**5. Gene expression profiles change related to hyperlipidemia**

110 Blood Cell – An Overview of Studies in Hematology

doi:10.1371/journal.pone.0019761.g005

(Figure 6C).

6D).

until 12 weeks of age. Their signal intensities stayed at constant levels after 12 weeks

Toll-like receptors (TLRs) are the principal pattern recognition receptors. With this innate immunity, the first immune response is mediated into reserved foreign patterns on recognition. TLRs recognize reserved molecular patterns, start rapid response to protect the host upon infection, and produce signals, such as cytokines and co-stimulatory molecules to activate the adaptive immune system [15-16]. Regulation of the TLR signaling cascade is important for inflammatory responses, innate host defense, and adaptive immune responses [17-18]. Most mammalian species are estimated to have between 10 and 15 types of TLRs. The Agilent porcine microarray has 10 types of TLRs probes. Among these TLRs, 5 of TLR genes fell under Category 2 (expressed until 12 weeks of age), 1 under Category 8, and 4 under Category 16. Their signal intensities remained constant after 12 weeks of age (Figure

**Figure 6. Signal intensity of major histocompatibility complex (MHC) genes.** (A) Swine leukocyte antigens (SLA) classical class I genes. (B) Swine leukocyte antigens (SLA) classical class II genes. (C) Interferon receptor genes. (D) Toll-like receptor (TLR) genes. Signal intensities were normalized using quantile normalization and log-transformed after excluded signals flagged as "absent." The category numbers are shown in graph legends. Genes in Categories 1, 2, and 4 are shown in the graph. To examine the usage of whole blood RNA analysis for the early diagnosis of the disease, we showed transitions in dietary induced hyperlipidemia gene expression profiles of whole blood RNA in miniature pigs.

Hyperlipidemia is well recognized as a risk factor for cardiovascular disease (CVD). As diet represents the most important determinant of hyperlipidemia, dietary animal models can be useful for the study of CVD progression [19]. High-fat, high-cholesterol, and high-sugar diets have been shown to induce hyperlipidemia, obesity, and insulin resistance in humans and rodents [20-22]. Dietary-induced hyperlipidemia pig models have also been established [23-29].

A high-fat and high-cholesterol diet (HFCD) as a typical dietary treatment were used for dietary-induced hyperlipidemia miniature pig models, by using specific pathogen-free (SPF) Clawn miniature pigs.

Eight 12-week-old, male Clawn miniature pigs were housed individually in cages of 1.5 m2 at the breeder's specific pathogen-free (SPF) facility (Japan Farm Co., Ltd, Kagoshima, Japan) for 27 weeks. Body weights at the beginning of the experiment were 5.1 (2.6) kg (mean (standard deviation; SD)). During this period, 5 pigs were fed with 450 g/day standard dry feed (Kodakara73, Marubeni Nisshin Feed Co., Ltd., Tokyo Japan), and had unlimited access to water (control group). Five pigs were fed a high-fat, high-cholesterol diet containing 15% lard and 2% cholesterol (HFCD group).

Almost no changes were observed in fasting plasma triglyceride levels. Fasting plasma total cholesterol concentrations had increased in the HFCD group by week 5 of the feeding period (P<0.001) and were maintained between 350 and 1150 mg/dL from weeks 10–27. Fasting plasma high-density lipoprotein cholesterol (HDL-C) concentrations increased and showed significant differences (P < 0.001) from weeks 10–27. Fasting plasma low-density lipoprotein cholesterol (LDL-C) concentrations also increased and showed significant differences from weeks 5–27. Fasting plasma glucose concentrations remained unchanged.
