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1.
Figure 2

Figure 2. Epitope mapping of PoAb anti-PPARγ peptide IgG. From: A putative role for platelet-derived PPAR? in vascular homeostasis demonstrated by anti-PPAR? induction of bleeding, thrombocytopenia and compensatory megakaryocytopoiesis.

Various peptides corresponding to the truncated portions of the peptide-immunogens were used in antigen-down ELISA to fine-point map the epitope reactivity of the PoAbs. A representative titration by ELISA is shown for one anti-14mer peptide rabbit (Rosita) in panel A and one anti-19mer rabbit (Patricia) in panel B.

Patricia J. Simpson-Haidaris, et al. J Biotechnol. ;150(3):417-427.
2.
Figure 3

Figure 3. Crossreactivity determination of anti-PPARγ MoAbs. From: A putative role for platelet-derived PPAR? in vascular homeostasis demonstrated by anti-PPAR? induction of bleeding, thrombocytopenia and compensatory megakaryocytopoiesis.

Conditioned media from two anti-19mer MoAbs, 6D4.H4 and 6B4.H4, were tested by ELISA for relative specificity for the following antigens: the original peptide-immunogen (MAP-19mer), a synthetic peptide corresponding to the C-terminal 12 residues of the original 19mer, rPPARγ1 isolated from E. coli as a fusion protein with MBP (MBP-rPPARγ1), and purified rPPARγ1 from Protein One (panel A). The crossreactivity of the two anti-19mer PPARγ peptide MoAbs against rPPARα and PPARβ was determined by Western immunodetection (panel B). An anti-PPARγ MoAb from Santa Cruz Biotechnology (sc-7273) was tested for specificity against rPPARα, PPARβ and PPARγ1 by ELISA (panel C, left) and Western (panel C, right) immunodetection.

Patricia J. Simpson-Haidaris, et al. J Biotechnol. ;150(3):417-427.
3.
Figure 1

Figure 1. Bleed-out ELISA titers and Western immunodetection of rPPAR isoforms. From: A putative role for platelet-derived PPAR? in vascular homeostasis demonstrated by anti-PPAR? induction of bleeding, thrombocytopenia and compensatory megakaryocytopoiesis.

The relative titer of each rabbit antiserum was determined against its respective immunogen by antigen-down ELISA. Four–fold dilutions of serum starting at 1:100 were tested in duplicate for each rabbit. A representative titration curve is shown (panel A). Recombinant human PPARα, β and γ1 were loaded at 1 μg/lane for Western immunodetection using commercially purchased rabbit anti-PPARα and anti-PPARβ and chicken anti-PPARγ (panel B). Ammonium sulfate IgG fractions of rabbit anti-PPARγ antisera were diluted 1:1000 for Western immunodetection. A representative blot is shown for one anti-14mer (Rosita) and one anti-19mer (Patricia) compared to a commercially available rabbit anti-PPARγ antibody, sc-7196 (panel C).

Patricia J. Simpson-Haidaris, et al. J Biotechnol. ;150(3):417-427.
4.
Figure 7

Figure 7. Passive administration of rabbit anti-platelet IgG and anti-PPARγ IgG induces megakaryocytopoiesis in mouse spleen. From: A putative role for platelet-derived PPAR? in vascular homeostasis demonstrated by anti-PPAR? induction of bleeding, thrombocytopenia and compensatory megakaryocytopoiesis.

The data is presented graphically (panel A) as the average number of megakaryocytes per 20X field (n=13 per condition), the positive control (anti-PT), the negative control (IgG), mice injected with anti-PPARγ-19mer (19mer, M1, M4, M5), and mice injected with anti-PPARγ-14mer (14mer, M3, M4). Statistical significance (*p < 0.0001) was achieved in all conditions compared to control IgG (panel A). Representative H&E stained spleen sections are shown for a negative control mouse injected with nonimmune IgG (panel B), a positive control mouse injected with anti-platelet IgG (panel C), and a mouse injected with anti-PPARγ-19mer (panel D). The bars in panels B-D represent 50 μm. The arrows in panels B-D denote spleen megakaryocytes.

Patricia J. Simpson-Haidaris, et al. J Biotechnol. ;150(3):417-427.
5.
Figure 6

Figure 6. Mouse models of acute and chronic immune-mediated thrombocytopenia. From: A putative role for platelet-derived PPAR? in vascular homeostasis demonstrated by anti-PPAR? induction of bleeding, thrombocytopenia and compensatory megakaryocytopoiesis.

Changes in platelet counts due to antibody treatment are presented as a percentage of the mean platelet ± SD of control (IgG) platelet counts at each time point. Relative platelet counts were reduced in response to high titer MoAb production of anti-PPARγ-14mer 3C11.E8.E1 but not in response to anti-human fibrinogen Bβ-chain D73H.7C3 in ascites mice (panel A) (sc2, subclone 2). Rat anti-GP1bα IgG induced acute thrombocytopenia (*p = 0.0134) in 1 hr that was sustained for 24 hr (#p = 0.0029) (panel B). No differences in platelet counts were observed in mice treated with either anti-19mer or anti-14mer IgG (panel B). Anti-platelet IgG induced significant thrombocytopenia 24 hr after the first injection (*p = 0.0111) that was sustained over nine days with injections of anti-platelet IgG repeated every 72 hr (#p = 0.0234) (panel C). Chronic administration of anti-PPARγ-19-mer or anti-PPARγ-14mer did not induce thrombocytopenia in naïve mice (panel C).

Patricia J. Simpson-Haidaris, et al. J Biotechnol. ;150(3):417-427.
6.
Figure 5

Figure 5. Enhanced megakaryocytopoiesis occurs in rabbit bone marrow in response to immunization with human PPARγ-synthetic peptides. From: A putative role for platelet-derived PPAR? in vascular homeostasis demonstrated by anti-PPAR? induction of bleeding, thrombocytopenia and compensatory megakaryocytopoiesis.

The number of megakaryocytes (MK) per 10X brightfield was quantified as described in Section 2.5. Statistical analysis was performed by paired Students t-test to compare control rabbits (Magnolia, */**) and (Petunia, #/##) to immunized rabbits with p-values as follows: (*) p = 0.0012; (#) p = 0.0160; (**) p < 0.0001; and (##) p < 0.0001. Representative H&E stained bone marrow sections are shown for a mock-immunized rabbit (panel B, Magnolia) and the rabbit showing sustained low platelet counts over the course of the immunization schedule (panels C and D, Alice). The bars in panels B and C represent 50 μm of images taken with the 20X objective; the bar in panel D represents 10 μm of images taken with the 100X objective. The arrows in panels B and C denote megakaryocytes; the asterisks in panel C denote probable megakaryocytes sectioned through portions of the cytoplasm only—these were not included in the quantitation. In panel D, the arrows point to neutrophils and the arrowhead points to lymphocytes engulfed by megakaryocytes (emperipolesis).

Patricia J. Simpson-Haidaris, et al. J Biotechnol. ;150(3):417-427.
7.
Figure 4

Figure 4. Unusual organ pathology is associated with mice and rabbits immunized with PPARγ synthetic peptides. From: A putative role for platelet-derived PPAR? in vascular homeostasis demonstrated by anti-PPAR? induction of bleeding, thrombocytopenia and compensatory megakaryocytopoiesis.

The upper panels (left to right panel A) show abdominal organs in situ of two Balb/c mice immunized with PPARγ MAP-14mer synthetic peptide-immunogen and one normal mouse; the lower panel A pictures show the gross pathology of the sensitized or normal spleen from each mouse shown in upper panel A. The thin arrows (upper left and middle panel A) point to the adhesions of abdominal fat/connective tissue around the intestines, liver, and spleen in the immunized mice (4MR and 5MP); the asterisks denote hemorrhagic blood vessels; in the normal mouse, the abdominal fat presents as a visceral deposit in the lower abdomen as denoted by the thick arrowhead (upper right panel A). The thick arrow indicates the tip of the spleen in each mouse. The spleens from the immunized mice (lower left and middle panel A) were encased in fatty tissue, which was not present around the spleen from a normal mouse. Furthermore, the spleens from the 5MP anti-14mer mouse (lower middle panel A) and 3MB anti-19mer mouse (not shown) were friable and hemorrhagic, which we have not observed in the sensitized spleens from other mice used by us for hybridoma production over the past 23 years. The black bars in upper right panel A represent 0.25 inches and in lower right panel A, 0.5 inches. The tumors in the peritoneal cavities of the ascites mice were encased in highly vascularized, hemorrhagic white fatty tissue (left and right panel B) and the lymph nodes (LN) were hemorrhagic (middle panel B). The white bars (left and right panel B) represent 0.25 inches and the black bar (middle panel B) represents 0.5 inches. A bleeding tendency, indicative of abnormal primary hemostasis (i.e., disrupted platelet aggregation), was observed in the toenail beds (arrows) of mice injected with purified rabbit IgG raised against rat platelets (panel C, anti-PT IgG) and IgG raised against human PPARγ synthetic peptides (panel C, anti-14mer and anti-19mer), but not in the toenail beds of mice treated with nonimmune rabbit IgG (panel C, NRS-IgG). The most striking pathology observed in the immunized rabbits was enlarged ovaries (Patricia, Alice and Rosita), which contained numerous necrotic and unruptured follicles compared to the ovaries from the mock-immunized rabbits (Petunia and Magnolia) (panel D). The black bars in each picture of panel D represent 1 inch and the length and weight of each ovary is indicated. Histomorphological examination of the ovaries (panel E) from mock-immunized (Magnolia) and rabbits immunized with PPARγ MAP-14mer (Alice and Rosita) or MAP-19mer (Patricia) peptide-immunogens confirmed the enhanced bleeding tendency observed in the immunized rabbits as denoted by increased eosinophilic staining of the tissue parenchyma indicating the presence of fibrinogen and/or fibrin, and by the old blood clots remaining in the enlarged follicles.

Patricia J. Simpson-Haidaris, et al. J Biotechnol. ;150(3):417-427.

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