The Toxicologist - Society of Toxicology

To validate the TIAR model, female rats were administered orally CPA (2 or 6
mg/kg/day) or CsA (10 or 25 mg/kg/day) once daily for 14 days (Day 1 to 14), and
immunized with TNP-Ficoll at 30 μg/rat on Day 8. Serum samples for measuring
anti-TNP antibodies were collected on Day 15.
In CPA groups, decreases in anti-TNP IgM and anti-TNP IgG were observed. The
lymphocyte subset analysis revealed decreases in mainly B cells in the peripheral
blood and spleen, and decreased T cells in the thymus. CPA also decreased white
blood cell parameters in hematology, and decreased spleen and thymus weights.
In CsA groups, decreases in anti-TNP IgM and anti-TNP IgG were also observed.
The lymphocyte subset analysis revealed decreased T cells in the peripheral blood
and thymus. In the hematology and organ weights, no significant changes were
observed.
In conclusion, this TIAR model in rats will be useful for evaluating the effects on
humoral immunity. Furthermore, a tiered approach using TDAR and TIAR may
provide a more detailed immunotoxicity profile of drugs. In addition, the results of
an additional study using other T-cell independent antigens will be reported.
650 ANALYTICAL VALIDATION OF
IMMUNOPHENOTYPING FOR CD5 IN PERIPHERAL
BLOOD, SPLEEN, AND THYMUS FOR CD 1 MICE.
P. Sims 2 , P. Joshi 2 , J. E. Arrington 1 and J. Puchalski 2 . 1 Toxicology Study Direction,
Covance, Madison, WI and 2 Immunotoxicology, Covance, Madison, WI.
The purpose of this study was to validate CD5 immunophenotyping in peripheral
blood, spleen, and thymus for naïve CD 1 mice so it could be used as a substitute
for CD3 when CD3 is the target molecule of immunomodulatory compounds.
Accuracy, precision, stability, and antibody cocktail optimization endpoints were
assessed for peripheral blood, spleen, and thymus for total T cells (CD5+/CD3+),
helper T cells (CD5+4+/CD3+4+), cytotoxic T cells (CD5+8+/CD3+8+), B cells
(CD5 19+/CD3-19+), natural killer (NK) cells (CD5 49b+/CD3-49b+), or thymus-specific
T cell subsets, as appropriate. Two to three replicates from each sample
were compared to evaluate inter and intra-assay precision and individual values
from each animal were compared to assess variability. The assay was precise and accurate
for blood, spleen, and thymus with most coefficient of variation (CV) values
less than 10%. Results of stability testing indicated that all stained samples of fixed
peripheral blood/single-cell suspensions were stable for up to 24 hours. The assay
was linear for two antibody concentrations tested (1X and 2X) with most CV values
less than 10%. Comparison of the immunophenotyping parameters for CD5 vs
CD3 associated T, B, and NK cell populations in blood and spleen showed excellent
correlation. Although the comparison in thymus was acceptable, the values
were not as close as noted for peripheral blood and spleen due to slight differences
in CD5 vs. CD3 ratios in the thymus for developing T cell populations. In summary,
peripheral blood and tissue immunophenotyping methods using CD5 have
been validated to accurately and precisely measure lymphocyte populations in peripheral
blood, spleen and thymus. Furthermore, using CD5 as a substitute marker
for CD3 yielded comparable lymphocyte populations in peripheral blood, spleen,
and thymus, which validated its use for risk assessment.
651 ANALYTICAL VALIDATION OF SPLEEN, THYMUS,
AND MESENTERIC AND MANDIBULAR LYMPH
NODE IMMUNOPHENOTYPING FOR SPRAGUE-
DAWLEY RATS.
J. E. Arrington and P. Joshi. Immunotoxicology, Covance, Madison, WI.
The purpose of this study was to develop and validate tissue immunophenotyping
procedures for Sprague Dawley rat spleen, thymus, and mesenteric and mandibular
lymph nodes. These procedures can and have been used to evaluate changes in lymphoid
tissue cell populations and were especially important when changes in lymphocyte
populations were not detected in peripheral blood. The accuracy, precision,
stability, and linearity of methods developed to measure total T cells (CD3+),
T helper cells (CD3+4+), cytotoxic T cells (CD3+8+), B cells (CD3+45RA+), natural
killer (NK) cells (CD3-161a+) in spleen and both lymph nodes and an array of
T cell subtypes in the thymus (combinations of CD3±, CD4±, and CD8±) by flow
cytometry were assessed. Three 1 cm3 sections of tissue were collected from each
animal to evaluate all the study parameters. The assay was linear for all cell concentrations
tested (10, 1, and 0.1 million input cells), except NK cells in the spleen and
B cells in mandibular lymph nodes. Three separate tissue samples/organ were compared
to determine accuracy and four replicate tests/sample were tested for precision.
The assay was accurate and precise for spleen, thymus, and lymph nodes evaluation
with most relative coefficient of variation values less than 10% (more
variability with lymph nodes). Results of stability testing for whole tissue samples
showed all parameters were stable up to at 24 hours, except cytotoxic T cells in
mesenteric lymph nodes (due to slight variability). Stability for fixed single-cell sus-
140 SOT 2011 ANNUAL MEETING
pensions was at least 24 hours for most parameters; CD3-4+8+ in the thymus and
relative NK cells in the mesenteric and mandibular lymph nodes were not considered
stable beyond the day of processing (linked to high initial values). In conclusion,
tissue immunophenotyping methods have been validated and accurately and
precisely measure lymphocyte populations in the spleen, thymus, and mandibular
and mesenteric lymph nodes (although more variability is present with lymph
nodes), but could be influenced by antibody concentration.
652 IN VITRO MODEL FOR IMMUNOTOXICITY: SURFACE
MARKER EXPRESSION AND CYTOKINE RELEASE IN
NORMAL HUMAN DENDRITIC CELLS.
T. Landry, M. Klausner, A. Hunter, J. Sheasgreen, P. J. Hayden and S.
Ayehunie. MatTek Corp, Ashland, MA.
Dendritic cells (DC) play a key role in the initiation of immune response by recognizing,
internalizing, processing, and presenting antigens to T cells. However, DC
function can be altered by environmental factors, pharmaceutical agents, and exogenous
chemicals. These perturbations to immune cell function, i.e. immunotoxicity,
can have significant effects on the natural defense mechanisms that ward off
pathogens. While animal based assays are commonly used for immunotoxicity assessment,
the Cosmetic Directive and REACH legislation bans the use of animals
for many such studies. To examine the potential use of DC for immunotoxicity
evaluation, large numbers of DC were generated from CD34+ progenitor cells and
characterized for their expression of HLA-DR, CD1a, langerin, Birbeck granules,
and co-stimulatory molecules (e.g. CD80, CD83, CD86, and CD40). In this
study, we evaluated the effect of 3 immunotoxic compounds (ITC) on DC function.
DC functionality was monitored by measuring surface marker expression and
cytokine secretion following exposure to lipopolysaccharide (LPS). DC were first
exposed to non-cytotoxic concentrations of the ITC for 24 hr and then to LPS for
an additional 18 hr. FACS analysis of ITC-exposed DC showed effects in the rank
order of immunotoxicity (severe to low effect): cyclosporine A > furosemide > verapamil
as follows: a) decreased CD86 expression and b) dose-dependent decreases in
the secretion of immuno-stimulatory cytokines including interleukin (IL)-12, IL-6,
and IL-1β. Conclusion: DC expression of CD86 and release of immuno-stimulatory
cytokines may serve as endpoints to predict immunotoxicity. Due to concerns
with animal models in terms of cost, ethical issues, and relevance to hazard assessment
in humans, the DC based assay could be an attractive in vitro model to predict
immunotoxicity of compounds.
653 USE OF AN ELECTROSPUN POLYCAPROLACTONE
NANOFIBROUS SCAFFOLD AS A POTENTIAL DRUG
DELIVERY SYSTEM FOR IMMUNOMODULATORY
COMPOUNDS.
C. E. McLoughlin, M. J. Smith, W. Auttachoat, G. L. Bowlin and K. L. White.
Virginia Commonwealth University, Richmond, VA.
Electrospun materials have potential use in many biomedical applications such as
soft tissue replacements or as scaffolds to target drug delivery to local sites.
Electrospinning is a polymer processing technique that can be used to create materials
composed of fibers with diameters ranging from the micron to the nanoscale.
Our laboratory has focused on investigating the effects of microfibrous and nanofibrous
electrospun polycaprolactone (EPCL) on the immune system. These results
have demonstrated that in both young (12 week) and old (6 month) mice, EPCL
has minimal effect on various immune parameters including innate, humoral, and
cell mediated immunity (CMI). With its lack of immunotoxicity, EPCL presents
an excellent polymer scaffold for use in delivering drugs to local sites. Our recent
studies focused on using EPCL nanofiber scaffolds with the known immunosuppressive
compound dexamethasone (DEX) incorporated within the matrix. The
ability of the EPCL-DEX scaffold to suppress CMI was evaluated using the delayed-type
hypersensitivity (DTH) response to Candida albicans . Preliminary
studies were conducted following subcutaneous implantation of a single disk (6mm
diameter) with 65, 100, or 200% w/w DEX in EPCL in the thigh region. Mice
were sensitized with C. albicans following implantation and 8 days later were challenged
with chitosan. Based on footpad swelling, dose responsive suppression of the
DTH was observed at all dose levels. The animals that received the high dose
(200%) had decreased spleen weights, however no change in spleen weight was observed
at the lower doses (65 and 100%). These preliminary results suggest that implantation
of a drug-containing electrospun scaffold may achieve local immunosuppression
without systemic toxicity. Furthermore, EPCL may be an advantageous
drug delivery system. Supported in part by NIEHS Contract ES 05454.