Chronic axial compression of the mouse tail segment induces MRI bone marrow edema changes that correlate with increased marrow vasculature and cellularity

Magnetic resonance imaging (MRI) of bone marrow edema (BME) has been found to be helpful in the diagnosis of back pain attributed to degenerative disk disease (DDD) and spondyloarthropathy (SA), but its interpretation is limited by a lack of knowledge of its nature and natural history. We assessed effects of compressive forces to mouse tail segments of WT and TNF‐Tg mice with SA, via contrast enhanced‐MRI and histology. Normalized marrow contrast enhancement (NMCE) of uninstrumented WT vertebrae significantly decrease, threefold (p < 0.01) from 8 to 12 weeks of age, while the NMCE of TNF‐Tg vertebrae remained elevated. Compressive loading (6× body weight) increased NMCE twofold (p < 0.02) within 2 weeks in WT tails, which was equal to 6× loaded TNF‐Tg tails within 4 weeks. Histology confirmed degenerative changes and that load‐induced NMCE corresponded to increased vascular sinus tissue (35 ± 3% vs. 19 ± 3%; p < 0.01) and cellularity (4,235 ± 886 vs.1,468 ± 320 cells/mm²; p < 0.01) for the loaded versus unloaded WT, respectively. However, micro‐computed tomography (CT) analyses failed to detect significant load‐induced changes to bone. While the bone marrow of loaded WT and TNF‐Tg vertebrae were similar, histology demonstrated mild cellular infiltrate and increased osteoclastic resorption in the WT tails versus severe inflammatory‐erosive arthritis in TNF‐Tg joints. Significant (p < 0.05) decreases in cortical and trabecular bone volume in uninstrumented TNF‐Tg versus WT vertebrae were confirmed by micro‐CT. Thus, chronic load‐induced DDD causes BME signals in vertebrae similar to those observed from SA, and both DDD and SA signals correlate with a conversion from yellow to red marrow, with increased vascularity. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1220–1228, 2010     

Targeting Notch‐Activated M1 Macrophages Attenuates Joint Tissue Damage in a Mouse Model of Inflammatory Arthritis

Expression levels of Notch signaling molecules are increased in synovium from patients with rheumatoid arthritis (RA). However, it is not known which cell type(s) in RA synovium have Notch activation or if they play a pathogenetic role in RA. Here, we used Hes1‐GFP/TNF‐transgenic (TNF‐Tg) mice to investigate the role of cells with active Notch signaling (GFP+) in RA. The number of GFP+ cells was significantly increased in synovium in Hes1‐GFP/TNF‐Tg mice and about 60% of them were F4/80+ macrophages expressing the inflammatory macrophage (M1) marker. TNF‐Tg mice transplanted with Hes1‐GFP/TNF‐Tg bone marrow (BM) had significantly more GFP+ cells in their synovium than in BM. Intraarticular injection of Hes1‐GFP/TNF‐Tg or Hes1‐GFP+ BM macrophages into WT and TNF‐Tg mice showed the highest synovial GFP+ cells in the TNF‐Tg mice that received Hes1‐GFP/TNF‐Tg cells. Thapsigargin (THAP), a Notch inhibitor, decreased TNF‐induced M1 and increased M2 numbers and reduced joint lesion, synovial M1s, and GFP+ cells in Hes1‐GFP/TNF‐Tg mice. THAP did not affect M1s from mice carrying a constitutively active Notch1. Thus, the main cells with activated Notch signaling in the inflamed synovium of TNF‐Tg mice are M1s derived from BM and targeting them may represent a new therapeutic approach for patients with inflammatory arthritis. © 2017 American Society for Bone and Mineral Research.     

Lipopolysaccharide-induced bone resorption is increased in TNF type 2 receptor-deficient mice in vivo

The release of tumor necrosis factor (TNF)-alpha from macrophages upon stimulation of lipopolysaccharide (LPS) is a major etiological factor of inflammatory bone disease and elicits the effects through TNF receptors type 1 and 2. Given the importance of TNF-alpha action on osteoclastic bone resorption, the role of TNF type 2 receptor (TNFR2) on bone resorption has not been elucidated well so far. The purpose of this study is to investigate the role of TNFR2 on LPS-induced inflammatory bone resorption in vivo. LPS at 10 mg/kg (Re 595) was injected subcutaneously on calvariae of wild-type (WT), TNF type 1 receptor (TNFR1)-deficient (KO), and TNFR2 KO mice, killed on day 5 after the LPS injection. The calvarial bone mineral density (BMD) was significantly decreased by LPS compared to the vehicle-injected control in WT mice, but not in TNFR1 KO mice. Interestingly, the decrease of calvarial BMD and the increase of the osteoclast number by LPS in TNFR2 KO mice seemed to be more than those in WT mice. Furthermore, the significant decrease by LPS on the BMD of tibiae, femurs, and lumber vertebrae were observed only in TNFR2 KO mice. Histomorphometric analysis of tibiae showed the significant increases of osteoclast number and surface in the LPS-injected TNFR2 KO mice, and the levels of urinary deoxypyridinoline reflected these increases of bone resorption parameters. The present data indicate that TNFR1 is critical for bone resorption at the site of LPS injection and that TNFR2 might have a protective role on the LPS-induced inflammatory bone resorption process.     

Hypochlorhydria‐induced calcium malabsorption does not affect fracture healing but increases post‐traumatic bone loss in the intact skeleton

Efficient calcium absorption is essential for skeletal health. Patients with impaired gastric acidification display low bone mass and increased fracture risk because calcium absorption is dependent on gastric pH. We investigated fracture healing and post‐traumatic bone turnover in mice deficient in Cckbr, encoding a gastrin receptor that affects acid secretion by parietal cells. Cckbr?/? mice display hypochlorhydria, calcium malabsorption, and osteopenia. Cckbr?/? and wildtype (WT) mice received a femur osteotomy and were fed either a standard or calcium‐enriched diet. Healed and intact bones were assessed by biomechanical testing, histomorphometry, micro‐computed tomography, and quantitative backscattering. Parathyroid hormone (PTH) serum levels were determined by enzyme‐linked immunosorbent assay. Fracture healing was unaffected in Cckbr?/? mice. However, Cckbr?/? mice displayed increased calcium mobilization from the intact skeleton during bone healing, confirmed by significantly elevated PTH levels and osteoclast numbers compared to WT mice. Calcium supplementation significantly reduced secondary hyperparathyroidism and bone resorption in the intact skeleton in both genotypes, but more efficiently in WT mice. Furthermore, calcium administration improved bone healing in WT mice, indicated by significantly increased mechanical properties and bone mineral density of the fracture callus, whereas it had no significant effect in Cckbr?/? mice. Therefore, under conditions of hypochlorhydria‐induced calcium malabsorption, calcium, which is essential for callus mineralization, appears to be increasingly mobilized from the intact skeleton in favor of fracture healing. Calcium supplementation during fracture healing prevented systemic calcium mobilization, thereby maintaining bone mass and improving fracture healing in healthy individuals whereas the effect was limited by gastric hypochlorhydria. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1914–1921, 2016.

     

The roles of TNFR1 in lipopolysaccharide‐induced bone loss: Dual effects of TNFR1 on bone metabolism via osteoclastogenesis and osteoblast survival

LPS (lipopolysaccharide), a major constituent of Gram‐negative bacteria, regulates proliferation and differentiation of osteoclasts directly or indirectly. This study sought to investigate the functions of the RANK/RANKL pathway in LPS‐induced bone loss in vivo. Wild‐type mice or TNFR1?/? mice were injected LPS with or without osteoprotegerin (OPG) and analyzed histologically. Bone volume was reduced by LPS injection in all groups, and OPG administration prevented the LPS‐induced bone loss regardless of genotypes. LPS‐induced enhancement of osteoclastogenesis in wild‐type mice was blocked by OPG administration. LPS or OPG did not affect osteoclastogenesis in TNFR1?/? mice. Interestingly, osteoblast surface was remarkably reduced in LPS‐treated TNFR1?/? mice as a result of enhanced osteoblast apoptosis. TRAIL, induced by TNF‐α in BMC, triggered apoptosis of primary osteoblast only when TNFR1 signal was ablated in vitro. In conclusion, RANK signaling plays a prominent role in osteoclastogenesis downstream of LPS. Furthermore, TNFR1 regulates bone metabolism through not only the regulation of osteoclast differentiation but also osteoblast survival. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:657–663, 2010     

One Year of Transgenic Overexpression of Osteoprotegerin in Rats Suppressed Bone Resorption and Increased Vertebral Bone Volume,Density, and Strength

RANKL is an essential mediator of bone resorption, and its activity is inhibited by osteoprotegerin (OPG). Transgenic (Tg) rats were engineered to continuously overexpress OPG to study the effects of continuous long‐term RANKL inhibition on bone volume, density, and strength. Lumbar vertebrae, femurs, and blood were obtained from 1‐yr‐old female OPG‐Tg rats (n = 32) and from age‐matched wildtype (WT) controls (n = 23). OPG‐Tg rats had significantly greater serum OPG (up to 260‐fold) and significantly lower serum TRACP5b and osteocalcin compared with WT controls. Vertebral histomorphometry showed significant reductions in osteoclasts and bone turnover parameters in OPG‐Tg rats versus WT controls, and these reductions were associated with significantly greater peak load in vertebrae tested through compression. No apparent differences in bone material properties were observed in OPG‐Tg rat vertebrae, based on their unchanged intrinsic strength parameters and their normal linear relationship between vertebral bone mass and strength. Femurs from OPG‐Tg rats were of normal length but showed mild osteopetrotic changes, including reduced periosteal perimeter (?6%) and an associated reduction in bending strength. Serum OPG levels in WT rats showed no correlations with any measured parameter of bone turnover, mass, or strength, whereas the supraphysiological serum OPG levels in OPG‐Tg rats correlated negatively with bone turnover parameters and positively with vertebral bone mass and strength parameters. In summary, low bone turnover after 1 yr of OPG overexpression in rats was associated with increased vertebral bone mass and proportional increases in bone strength, with no evidence for deleterious effects on vertebral material properties.     

Innate immune molecule surfactant protein D attenuates sepsis‐induced acute kidney injury through modulating apoptosis and NFκB‐mediated inflammation

The objective of this study is to investigate the mechanism whereby innate immune molecule surfactant protein D (SP‐D) attenuates sepsis‐induced acute kidney injury (AKI) through modulating apoptosis and nuclear factor kappa‐B (NFκB)‐mediated inflammation. In the present study, a mouse sepsis model was established by cecal ligation and puncture in SP‐D knockout (KO) mice and wild‐type (WT) mice. A sham‐operated group was included as the control. The experimental materials were extracted 6 and 24 hours postoperatively. The plasma levels of tumour necrosis factor alpha (TNF‐α) and MCP‐1 were determined by enzyme‐linked immunosorbent assay (ELISA). Apoptosis was measured by double staining with Annexin V/propidium iodide and flow cytometry. The levels of NFκB in renal tissues were measured by ELISA and Western blotting assay. Apoptosis was detected by TUNEL assays. There were no significant differences in plasma TNF‐α levels between the WT sham group and the KO sham group at 6 and 24 hours postoperatively (P < .05), but the levels of TNF‐α in the WT sepsis and KO sepsis groups were significantly higher than those in controls (P < .05). The levels of TNF‐α in the KO sepsis group were significantly higher than those of the WT sepsis group (P < .05). TNF‐α levels in the WT sepsis group and the KO sepsis group at 24 hours postoperatively were significantly higher than those at 6 hours postoperatively (P < .05). The levels of MCP‐1 in the WT sepsis group and the KO sepsis group at 6 and 24 hours postoperatively were significantly higher than those in the control group (P < .05), and MCP‐1 levels in the KO sepsis group were significantly higher than those in the WT sepsis group (P < .05). MCP‐1 levels in the WT sepsis group and the KO sepsis group at 24 hours postoperatively were significantly higher than those at 6 hours postoperatively (P < .05). The expression of SP‐D in WT kidneys was significantly lower at 6 and 24 hours postoperatively (P < .05). The number of TUNEL‐positive cells in the kidneys from septic SP‐D KO mice was significantly higher (P < .05). The levels of NFκB in septic mice were significantly increased at 6 and 24 hours after induction of sepsis compared with the sham‐operated group compared with those of septic SP‐D KO mice and WT mice (P < .05). Innate immune molecule SP‐D significantly decreased plasma levels of inflammatory cytokines in mice and attenuated sepsis‐induced AKI by inhibiting NFκB activity and apoptosis.     

Spermatogenesis‐associated 48 is essential for spermatogenesis in mice

Azoospermia, oligospermia and teratozoospermia all seriously impact male reproductive health. Spermatogenesis is a complex and precisely regulated process in which germ cells proliferate and differentiate and involves the regulation of multiple testis‐specific genes. Here, we identified testis‐specific gene spermatogenesis‐associated 48 (SPATA48), the expression of which was age‐dependent, indicating that it is involved in spermatogenesis. In humans and mice with azoospermia, expression of SPATA48 disappeared in the testis. Spata48?/? knockout male mice had smaller testis and defective spermatogenesis compared to wild‐type (WT) mice. This study can help in the exploration of the genetic basis of male infertility and identify new targets for the diagnosis and treatment of male infertility.     

TNF Receptors Differentially Signal and Are Differentially Expressed and Regulated in the Human Heart

Tumor necrosis factor (TNF) utilizes two receptors, TNFR1 and 2, to initiate target cell responses. We assessed expression of TNF, TNFRs and downstream kinases in cardiac allografts, and compared TNF responses in heart organ cultures from wild‐type (^WTC57BL/6), TNFR1‐knockout (^KO), TNFR2^KO, TNFR1/2^KO mice. In nonrejecting human heart TNFR1 was strongly expressed coincidentally with inactive apoptosis signal‐regulating kinase‐1 (ASK1) in cardiomyocytes (CM) and vascular endothelial cells (VEC). TNFR2 was expressed only in VEC. Low levels of TNF localized to microvessels. Rejecting cardiac allografts showed increased TNF in microvessels, diminished TNFR1, activation of ASK1, upregulated TNFR2 co‐expressed with activated endothelial/epithelial tyrosine kinase (Etk), increased apoptosis and cell cycle entry in CM. Neither TNFR was expressed significantly by cardiac fibroblasts. In ^WTC57BL/6 myocardium, TNF activated both ASK1 and Etk, and increased both apoptosis and cell cycle entry. TNF‐treated TNFR1^KO myocardium showed little ASK1 activation and apoptosis but increased Etk activation and cell cycle entry, while TNFR2^KO myocardium showed little Etk activation and cell cycle entry but increased ASK1 activation and apoptosis. These observations demonstrate independent regulation and differential functions of TNFRs in myocardium, consistent with TNFR1‐mediated cell death and TNFR2‐mediated repair.     

Immune molecular profiling of whole blood drawn from a non‐human primate cardiac xenograft model treated with anti‐CD154 monoclonal antibodies

Most studies of xenografts have been carried out with complex immunosuppressive regimens to prevent immune rejection; however, such treatments may be fatal owing to unknown causes. Here, we performed immune molecular profiling following anti‐CD 154 monoclonal antibody (mA b) treatment in heterotopic abdominal cardiac xenografts from α‐1,3‐galactosyltransferase‐knockout pigs into cynomolgus monkeys to elucidate the mechanisms mediating the undesirable fatal side effects of immunosuppressive agents. Blood samples were collected from healthy monkeys as control and then at 2 days after xenograft transplantation and just before humane euthanasia; 94 genes related to the immune system were analyzed. The basic immunosuppressive regimen included cobra venom factor, anti‐thymocyte globulin, and rituximab, with and without anti‐CD 154 mA bs. The maintenance therapy was followed with tacrolimus, MMF , and methylprednisolone. The number of upregulated genes was initially decreased on Day 2 (?/+ anti‐CD 154 mA b, 22/13) and then increased before euthanasia in recipients treated with anti‐CD 154 mA bs (?/+ anti‐CD 154 mA b, 30/37). The number of downregulated genes was not affected by anti‐CD 154 mA b treatment. Additionally, the number of upregulated genes increased over time for both groups. Interestingly, treatment with anti‐CD 154 mA bs upregulated coagulation inducers (CCL 2/IL 6 ) before euthanasia. In conclusion, immunosuppressive regimens used for cardiac xenografting affected upregulation of 6 inflammation genes (CXCL 10, MPO , MYD 88, NLRP 3, TNF α, and TLR 1) and downregulation of 8 genes (CCR 4 , CCR 6 , CD 40 , CXCR 3 , FOXP 3 , GATA 3 , STAT 4 , and TBX21 ).     

Serum Biomarkers Related to Glucocorticoid‐Induced Osteonecrosis of the Femoral Head: A Prospective Nested Case‐Control Study

Early diagnosis and prevention of glucocorticoid (GC)‐induced osteonecrosis of the femoral head (ONFH) continues to be a challenging problem for clinicians and researchers. However, the role of circulating biomarkers for GC‐induced ONFH, which may reveal individual susceptibility and facilitate earlier diagnosis, remains to be determined. The aim of this study was to identify potential biomarkers that may predict early GC‐induced ONFH. A total of 123 patients scheduled for initial systemic GC therapy were enrolled in this prospective nested case–control study. The serum concentrations of 13 potential biomarkers were measured in seven patients with GC‐induced ONFH, diagnosed instantly after short‐term use of GCs and in 20 controls who did not develop osteonecrosis despite similar GC therapy. Biomarkers were measured both before and after taking GCs to identify any differences in marker levels and the changes during GC therapy between two groups. Type I collagen cross‐linked C‐telopeptide (β‐CTX; p = 0.000) was significantly lower, high‐density lipoprotein cholesterol (p = 0.092) and apolipoprotein (apo)‐B/apo‐A1 (p = 0.085) tended to be lower and higher, respectively, before GC treatment in osteonecrosis group. After GC therapy, β‐CTX (p = 0.014) was significantly lower and amino terminal telopeptide of procollagen type I (PINP; p = 0.068) tended to be lower in the osteonecrosis group. As secondary outcomes, we observed remarkable changes in nine potential biomarkers following short‐term GC therapy in both groups. In conclusion, we found that β‐CTX, could potentially be used to predict GC‐induced ONFH before GC therapy. Lower β‐CTX and PINP are promising biomarkers for the early diagnosis of GC‐induced ONFH. These findings need to be confirmed in large‐scale prospective studies. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2348–2357, 2019     

NAD(P)H‐quinone oxidoreductase 1 silencing aggravates hormone‐induced prostatic hyperplasia in mice

NAD(P)H‐quinone oxidoreductase 1 (NQO1) is a highly inducible flavoprotein known to involve in various cellular defence mechanisms. In this study, we explored whether NQO1 deletion affects hormone‐induced prostatic hyperplasia. Testosterone propionate (3 mg/kg, IP) was injected into wild‐type (WT) and NOQ1 knockout C57BL/6 mice (NQO1^?/?) for 14 consecutive days, and the samples were collected for biological and histochemical studies. The testosterone‐treated NQO1^?/? showed about 140% higher prostate weight than the testosterone‐treated WT, with enhanced connective tissue and hyperplastic glands formations. However, increased dihydrotestosterone level after testosterone treatment was not significantly different between the WT and NQO1^?/?. In contrast, the enhanced nuclear expression of proliferating cell nuclear antigen in NQO1^?/? prostate confirmed aggravated prostatic hyperplasia in NQO1^?/?. Moreover, the expression of heat shock protein (HSP) 90‐α was markedly increased in the NQO1^?/?, and this was supported by increased testosterone‐induced nuclear androgen receptor expression in NQO1‐silenced LNCaP cells. Testosterone‐induced prostate‐specific antigen expression was not reversed in NOQ1‐silenced cells after finasteride treatment. Although the exact role of NQO1 in prostatic hyperplasia remains unclear, the hyperplasia exacerbation due to NQO1 deletion might be independent of type 2 5α‐reductase and might be related to enhanced androgen receptor affinity due to enhanced HSP90‐α expression.     

Interleukin‐17 synergizes with IFNγ or TNFα to promote inflammatory mediator release and intercellular adhesion molecule‐1 (ICAM‐1) expression in human intervertebral disc cells

Interleukin‐17 (IL‐17) is a cytokine recently shown to be elevated, along with interferon‐γ (IFNγ) and tumor necrosis factor (TNFα), in degenerated and herniated intervertebral disc (IVD) tissues, suggesting a role for these cytokines in intervertebral disc disease. The objective of our study was to investigate the involvement of IL‐17 and costimulants IFNγ and TNFα in intervertebral disc pathology. Cells were isolated from anulus fibrosus and nucleus pulposus tissues of patients undergoing surgery for intervertebral disc degeneration or scoliosis. The production of inflammatory mediators, nitric oxide (NOx), prostaglandin E2 (PGE2) and interleukin‐6 (IL‐6), as well as intercellular adhesion molecule (ICAM‐1) expression, were quantified for cultured cells following exposure to IL‐17, IFNγ, and TNFα. Intervertebral disc cells exposed to IL‐17, IFNγ, or TNFα showed a remarkable increase in inflammatory mediator release and ICAM‐1 expression (GLM and ANOVA, p < 0.05). Addition of IFNγ or TNFα to IL‐17 demonstrated a synergistic increase in inflammatory mediator release, and a marked increase in ICAM‐1 expression. These findings suggest that IVD cells not only respond with a catabolic phenotype to IL‐17 and costimulants IFNγ and TNFα, but also express surface ligands with consequent potential to recruit additional lymphocytes and immune cells to the IVD microenvironment. IL‐17 may be an important regulator of inflammation in the IVD pathologies. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:1–7, 2011     

Differential effects between the loss of MMP‐2 and MMP‐9 on structural and tissue‐level properties of bone

Matrix metalloproteinases (MMPs) are capable of processing certain components of bone tissue, including type 1 collagen, a determinant of the biomechanical properties of bone tissue, and they are expressed by osteoclasts and osteoblasts. Therefore, we posit that MMP activity can affect the ability of bone to resist fracture. To explore this possibility, we determined the architectural, compositional, and biomechanical properties of bones from wild‐type (WT), Mmp2^?/?, and Mmp9^?/? female mice at 16 weeks of age. MMP‐2 and MMP‐9 have similar substrates but are expressed primarily by osteoblasts and osteoclasts, respectively. Analysis of the trabecular compartment of the tibia metaphysis by micro–computed tomography (µCT) revealed that these MMPs influence trabecular architecture, not volume. Interestingly, the loss of MMP‐9 improved the connectivity density of the trabeculae, whereas the loss of MMP‐2 reduced this parameter. Similar differential effects in architecture were observed in the L₅ vertebra, but bone volume fraction was lower for both Mmp2^?/? and Mmp9^?/? mice than for WT mice. The mineralization density and mineral‐to‐collagen ratio, as determined by µCT and Raman microspectroscopy, were lower in the Mmp2^?/? bones than in WT control bones. Whole‐bone strength, as determined by three‐point bending or compression testing, and tissue‐level modulus and hardness, as determined by nanoindentation, were less for Mmp2^?/? than for WT bones. In contrast, the Mmp9^?/? femurs were less tough with lower postyield deflection (more brittle) than the WT femurs. Taken together, this information reveals that MMPs play a complex role in maintaining bone integrity, with the cell type that expresses the MMP likely being a contributing factor to how the enzyme affects bone quality. © 2011 American Society for Bone and Mineral Research.     

Adalimumab treatment leads to reduction of tissue tumor necrosis factor‐alpha correlated with venous leg ulcer improvement: a pilot study

Venous leg ulcers (VLUs) have higher tumor necrosis factor‐α (TNF‐α) levels compared with normal skin. Refractory VLUs of long duration have higher TNF‐α levels compared with VLUs of shorter duration. As up to 75% of VLUs fail to heal with standard care, we sought to evaluate the role of anti‐TNF‐α therapy for patients with refractory VLUs. Evaluable data were obtained in four of five subjects with recalcitrant VLUs treated with 80 mg of subcutaneous adalimumab at week 0 and with 40 mg at week 2 along with compression therapy and were followed‐up for 6 weeks. Wound biopsies taken at weeks 0 and 4 were stained with anti‐TNF‐α antibodies. Average 4‐week percent wound size reduction was 20.5% ± 6.4%. Two patients had wound size reduction more than 25%, and their percent wound size reduction correlated to percent TNF‐α staining score reductions (P = 0.02, R² = 0.999). VLU TNF‐α level decrease 4 weeks post‐adalimumab treatment correlated with wound healing.     

Pig kidney graft survival in a baboon for 136 days: longest life‐supporting organ graft survival to date

The longest survival of a non‐human primate with a life‐supporting kidney graft to date has been 90 days, although graft survival > 30 days has been unusual. A baboon received a kidney graft from an α‐1,3‐galactosyltransferase gene‐knockout pig transgenic for two human complement‐regulatory proteins and three human coagulation‐regulatory proteins (although only one was expressed in the kidney). Immunosuppressive therapy was with ATG+anti‐CD20mAb (induction) and anti‐CD40mAb+rapamycin+corticosteroids (maintenance). Anti‐TNF‐α and anti‐IL‐6R were administered. The baboon survived 136 days with a generally stable serum creatinine (0.6 to 1.6 mg/dl) until termination. No features of a consumptive coagulopathy (e.g., thrombocytopenia, decreased fibrinogen) or of a protein‐losing nephropathy were observed. There was no evidence of an elicited anti‐pig antibody response. Death was from septic shock (Myroides spp). Histology of a biopsy on day 103 was normal, but by day 136, the kidney showed features of glomerular enlargement, thrombi, and mesangial expansion. The combination of (i) a graft from a specific genetically engineered pig, (ii) an effective immunosuppressive regimen, and (iii) anti‐inflammatory agents prevented immune injury and a protein‐losing nephropathy, and delayed coagulation dysfunction. This outcome encourages us that clinical renal xenotransplantation may become a reality.     

LPS‐Induced Inhibition of Osteogenesis Is TNF‐α Dependent in a Murine Tooth Extraction Model

TNF‐α is a major etiologic factor of inflammatory bone diseases such as periodontitis and rheumatoid arthritis. In addition, patients with metabolic diseases such as chronic heart disease and diabetes have significantly increased plasma levels of TNF‐α. Several lines of evidence show inhibition of osteoblastogenesis by TNF‐α in vitro. Therefore, bone formation and osteogenesis in these patients might be inhibited because of TNF‐α. However, little is known about the inhibitory role of TNF‐α in bone formation/osteogenesis in vivo. The purpose of this study was to investigate the role of TNF‐α in osteogenesis using a murine tooth extraction model. Lipopolysaccharide (LPS) was injected subcutaneously into the calvariae of either wildtype (WT) or TNF‐α–deficient (KO) mice. The left incisor was extracted 4 days after LPS injection. The measuring area was established as the tooth socket under the mesial root of the first molar. A significant increase in serum TNF‐α levels after LPS injection was observed in WT mice. The BMD of the tooth socket was significantly decreased by LPS injection 21 days after extraction in WT but not in KO mice. Histomorphometric analysis showed a significant decrease in the mineral apposition rate after LPS injection, which appeared at an early stage in WT but not in KO mice. Injection of a peptide that blocked the TNF‐α signaling pathway by preventing transmission of the NF‐κB signal recovered the inhibition of osteogenesis observed after LPS injection. In conclusion, TNF‐α might play a major role in LPS‐induced inhibition of osteogenesis under inflammatory conditions.     

SH3BP2 Cherubism Mutation Potentiates TNF‐α–Induced Osteoclastogenesis via NFATc1 and TNF‐α–Mediated Inflammatory Bone Loss

Cherubism (OMIM# 118400) is a genetic disorder with excessive jawbone resorption caused by mutations in SH3 domain binding protein 2 (SH3BP2), a signaling adaptor protein. Studies on the mouse model for cherubism carrying a P416R knock‐in (KI) mutation have revealed that mutant SH3BP2 enhances tumor necrosis factor (TNF)‐α production and receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclast differentiation in myeloid cells. TNF‐α is expressed in human cherubism lesions, which contain a large number of tartrate‐resistant acid phosphatase (TRAP)‐positive multinucleated cells, and TNF‐α plays a critical role in inflammatory bone destruction in homozygous cherubism mice (Sh3bp2^KI/KI). The data suggest a pathophysiological relationship between mutant SH3BP2 and TNF‐α–mediated bone loss by osteoclasts. Therefore, we investigated whether P416R mutant SH3BP2 is involved in TNF‐α–mediated osteoclast formation and bone loss. Here, we show that bone marrow–derived M‐CSF–dependent macrophages (BMMs) from the heterozygous cherubism mutant (Sh3bp2^KI/+) mice are highly responsive to TNF‐α and can differentiate into osteoclasts independently of RANKL in vitro by a mechanism that involves spleen tyrosine kinase (SYK) and phospholipase Cγ2 (PLCγ2) phosphorylation, leading to increased nuclear translocation of NFATc1. The heterozygous cherubism mutation exacerbates bone loss with increased osteoclast formation in a mouse calvarial TNF‐α injection model as well as in a human TNF‐α transgenic mouse model (hTNFtg). SH3BP2 knockdown in RAW264.7 cells results in decreased TRAP‐positive multinucleated cell formation. These findings suggest that the SH3BP2 cherubism mutation can cause jawbone destruction by promoting osteoclast formation in response to TNF‐α expressed in cherubism lesions and that SH3BP2 is a key regulator for TNF‐α–induced osteoclastogenesis. Inhibition of SH3BP2 expression in osteoclast progenitors could be a potential strategy for the treatment of bone loss in cherubism as well as in other inflammatory bone disorders. © 2014 American Society for Bone and Mineral Research.