Non‐invasive assessment of kidney allograft fibrosis with shear wave elastography: A radiological‐pathological correlation analysis

Objectives

To evaluate the use of shear wave elastography in assessment of kidney allograft tubulointerstitial fibrosis.

Methods

Shear wave elastography assessment was carried out by two independent operators in kidney transplant recipients who underwent allograft biopsy for clinical indications (i.e. rising creatinine >15% or proteinuria >1 g/day). Allograft biopsies were interpreted by the same pathologist according to the 2013 Banff Classification.

Results

A total of 40 elastography scans were carried out (median creatinine 172.5 μmol/L [interquartile range 133.8–281.8 μmol/L]). Median tissue stiffness at the cortex (22.6 kPa [interquartile range 18.8–25.7 kPa] vs 22.3 kPa [interquartile range 19.0–26.5 kPa], P = 0.70) and medulla (15.0 kPa [interquartile range 13.7–18.0 kPa] vs 15.6 kPa [interquartile range 14.4–18.2 kPa]) showed no significant differences between the two observers. Interobserver agreement was satisfactory (intraclass correlation coefficient of the cortex 0.84, 95% CI 0.70–0.92 and intraclass correlation coefficient of the medulla 0.88, 95% CI 0.78–0.94). The areas under the receiver operating characteristic curves for detection of tubulointerstitial fibrosis were estimated to be 0.75 (95% CI 0.61–0.89), 0.85 (95% CI 0.75–0.95) and 0.65 (95% CI 0.53–0.78) for cortical, medullary tissue stiffness and serum creatinine, respectively.

Conclusions

Shear wave elastography can be used as a non‐invasive tool to evaluate kidney allograft fibrosis with reasonable interobserver agreement and superior test performance to serum creatinine in detecting early tubulointerstitial fibrosis.     

三七总皂甙对人肝癌细胞缝隙连接细胞间通讯的调节机制

目的：观察三七总皂甙对人肝癌细胞SMMC-7721缝隙连接细胞间通讯、以及缝隙连接蛋白32mRNA和磷酸化细胞外信号调节激酶1，2表达的影响。方法：实验于2005—08／2006—03在江西省分子医学重点实验室完成。①实验操作：SMMC-7721细胞用含100mg／L，200mg／L，400mg／L三七总皂甙的培养液预处理，对照组SMMC-7721细胞不加三七总皂甙。②实验评估：采用划痕标记，染料示踪技术检测缝隙连接细胞间通讯的变化；采用反转录-聚合酶链反应检测缝隙连接蛋白32mRNA水平；采用流式细胞仪检测磷酸化细胞外信号调节激酶1／2的表达。结果：①对照组染料局限于划痕两侧的细胞内，无明显的荧光染料传输，GJIC阴性；三七总皂甙可上调染料传输范围，且随三七总皂甙剂量增高，染料传输范围逐渐增大，最远可达三四层细胞。②反转录-聚合酶链反应结果显示，各组细胞缝隙连接蛋白32mRNA水平差异无显著性意义（P〉0．05）。③流式细胞仪结果显示，对照组SMMC-7721细胞磷酸化细胞外信号调节激酶1／2表达的阳性率较高，伴随三七总皂甙剂量增加磷酸化细胞外信号调节激酶1／2表达的阳性率逐渐下降，呈一定的剂量依赖效应。结论：三七总皂甙可能通过抑制SMMC-7721细胞磷酸化细胞外信号调节激酶1／2的活化减少缝隙连接蛋白32的磷酸化，进而上调SMMC-7721细胞的缝隙连接细胞间通讯功能。     

The tyrosine kinase inhibitor dasatinib dysregulates bone remodeling through inhibition of osteoclasts in vivo

Dasatinib is a potent tyrosine kinase inhibitor that is used to treat chronic myeloid leukemia in patients resistant or intolerant to imatinib mesylate. While designed to inhibit Abl and Src kinases, dasatinib shows multitarget effects, including inhibition of the macrophage colony‐stimulating factor (M‐CSF) receptor c‐fms. We have shown previously that dasatinib abrogates osteoclast formation and activity in vitro owing, in part, to its specificity for c‐fms. In this study we examined whether dasatinib could significantly alter bone volume in a model of physiologic bone turnover. Sprague‐Dawley rats were administered dasatinib (5 mg/kg/day) or vehicle by gavage or zoledronic acid (ZOL; 100 µg/kg/6 weeks) subcutaneously. Following 4, 8, and 12 weeks of treatment, serum biochemical, bone morphometric, and histologic analyses were performed. Whole‐body bone mineral density and tibial cortical thickness where unchanged in the dasatinib‐ or ZOL‐treated animals relative to controls. However, micro–computed tomographic (µCT) analysis of cancellous bone at the proximal tibias showed that trabecular volume (BV/TV) and thickness (Tb.Th) were increased in dasatinib‐treated animals at levels comparable with those of the ZOL‐treated group. These changes were associated with a decrease in osteoclast numbers (N.Oc/B.Pm) and surface (Oc.S/BS) and decreased serum levels of the osteoclast marker c‐terminal collagen crosslinks (CTX‐1). Mineral apposition rate (MAR), bone‐formation rate (BFR), and levels of the serum osteoblast markers osteocalcin and N‐terminal propeptide of type I procollagen (P1NP) were not altered significantly in the dasatinib‐treated animals relative to controls. These studies show that dasatinib increases trabecular bone volume at least in part by inhibiting osteoclast activity, suggesting that dasatinib therapy may result in dysregulated bone remodeling. © 2010 American Society for Bone and Mineral Research     

Targeted Disruption of the CXCL12/CXCR4 Axis Inhibits Osteolysis in a Murine Model of Myeloma‐Associated Bone Loss

The plasma cell (PC) malignancy, multiple myeloma (MM), is unique among hematological malignancies in its capacity to cause osteoclast (OC)‐mediated skeletal destruction. We have previously shown that elevated plasma levels of PC‐derived CXCL12 are associated with presence of X‐ray detectable osteolytic lesions in MM patients. To further investigate this relationship, plasma levels of CXCL12 and βCrossLaps, a marker of bone loss, were measured. A strong correlation between levels of CXCL12 and OC‐mediated bone resorption was identified. To confirm the OC‐activating potential of MM PC‐derived CXCL12 in vivo, we established a model of MM‐mediated focal osteolysis, wherein MM PC lines, such as RPMI‐8226, were injected into the tibias of nude mice. Implanting RPMI‐8226 gave rise to osteolytic lesions proximal to the tumor, resulting in a 5% decrease in bone volume (BV) compared with vehicle control. Importantly, bone loss was significantly inhibited with systemic administration of the CXCL12/CXCR4 antagonist T140. Furthermore, implanting CXCL12‐overexpressing RPMI‐8226 cells resulted in a 13% decrease in BV and was associated with increased OC recruitment proximal to the tumor, increased serum matrix metalloproteinase activity, and increased levels of collagen I degradation products. These findings confirm our hypothesis that MM PC‐derived CXCL12 stimulates the recruitment and activity of OC, thereby contributing to the formation of MM osteolytic lesions.     

CD33+ CD14− Phenotype Is Characteristic of Multinuclear Osteoclast‐Like Cells in Giant Cell Tumor of Bone

Giant cell tumor of bone (GCTB) is a benign bone tumor with a shown clinical behavior of local recurrences and rare distant metastases. GCTB is composed of uniformly distributed osteoclastic giant cells, thought to originate from the fusion of monocyte–macrophage lineage cells, in a background consisting of mononuclear rounded cells and spindle‐shaped cells. Several reports showed the specific expression of markers, such as CD14 on the mononuclear rounded cell population, however, lacking osteoclastic giant cells. Blood monocytes that were CD14+, CD33+, or CD14+/CD33+ have also been shown to be programmed as pre‐osteoclasts. The macrophage marker CD33 is expressed earlier than CD14 in macrophage maturation, whereas CD14 is expressed longer than CD33. The aim of this study was to investigate CD14/CD33 expression profiles in GCTB. Nineteen GCTB tumor samples of 19 patients were studied. Immunofluorescent analyses were performed with monoclonal antibodies against CD14, CD33, RANK, and CD51. To unambiguously further prove the expression of these molecules, quantitative RT‐PCR was used with subsequent sequencing of its products. All samples showed similar immunoreactivity profiles. The mononuclear rounded cell population was positive for RANK, CD51, CD14, and CD33. The osteoclastic giant cell population expressed RANK and CD51, as well as CD33, but was consistently negative for CD14 expression. The CD14 and CD33 profiles were confirmed by quantitative RT‐PCR. These RT‐PCR products were sequence verified. Osteoclasts in GCTB are the result of fusion of CD33‐expressing pre‐osteoclasts that further fuse with CD14+ mononuclear cells. Although these results reflect a static rather than a dynamic spectrum, we strongly believe that osteoclastogenesis seems not to be the exclusive result of fusion of intratumoral CD14+ mononuclear cells. Moreover, CD33‐modulated osteoclastogenesis opens up the possibility for novel therapeutic directions.     

Metaanalysis of randomized controlled trials of endoscopic retrograde cholangiography and endoscopic sphincterotomy for the treatment of acute biliary pancreatitis

OBJECTIVE: Endoscopic retrograde cholangiography with endoscopic sphincterotomy (ERC+ES) has been advocated for the management of acute biliary pancreatitis. However, it is also viewed as dangerous. Our objective was to review published randomized, controlled trials (RCTs) of ERC+ES in patients with acute biliary pancreatitis and, by metaanalysis, to estimate the overall efficacy and safety of this approach. METHODS: We performed a fully recursive literature search for published RCTs of ERC+ES in gallstone-related acute pancreatitis. RCTs were pooled. Individual and overall mortality and complication rates were calculated, together with their 95% confidence intervals (CI), absolute risk reduction (ARR), relative risk reduction (RRR), and numbers needed to treat (NNT) for avoidance of complications or death. RESULTS: Four published RCTs had a numerically lower complication rate, and three had a numerically lower mortality rate, in the treated groups than in controls. After pooling, there were 460 treated patients and 374 controls. Complications occurred in 115 (25.0%) treated patients and 143 (38.2%) controls (z = 4.10; p < 0.001). Twenty-four treated patients (5.2%) and 34 controls (9.1%) died (z = 2.15; p < 0.05). ERC+ES had a 34.6% RRR for complications and a 42.9% RRR for death; ARR for complications and death was 13.2% (95% CI: 6.9-19.5%) and 3.9% (95% CI: 0.35-7.45%), respectively. The NNT for avoidance of complications and death was 7.6 and 25.6, respectively. CONCLUSIONS: ERC+ES reduces morbidity and mortality in patients with acute biliary pancreatitis. Treating 26 such patients with ERC+ES is predicted to save one life.     

Adapting agriculture to climate change

The strong trends in climate change already evident, the likelihood of further changes occurring, and the increasing scale of potential climate impacts give urgency to addressing agricultural adaptation more coherently. There are many potential adaptation options available for marginal change of existing agricultural systems, often variations of existing climate risk management. We show that implementation of these options is likely to have substantial benefits under moderate climate change for some cropping systems. However, there are limits to their effectiveness under more severe climate changes. Hence, more systemic changes in resource allocation need to be considered, such as targeted diversification of production systems and livelihoods. We argue that achieving increased adaptation action will necessitate integration of climate change-related issues with other risk factors, such as climate variability and market risk, and with other policy domains, such as sustainable development. Dealing with the many barriers to effective adaptation will require a comprehensive and dynamic policy approach covering a range of scales and issues, for example, from the understanding by farmers of change in risk profiles to the establishment of efficient markets that facilitate response strategies. Science, too, has to adapt. Multidisciplinary problems require multidisciplinary solutions, i.e., a focus on integrated rather than disciplinary science and a strengthening of the interface with decision makers. A crucial component of this approach is the implementation of adaptation assessment frameworks that are relevant, robust, and easily operated by all stakeholders, practitioners, policymakers, and scientists.     

Site-specific, Rep-mediated integration of the intact beta-globin locus in the human erythroleukaemic cell line K562

The stable, regulated and tissue-specific expression of a therapeutic transgene can be best achieved by the transfer of a complete genomic locus, which will include the short- and long-range regulatory elements that are critical for the accurate control of gene expression. However, when techniques that rely on the random integration of exogenous DNA into the human genome are used for gene transfer, the risk of insertional mutagenesis remains a major issue. Using components derived from the adeno-associated virus (AAV), we have successfully targeted the integration of 200 kb bacterial artificial chromosomes containing the entire beta-globin locus into the AAVS1 site on human chromosome 19. We show that transient expression of the AAV Rep proteins in K562 cells facilitated site-specific transgene integration in 17% (6 of 36) of all analysed integration sites. Southern blot analysis revealed the locus had integrated into AAVS1 as an intact, functional unit in five of the six clones generated. Furthermore, each of the site-specific integrants exhibited sustained and appropriately regulated transgene gene expression over a period of 8 months of continuous culture in the absence of selective pressure. We anticipate that the approach developed in this study may be suitable for facilitating targeted integration of intact genomic loci in adult and embryonic stem cells, and therefore provide a powerful tool not just for functional studies but in establishing model systems for the ex vivo correction of genetic disorders.