CRO Expertise
EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS
(EAE) MODEL FOR MULTIPLE SCLEROSIS
Multiple sclerosis (MS) is a major demyelinating disease in humans. Autoreactive immune cells recognize the myelin sheath and axon as foreign. The infi ltrating immune cells trigger episodes of damage and regeneration to the myelin sheath and nerve axon. Clinical symptoms of neurological dysfunction are concomitant with pathology.
KEY POINTS IN RODENTS
Mice and rats are the two commonly used animal models of EAE, in addition to NHPs. EAE is greatly influenced by several factors including: gender, strain, (MHC haplotype), and age.
In rats, EAE is typically monophasic while mice exhibit chronic or remitting relapsing disease, pending on the mouse strains and the induction material. Induction with myelin-oligodendrocyte-glycoprotein (MOG) in C57BL mice resulted in chronic EAE while proteo-lipoprotein (PLP) in SJL mice resulted in remitting relapsing EAE. The severity of actively induced disease is correlated with strain MHC haplotype and encephalitogenic peptide sequence.
TREATMENT OPTIONS
- Prophylactic
- Therapeutic at peak of disease
- Therapeutic at remission phase
DISEASE EVALUATION
- Clinical scoring system of mice (0-4)
- Body weight
- Relapse rate
- Histopathology
- Flow cytometry
- ELISA
KEY POINTS IN Non-Human Primates (NHPS)
The common marmoset is susceptible to a form of EAE that resembles MS in humans. The common marmoset is a unique model for evaluating potential MS treatment because of the molecular and functional similarity of its immune system to humans. The topography of lesions in the marmoset brain, which are remarkably similar to the human MS pattern, can be identified using diagnostic tools used in humans, including MRI.
CLINICAL SCORING IN NHPS (0-5)
0 = No clinical signs
0.5 = Apathy, loss of appetite, vomiting, altered walking with ataxia
1 = Lethargy, anorexia, tail paralysis, tremor
2 = Ataxia, optic disease (vision problems)
2.5 = Incomplete paralysis of one or both sides, sensory loss, brainstem syndrome
3 = Complete paralysis of one or two sides
4 = Complete paralysis
5 = Moribund or spontaneous death attributable to EAE
T-CELL-DEPENDENT ANTIBODY RESPONSE (TDAR)
OVERVIEW
The immune system has been identified as a target organ for adverse effects caused by test material administration. Because of this, the T-Cell-Dependent Antibody Response (TDAR) assay is used to assess potential immunotoxicity and to determine the impact of a test material on the immune response at the preclinical stage of drug discovery. This assay is the gold-standard for studying these effects due to the requirement of multiple immune functions and cell types to generate an antibody response including antigen uptake and presentation, CD4 T cell help, B cell activation and antibody production.
Biomere has validated multiple TDAR models including non-human primates and rodent models.
TDAR ASSAY ANIMAL MODELS AVAILABLE
Non-Human Primates
- KLH-peptide immunization in cynomolgus macaques
Rodents
- KLH-peptide immunization in C57BL/6 female mice
- OVA-peptide in alhydrogel immunizations in C57BL/6 female mice
TDAR ASSAY READOUTS
- IgG and IgM serum levels by ELISA
- Immune response analysis by flow cytometry
- Hematology analysis by complete blood count (CBC)
Female C57BL/6 mice from JAX were treated with either vehicle or anti-CD154 mAb intraperitoneally (IP) on Day -1. On Day 0, mice were given NP-KLH IP. Serum was collected from mice on Day -1, 7, and 14 and (A) anti-NP IgM and (B) anti-NP IgG ELISAs were performed.
Naïve male and female cynomolgus macaques of Chinese origin were treated with vehicle or anti-CD154 mAb IV on Day 0, 14, and 28. On Day 3 and 31, NHPs received KLH intramuscularly (IM). Serum was collected from animals twice weekly through Day 56 and (A) anti-KLH IgM and (B) anti-KLH IgG ELISAs were performed on serum samples. (C-I) Flow cytometry was performed on blood samples on Day 49. Frequency of (C) CD4+ TH cells, (D) IFNγ+ TH cells, and (E) TNFα+ TH cells. Frequency of (F) CD20+ B cells, (G) Naïve B Cells, (H) Germinal Center B Cells, and (I) Memory B Cells.
Summary
Significant decreases were observed in IgM and IgG antibodies generated in both the mouse and NHPTDAR assays in immune suppressive treatment (α-CD40L) compared to vehicle control. A proportional decrease in germinal center B cells compared to other B cell groups, as well as a decrease in B cells as a total population in α-CD40L versus control were observed. Decreases in the proportion of CD4+ TH cells were also observed in α-CD40L treated versus control, with a decrease in the proportion of IFNγ+ TH cells. From these results, we can see α-CD40L was able to suppress the generation of antibodies against the target antigen and impacted the TH cell and germinal center B cell response.
MOUSE MODELS OF SYSTEMIC LUPUS
ERYTHEMATOSUS (SLE)
NZB/W F1 MOUSE MODEL OF SLE
Characteristics
- Most commonly used preclinical model for SLE
- Model developed by Helyer and Howie in 1963 and subsequently transferred to Jackson Laboratories
- Develops anti-dsDNA (auto) antibodies at ≥ 16 weeks of age
- Develops 3+ proteinuria after 20 weeks of age
- NZM lines: Genetic susceptibility loci (sle1,2,3)
Clinical Measures
- Proteinuria
- Weekly body weights
- Anti-dsDNA antibody ELISA
- Glomerular filtration rate
- IDEXX clinical analyzer
FLOW CYTOMETRY
- B cells, T cells, dendritic cells, NK cells, NKT cells, apoptosis
- Experienced with multicolor (up to 10 color) panel design
- Validation with human (PMBCs), rat and mouse cells (whole blood, splenocytes and lymph node cells)
HISTOPATHOLOGY
- Glomerulonephropathy
- Dilated tubules
- Degenerate tubules
- Lymphocyte aggregates
NZB/W F1 SLE MOUSE PREVENTION STUDY
- N=10/group with proteinuria = 0+ at 20 weeks of age
- Dose once every two weeks from 20 – 46 weeks of age
- Measure body weights once weekly and proteinuria once every two weeks
- Humane survival end points: ≥ 3+ proteinuria on two consecutive weeks
NZB/W F1 SLE MOUSE REMISSION STUDY
- NZB/W F1 female mice with moderate proteinuria (1 – 2+) were entered into groups at 28 weeks of age (n = 10/group) and initiated treatment (bar)
- Proteinuria was measured once every two weeks (the following week to confirm a ≥ 3+ reading)
- Humane survival end points: ≥ 3+ proteinuria on two consecutive weeks
Both mice reached the same humane endpoint, but lymphocyte aggregates were noticeably more severe in MRL/lpr mice (mean score = 3) compared to NZB/W F1 mice (mean score = 2)
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