was based on commercially available reagents directed specifically against porcine proteins and background literature (Gerner et al., 2009; Haverson et al., 2001; Hernandez et al., 2001). When planning the panel, precaution was taken to select the brightest fluorochromes for the most infrequent cellular subsets (Mahnke and Roederer, 2007). In this case, antibodies directed against B cells were conjugated to PE-Cy7. Each antibody was titrated to evaluate best positive fluorescent signal relative to concentration (Cunliffe et al., 2009; Hulspas et al., 2009; Maecker and Trotter, 2006). Fluorescence minus one control strategy was utilized to ascertain negative threshold levels for each reagent (Maecker and Trotter, 2006). The final six-color flow cytometry panel is shown in Fig. 5. In order to identify leukocyte population and exclude debris, we initially gated on SSC and CD45þ (Fig. 5A). From this leukocyte gate, monocytes were selected from

Fig. 3. IL-8 immunohistochemical staining of colon from animals at study termination (100). A) Photomicrograph of colon from PBS-dosed negative control animal stained at 1:800 dilution with IL-8 antibody. B) Photomicrograph of colon from DSS-dosed positive control animal stained at 1:800 dilution with IL-8 antibody.

CD14þCD45þ events (median: 7.0% of leukocytes; SD: 2.2% N ¼ 6). Also from the leukocyte gate, lymphocytes were positively identified based on their forward and side scatter characteristics (median: 28.9% of leukocyte; SD: 4.8%, N ¼ 20) (Fig. 5B). The lymphocyte populations were divided into B cells CD20þCD45þ (median: 14.6% of lymphocytes SD: 5.8% N ¼ 6), NK Cells CD45þCD8þCD3CD4 (median: 8.5% of lymphocytes SD: 7.5% N ¼ 6) and mature T cells CD3þCD45þ (median: 65.3% of lymphocytes SD: 8.1% N ¼ 6) (Fig. 5D and F). CD3þCD45þ mature T cells were further separated into Helper T cells CD4þCD3þCD45þ (median: 33.8% of lymphocytes SD: 9.6% N ¼ 6) and Cytotoxic T cells CD8þCD3þCD45þ (median: 12.4% of lymphocytes SD: 2.3% N ¼ 6) (Fig. 5E). There was clear separation of negative and positive populations within each plot, so appropriate gating of each population was accurate. Backgating the subsets to a SSC/FSC plot confirmed that each cellular subset's size and granularity profile was similar to past literature. The overall summary of the gated percentage data generated over the course of the validation is shown in Table 4.

3.4. Immunophenotyping panel validation criteria Using the one panel method, staining of recently collected piglet whole blood was compared with staining of whole blood stored at RT for 24 h. The percentages and absolute cell counts of all cellular

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24 h can be processed and still generate statistically equivalent data to samples processed immediately after blood collection. Samples analyzed immediately after fixation were compared with fixed cells stored 24 and 48 h at 2e8
C. The gated percentages and absolute cell counts of all cellular subsets at each post-fixation time intervals were under 20% CVperiod (Table 4). The monocyte cell population showed the greatest variance (CVperiod of 19.1%), indicating that these cells may be more fragile under the storage conditions outlined in the method. The remainder of the endpoints demonstrated a period coefficient of variation CVperiod of less than 8% indicating a highly precise assay. There was no statistical significance among the different intervals according to the CVperiod statistic. Therefore, fixed, stained cells can be stored in the dark at 2e8
C up to 48 h prior to analysis and still be statistically equivalent to samples immediately analyzed after sample fixation. Whole blood samples were stained, fixed, and run in triplicate on the LSR to asses for intra-sample variance. For all samples, the within animal coefficient of variation (CVw values in Table 4) for all cellular subsets was less than 6% for both cell count and gated subpopulation percentage, indicating a high degree of reproducibility within the replicates. This data also denote that study samples only have to be run in singlet to obtain reliable data. Sample percentages and absolute cell counts were compared between Study Day 13, 14, 28 and 29 to determine if piglet developmental time would be a source of variance (Table 4). The percentages of lymphocytes, B cells, mature T cells, cytotoxic T cells, helper T cells, and monocytes across these time intervals all had cellular percentage and absolute cell count values demonstrating a CVp of below 30% indicating low variance over time. The NK cell did not meet acceptance criteria as all data displayed CVp values that were above 30%. To further ascertain if developmental time of whole blood collection was a source of variance, the data from Study Day 13 and 14 as well as Study Day 28 and 29 were separately compared statistically for the same patterns of variance. The CVp values for the cell populations, with the exception of NK cells, are all below a CVp of 30% indicating the piglet developmental time was not a significant source of variance. Since a large CV was observed it is recommended to run a control group in parallel to monitor the lymphocyte fluctuations due to piglet age. To monitor inter-analyst precision, two analysts ran samples on separate days of the study and the variance was compared between Study Day 28 and Study Day 29 (Table 4). For all cellular subsets, except the NK cell subset, the analyst variance data displayed a CVp of less than 13% for the cellular percentage data and less than 24% for absolute cell counts indicating that there is a low degree of variance between different laboratory staff conducting the assay (Table 4). This data is within the acceptance criteria of less than 30% CVp per parameter. This demonstrates that the assay is robust enough to be performed by different technicians within the laboratory. Fig. 4. TNF-a immunohistochemical staining of colon from animals at study termination (100). A) Photomicrograph of colon from PBS-dosed negative control animal stained at 1:800 dilution with TNF-a antibody. B) Photomicrograph of colon from DSSdosed positive control animal stained at 1:800 dilution with TNF-a antibody. C) Photomicrograph of colon from control animal injected with recombinant TNF-a and stained at 1:800 dilution with TNF-a antibody.

subsets period coefficient of variation was CVperiod less than 23% (Table 4). The monocyte cell population showed the greatest variance (CVperiod of 22.9%), indicating that these cells may be more fragile under the storage conditions outlined in the method. The remainder of the endpoints demonstrated a period coefficient of variation CVperiod of less than 8% indicating a highly precise assay. Therefore, blood samples stored protected from light at RT up to

3.5. CCE assay development and validation For the method validation IL-1 beta, IL-6, IL-8, and TNF-a kits from R&D systems were evaluated. The working range of the assay was first established by evaluating the performance of the calibrators standards prepared in diluent buffer. In six independent runs by a minimum of two analysts, seven non-zero calibrator levels in assay diluent were used to span the working range of the assay. A standard curve was considered acceptable if the mean of the replicates of each standard (calibrator) level had a precision of 20% CV and accuracy within ±20% RE compared with nominal values. Performance characteristics of the calibrator's standards demonstrated acceptable accuracy and precision for the four cytokine assays (Table 5). Recovery was lower within the IL-6 assay,

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Fig. 5. Six color flow cytometry porcine panel. Whole blood was analyzed from recently processed and fixed (within 2 h) samples. First, leukocytes were gated via an anti CD45þ marker and side scatter profile (A). From the Leukocyte gate, Lymphocytes were evaluated by forward and side scatter characteristics (B) and monocytes were positively identified based on CD45þCD14þ expression (C). From the Lymphocyte gate, B cells and mature T cells were separated via CD21þ and CD3þ, respectively (D). From the mature T cell population were separated with anti-porcine CD8þ T cells and anti-porcine CD4þ T cells (E). From the lymphocyte gate, CD4þ cell populations were excluded and then NKs were isolated from CD3CD8þ (F).

but the reduced recovery was consistent between two analysts running separate assays. The working ranges for the assays based on calibrators were: 39.1e2500 pg/mL, 18.8e1200 pg/mL, 62.5e4000 pg/mL, and 23.4e1500 pg/mL for the IL-1 beta, IL-6, IL8, and TNF-a, respectively. The lower limit of quantification (LLOQ) of analyte concentration for each kit was considered the assay's lower limits. LLOQ was determined by the lowest concentration of analyte which had intra- and inter-assay precision of 25% CV and accuracy of ±25% RE. The sensitivities for the assays were 39.1 pg/ mL, 19.2 pg/mL, 193 pg/mL, and 151 pg/mL for the IL-1 beta, IL-6, IL8, and TNF-a, respectively. The intra-assay performance of the assay was evaluated in six independent runs with two replicates at each level of the standard curve. Also spiked samples were prepared in a matrix at three QC concentration levels (High, Mid, Low) and stored at 70
C. Inter-assay precision was considered acceptable, if across all runs, the mean precision was <20% CV and accuracy within ±20% RE for each standard level and each spiked sample compared to a nominal value. The intra assay precision for all kits tested using this criteria were deemed fit for use on study. Dilution linearity was performed with all kits to evaluate the linearity of quantitation upon sample dilution. Two-fold serial dilutions were performed out to a dilution factor of 1:16 using the calibrator diluent (Table 5). The dilution schemes were designed so that the resulting signal would fall within the quantitation range of the assay. Specific analyte concentrations were reported as the mean of the duplicate determinations at each dilution of sample tested. Linearity was assessed by plotting the observed analyte concentrations versus the dilution factor and accuracy to the 100% value of the initial neat

matrix spike. The results showed that all kits had analyte recovery ±25% RE after a 1:16 dilution. Stability testing was performed on the recombinant porcine cytokines under the following conditions: at ambient temperatures for 5 h, three freeze/thaw cycles, and long term frozen storage (70
C). Long term freeze stability was evaluated at 1 week, 3 weeks, and 13weeks using calibrator spiked into matrix. The stability results indicated that there was no significant degradation of the protein after freeze/thaw cycles or long term freezer storage for each cytokine evaluated (Table 5). Cross reactivity was examined and defined as the ability of the assays to detect and give false positive readings to other cytokines used in the study. For instance in the IL-6 kit, porcine TNF-a; porcine IL-1b, porcine TNF-a and porcine IL-8 were monitored. A single concentration of each protein that was within the quantification working range of each assay was evaluated in duplicate. The cross reactivity evaluation determined that each kit did not cross react with the three other cytokines of interest. 3.6. CCE functional assessment After each assay had been validated an in vivo functional assay was performed to demonstrate that the kits could detect an upregulation of each of the cytokines (Carroll et al., 2005). Table 5 summarizes the CCE. Animals aged 14 and 29 days were dosed with 0.05 mg/kg of LPS and after 4 h whole blood was collected and serum processed. Animals dosed with LPS (positive control) showed significant increases in the concentrations of the each circulating cytokine evaluated, as compared to controls (Table 5).

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205

Table 4 Immunophenotyping validation of each cell parameter. NK Cell, Cytotoxic T cell, Helper T cell, Mature T Cell and B cell are based upon percentage of lymphocytes, Monocytes are percentage of Leukocytes. Panel Testing CVw within animal coefficient of variation, CVp period coefficient of variation. NA, Not applicable. N ¼ 6. Parameter

Interval comparison

B Cells

Percent of gated

Intra assay precision 24 h post collection precision 24 h post fixation precision 48 h post fixation precision Inter assay precision Inter analyst precision Inter age precision (2v4week) Intra assay precision 24 h post collection precision 24 h post fixation precision 48 h post fixation precision Inter assay precision Inter analyst precision Inter age precision (2v4week) Intra assay precision 24 h post collection precision 24 h post fixation precision 48 h post fixation precision Inter assay precision Inter analyst precision Inter age precision (2v4week) Intra assay precision 24 h post collection precision 24 h post fixation precision 48 h post fixation precision Inter assay precision Inter analyst precision Inter age precision (2v4week) Intra assay precision 24 h post collection precision 24 h post fixation precision 48 h post fixation precision Inter assay precision Inter analyst precision Inter age precision (2v4week) Intra assay precision 24 h post collection precision 24 h post fixation precision 48 h post fixation precision Inter assay precision Inter analyst precision Inter age precision (2v4week)

Monocytes

NK Cells

Cytotoxic T Cells

Helper T Cells

Mature T Cells

Absolute cell count

Mean

CVw %

CVp %

Mean

CVp %

14.56 16.34 17.8 17.76 15.83 18.25 17.04 7.01 9.34 8.16 8.56 7.43 9.25 8.34 8.53 13.56 13.56 13.21 10.95 6.43 8.69 11.58 12.43 12.05 12.02 11.99 12.36 12.18 33.76 29.74 27.93 27.99 31.28 23.46 27.37 65.28 63.02 61.35 61.75 63.63 62.71 63.17

4.62 1.37 1.49 1.43 3.16 1.57 5.25 3.51 2.68 1.64 1.64 2.73 1.91 4.91 2.97 1.18 1.53 1.24 1.98 3.5 33.4 2.66 1.55 2.71 1.45 2.15 2.31 3.1 3.81 0.83 1.86 1.37 2.96 1.79 6.02 0.81 0.54 0.64 0.62 0.64 1.16 3.07

NA 7.97 6.52 7.45 19.01 12.59 14.07 NA 22.93 14.09 19.09 20.2 9.32 19.98 NA 4.26 2.11 3.07 80.28 133.04 36.38 NA 0.8 5.53 5.96 7.53 9.21 5.95 NA 5.36 6.25 7.64 20.89 10.85 22.21 NA 2.41 3.09 3.85 10.06 8.86 4.95

742.17 750 817.42 814.75 763.42 878.17 820.79 609.17 876.25 769.83 804.5 671.17 831.5 751.33 490.83 659.25 660.17 642.75 571 417.83 494.42 579.5 567 550.33 548.58 572 631.58 601.79 1623.17 1357.5 1271.92 1275.83 1468.67 1157.75 1313.21 3160.17 2864.42 2783.92 2805.83 2988.58 3132.5 3060.54

NA 7.84 6.87 8.26 24.68 20.99 21.9 NA 24.46 14.35 20.69 29.09 19.69 28.85 NA 3.84 2.42 3.84 80.09 180.51 107.34 NA 1.93 5.4 6.22 19.32 23.18 19.77 NA 5.46 6.73 8.58 20.27 9.89 23.03 NA 2.48 3.49 4.42 14.6 12.38 15.29

Two kits IL-1b and IL-6 could detect the positive LPS signal, but the control levels of the analyte were below the level of assay detection. No statistical difference in the cytokine levels of the LPS treated day 14 piglets versus day 29 was observed (data not shown). 4. Discussion Validating assays is essential for the collection of quality data acceptable for review by regulatory agencies. For instance, the ability to store collected and processed samples gives the lab a

contingency plan for unexpected scenarios, like short-term instrument malfunction, without affecting data integrity. In addition, demonstrating stability for an extended time frame provides the laboratory with the ability to evaluate blood samples shipped overnight for cross site analyses. Sample preparation and storage conditions are critical to performing GLP compliant laboratory experiments. Regulatory blood or plasma volume level restrictions may limit sample volume, making extended stability windows essential for potential reanalysis without resorting to additional blood collections that could be deleterious to the health of the test

Table 5 Cytokine assay validation summary. Cytokine Range of calibrators (pg/mL) IL-1b IL-6 IL-8 TNF-a a b c

LPS treated Assay Lower limit of Dilutional 70
C stability Bench top Assay Freeze/thaw Assay linearity detection 15 weeks %RE stability %RE 3 cycles %RE variance variance variance animalsa (pg/mL) at 1:16%RE (pg/mL) QCL %CV QCM %CV QCH %CV

39.1e2500 39.1 18.8e1200 19.2 62.5e4000 193 23.4e1500 23.4

18 24 1 15

92 94 104 101

93 49 99 87

98 64 97 101

15 54 11 21

10 37 11 16

Animals were dosed with 0.05 mg/kg of LPS. Whole blood was drawn and processed into serum 4 h post dose, n ¼ 6. Animals were dosed with PBS. Whole blood was drawn and processed into serum 4 h post dose, n ¼ 10. For IL-1b and IL-6 concentrations of both cytokines were below the level of detection for the respective kits.

4 36 8 18

247 (SD ± 190) 569 (SD ± 376) 3220 (SD ± 1288) 1418 (SD ± 156)

Controlb (pg/mL)

<39.1c <19.2c 258 (SD ± 126) 44 (SD ± 15)

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subjects. In addition, inter-analyst precision is an indication that the assay is robust and verifies if the data is reproducible, ensuring that multiply analysts can perform this assay during the course of a toxicology study. The results of the validation demonstrated that the various leukocyte subsets were able to be enumerated using a newly developed eight parameter IP panel. Whole blood samples may be stored up to 24 h at ambient room temperature before processing procedures are performed and still be statistically reproducible. Furthermore, the fixed samples stored at 2e8
C, protected from light, can be analyzed up to 48 h after fixation and still be statistically comparable to samples immediately analyzed after sample fixation. The new immunophenotyping method that was developed and validated is able to monitor the inflammatory response of porcine cells during regulatory pre-clinical drug and nutritional supplement assessment. A limitation of the IP panel is that not all leukocyte subsets passed the acceptance criteria. The NK cell precision values did not meet acceptance criteria as inter assay precision and inter analyst precision displayed CVp values that were above 30%. The variance caused by intra assay, post collection and fixation stability statistics were low; therefore it is unlikely that assay robustness, whole blood sample stability or fixation methodology are the underlying cause of the high NK cell variance observed. The fluctuations in NK cell levels may be due to the developing piglet immune system (Worliczek et al., 2010). It has been observed by previous literature that the immune system of the piglet goes through alterations in cell surface markers of hematopoietic cells (Gerner et al., 2009; Worliczek et al., 2010). Another explanation for the high variance is the lack of information concerning porcine NK cell markers or receptors. For instance, there are no commercially available reagents specific to porcine NK cell; therefore in our assay we used a negative gating strategy to enumerate this cell type, CD45þCD8þCD3CD4. While this may prove a robust method for NK cell detection in the adults, our research suggests that it is not a reliable detection technique for piglet system (Gerner et al., 2009). With additional research efforts and commercial interests, reagents to track NK cells could be discovered and developed for future NK cell evaluation within the peripheral whole blood of piglets. The commercially acquired ELISA kits for porcine IL-1b, IL-6, IL8, and TNF-a were evaluated for inter and intra assay precision and accuracy, stability, and dilution linearity. The assays were considered fit-for-use and the (LPS) positive control confirmed upregulation of circulating cytokines in neonatal pigs. The IL-6 kit had lower recovery than the other assays. Although the kit detected recombinant IL-6 at lower than nominal levels this detection was consistant across multiple runs and multiple analysts. This indicates that while the accuracy was low, the precision was at acceptable levels; therefore, it was fit for use on study. Furthermore the IL-6 could accurately detect a positive versus a negative response in vivo. All six animals dosed with LPS, demonstrated an increase in IL-6 levels, while the ten animals not dosed with LPS had no analyte detected (Table 5). This finding indicates that the IL6 assay is specific and can detect an immunotoxic response induced by a chemical compound. The results of the IHC validation were confirmed by histopathology assessment. DSS-treated animals had findings in both the proximal and distal colon that were recognizable on H&E-stained tissue, with accompanying increased cytokine production and staining seen for IL-8 and TNF-a. Increased IL-8 and TNF-a staining in representative DSS-treated animals is clearly associated with light microscopic findings of inflammation and edema within the colon. Assessment of both H&E stained sections and IHC-stained sections allowed clear linkages to be made regarding the presence of the IL-8 and TNF-a inflammatory cytokines in the affected

areas of the intestinal tract. While other immunohistochemical assays were attempted (specifically, IL-6), those were not responsive to the induced damage created by DSS and seemed instead to have an overall homogenous expression in the neonatal swine intestine. Characterization of neonatal histology combined with localized cytokine production in GIT lesions is not clearly established, particularly in swine. Careful evaluation of the GIT response to DSS, an irritant, allows for clear establishment of a positive control for future assessment of toxicologic pathology and immunopathology of the GIT in neonatal pigs, an important species in human infant formula assessment. Development of the baseline histopathological, immunohistochemical, cytokine, and flow cytometry assays provides a strong basis for a weight of evidence approach towards immunotoxicological evaluation of food additives and potential gastrointestinal irritants in the neonatal pig model of human infant nutrition. Weiner et al. (2015) employed the methods developed in this paper in a study of the effects of the common food additive, carrageenan, on the neonatal GIT and immune system when dosed in infant formula. Development of further immunohistochemical assays would benefit evaluation and assessment of localized responses to toxic insults and/or irritants in the gastrointestinal tract of this important model species. Conflicts of interest MPI research performed this research under paid contract by FMC Corporation and Abbott Laboratories. The FMC Corporation and Abbott Laboratories were given the opportunity to review the paper and offer comments on the manuscript. Those comments did not alter the professional opinions of the co-authors. Acknowledgments The authors would like to thank Deanna Falzone, Hoan Nguyen, Shiqing Wang and Rossi Loker for their assistance on the immunophenotyping, IHC and cytokine validation and Mark Wolfe for providing departmental resources to make this publication possible. The authors wish to thank Dr. Heather Ferguson of Abbott Laboratories for scientific support and input, and to thank Ms. Allison Cooke, the Infant Nutrition Council of America and Ms. Eunice Cuirle, FMC Corporation, for their regulatory input. The authors also thank Mr. William Blakemore, Celtic Colloids, and Dr. James M. McKim, IONTOX, LLC, for their thoughtful comments and assistance during the study. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fct.2015.08.027. References Barrow, P.C., 2012. Pediatric Non-clinical Drug Testing: Principles, Requirements, and Practice. John Wiley and Sons. Bassaganya-Riera, J., Hontecillas, R., 2006. CLA and n-3 PUFA differentially modulate clinical activity and colonic PPAR-responsive gene expression in a pig model of experimental IBD. Clin. Nutr. 25, 454e465. Carroll, J.A., Carter, D.B., Korte, S.W., Prather, R.S., 2005. Evaluation of the acute phase response in cloned pigs following a lipopolysaccharide challenge. Domest. Anim. Endocrinol. 29, 564e572. (CLSI) C.a.L.S.I, 2011. Quality Assurance for Design Control and Implementation of Immunohistochemistry Assays; Approved Guideline, second ed. National Institutes of Health. Crissman, J.W., Goodman, D.G., Hildebrandt, P.K., Maronpot, R.R., Prater, D.A., Riley, J.H., Seaman, W.J., Thake, D.C., 2004. Best practices guideline: toxicologic histopathology. Toxicol. Pathol. 32, 126e131. Cunliffe, J., Derbyshire, N., Keller, S., Coldwell, R., 2009. An approach to the

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