The Toxicologist - Society of Toxicology

The purpose of this project was to develop quality control measures, method qualification,
and performance guidelines for the enumeration of lymphocyte cell types
in the peripheral blood of cynomolgus monkeys using flow cytometry. Here we
outline the need for both horizontal and vertical control of instrument and
methodology as well as reagents to ensure integrity of all data generated. Though
the guidelines established here are widely applicable, the scope of this poster will
focus on a wide variety of cell types and subpopulations demonstrating the changes
for each and in addition the challenges of multi-site operation of machines to
match data sets. Consistent interpretation of flow cytometry data relies on standarization
and validation of the instrument, the reagents and the analytical procedure.
To achieve this, we assessed single vs. multicolor staining, inter- and intraassay
precision and sample stability for each lymphocyte population. Additionally,
QC samples and population summations were performed to assure accuracy.
According to the GLP regulations, each laboratory is ultimately responsible for developing
methods for its own equipment use and analytical procedures; this poster
reviews the validation and quality control of all aspects of the operation of flow cytometry
assays in non-human primates
1145 DELAYED-TYPE HYPERSENSITIVITY (DTH)
REACTION WITH TETANUS TOXOID (TTX) IN
CYNOMOLGUS MONKEYS.
Y. Takahashi, Y. Otsubo, K. Nagatomo, T. Nakamura, T. Sukamoto, K.
Fukuzaki and R. Nagata. Shin Nippon Biomedical Laboratories (SNBL), Ltd.,
Kagoshima, Japan.
Purpose: DTH reactions are mentioned in the ICH S8 immunotoxicity guideline
as difficult to reproduce and ascertain in monkeys. We investigated sensitization periods
and frequencies, and challenge volumes to establish an appropriate design for
a DTH reaction test with TTx in cynomolgus monkeys. Method: Three male
cynomolgus monkeys per group (total 5 groups) were sensitized one (Groups 1 and
2), two (Group 3), three (Group 4), or four (Group 5) times. They were challenged
at three (Groups 1, and 3 to 5) or four (Group 2) weeks after the final sensitization
with TTx at 10, 20, and 30 μL/site (Groups 1 and 2; 10 Lf/mL) or at 30 μL/site
(Groups 3 to 5; 10, 3, and 1 Lf/mL). The sensitization was performed by injecting
TTx (10 Lf/mL) intramuscularly into the femoral region (0.6 mL) and intradermally
into the dorsal region (50 μL/site × 12) under anesthesia. The challenge with
TTx or physiological saline was made by intradermal injection into the thoracic region
(each volume × 3 sites) under anesthesia. Skin reactions (erythema and edema)
were evaluated in accordance with the Draize method at approximately 24 and 48
hours after challenge. Result: The mean reaction scores at 24 hours after challenge
for TTx (10 Lf/mL) at 30 μL/site in Groups 1, 2, 3, 4 and 5, respectively, were
3.77, 3.06, 2.90, 3.77, and 2.80. The mean reaction scores at 24 hours after challenge
for TTx (10 Lf/mL) at 20 and 10 μL/site, respectively, were 3.30 and 2.56 in
Group 1 and 3.10 and 2.00 in Group 2. In Groups 3, 4, and 5, respectively, the
mean reaction scores at 24 hours after challenge at 30 μL/site were 2.67, 2.97, and
2.10 for 3 Lf/mL TTx, and 1.93, 1.47, and 1.37 for 1 Lf/mL TTx. Mean scores
were lower at 48 than 24 hours after challenge. Conclusion: We propose a
cynomolgus monkey DTH reaction test with a three-week period and one-time frequency
of TTx sensitization, and challenge volume of 30 μL/site for TTx (10
Lf/mL).
1146 IMMUNOTOXICITY ASSESSMENT IN INFANT
CYNOMOLGUS MONKEYS BY MEASUREMENT OF
HUMORAL IMMUNITY (TDAR) TO KLH AND INNATE
IMMUNITY (NATURAL KILLER (NK) CELL ACTIVITY).
F. G. Burleson 1 , N. Makori 2 , N. Lalayeva 2 , G. R. Burleson 1 and H. Tsusaki 2 .
1 BRT-Burleson Research Technologies, Inc., Morrisville, NC and 2 SNBL, Everett, WA.
Immunotoxicity assessment of biologics that do not have activity in rodents must
be evaluated in non-human primates. Developmental immunotoxicity evaluations
are performed to determine if the test article has an immunotoxic effect on the developing
immune system. Infant Cynomolgus Monkeys are now routinely used in
evaluation of (1) humoral-mediated immunity using a T-dependent antibody response
(TDAR) to KLH, and (2) evaluation of innate immunity with NK cell activity.
In continuing efforts to standardize methods, enzyme-linked immunoassays
were validated to measure KLH-specific IgM and IgG in monkey serum samples.
The validation confirmed suitability by evaluation of recovery, dilutional linearity,
intra-assay and inter-assay precision and accuracy, and freeze-thaw sample stability.
Control background data is obtained from infant Cynomolgus Monkeys starting at
120 days of age by immunization with KLH on DB120 and DB180. Serum is collected
prior to the primary KLH immunization on DB120; and 7, 14, 21, and 28
days post immunization to evaluate the primary antibody response to KLH. Serum
is also collected on DB180 (prior to KLH boost), and 7, 14, 21, and 30 days post
KLH boost to evaluate the secondary antibody response. KLH-specific IgM and
IgG antibody concentrations are measured in the serum samples. It is shown that
anti-KLH IgG antibody concentrations are elevated 14-30 days post-immunization
in individual infants during the primary response. A strong secondary response is
observed 7 days after the second KLH immunization. Innate immunity is evaluated
by measuring NK cell activity in 100-195 days old infant Cynomolgus Monkeys.
PBMC are obtained from whole blood using Ficoll-Hypaque density gradient centrifugation
and tested using a minimum of 3 effector:target (E:T) cell ratios. Lysis
of K562 target cells is measured by release of 51Cr.
1147 REPRODUCTIVE TOXICOLOGY AND INFANT
IMMUNOPHENOTYPING: REFERENCE RANGES FOR
T-CELL ASSESSMENT IN INFANTS.
C. Cornwall, A. R. Mcintyre, T. Salewsky, T. Warren, N. Pratt, R. Eyre and H.
Tsusaki. SNBL USA, Ltd., Everett, WA.
Reproductive toxicology is a necessary part of any drug assessment program intended
to evaluate toxicological effects on the mother and infant. Data are available
referencing the mature immune systems, which can be used as comparison for data
interpretation. However, reference ranges for the developing immune system are
not easily accessible due to the rarity of infant animals and the small blood collection
volumes possible. Normal ranges are vital to the interpretation and final conclusions
made for any data set. Without baseline data conclusions derived may be
flawed. Presented is a compiled set of infant reference ranges of T-lymphocytes (Thelper
cells, T-cytotoxic cells, as well as rare lymphocyte populations). Samples obtained
from 82 cynomolgus monkey neonates (ages ranging between 25 and 35
days) were stained for CD3, CD4 and CD8 cell surface markers. The maximum,
minimum, mean, standard deviation and confidence intervals were calculated for
the relative percentages of these populations. The mean relative percentage for T-cytotoxic
cells (CD3+, CD8+) was 19.0%. The more rare lymphocyte populations
(CD3+CD8-, CD3-CD8+, and CD3-CD8-) were 18.7%, 15.0%, and 47.2% respectively.
This combined information allows for an accurate assessment of changes
in the developing T-lymphocyte population and confidence in data sets with large
variation.
1148 DEVELOPMENT OF AN ASSAY TO DETECT CMV AND
LCV SPECIFIC T-CELLS TO EVALUATE IMMUNE
STATUS IN CYNOMOLOGUS MONKEYS.
P. A. Schneider, L. O’Donnell, T. T. Kawabata and C. Kamperschroer. Pfizer,
Groton, CT.
The majority of people are chronically infected with the herpesviruses cytomegalovirus
(CMV) and Epstein-Barr virus (EBV). These infections are asymptomatic
in healthy individuals but can cause serious disease in immune compromised
individuals. Cynomolgus macaques harbor both CMV as well as
lymphocryptovirus (LCV), the equivalent of human EBV, and immunosuppression
can lead to disease similar to that observed in humans. Because chronic CMV, LCV
and EBV infections are controlled primarily by T cells, it is useful to measure T cells
specific for these viruses in order to assess the risk of viral reactivation. To determine
whether candidate drugs affect T cell responses to CMV or LCV, we developed an
INF-γ ELISpot assay to enumerate CMV- and LCV-specific T cells in macaques.
This is done by incubating cynomologus peripheral blood mononuclear cells with
viral lysates and then measuring the number of cells that respond by secreting IFNγ.
Responding cells were confirmed to be T cells and specific for the antigen of interest.
Multiple parameters were evaluated in an effort to optimize the response
and decrease background. In addition, a 3 month longitudinal study was completed
to evaluate variability of the T cell responses to LCV and CMV. Although there is
variability in the frequency of responding cells over time, power calculations suggest
that it should not preclude using the assay for detecting T cell suppression. Initial
in-vitro experiments indicate that cyclosporine reduces the frequency of CMV specific
T cells, and future experiments are designed to determine whether this assay is
capable of detecting suppression of CMV or LCV-specific T cell responses in immune
compromised monkeys. Our ability to monitor LCV and CMV specific T
cell function in macaques should greatly improve our ability to assess whether drugs
affect immune control of these viral pathogens.
1149 VIRAL LOAD AND LYTIC GENE EXPRESSION ASSAYS
TO DETECT REACTIVATION OF LCV IN
CYNOMOLGUS MACAQUES.
K. R. Tartaro 1 , S. W. Kumpf 2 , T. T. Kawabata 1 and C. Kamperschroer 1 .
1 Immunotoxicology, Pfizer, Groton, CT and 2 DART, Pfizer, Groton, CT.
Epstein-Barr virus (EBV) is an important pathogen associated with the development
of several malignancies in humans. Monitoring of EBV load is commonly
used in immunosuppressed transplant patients as a way to assess their risk for developing
post-transplant lymphoproliferative disorder (PTLD) or lymphoma.
SOT 2011 ANNUAL MEETING 245