Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2016 Jan 31.
Published in final edited form as: Bone. 2014 Oct 31;71:201–209. doi: 10.1016/j.bone.2014.10.021

Bone Marrow Transplantation Improves Autoinflammation and Inflammatory Bone Loss in SH3BP2 Knock-In Cherubism Mice

Teruhito Yoshitaka a, Mizuho Kittaka a, Shu Ishida a,b, Noriyoshi Mizuno a,b, Tomoyuki Mukai a, Yasuyoshi Ueki a,*
PMCID: PMC4274253  NIHMSID: NIHMS647525  PMID: 25445458

Abstract

Cherubism (OMIM#118400) is a genetic disorder in children characterized by excessive jawbone destruction with proliferation of fibro-osseous lesions containing a large number of osteoclasts. Mutations in the SH3-domain binding protein 2 (SH3BP2) are responsible for cherubism. Analysis of the knock-in (KI) mouse model of cherubism showed that homozygous cherubism mice (Sh3bp2KI/KI) spontaneously develop systemic autoinflammation and inflammatory bone loss and that cherubism is a TNF-α-dependent hematopoietic disorder. In this study, we investigated whether bone marrow transplantation (BMT) is effective for the treatment of inflammation and bone loss in Sh3bp2KI/KI mice. Bone marrow (BM) cells from wild-type (Sh3bp2+/+) mice were transplanted to 6-week-old Sh3bp2KI/KI mice with developing inflammation and to 10-week-old Sh3bp2KI/KI mice with established inflammation. Six-week-old Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells exhibited improved body weight loss, facial swelling, and survival rate. Inflammatory lesions in the liver and lung as well as bone loss in calvaria and mandibula were ameliorated at 10 weeks after BMT compared to Sh3bp2KI/KI mice transplanted with Sh3bp2KI/KI BM cells. Elevation of serum TNF-α levels was not detected after BMT. BMT was effective for up to 20 weeks in 6-week-old Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells. BMT also ameliorated the inflammation and bone loss in 10-week-old Sh3bp2KI/KI mice. Thus our study demonstrates that BMT improves the inflammation and bone loss in cherubism mice. BMT may be effective for the treatment of cherubism patients.

Keywords: Cherubism, SH3BP2, Autoinflammation, Bone loss, Bone marrow transplantation

Introduction

Cherubism (OMIM#118400) is an autosomal dominant disorder characterized by bilateral multilocular cystic bone destruction in the maxilla and mandibula. The cystic bone lesions are filled with proliferative fibro-osseous tissues containing a large number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated giant cells, which lead to a disfiguring swelling of the lower face. Periodontal bone destruction causes “free-floating” teeth and chewing problems [1]. Swelling of submandibular lymph nodes is also observed [2]. Affected children seem normal at birth and manifest the characteristic facial appearance at 2 to 5 years old, and the cherubism lesions usually begin to regress after puberty by mechanisms that may involve Toll-like receptor (TLR)-MYD88 pathway in macrophages [3]. In some severe cases, the lesions expand into the orbital walls or upper airway [4–7], resulting in upward gazing, optic neuropathy [7], or respiratory disturbance [8]. Currently there is no established protocol for cherubism treatment.

We have previously discovered gain-of-function heterozygous mutations in the SH3-domain binding protein 2 (SH3BP2) responsible for cherubism [9]. SH3BP2 is a signaling adaptor protein that interacts with various proteins, such as SYK [10], VAV [11], PLCγ1 and PLCγ2 [10, 12]. SH3BP2 is expressed in hematopoietic cells including T and B lymphocytes, macrophages, neutrophils, mast cells, and osteoclasts [13–17]. We created a knock-in (KI) mouse model of cherubism by introducing the most common P418R amino acid substitution in cherubism patients into the mouse Sh3bp2 gene (P416R in mice) [18]. The Sh3bp2KI/KI mice spontaneously develop systemic inflammation including in liver and lung with elevation of serum TNF-α levels and show periodontal and calvarial bone erosion caused by the inflammatory infiltrates, suggesting that cherubism is an inflammatory disorder. Inflammation in Sh3bp2KI/KI mice, which develops independently of T and B cells, is regarded as a new form of autoinflammation characterized by increased cytokine responsiveness of macrophages [19], and it starts to develop at around 1 week of age [20]. Genetic deletion of TNF-α in Sh3bp2KI/KI mice shows a significant rescue of inflammation, suggesting that TNF-α plays a key role in the pathogenesis of inflammation in Sh3bp2KI/KI mice. In fact, TNF-α is expressed in both multinucleated giant cells and stromal cells in human cherubism lesions [21, 22]. These results led us to investigate whether postnatal administration of anti-TNF-α drug is able to prevent or ameliorate the symptoms in Sh3bp2KI/KI mice, which is more relevant to clinical cases of human cherubism. We found that postnatal administration of a TNF-α blocker, etanercept, is able to prevent the development of inflammation when administered to the inflammation-free neonatal (1 week old) Sh3bp2KI/KI mice [20]. However, inflammation recurs after discontinuation of etanercept treatment. In contrast, administration of etanercept to 10-week-old Sh3bp2KI/KI mice with fully active inflammation showed only a limited effect [20]. These results suggest that anti-TNF-α therapy may be effective in young cherubism patients at the early stages of the disease before the onset of lesion formation, but not in patients after the onset of active lesion formation.

Bone marrow transplantation (BMT) has been used for the treatment of a wide range of human diseases such as leukemia [23], lymphoma [24], aplastic anemia [25], Krabbe disease [26], Hurler syndrome [27–29], Gaucher disease [28], and Wiskott-Aldrich syndrome [30, 31]. Since our previous study on the Sh3bp2KI/KI mice demonstrated that cherubism is a hematopoietic disorder of myeloid lineage cells that causes autoinflammation [18] and abundant active inflammatory cells in human cherubism lesions are reported [32], we hypothesized that transplantation of wild-type (Sh3bp2+/+) bone marrow (BM) cells may be able to ameliorate the actively growing or established inflammation in Sh3bp2KI/KI mice that cannot be ameliorated by etanercept treatment. Here, we show that transplantation of Sh3bp2+/+ BM cells to Sh3bp2KI/KI mice rescues systemic inflammation and inflammatory bone loss in Sh3bp2KI/KI cherubism mice with actively growing and established inflammatory lesions.

Materials and Methods

Animals

Cherubism mice (Sh3bp2KI/KI) in C57BL/6 background were described previously [18]. Briefly, the most common mutation in human cherubism patients (P418R) was knocked-in to the mouse Sh3bp2 gene (P416R in mouse) by homologous recombination. The Sh3bp2+/+ or Sh3bp2KI/KI mice in C57BL/6 background mice were used as donors and recipients of BMT. All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Missouri-Kansas City.

Bone marrow (BM) cell transplantation

Twenty-four hours before transplantation, recipient mice were intraperitoneally injected with busulfan (25 mg/kg in 1:1 ratio of PBS/DMSO for 10-week-old recipients and 50 mg/kg for 6-week-old recipients, Sigma-Aldrich, St. Louis, MO) for myeloablation. BM cells were flushed out with Hank’s Balanced Salt Solution (HBSS) from tibias and femurs of 6-week-old donor mice. The cells were filtrated with 70 μm nylon mesh (BD Biosciences, San Jose, CA), and red blood cells were lysed with RBC lysis solution (eBioscience, San Diego, CA). 1 × 107 cells in HBSS were injected intravenously to the busulfan-conditioned recipient mice. Water supplemented with sulfamethoxazole/trimethoprim (800 and 160 mg/l, respectively, Hi-Tech Pharmacal, Amityville, NY) was given for 2 weeks after busulfan treatment to prevent infections in the recipient mice.

Body weight measurement, assessment of facial swelling, and enzyme-linked immunosorbent assay (ELISA)

Body weights were recorded after BMT. At 10 or 20 weeks after transplantation, facial swelling was assessed by two independent observers in a blinded manner as previously described [20]. Blood samples were collected at the time of necropsy. Serum TNF-α levels were measured by ELISA in accordance with the manufacturer’s instruction (R&D, Minneapolis, MN).

Evaluation of chimerism

Chimerism of BM cells in recipient mice at the time of analysis was determined by semi-quantitative genomic PCR detecting mouse Sh3bp2 using genomic DNAs isolated from whole BM cells. Primers for Sh3bp2 are as follows: Forward: 5′-CTTGGAGACTGGGCTTAAGAGGAC - 3′ and Reverse: 5′-TAATACTGGTAAGCAGGGGTGCTG - 3′ (product size: 262bp for wild-type, 303bp for mutant). Control samples for the standard curve creation were prepared by mixing homozygous mutant and wild-type genomic DNAs at various ratios (0 to 100% of homozygous mutant DNA with 10% increments). PCR products were separated on an agarose gel and a digital image was taken. The intensity of DNA bands (INT) for mutant (INTKI), wild-type (INTWT), and background (INTBgd) were acquired using ImageJ (NIH). The ratio (R) of adjusted INT for sample (INTRSample) and control (INTRControl) were calculated by the following equation: INTR = (INTKI – INTBgd)/(INTWT – INTBgd). Standard curves were made using values of INTRControl at different mix ratios. Percentage of chimerism in each genomic DNA from bone marrow cells was calculated from the standard curve and INTRSample. Sh3bp2KI/KI recipient mice transplanted with Sh3bp2+/+ BM cells with less than 70% chimerism at the time of analysis were excluded from the study. When genotype of recipients is identical to donor cells, the chimerism percentage was regarded as 100%.

Tissue preparation, staining, and histomorphometric analysis

Liver and lung tissues were fixed with 4% paraformaldehyde/PBS, embedded in paraffin, and 6-μm-thick sections were stained by hematoxylin and eosin (H&E). To quantify the inflammation in liver tissues, digital images of the stained liver sections were taken with a 4x objective lens. Areas occupied by inflammatory cells (AreaInf) and by blood vessels (AreaVessels) were measured with ImageJ software as pixels. Inflammatory area in the liver (%) was calculated by the following equation: (total inflammatory area) = 100 x (total AreaInf)/((total image area) – (total AreaVessels)). An averaged value from 3 to 4 images per sample was regarded as the representative value for each individual sample. Inflammatory lung lesions on H&E image were measured by ImageJ as pixels. The equation used for the calculation of inflammatory lung area (%) is as follows: (inflammatory area in lung) = 100 x (total inflammatory area)/(total lung area).

MicroCT analysis

Paraformaldehyde-fixed mandibulae, calvariae, and tibiae were scanned with microCT (vivaCT 40, Scanco Medical, PA) with an X-ray energy of 55 kVp (145 μA). Resulting images were used to quantify bone loss. Two-dimensional images were taken with following thresholds: 300 (equivalent to 471.1 mg hydroxyapatite (HA)/cm3) for jaw and calvarial bones, 220 (equivalent to 298.2 mg HA/cm3) for trabecular bones to distinguish mineralized tissues from non-mineralized tissues, and they were processed for three-dimensional (3D) image reconstruction with a spatial resolution of 10 μm. To assess the proportion of erosion area on calvaria (%), microCT images (6 × 6 mm square) were used. The intersections of the coronal and sagittal sutures (bregma) were set as the reference positions of the square center [20]. Total area of bone erosions, including suture areas in the 6 mm × 6 mm square on calvaria, was measured as pixels with ImageJ and divided by the total pixel number of the square. Distance between the cemento-enamel junction (CEJ) and alveolar bone crest (ABC) at the distal lingual surface of the mandibular first molar was measured using reconstructed 3D images according to our previous report [20] to assess periodontal bone loss. The region of trabecular bone comprised 100 slices of secondary spongiosa beginning just below primary spongiosa. Bone properties were analyzed using Scanco bone evaluation software. Micro-CT assessments were performed according to the international guidelines [33].

Statistical analysis

Statistical analysis was performed using SPSS ver. 20 (IBM, Chicago, IL). One-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was applied for the comparisons among groups. Statistical significance was defined as P < 0.05.

RESULTS

Transplantation of Sh3bp2+/+ bone marrow cells to 6-week-old Sh3bp2KI/KI mice with actively growing inflammation rescues facial swelling, body weight loss, mortality, and elevation of serum TNF-α levels

We examined whether BMT rescues the Sh3bp2KI/KI mice that have actively developing inflammation. Six-week-old Sh3bp2KI/KI mice were selected as recipient mice, because Sh3bp2KI/KI mice start to die after 8 weeks of age due to severe systemic inflammation, which is supposed to occur after reaching the full active inflammatory phase [18]. We intravenously injected wild-type (Sh3bp2+/+) BM cells from 6-week-old mice to 6-week-old Sh3bp2KI/KI mice (Fig. 1A). The Sh3bp2KI/KI recipients transplanted with Sh3bp2+/+ BM cells were followed up for 10 or 20 weeks and compared to Sh3bp2+/+ mice transplanted with Sh3bp2+/+ BM cells (non-inflamed controls) and Sh3bp2KI/KI inflamed control mice. As inflamed controls, we used Sh3bp2KI/KI mice transplanted with Sh3bp2KI/KI BM cells for a 10-week follow-up and Sh3bp2KI/KI mice without treatment for a 20-week follow-up because our pilot study showed that all three Sh3bp2KI/KI mice transplanted with Sh3bp2KI/KI BM cells died during the 20-week follow-up period (Fig. 1D), which is consistent with the previous report that more than 70% of Sh3bp2KI/KI mice die by 30 weeks of age due to systemic inflammations [18].

Fig. 1.

Fig. 1

Open in a new tab

Transplantation of Sh3bp2+/+ bone marrow (BM) cells to 6-week-old Sh3bp2KI/KI mice improves facial swelling, loss of body weight, survival rate, and elevation of serum TNF-α levels. (A) Experimental procedure of bone marrow transplantation (BMT). Six-week-old Sh3bp2+/+ and Sh3bp2KI/KI mice were intravenously injected with BM cells isolated from 6-week-old mice. Ten or 20 weeks after BMT, namely at 16- or 26-week-old, respectively, recipient mice were analyzed. (B) Facial appearance of the recipient mice at 10 (top) or 20 weeks (bottom) after BMT. Twenty-six-week-old Sh3bp2KI/KI mice without BMT were used as controls for Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells that were followed-up for 20 weeks (center, bottom). (C) Body weight changes after BMT. (D) Survival rate after BMT. (E) TNF-α levels in serum from recipient mice followed up for 10 weeks (left) or for 20 weeks (right). Numbers in parentheses represent the number of mice measured. Error bars in (C) represent ± SEM. Horizontal bars in (E) represent the mean values. *P < 0.05. WT to WT: Sh3bp2+/+ mice transplanted with Sh3bp2+/+ BM cells. KI to KI: Sh3bp2KI/KI mice transplanted with Sh3bp2KI/KI BM cells. WT to KI: Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells.

At 10 weeks after BMT, all Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells showed a rescue of facial swelling with open eyelids (Fig. 1B right, top, n = 9/9), while none of Sh3bp2KI/KI mice transplanted with Sh3bp2KI/KI BM cells showed a similar rescue (Fig. 1B center, top, n = 0/8). The rescue of facial swelling was observed at 20 weeks after the BMT in 88.9% of Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells (Fig. 1B right, bottom, n = 8/9), while none of 26-week-old Sh3bp2KI/KI mice without BMT exhibited an improvement of severe eyelid closure (Fig. 1B center, bottom, n = 0/9). Consistent with these observations, loss of body weight was improved in the Sh3bp2KI/KI mutants transplanted with Sh3bp2+/+ BM cells in both males and females (Fig. 1C, red lines) compared to Sh3bp2KI/KI mice without BMT (Fig. 1C, green lines).

All Sh3bp2KI/KI mutants transplanted with Sh3bp2+/+ BM cells survived for 20 weeks after BMT (26 weeks of age, n = 9/9, Fig. 1D, red line), while 56.3 % of non-transplanted Sh3bp2KI/KI mice survived to 26 weeks of age (n = 9/16, Fig. 1D, green line). Serum TNF-α levels in Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells at 10 and 20 weeks after BMT were significantly reduced and practically undetectable in all cases compared to Sh3bp2KI/KI mice transplanted with Sh3bp2KI/KI BM cells or Sh3bp2KI/KI mice without transplantation, respectively (Fig. 1E). Taken together, these results demonstrate that transplantation of wild-type BM cells rescues facial swelling, body weight loss, reduction in survival rate, and elevation of serum TNF-α levels in Sh3bp2KI/KI mice by 10 weeks after BMT, and the rescue continues for at least 20 weeks after BMT.

Transplantation of Sh3bp2+/+ bone marrow cells to 6-week-old Sh3bp2KI/KI mice with actively growing inflammation improves liver and lung inflammation

To evaluate whether inflammation is rescued in internal organs in Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells, liver and lung tissue sections were stained by H&E, and the inflammatory lesions were quantified at 10 and 20 weeks after BMT. Liver inflammatory lesions in Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells were significantly reduced at 10 weeks after BMT (Fig. 2A, right, top) compared to Sh3bp2KI/KI mice transplanted with Sh3bp2KI/KI BM cells (Fig. 2A, center, top), and the reduced liver inflammation continued until 20 weeks after transplantation (Fig. 2A, right, bottom). Histomorphometric analysis confirmed the reduction of liver inflammation (Fig. 2B). Consistent with the improvement of liver lesions, inflammatory lesions in lung tissues were significantly reduced in Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells (Fig. 2C), which was verified by histomorphometric analysis (Fig. 2D). These results indicate that transplantation of Sh3bp2+/+ BM cells is effective for the reduction of inflammatory lesions in liver and lung in Sh3bp2KI/KI mice.

Fig. 2.

Fig. 2

Open in a new tab

Transplantation of Sh3bp2+/+ BM cells to 6-week-old Sh3bp2KI/KI mice reduces infiltration of inflammatory cells in liver and lung. (A) Images of liver tissue in recipient mice at 16-week-old (10 weeks after BMT, top) and 26-week-old (20 weeks after BMT, bottom). Arrowheads indicate the inflammatory cell infiltration. Scale bar = 500 μm. H&E staining. (B) Quantitative analysis of inflammation in liver. Data from 10-week follow-up (left) and 20-week follow-up (right) (n = 7–9). (C) Images of lung tissue. Panel order corresponds to (A). Arrowheads indicate the nodule formation by inflammatory cells. Scale bar = 500 μm. H&E staining. (D) Quantitative analysis of inflammation in lung. Data from 10-week follow-up (left) and 20-week follow-up (right) (n = 7–9). Error bars represent SD. *P < 0.05. WT to WT: Sh3bp2+/+ mice transplanted with Sh3bp2+/+ BM cells. KI to KI: Sh3bp2KI/KI mice transplanted with Sh3bp2KI/KI BM cells. WT to KI: Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells.

BMT with Sh3bp2+/+ cells to 6-week-old Sh3bp2KI/KI mice rescues inflammatory bone loss

To examine the effect of BMT on bone loss in Sh3bp2KI/KI mice, we evaluated calvarial and periodontal bones using 3D reconstructed microCT images. Inflammatory bone loss on the surface of calvaria was detected as multiple erosion pit formation in Sh3bp2KI/KI mice transplanted with Sh3bp2KI/KI BM cells (Fig. 3A, center panel, arrowheads). Quantitation of areas of calvarial bone erosion showed that Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells exhibit a significant reduction in erosion pit formation at both 10 and 20 weeks after BMT (Fig. 3B).

Fig. 3.

Fig. 3

Open in a new tab

Transplantation of Sh3bp2+/+ BM cells to 6-week-old Sh3bp2KI/KI mice improves inflammatory bone loss. (A) MicroCT images of calvaria. Upper panels: gross images of dorsal skull of the recipient mice at 10 weeks after BMT (16-week-old). Lower panels: enlarged 6 × 6 mm area on calvaria with the bregma at center. Arrowheads indicate erosion pits. (B) Quantitative measurement of calvarial bone defects. Data from 10-week follow-up (left) and 20-week follow-up (right) (n = 6–9). (C) MicroCT images. Top: Gross image of jaw and craniofacial bones from lingual side (left) and location of the CEJ and ABC (right) (16-week-old Sh3bp2+/+ mouse). Bottom: Images of periodontal bone in 16-week-old recipient mice (10 weeks after BMT). Arrowheads indicate erosion pits. Scale bar = 500 μm. (D) Quantitative measurement of periodontal bone loss. Distance between CEJ and ABC at the distal lingual side of the first molar was measured. Data from 10-week follow-up (left) and 20-week follow-up (right) (n = 6–9). (E) Reconstructed microCT images of trabecular bone and quantitative measurement of trabecular bone volume per total volume (BV/TV) at proximal tibia at 20 weeks after BMT (n = 3–4). Scale bar = 1 mm. Error bars represent SD. *P < 0.05. WT to WT: Sh3bp2+/+ mice transplanted with Sh3bp2+/+ BM cells. KI to KI: Sh3bp2KI/KI mice transplanted with Sh3bp2KI/KI BM cells. WT to KI: Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells. CEJ: cementoenamel junction. ABC: alveolar bone crest.

Next, we assessed the periodontal bone loss in Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells using reconstructed 3D microCT images. Consistent with the rescue from calvarial bone erosion in Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells, transplantation of Sh3bp2+/+ BM cells reduced erosion pit formation on the surface of mandibula at 10 weeks after BMT (Fig. 3C, right, bottom). To quantitate the periodontal bone loss, the distance between CEJ and ABC at the distal lingual surface of the first molar was measured. Average CEJ-ABC distance in Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells at 10 and 20 weeks after BMT was also reduced compared to 16-week-old Sh3bp2KI/KI mice transplanted with Sh3bp2KI/KI BM cells (10 weeks after BMT) or 26-week-old Sh3bp2KI/KI mice without transplantation (20 weeks after BMT), respectively (Fig. 3D). Consistent with these results, transplantation of wild-type BM cells partially, but effectively, prevented trabecular bone loss at proximal tibia (bone volume per total volume: BV/TV) in Sh3bp2KI/KI mice at 20 weeks after BMT (Fig. 3E).

Taken together, these results indicate that transplantation of Sh3bp2+/+ BM cells is effective for improving inflammatory bone loss in Sh3bp2KI/KI mice that have actively growing inflammatory lesions.

Transplantation of Sh3bp2+/+ bone marrow cells to 10-week-old Sh3bp2KI/KI mice with fully established inflammation rescues facial swelling, body weight loss, mortality, and elevation of serum TNF-α levels

In human, cherubism is usually diagnosed during the full active phase in which rapid expansion of jaws occurs. Therefore, we questioned whether BMT is able to relieve the fully established phenotypes in Sh3bp2KI/KI mice, which presumably simulate the situation in human cherubism treatment. For this aim, 10-week-old Sh3bp2KI/KI mice were used as recipients for BMT, and they were followed up for 10 weeks after BMT (Fig. 4A). At 10 weeks of age, Sh3bp2KI/KI mice show swelling in the face with closed eyelids, which becomes more severe at 20 weeks of age (Fig. 4B, center). In contrast, transplantation of Sh3bp2+/+ BM cells to 10-week-old Sh3bp2KI/KI mice resulted in a partial rescue of facial swelling after 10 weeks of BMT in 42.9% (n = 3/7) of Sh3bp2KI/KI mice (Fig. 4B, right). Furthermore, 10-week-old Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells showed a significant improvement in a gain of body weight in both males and females (Fig. 4C, red lines) compared to Sh3bp2KI/KI mice without BMT (Fig. 4C, green lines). The improved weight gain in 10-week-old Sh3bp2KI/KI mice started at around 4 weeks after BMT, whereas such improvement started at around 2 weeks after BMT in 6-week-old Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells. All Sh3bp2KI/KI mice (n = 7) transplanted with Sh3bp2+/+ BM cells survived for 10 weeks after BMT (data not shown). Significant reduction of serum TNF-α levels was observed in Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells at 10 weeks after BMT compared to Sh3bp2KI/KI mice without transplantation (Fig. 4D).

Fig. 4.

Fig. 4

Open in a new tab

Transplantation of Sh3bp2+/+ BM cells to 10-week-old fully inflamed Sh3bp2KI/KI mice improves facial swelling, loss of body weight, and elevated serum TNF-α levels. (A) Experimental procedure of BMT. Ten-week-old Sh3bp2+/+ or Sh3bp2KI/KI mice were transplanted with BM cells and analyzed 10 weeks later. (B) Facial appearance of recipient mice (WT to WT, and WT to KI) before (top) and after (bottom) BMT. Sh3bp2KI/KI mouse transplanted with Sh3bp2+/+ BM cells (right) shows a partial rescue of the inflammation around eyelids compared to Sh3bp2KI/KI mouse without BMT (center). (C) Body weight changes after BMT. Numbers in parentheses represent the number of mice measured. Error bars represent ± SEM. (D) Serum TNF-α levels at 10 weeks after BMT (20 weeks old). Transplantation of Sh3bp2+/+ BM cells to fully inflamed Sh3bp2KI/KI mice reduces the TNF-α levels to undetectable levels comparable to those in “WT to WT” mice. *P < 0.05. WT to WT: Sh3bp2+/+ mice transplanted with Sh3bp2+/+ BM cells. WT to KI: Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells.

Transplantation of Sh3bp2+/+ bone marrow cells to 10-week-old Sh3bp2KI/KI mice with fully established inflammation improves inflammation in liver and lung as well as inflammatory bone loss

We examined inflammation in liver and lung in Sh3bp2KI/KI mice that were transplanted with Sh3bp2+/+ BM cells at 10 weeks of age. The transplantation greatly reduced the infiltration of inflammatory cells in both liver and lung at 10 weeks after BMT (Fig. 5A, right), whereas Sh3bp2KI/KI mice without BMT exhibited inflammatory cell accumulation (Fig. 5A, center, arrowheads). Histomorphometric analysis confirmed the significant rescue of liver and lung inflammation in Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells (Fig. 5B).

Fig. 5.

Fig. 5

Open in a new tab

Transplantation of Sh3bp2+/+ BM cells to 10-week-old fully inflamed Sh3bp2KI/KI mice reduces inflammation in liver and lung, and improves inflammatory calvarial and periodontal bone loss. (A) Liver (top) and lung (bottom) tissue images at 10 weeks after BMT. Arrowheads indicate the inflammatory cell infiltration. Scale bar = 500 μm. H&E staining. (B) Quantitation of inflammation in liver (left, n = 6–9) and lung (right, n = 6–9) tissues. (C) MicroCT images of calvarial (top) and periodontal (bottom) bones after BMT. Arrowheads indicate calvarial and jaw bone erosions. Scale bar = 500 μm. (D) Quantitative measurement of calvarial (left, n = 5–7) and periodontal (right, n = 5–7) bone loss. Distance between CEJ and ABC at the distal end of the first molar on lingual side was measured for the evaluation of periodontal bone loss. (E) Reconstructed microCT images of trabecular bone and quantitative measurement of trabecular bone volume per total volume (BV/TV) at proximal tibia at 10 weeks after BMT (n = 3–4). Scale bar = 1 mm. Error bars represent SD. *P < 0.05. WT to WT: Sh3bp2+/+ mice transplanted with Sh3bp2+/+ BM cells. WT to KI: Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells. CEJ: cementoenamel junction. ABC: alveolar bone crest.

Next, we investigated whether transplantation of Sh3bp2+/+ BM cells to 10-week-old Sh3bp2KI/KI mice was able to rescue the bone destruction in calvarial and periodontal regions. MicroCT images showed that the number of erosion pits on calvaria was reduced in Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells (Fig. 5C, right, top) compared to Sh3bp2KI/KI mice without transplantation (Fig. 5C, center, top). Erosion pit formation on the surface of mandibula was reduced in the Sh3bp2KI/KI mice (Fig. 5C, right, bottom). Quantitative analysis revealed that the rescue of calvarial bone loss in Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells is not statistically significant, but showed a trend of reduction (P = 0.11), compared to Sh3bp2KI/KI mice without transplantation. (Fig. 5D, left). Measurement of the CEJ-ABC distance showed a significant rescue of periodontal bone loss in Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells compared to Sh3bp2KI/KI mice without transplantation (Fig. 5D, right). Trabecular bone loss was also ameliorated in Sh3bp2KI/KI mice that underwent wild-type BMT (Fig. 5E). Collectively, these results suggest that transplantation of Sh3bp2+/+ BM cells effectively ameliorates the systemic inflammation associated with elevated serum TNF-α levels as well as inflammatory bone loss in Sh3bp2KI/KI mice that have fully established inflammation.

DISCUSSION

At present, standard management of cherubism symptoms is generally limited to supportive care including observation of the disease course with the expectation that lesions may regress after puberty [34]. While lesions in human cherubism are usually localized to the jaw region [35–37] and functional or cosmetic problems during the active phase of the disease differ between affected children, aggressive cases involving nasal obstruction, proptosis, or severe facial deformities have been reported [38–42]. To correct upward gazing and visional disturbance, orbital surgery may be required [43]. Corrective surgery by curettage with or without bone grafting is sometimes performed for extensively enlarged jaws [44]. Generally, such surgical intervention is considered only in severe cases and most of the surgical interventions are performed after puberty when cherubic lesions may become inactive, although mechanisms of the age-dependent phenotype regression in human cherubism patients, but not in cherubism mice, remain to be further elucidated [3]. Age-associated change in immune responses by TLRs [45, 46] might be a cause of the regression [3]. Prosthetic treatment could be applied for the missing or floating teeth [47].

Currently there is no established pharmacological treatment for cherubism, but administration of calcitonin or a TNF-α antagonist has been applied to control the disease progression during the active phase. de Lange and colleagues have reported a considerable regression of the cherubism lesions in one 11-year-old boy after daily administration of calcitonin for 15 months [48]. Etoz and colleagues have also reported one case in which cherubism lesions in the mandible significantly regressed after 30 months of calcitonin therapy [49]. On the other hand, Lannon and Earley have reported no obvious improvement by calcitonin administration [38]. Therefore, efficacy of calcitonin for cherubism treatment seems to be still controversial. Our recent study in Sh3bp2KI/KI mice demonstrated that the anti-TNF-α drug, etanercept, is able to prevent disease manifestation when administration begins at 1 week of age [20]. However, symptoms recurred after discontinuation of the etanercept treatment, suggesting that long-term anti-TNF-α therapy will be required until puberty to keep the non-active status in human cherubism patients, which may adversely cause side effects of anti-TNF-α treatment [50]. In addition, anti-TNF-α treatment was not effective for 10-week-old Sh3bp2KI/KI mice with fully active inflammation, suggesting that anti-TNF-α treatment initiated after manifestation of the symptoms may not be effective in cherubism treatment and that inflammatory cytokines other than TNF-α such as IL-1α/β are involved in the later phase of the disease [20]. In agreement with the results in Sh3bp2KI/KI mice, Hero and colleagues have reported that treatment with a TNF-α inhibitor, adalimumab, failed to introduce the regression of lesions in two cherubism patients with actively growing lesions [21]. Likewise, a case of unsuccessful treatment with adalimumab in combination with bisphosphonate has been reported [51]. Therefore, besides current treatment options, alternative approaches have been demanded.

In this study, we elucidated that BMT can be a future potential treatment option for cherubism by using a cherubism mouse model. We found that transplantation of Sh3bp2+/+ BM cells to Sh3bp2KI/KI recipient mice is able to cure both inflammation and bone loss in Sh3bp2KI/KI mice. Notably, a single injection of wild-type BM cells could induce this remission. Furthermore, BMT was effective even in treating fully inflamed Sh3bp2KI/KI mice, which were not ameliorated with anti-TNF-α therapy in mice and humans [20, 21], suggesting that wild-type BMT also corrected inflammatory cytokine levels other than TNF-α. We have previously reported that cherubism is a hematopoietic disorder of myeloid lineage cells caused by hyperactive macrophages and osteoclasts [3, 18, 22, 52]. Therefore, we assume that replacement of the mutant macrophages and osteoclasts with those carrying wild-type SH3BP2 is a main cause of the improved symptoms in Sh3bp2KI/KI mice, which resulted in the comprehensive reconstitution of the expression profile of inflammatory cytokines and osteoclast-mediated bone resorption activity in Sh3bp2KI/KI recipients. Effective rescue by BMT further suggests that contribution of non-hematopoietic Sh3bp2KI/KI stromal cells to the inflammation and bone loss is limited. Interestingly, improvement in the trabecular bone loss appears to be smaller than that in calvarial bone and jawbone loss, indicating that dysfunction of osteoblasts that is responsible for impaired mineral and matrix properties of trabecular bone in Sh3bp2KI/KI mice [53] was not rescued by BMT and that BMT primarily rescues bone loss that is associated with inflammation in Sh3bp2KI/KI mice.

We found that BMT in 6-week-old Sh3bp2KI/KI mice has more therapeutic effect when compared to 10-week-old Sh3bp2KI/KI mice in the improvement of body weight gain and liver inflammation, suggesting that BMT in the earlier phase of cherubism may be more effective. We did not see any gender-dependent differences in the BMT responses (data not shown). Because of the potential adverse effects of BMT such as graft-versus-host or graft-versus-leukemia disease [54, 55], infections [56], thrombocytopenic hemorrhage [57], and toxicities of busulfan [58], current application of BMT to humans is limited to malignant hematopoietic or severe life-threatening genetic disorders. Therefore, it is important to weigh the risks and benefits if BMT is applied to cherubism patients. Since high levels of serum TNF-α increase the risk of complications of BMT [59], a combination of BMT and anti-TNF-α therapy may also contribute to the successful treatment of cherubism. With novel genome editing technologies [60, 61], we may be able to correct the cherubism mutation in induced pluripotent stem (iPS) cells [62, 63] that are derived from cherubism patients. Transplantation of hematopoietic stem cells differentiated from patient’s own iPS cells in which cherubism mutation has been corrected would reduce the risk of graft-versus-host disease and contribute to the improvement of the benefit/risk balance in the treatment of cherubism.

In conclusion, we showed that transplantation of Sh3bp2+/+ BM cells to Sh3bp2KI/KI mice is able to improve inflammation and inflammatory bone loss in Sh3bp2KI/KI mice with developing inflammation as well as Sh3bp2KI/KI mice with fully established inflammation. Our results propose that BMT is a future potential therapeutic option for human cherubism patients, particularly for those individuals with aggressive and severe symptoms.

Highlights.

  • Bone marrow transplantation rescues adult cherubism mice from autoinflammation

  • Bone marrow transplantation improves inflammatory bone loss in adult cherubism mice

  • Bone marrow transplantation may be a therapeutic option for cherubism treatment

Acknowledgments

We thank members in the Department of Oral and Craniofacial Sciences at the University of Missouri-Kansas City, School of Dentistry for critical discussion and helpful suggestions. We are also grateful to M. Dallas and Y. Xie for their technical assistance and to R. Gallant and S. Masrani for critical reading of the manuscript. This work was supported by a grant from the National Institutes of Health (R01DE020835 to YU).

Footnotes

Conflict of interest statement

The authors have no conflicting financial interests to declare.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Contributor Information

Teruhito Yoshitaka, Email: yoshitakat@umkc.edu.

Mizuho Kittaka, Email: kittakam@umkc.edu.

Shu Ishida, Email: shu1222@hotmail.co.jp.

Noriyoshi Mizuno, Email: mizuno@hiroshima-u.ac.jp.

Tomoyuki Mukai, Email: mukait@umkc.edu.

References

  • 1.Kalantar Motamedi MH. Treatment of cherubism with locally aggressive behavior presenting in adulthood: report of four cases and a proposed new grading system. J Oral Maxillofac Surg. 1998;56:1336–42. doi: 10.1016/s0278-2391(98)90618-8. [DOI] [PubMed] [Google Scholar]
  • 2.Hyckel P, Berndt A, Schleier P, Clement JH, Beensen V, Peters H, Kosmehl H. Cherubism - new hypotheses on pathogenesis and therapeutic consequences. J Craniomaxillofac Surg. 2005;33:61–8. doi: 10.1016/j.jcms.2004.07.006. [DOI] [PubMed] [Google Scholar]
  • 3.Yoshitaka T, Mukai T, Kittaka M, Alford LM, Masrani S, Ishida S, Yamaguchi K, Yamada M, Mizuno N, Olsen BR, Reichenberger EJ, Ueki Y. Enhanced TLR-MYD88 Signaling Stimulates Autoinflammation in SH3BP2 Cherubism Mice and Defines the Etiology of Cherubism. Cell Rep. 2014;8:1752–1766. doi: 10.1016/j.celrep.2014.08.023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Hawes MJ. Cherubism and its orbital manifestations. Ophthal Plast Reconstr Surg. 1989;5:133–40. doi: 10.1097/00002341-198906000-00010. [DOI] [PubMed] [Google Scholar]
  • 5.Colombo F, Cursiefen C, Neukam FW, Holbach LM. Orbital involvement in cherubism. Ophthalmology. 2001;108:1884–8. doi: 10.1016/s0161-6420(01)00757-6. [DOI] [PubMed] [Google Scholar]
  • 6.Carroll AL, Sullivan TJ. Orbital involvement in cherubism. Clin Experiment Ophthalmol. 2001;29:38–40. doi: 10.1046/j.1442-9071.2001.00363.x. [DOI] [PubMed] [Google Scholar]
  • 7.Ahmadi AJ, Pirinjian GE, Sires BS. Optic neuropathy and macular chorioretinal folds caused by orbital cherubism. Arch Ophthalmol. 2003;121:570–3. doi: 10.1001/archopht.121.4.570. [DOI] [PubMed] [Google Scholar]
  • 8.Battaglia A, Merati A, Magit A. Cherubism and upper airway obstruction. Otolaryngol Head Neck Surg. 2000;122:573–4. doi: 10.1067/mhn.2000.103534. [DOI] [PubMed] [Google Scholar]
  • 9.Ueki Y, Tiziani V, Santanna C, Fukai N, Maulik C, Garfinkle J, Ninomiya C, doAmaral C, Peters H, Habal M, Rhee-Morris L, Doss JB, Kreiborg S, Olsen BR, Reichenberger E. Mutations in the gene encoding c-Abl-binding protein SH3BP2 cause cherubism. Nat Genet. 2001;28:125–6. doi: 10.1038/88832. [DOI] [PubMed] [Google Scholar]
  • 10.Deckert M, Tartare-Deckert S, Hernandez J, Rottapel R, Altman A. Adaptor function for the Syk kinases-interacting protein 3BP2 in IL-2 gene activation. Immunity. 1998;9:595–605. doi: 10.1016/s1074-7613(00)80657-3. [DOI] [PubMed] [Google Scholar]
  • 11.Foucault I, Le Bras S, Charvet C, Moon C, Altman A, Deckert M. The adaptor protein 3BP2 associates with VAV guanine nucleotide exchange factors to regulate NFAT activation by the B-cell antigen receptor. Blood. 2005;105:1106–13. doi: 10.1182/blood-2003-08-2965. [DOI] [PubMed] [Google Scholar]
  • 12.Jevremovic D, Billadeau DD, Schoon RA, Dick CJ, Leibson PJ. Regulation of NK cell-mediated cytotoxicity by the adaptor protein 3BP2. J Immunol. 2001;166:7219–28. doi: 10.4049/jimmunol.166.12.7219. [DOI] [PubMed] [Google Scholar]
  • 13.de la Fuente MA, Kumar L, Lu B, Geha RS. 3BP2 deficiency impairs the response of B cells, but not T cells, to antigen receptor ligation. Mol Cell Biol. 2006;26:5214–25. doi: 10.1128/MCB.00087-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Chen G, Dimitriou ID, La Rose J, Ilangumaran S, Yeh WC, Doody G, Turner M, Gommerman J, Rottapel R. The 3BP2 adapter protein is required for optimal B-cell activation and thymus-independent type 2 humoral response. Mol Cell Biol. 2007;27:3109–22. doi: 10.1128/MCB.01014-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Levaot N, Voytyuk O, Dimitriou I, Sircoulomb F, Chandrakumar A, Deckert M, Krzyzanowski PM, Scotter A, Gu S, Janmohamed S, Cong F, Simoncic PD, Ueki Y, La Rose J, Rottapel R. Loss of Tankyrase-mediated destruction of 3BP2 is the underlying pathogenic mechanism of cherubism. Cell. 2011;147:1324–39. doi: 10.1016/j.cell.2011.10.045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Chen G, Dimitriou I, Milne L, Lang KS, Lang PA, Fine N, Ohashi PS, Kubes P, Rottapel R. The 3BP2 adapter protein is required for chemoattractant-mediated neutrophil activation. J Immunol. 2012;189:2138–50. doi: 10.4049/jimmunol.1103184. [DOI] [PubMed] [Google Scholar]
  • 17.Ainsua-Enrich E, Alvarez-Errico D, Gilfillan AM, Picado C, Sayos J, Rivera J, Martin M. The adaptor 3BP2 is required for early and late events in FcepsilonRI signaling in human mast cells. J Immunol. 2012;189:2727–34. doi: 10.4049/jimmunol.1200380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Ueki Y, Lin CY, Senoo M, Ebihara T, Agata N, Onji M, Saheki Y, Kawai T, Mukherjee PM, Reichenberger E, Olsen BR. Increased myeloid cell responses to M-CSF and RANKL cause bone loss and inflammation in SH3BP2 “cherubism” mice. Cell. 2007;128:71–83. doi: 10.1016/j.cell.2006.10.047. [DOI] [PubMed] [Google Scholar]
  • 19.Masters SL, Simon A, Aksentijevich I, Kastner DL. Horror autoinflammaticus: the molecular pathophysiology of autoinflammatory disease (*) Annu Rev Immunol. 2009;27:621–68. doi: 10.1146/annurev.immunol.25.022106.141627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Yoshitaka T, Ishida S, Mukai T, Kittaka M, Reichenberger E, Ueki Y. Etanercept Administration to Neonatal SH3BP2 Knock-In Cherubism Mice Prevents TNF-α-induced Inflammation and Bone Loss. J Bone Miner Res. 2014;29:1170–1182. doi: 10.1002/jbmr.2125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Hero M, Suomalainen A, Hagstrom J, Stoor P, Kontio R, Alapulli H, Arte S, Toiviainen-Salo S, Lahdenne P, Makitie O. Anti-tumor necrosis factor treatment in cherubism--clinical, radiological and histological findings in two children. Bone. 2013;52:347–53. doi: 10.1016/j.bone.2012.10.003. [DOI] [PubMed] [Google Scholar]
  • 22.Mukai T, Ishida S, Ishikawa R, Yoshitaka T, Kittaka M, Gallant R, Lin YL, Rottapel R, Brotto M, Reichenberger EJ, Ueki Y. SH3BP2 Cherubism Mutation Potentiates TNF-alpha-Induced Osteoclastogenesis Via NFATc1 and TNF-alpha-Mediated Inflammatory Bone Loss. J Bone Miner Res. 2014 doi: 10.1002/jbmr.2295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Burnett AK, Goldstone AH, Stevens RM, Hann IM, Rees JK, Gray RG, Wheatley K. Randomised comparison of addition of autologous bone-marrow transplantation to intensive chemotherapy for acute myeloid leukaemia in first remission: results of MRC AML 10 trial. UK Medical Research Council Adult and Children’s Leukaemia Working Parties. Lancet. 1998;351:700–8. doi: 10.1016/s0140-6736(97)09214-3. [DOI] [PubMed] [Google Scholar]
  • 24.Philip T, Guglielmi C, Hagenbeek A, Somers R, Van der Lelie H, Bron D, Sonneveld P, Gisselbrecht C, Cahn JY, Harousseau JL, et al. Autologous bone marrow transplantation as compared with salvage chemotherapy in relapses of chemotherapy-sensitive non-Hodgkin’s lymphoma. N Engl J Med. 1995;333:1540–5. doi: 10.1056/NEJM199512073332305. [DOI] [PubMed] [Google Scholar]
  • 25.Young NS, Bacigalupo A, Marsh JC. Aplastic anemia: pathophysiology and treatment. Biol Blood Marrow Transplant. 2010;16:S119–25. doi: 10.1016/j.bbmt.2009.09.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Escolar ML, Poe MD, Provenzale JM, Richards KC, Allison J, Wood S, Wenger DA, Pietryga D, Wall D, Champagne M, Morse R, Krivit W, Kurtzberg J. Transplantation of umbilical-cord blood in babies with infantile Krabbe’s disease. N Engl J Med. 2005;352:2069–81. doi: 10.1056/NEJMoa042604. [DOI] [PubMed] [Google Scholar]
  • 27.Hobbs JR, Hugh-Jones K, Barrett AJ, Byrom N, Chambers D, Henry K, James DC, Lucas CF, Rogers TR, Benson PF, Tansley LR, Patrick AD, Mossman J, Young EP. Reversal of clinical features of Hurler’s disease and biochemical improvement after treatment by bone-marrow transplantation. Lancet. 1981;2:709–12. doi: 10.1016/s0140-6736(81)91046-1. [DOI] [PubMed] [Google Scholar]
  • 28.Krivit W, Peters C, Shapiro EG. Bone marrow transplantation as effective treatment of central nervous system disease in globoid cell leukodystrophy, metachromatic leukodystrophy, adrenoleukodystrophy, mannosidosis, fucosidosis, aspartylglucosaminuria, Hurler, Maroteaux-Lamy, and Sly syndromes, and Gaucher disease type III. Curr Opin Neurol. 1999;12:167–76. doi: 10.1097/00019052-199904000-00007. [DOI] [PubMed] [Google Scholar]
  • 29.Souillet G, Guffon N, Maire I, Pujol M, Taylor P, Sevin F, Bleyzac N, Mulier C, Durin A, Kebaili K, Galambrun C, Bertrand Y, Froissart R, Dorche C, Gebuhrer L, Garin C, Berard J, Guibaud P. Outcome of 27 patients with Hurler’s syndrome transplanted from either related or unrelated haematopoietic stem cell sources. Bone Marrow Transplant. 2003;31:1105–17. doi: 10.1038/sj.bmt.1704105. [DOI] [PubMed] [Google Scholar]
  • 30.Bach FH, Albertini RJ, Joo P, Anderson JL, Bortin MM. Bone-marrow transplantation in a patient with the Wiskott-Aldrich syndrome. Lancet. 1968;2:1364–6. doi: 10.1016/s0140-6736(68)92672-x. [DOI] [PubMed] [Google Scholar]
  • 31.Ozsahin H, Cavazzana-Calvo M, Notarangelo LD, Schulz A, Thrasher AJ, Mazzolari E, Slatter MA, Le Deist F, Blanche S, Veys P, Fasth A, Bredius R, Sedlacek P, Wulffraat N, Ortega J, Heilmann C, O’Meara A, Wachowiak J, Kalwak K, Matthes-Martin S, Gungor T, Ikinciogullari A, Landais P, Cant AJ, Friedrich W, Fischer A. Long-term outcome following hematopoietic stem-cell transplantation in Wiskott-Aldrich syndrome: collaborative study of the European Society for Immunodeficiencies and European Group for Blood and Marrow Transplantation. Blood. 2008;111:439–45. doi: 10.1182/blood-2007-03-076679. [DOI] [PubMed] [Google Scholar]
  • 32.Penarrocha M, Bonet J, Minguez JM, Bagan JV, Vera F, Minguez I. Cherubism: a clinical, radiographic, and histopathologic comparison of 7 cases. J Oral Maxillofac Surg. 2006;64:924–30. doi: 10.1016/j.joms.2006.02.003. [DOI] [PubMed] [Google Scholar]
  • 33.Bouxsein ML, Boyd SK, Christiansen BA, Guldberg RE, Jepsen KJ, Muller R. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J Bone Miner Res. 2010;25:1468–86. doi: 10.1002/jbmr.141. [DOI] [PubMed] [Google Scholar]
  • 34.Silva GC, Gomez RS, Vieira TC, Silva EC. Cherubism: long-term follow-up of 2 patients in whom it regressed without treatment. Br J Oral Maxillofac Surg. 2007;45:567–70. doi: 10.1016/j.bjoms.2006.08.013. [DOI] [PubMed] [Google Scholar]
  • 35.Von Wowern N. Cherubism: a 36-year long-term follow-up of 2 generations in different families and review of the literature. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2000;90:765–72. doi: 10.1067/moe.2000.108438. [DOI] [PubMed] [Google Scholar]
  • 36.Papadaki ME, Lietman SA, Levine MA, Olsen BR, Kaban LB, Reichenberger EJ. Cherubism: best clinical practice. Orphanet J Rare Dis. 2012;7(Suppl 1):S6. doi: 10.1186/1750-1172-7-S1-S6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Prescott T, Redfors M, Rustad CF, Eiklid KL, Geirdal AO, Storhaug K, Jensen JL. Characterization of a Norwegian cherubism cohort; molecular genetic findings, oral manifestations and quality of life. Eur J Med Genet. 2013;56:131–7. doi: 10.1016/j.ejmg.2012.12.008. [DOI] [PubMed] [Google Scholar]
  • 38.Lannon DA, Earley MJ. Cherubism and its charlatans. Br J Plast Surg. 2001;54:708–11. doi: 10.1054/bjps.2001.3701. [DOI] [PubMed] [Google Scholar]
  • 39.Wang CN, Song YL, Peng B, Lu DH, Fan MW, Li J, Ye XQ, Fan HL, Bian Z. The aggressive form of cherubism: report of two cases in unrelated families. Br J Oral Maxillofac Surg. 2006;44:322–4. doi: 10.1016/j.bjoms.2005.10.003. [DOI] [PubMed] [Google Scholar]
  • 40.Pontes FS, Ferreira AC, Kato AM, Pontes HA, Almeida DS, Rodini CO, Pinto DS., Jr Aggressive case of cherubism: 17-year follow-up. Int J Pediatr Otorhinolaryngol. 2007;71:831–5. doi: 10.1016/j.ijporl.2007.01.017. [DOI] [PubMed] [Google Scholar]
  • 41.Carvalho VM, Perdigao PF, Pimenta FJ, de Souza PE, Gomez RS, De Marco L. A novel mutation of the SH3BP2 gene in an aggressive case of cherubism. Oral Oncol. 2008;44:153–5. doi: 10.1016/j.oraloncology.2007.01.012. [DOI] [PubMed] [Google Scholar]
  • 42.Reddy G, Reddy GS, Reddy NS, Badam RK. Aggressive form of cherubism. J Clin Imaging Sci. 2012;2:8. doi: 10.4103/2156-7514.93275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Raposo-Amaral CE, de Campos Guidi M, Warren SM, Almeida AB, Amstalden EM, Tiziane V, Raposo-Amaral CM. Two-stage surgical treatment of severe cherubism. Ann Plast Surg. 2007;58:645–51. doi: 10.1097/01.sap.0000248141.36904.19. [DOI] [PubMed] [Google Scholar]
  • 44.Meng XM, Yu SF, Yu GY. Clinicopathologic study of 24 cases of cherubism. Int J Oral Maxillofac Surg. 2005;34:350–6. doi: 10.1016/j.ijom.2004.09.006. [DOI] [PubMed] [Google Scholar]
  • 45.Kollmann TR, Levy O, Montgomery RR, Goriely S. Innate immune function by Toll-like receptors: distinct responses in newborns and the elderly. Immunity. 2012;37:771–83. doi: 10.1016/j.immuni.2012.10.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Shaw AC, Goldstein DR, Montgomery RR. Age-dependent dysregulation of innate immunity. Nat Rev Immunol. 2013;13:875–87. doi: 10.1038/nri3547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Yilmaz B, Ozan O, Karaagaclioglu L, Ersoy AE. A prosthetic treatment approach for a cherubism patient: A clinical report. J Prosthet Dent. 2006;96:313–6. doi: 10.1016/j.prosdent.2006.09.014. [DOI] [PubMed] [Google Scholar]
  • 48.de Lange J, van den Akker HP, Scholtemeijer M. Cherubism treated with calcitonin: report of a case. J Oral Maxillofac Surg. 2007;65:1665–7. doi: 10.1016/j.joms.2006.06.266. [DOI] [PubMed] [Google Scholar]
  • 49.Etoz OA, Dolanmaz D, Gunhan O. Treatment of cherubism with salmon calcitonin: a case report. Eur J Dent. 2011;5:486–91. [PMC free article] [PubMed] [Google Scholar]
  • 50.Scheinfeld N. A comprehensive review and evaluation of the side effects of the tumor necrosis factor alpha blockers etanercept, infliximab and adalimumab. J Dermatolog Treat. 2004;15:280–94. doi: 10.1080/09546630410017275. [DOI] [PubMed] [Google Scholar]
  • 51.Pagnini I, Simonini G, Mortilla M, Giani T, Pascoli L, Cimaz R. Ineffectiveness of tumor necrosis factor-alpha inhibition in association with bisphosphonates for the treatment of cherubism. Clin Exp Rheumatol. 2011;29:147. [PubMed] [Google Scholar]
  • 52.Mukai T, Gallant R, Ishida S, Yoshitaka T, Kittaka M, Nishida K, Fox DA, Morita Y, Ueki Y. SH3BP2 Gain-Of-Function Mutation Exacerbates Inflammation and Bone Loss in a Murine Collagen-Induced Arthritis Model. PLoS One. 2014;9:e105518. doi: 10.1371/journal.pone.0105518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Wang CJ, Chen IP, Koczon-Jaremko B, Boskey AL, Ueki Y, Kuhn L, Reichenberger EJ. Pro416Arg cherubism mutation in Sh3bp2 knock-in mice affects osteoblasts and alters bone mineral and matrix properties. Bone. 2010;46:1306–15. doi: 10.1016/j.bone.2010.01.380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Horowitz MM, Gale RP, Sondel PM, Goldman JM, Kersey J, Kolb HJ, Rimm AA, Ringden O, Rozman C, Speck B, et al. Graft-versus-leukemia reactions after bone marrow transplantation. Blood. 1990;75:555–62. [PubMed] [Google Scholar]
  • 55.Ferrara JL, Levine JE, Reddy P, Holler E. Graft-versus-host disease. Lancet. 2009;373:1550–61. doi: 10.1016/S0140-6736(09)60237-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Mossad SB, Longworth DL, Goormastic M, Serkey JM, Keys TF, Bolwell BJ. Early infectious complications in autologous bone marrow transplantation: a review of 219 patients. Bone Marrow Transplant. 1996;18:265–71. [PubMed] [Google Scholar]
  • 57.Roy V, Rizvi MA, Vesely SK, George JN. Thrombotic thrombocytopenic purpura-like syndromes following bone marrow transplantation: an analysis of associated conditions and clinical outcomes. Bone Marrow Transplant. 2001;27:641–6. doi: 10.1038/sj.bmt.1702849. [DOI] [PubMed] [Google Scholar]
  • 58.Ohnishi K, Ohno R, Tomonaga M, Kamada N, Onozawa K, Kuramoto A, Dohy H, Mizoguchi H, Miyawaki S, Tsubaki K, et al. A randomized trial comparing interferon-alpha with busulfan for newly diagnosed chronic myelogenous leukemia in chronic phase. Blood. 1995;86:906–16. [PubMed] [Google Scholar]
  • 59.Holler E, Kolb HJ, Moller A, Kempeni J, Liesenfeld S, Pechumer H, Lehmacher W, Ruckdeschel G, Gleixner B, Riedner C, et al. Increased serum levels of tumor necrosis factor alpha precede major complications of bone marrow transplantation. Blood. 1990;75:1011–6. [PubMed] [Google Scholar]
  • 60.Bogdanove AJ, Voytas DF. TAL effectors: customizable proteins for DNA targeting. Science. 2011;333:1843–6. doi: 10.1126/science.1204094. [DOI] [PubMed] [Google Scholar]
  • 61.Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. RNA-guided human genome engineering via Cas9. Science. 2013;339:823–6. doi: 10.1126/science.1232033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663–76. doi: 10.1016/j.cell.2006.07.024. [DOI] [PubMed] [Google Scholar]
  • 63.Cherry AB, Daley GQ. Reprogrammed cells for disease modeling and regenerative medicine. Annu Rev Med. 2013;64:277–90. doi: 10.1146/annurev-med-050311-163324. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES