Reg4 and its downstream transcriptional activator CD44ICD in stage II and III colorectal cancer

Reg4 is highly expressed in gastrointestinal malignancies and acts as a mitogenic and pro-invasive factor. Our recent works suggest that Reg4 binds with CD44 and induces its proteolytic cleavage to release intra-cytoplasmic domain of CD44 (CD44ICD). The goal of this study is to demonstrate clinical significance of the Reg4-CD44/CD44ICD pathway in stage II/III colon cancer and its association with clinical parameters of aggression. We constructed a tissue microarray (TMA) of 93 stage II/III matched colon adenocarcinoma patients, 23 with recurrent disease. The TMA was immunohistochemically stained for Reg4, CD44, and CD44ICD proteins and analyzed to identify associations with tumor characteristics, recurrence and overall survival. The TMA data analysis showed a significant correlation between Reg4 and CD44 (r² = 0.23, P = 0.028), CD44 and CD44ICD (r² = 0.36, p = 0.0004), and Reg4 and CD44ICD (r² = 0.45, p ≤ 0.0001). Reg4 expression was associated with larger tumor size (r² = 0.23, p = 0.026). Although, no association was observed between Reg4, CD44, or CD44ICD expression and disease recurrence, Reg4-positive patients had a median survival of 4 years vs. 7 years for Reg4-negative patients (p = 0.04) in patients who recurred. Inhibition of the Reg4-CD44/CD44ICD pathway may be a future therapeutic target for colon cancer patients.     

Frederick Banting's observations leading to the potential for islet neogenesis without transplantation

On 31 October 1920, Sir Frederick Banting, while preparing for a medical student lecture on diabetes, a topic that he knew little about, learned how pancreatic stones resulted in the formation of new islets of Langerhans. He then scribbled down a potential research study of tying off the ducts of the pancreas and collecting the secretions to improve diabetes. These secretions became known as insulin. A century later, 60 different oral medications and 20 different insulins are available for the treatment of diabetes, yet none stimulate new islet formation. One hundred years later, after the discovery of insulin, more than a dozen research teams from around the world have demonstrated that similar studies to Banting''s pancreatic ligation studies have resulted in upregulation of the REG gene. There are now more than 200 publications on the role of Reg gene proteins and shorter Reg peptides in initiating new islet formation islet from exocrine pancreatic ducts and protecting against inflammation to islets resulting in islet death. Human data through Phase 2b in both type 1 and 2 diabetes patients with diabetes for an average of 20 years have demonstrated that the use of a shorter bioactive Reg peptide can generate new endogenous insulin production, resulting in significant reductions in hemoglobin A1C and increases in stimulated C‐peptide. The observations of Frederick Banting, one century ago, may now lead to the generation of therapeutics that form new islets without the need for transplantation.     

Cyclic ADP-ribose-mediated insulin secretion and Reg, regenerating gene

Glucose is the primary stimulus of insulin secretion in pancreatic β-cells of the islets of Langerhans. CD38 has both ADP-ribosyl cyclase, which catalyzes the formation of cyclic ADP-ribose from NAD⁺, and cyclic ADP-ribose hydrolase, which converts cyclic ADP-ribose to ADP-ribose. ATP, produced by glucose metabolism, inhibits the cyclic ADP-ribose hydrolase of CD38 and therefore causes cyclic ADP-ribose accumulation in β-cells. Then, cyclic ADP-ribose acts as a second messenger for Ca²⁺ mobilization from the endoplasmic reticulum to secrete insulin. The mechanism of insulin secretion as described above is completely different from the conventional hypothesis in which Ca²⁺ influx from extracellular sources was assumed to play a role in insulin secretion by glucose. On the other hand, strategies for influencing the replication of islet β-cells and the growth of the β-cell mass may be more important for ameliorating diabetes. Reg, regenerating gene, is involved in the growth of the β-cell mass, and Reg protein has been shown to increase the β-cell mass in a 90% depancreatized diabetic rat model, thereby ameliorating the diabetes. CD38 is involved in the formation of cyclic ADP-ribose and is essential for the glucose sensitivity of β-cells for insulin secretion. Therefore, CD38 gene and Reg gene will become targets for genetic engineering for diabetic β-cells.     

The Neuroprotective Effects of Reg‐2 Following Spinal Cord Transection Injury

This study was designed to elucidate the potential neuroprotective effects of Reg‐2 (regeneration gene protein 2) in a rodent model of spinal cord transection injury at the ninth thoracic level. Reg‐2 at 100 and 500 μg, recombinant rat ciliary neurotrophic factor, or vehicle were delivered intrathecally using Alzet miniosmotic pumps. We found that Reg‐2 treatment significantly reduced neuronal death in the spinal cord. There was also an attenuation of inflammation at the injury site and an increase in white matter sparing and retained myelination. Retrograde tracing revealed that Reg‐2 protected axons of long descending pathways at 6 weeks post‐SCI, and the number of FluoroGold‐labeled neurons in spinal and supraspinal regions was also significantly increased. Immunofluorescent staining confirmed that the spared white matter contained neurofilament‐positive axons. Moreover, behavioral improvements were revealed by Basso Beattie Bresnahan locomotor rating scores and grid‐walk analysis. These results suggest that Reg‐2 might promote functional recovery by increasing axonal growth, inhibiting neuronal apoptosis, and attenuating spinal cord secondary injury after SCI. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

五脉绿绒蒿超临界二氧化碳萃取工艺及其成分分析

目的 研究五脉绿绒蒿超临界二氧化碳萃取挥发油化学成分,同时对萃取工艺进行优化.方法 应用超临界二氧化碳萃取法对五脉绿绒蒿挥发油部分进行萃取,采用正交试验,考察萃取压力、萃取温度和萃取时间三因素对五脉绿绒蒿超临界二氧化碳萃取物得率的影响,并应用气相色谱-质谱联用（GC-MS）技术分析萃取物的化学成分,用峰面积归一法测定各化合物的相对含量.结果 确定优选工艺条件为：萃取温度45℃,萃取时间2.5 h,萃取压强25 mPa,五脉绿绒蒿药材中萃取物经GC-MS分析,通过美国国家标准与技术局（NIST）检索一共鉴定出66个化合物的具体结构,其中正十六烷酸、亚油酸、正二十九烷、新植二烯、芳樟醇等在该药材萃取物中占的比例较高.结论 该方法提取条件优良,初步揭示了该药材萃取物成分,对进一步研究其化学成分打下基础,为药材的质量评价提供科学参考.     

泽兰有效部分对血小板聚集和血栓形成的影响

目的 研究泽兰有效部分L．F04：对血小板聚集和血栓形成的影响，探讨其活血化瘀作用机制。方法用高分子右旋糖酐静脉推注造成大鼠血瘀证模型，观察泽兰L.F04对二磷酸腺苷(ADP)诱导的大鼠体内血小板聚集以及体内动静脉旁路血栓、体外旋转环内血栓形成的影响。结果 L．F04 0．408，0．204g／kg对模型组大鼠ADP诱导的体内血小板最大聚集率明显增加皆有显著的抑制。且呈剂量依赖关系；与对照组相比，血瘀模型大鼠体外血栓质量明显增加，长度仅有增加趋势，L．F04高、低剂量(0．408，0．204g／kg)皆有抗血栓形成作用，L．F04高剂量对血栓干质量、湿质量的减轻尤为明显；L．F04高、低剂量对实验性动静脉旁路血栓形成均有明显的抑制作用，抑制率分别为27．41％，27．14％。结论泽兰L．F04可显著抑制血小板聚集及体内、外血栓形成。     

Pretransplant Serum Citrulline Predicts Acute Graft-versus-Host Disease

Post-transplant biomarkers of acute graft-versus-host disease (aGVHD) and nonrelapse mortality (NRM) after allogeneic hematopoietic cell transplantation (allo-HCT) have been extensively studied. However, pretransplant biomarkers may provide a greater window of opportunity to intervene. We measured serum biomarkers of various aspects of gut barrier physiology before HCT (median, day –7) and 7 and 28 days post-HCT in 95 consecutive allo-HCT recipients enrolled in an open-label biorepository protocol. Biomarkers included citrulline for total functional enterocyte mass, Reg3a for antibacterial activity of the gut, and intestinal fatty acid binding protein (I-FABP) for enterocyte turnover. Compared to 16 healthy control subjects, we demonstrated that patients came to transplant with abnormal levels of all 3 biomarkers (P < .05), reflecting residual damage from prior chemotherapy. All 3 biomarkers initially declined from pre-HCT to day +7 (more pronounced after myeloablative than reduced-intensive conditioning) followed by a recovery phase and return toward pre-HCT values by day +28. A lower pre-HCT citrulline was independently associated with a higher risk of aGVHD grades II to IV (hazard ratio, 1.32; 95% confidence interval, 1.03 to 1.69; P?=?.02), and this association was not specific to gut GVHD. The strongest correlate of NRM was a higher level of Reg3a at day +7 (P < .001). I-FABP did not predict transplant outcomes. In conclusion, pre-HCT serum citrulline levels identify patients at high risk for developing aGVHD. Our results suggest that pre-HCT interventions to augment the gut barrier may decrease the risk of aGVHD.     

Temporal overexpression of IL‐22 and Reg3γ differentially impacts the severity of experimental autoimmune encephalomyelitis

IL‐22 is an alpha‐helical cytokine which belongs to the IL‐10 family of cytokines. IL‐22 is produced by RORγt+ innate and adaptive lymphocytes, including ILC3, γδ T, iNKT, Th17 and Th22 cells and some granulocytes. IL‐22 receptor is expressed primarily by non‐haematopoietic cells. IL‐22 is critical for barrier immunity at the mucosal surfaces in the steady state and during infection. Although IL‐22 knockout mice were previously shown to develop experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), how temporal IL‐22 manipulation in adult mice would affect EAE course has not been studied previously. In this study, we overexpressed IL‐22 via hydrodynamic gene delivery or blocked it via neutralizing antibodies in C57BL/6 mice to explore the therapeutic impact of IL‐22 modulation on the EAE course. IL‐22 overexpression significantly decreased EAE scores and demyelination, and reduced infiltration of IFN‐γ+IL‐17A+Th17 cells into the central nervous system (CNS). The neutralization of IL‐22 did not alter the EAE pathology significantly. We show that IL‐22‐mediated protection is independent of Reg3γ, an epithelial cell‐derived antimicrobial peptide induced by IL‐22. Thus, overexpression of Reg3γ significantly exacerbated EAE scores, demyelination and infiltration of IFN‐γ+IL‐17A+ and IL‐17A+GM‐CSF+Th17 cells to CNS. We also show that Reg3γ may inhibit IL‐2‐mediated STAT5 signalling and impair expansion of Treg cells in vivo and in vitro. Finally, Reg3γ overexpression dramatically impacted intestinal microbiota during EAE. Our results provide novel insight into the role of IL‐22 and IL‐22‐induced antimicrobial peptide Reg3γ in the pathogenesis of CNS inflammation in a murine model of MS.     

RegⅠ在胃泌素促胃癌细胞体外增殖通路中的作用

目的 探讨再生蛋白I (RegⅠ)在胃泌素刺激胃癌细胞增殖通路中的作用.方法 根据RegⅠ cDNA已知序列,在线设计3个小干扰RNA,并经基因库同源性比较,构建其RegⅠ-shRNA表达载体(pSUPER-EGFP-REG/1、pSUPER-EGFP-REG/2和pSUPER-EGFP-REG/3),利用脂质体2000转染试剂分别转染胃癌细胞BGC823细胞株、SGC7901细胞株,同时设转染空质粒的实验对照组和无质粒转染的空白细胞组.后经G418筛选建立稳定转染细胞株.RT-PCR检测RegⅠ基因mRNA的转录水平,四甲基偶氮唑盐(MTT)法检测胃泌素孵育对胃癌细胞增殖的效应.结果 酶切及测序鉴定,插入片段正确,3个RegⅠ-shRNA表达载体构建成功;RT-PCR显示,pSUPER-EGFP-REG/1可有效抑制RegⅠ mRNA在胃癌细胞BGC823、SGC7901的转录.Western boltting结果显示,BGC823和SGC7901细胞的RegⅠ蛋白表达率分别下调到空载体对照组的(45±4)%和(53±4)%;MTT结果显示,RegⅠ基因表达抑制胃癌细胞系的增殖效率均降低(P＜0.05).胃泌素孵育后,胃癌细胞BGC823、SGC7901的增殖效率均增加(P＜0.05),而RegⅠ基因表达抑制的胃癌细胞系增殖效率则无明显改变(P＞0.05).结论 实验成功建立了RegⅠ基因表达抑制的胃癌细胞系; RegⅠ基因可能对胃泌素促胃癌细胞的增殖有促进的作用.