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.     

Combined heart–liver transplantation: a single‐center experience

Combined orthotopic heart and liver transplantation (CHLT) is a lifesaving procedure for patients with end‐stage heart–liver disease. We reviewed the long‐term outcome of patients who have undergone CHLT at the University of Bologna, Italy. Fifteen patients with heart and liver failure were placed on the transplant list between November 1999 and March 2012. The pretransplant cardiac diagnoses were familial amyloidosis in 14 patients and chronic heart failure due to chemotherapy with liver failure due to chronic hepatitis in one patient. CHLT was performed as a single combined procedure in 14 hemodynamically stable patients; there was no peri‐operative mortality. The survival rates for the CHLT recipients were 93%, 93%, and 82% at 1 month and 1 and 5 years, respectively. Freedom from graft rejection was 100%, 90%, and 36% at 1, 5, and 10 years, respectively, for the heart graft and 100%, 91%, and 86% for the liver graft. The livers of eight recipients were transplanted as a “domino” with mean overall 1‐year survival of 93%. Simultaneous heart and liver transplantation is feasible and was achieved in this extremely sick cohort of patients. By adopting the domino technique, we were able to enlarge the donor cohort and include high‐risk patients.     

Reconstitution of T and NK cells after haploidentical hematopoietic cell transplantation using αβ T cell–depleted grafts and the clinical implication of γδ T cells

To investigate reconstitution of T and NK cells after αβ T lymphocyte–depleted haploidentical hematopoietic cell transplantation (HHCT) and the clinical implications of γδ T cells, we analyzed 50 pediatric patients who received 55 HHCTs using αβ T cell–depleted grafts. The number of CD3+ T cells and CD8+ T cells recovered rapidly and reached donor levels at days 180 and 60, respectively. Recovery of NK cells was rapid, and the median of NK cells at day 14 was comparable to the donor level. At day 14, median percentage of γδ T lymphocytes was 70.5%. After day 14, the percentage of γδ T cells gradually decreased, while the percentage of αβ T cells gradually increased. Patients with a low percentage (≤21%) of γδ T cells at day 30 had significantly higher incidence of cytomegalovirus (CMV) reactivation compared to patients with a high percentage (>70%) of γδ T cells (P < .01). In patients with acute leukemia, patients with high percentage of γδ T cells at day 30 showed significantly higher relapse‐free survival compared to those with low percentage of γδ T cells (P = .02). Data suggest that early recovery of γδ T cells decreases the risk of CMV reactivation and leukemia relapse.     

Quantitative flow cytometry to measure the TNF‐α and IL‐2 system after heart transplantation

Abstract After heart transplantation a high incidence of infections and malignancies is found. Not only immunosuppression, but also intrinsic cytokine systems with some unbalance, e.g. TNF‐α and IL‐2, can result in impaired immune competence and may have a role in these complications. The aim of this study was to assess the activity of the TNF‐α and IL‐2 systems after heart transplantation. In peripheral blood we measured expression of activation markers of TNF‐α (TNF‐R2) and IL‐2 (IL‐2Rα, IL‐2Rβ‐chain) on monocytes and lymphocytes using quantitative flow‐cytometric analysis. TNF‐R2 expression was significantly enhanced on monocytes and lymphocytes in patients after heart transplantation, while the expression of IL‐2Rα and IL‐2Rβ was not elevated. Increased TNF‐R2 expression in peripheral blood after heart transplantation reflects an activated TNF‐α system, leading to high levels of actives TNF‐R, which impairs TNF‐α bioavailibility and consequently leads to immune incompetence.     

Interleukin‐1β stimulates stromal‐derived factor‐1α expression in human subacromial bursa

Chemokines produced by synoviocytes of the subacromial bursa are up‐regulated in subacromial bursitis and rotator cuff disease. We hypothesized that SDF‐1α production in bursal synoviocytes may be induced by local cytokines such as interleukin IL‐1β and IL‐6. Subacromial bursa specimens were obtained from patients undergoing shoulder surgery. Bursal specimens were stained with anti‐human antibodies to IL‐1, IL‐6, and SDF‐1α by immunohistochemistry and compared to normal and rheumatoid controls. Bursal cells were also isolated from specimens and cultured. Early passaged cells were then treated with cytokines (IL‐1β and IL‐6) and SDF‐1α expression was measured by ELISA and RT‐PCR. SDF‐1α, IL‐1β, and IL‐6 were expressed at high levels in bursitis specimens from human subacromial bursa compared to normal controls. In cultured bursal synoviocytes, there was a dose‐dependent increase in SDF‐1α production in the supernatants of cells treated with IL‐1β. SDF‐1α mRNA expression was also increased in bursal cells treated with IL‐1β. IL‐6 caused a minimal but not statistically significant increase in SDF‐1α expression. SDF‐1α, IL‐1β, and IL‐6 are expressed in the inflamed human subacromial bursal tissues in patients with subacromial bursitis. In cultured bursal synoviocytes, SDF‐1α gene expression and protein production are stimulated by IL‐1β. IL‐1β produced by bursal syvoviocytes and inflammatory cells in the human subacromial bursa is an important signal in the inflammatory response that occurs in subacromial bursitis and rotator cuff disease. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1695–1699, 2011     

Autologous solution protects bovine cartilage explants from IL‐1α‐ and TNFα‐induced cartilage degradation

Osteoarthritis (OA) is characterized by deterioration of articular cartilage driven by an imbalance of pro‐ and anti‐inflammatory cytokines. To address the cartilage deterioration observed in OA, an autologous protein solution (APS) has been developed which has been shown to inhibit the production of destructive proteases and inflammatory cytokines from chondrocytes and monocytes, respectively. The purpose of this study was to determine the chondroprotective effect of APS on IL‐1α‐ or TNFα‐challenged bovine articular cartilage explants. Cartilage explants were cultured in the presence or absence of recombinant inflammatory cytokines, IL‐1α and TNFα. Explants under equivalent inflammatory conditions were pretreated with recombinant antagonists IL‐1ra, sTNF‐RI, or APS to measure their inhibition of matrix degradation. Explants were further evaluated with Safranin‐O, Masson's Trichrome, and Hematoxylin and Eosin histological staining. APS was more effective than recombinant antagonists in preventing cartilage matrix degradation and inhibited any measurable IL‐1α‐induced collagen release over a 21‐day culture period. APS treatment reduced the degree of Safranin‐O staining loss when cartilage explants were cultured with IL‐1α or TNFα. Micrographs of APS treated cartilage explants showed an increase in observed cellularity and apparent cell division. APS may have the potential to prevent cartilage loss associated with early OA. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1929–1935, 2013     

Apolipoprotein A‐Ib as a biomarker of focal segmental glomerulosclerosis recurrence after kidney transplantation: diagnostic performance and assessment of its prognostic value – a multi‐centre cohort study

Recurrence of idiopathic focal segmental glomerulosclerosis (FSGS) is a serious complication after kidney transplantation. FSGS relapse is suspected by a sudden increase in proteinuria but there is not an accurate noninvasive diagnostic tool to confirm this entity or to detect patients at risk. We aimed to validate the diagnostic performance of ApoA‐Ib to detect FSGS relapses by measuring urinary ApoA‐Ib in a retrospective cohort of 61 kidney transplanted patients (37 FSGS and 24 non‐FSGS). In addition, to assess the ApoA‐Ib predictive ability, ApoA‐Ib was measured periodically in a prospective cohort of 13 idiopathic FSGS patients who were followed during 1 year after transplantation. ApoA‐Ib had a sensitivity of 93.3% and a specificity of 90.9% to diagnose FSGS relapses, with a high negative predictive value (95.2%), confirming our previous results. In the prospective cohort, ApoA‐Ib predated the recurrence in four of five episodes observed. In the nonrelapsing group (n = 9), ApoA‐Ib was negative in 37 of 38 samples. ApoA‐Ib has the potential to be a good diagnostic biomarker of FSGS relapses, providing a confident criterion to exclude false positives even in the presence of high proteinuria. It has also the potential to detect patients at risk of relapse, even before transplantation.     

Adhesion molecules α, β and γ‐catenin as prognostic factors of tumour progression in upper urinary tract urothelial tumours: the role of AKT‐P/GSK‐3β/β‐catenin pathway

OBJECTIVES

To evaluate α, β and γ‐catenin expression in upper urinary tract urothelial tumours (UUTC) and determine their value as prognostic factors; to investigate the correlation between the catenin complex and the AKT pathway.

PATIENTS AND METHODS

We retrospectively analysed 114 consecutive patients treated at our institution from 1990 to 2004; the mean follow‐up was 54 months. Tumour samples were available from 70 patients, and included in tissue microarrays for immunohistochemical analysis. The antibodies used were anti‐α, ‐β and γ‐catenin, and antiphospho‐AKT. The prognostic value of the expression of these molecules was analysed using tumour progression and cancer‐specific survival as end‐points.

RESULTS

Of the 114 patients, 27% developed tumour progression; the cancer‐specific and overall survival were 77% and 60.6%, respectively. Abnormal α, β and γ‐catenin expression was found in 44 (63%), 22 (31%) and 28 (41%) patients, respectively; the abnormal catenin expression patterns correlated with each other. Positive cytoplasm phospho‐AKT expression was found in 27 (39%) patients. Three of them were found to have cytoplasmic β‐catenin accumulation and none of them nuclear expression. β‐catenin expression was the only one that was an independent marker of tumour progression, with a hazard ratio (95% confidence interval) of 3.1(1.2–8.6), together with grade (7.1, 1.2–55.8) and stage (4.6, 2.1–10). In the cancer‐specific survival analysis, again β‐catenin was an independent prognostic factor (3.4, 1–11.5) together with stage (4.6, 2.2–9.8).

CONCLUSIONS

The loss of the normal membrane β‐catenin expression constitutes an independent factor of tumour progression and cancer‐specific survival. Our data suggest that the AKT/GSK3β/β‐catenin signalling pathway is not activated in the UUTC carcinogenesis.     

肾移植后继发移植肾淀粉样变一例

淀粉样变病是一种以淀粉样蛋白物质沉积于组织器官的细胞间引起的少见疾病.我院自1977年7月至今共施行同种异体肾移植2400余例,其中并发移植肾淀粉样变1例,发生率为0.04%.现结合文献报告如下.