Preclinical studies for measuring beta cell mass in vivo

这份报告中描述的大部分工作来自哥伦比亚大学的Paul Harris博士的观察，他使用差异基因表达研究指出，β细胞(比其他胰腺细胞在更大程度上)表达囊泡单胺转运体-2(VMAT-2)。Harris博士和他的同事继续使用名为11C-二氢四苯并嗪(11C-DTBZ)的VMAT-2放射性标记配体作为显像剂，在T1 DM大鼠模型上进行正电子发射断层扫描(PET)研究，结果表明，随着β细胞质量在自身免疫过程中下降，11C-DTBZ-PET胰腺信号也随之下降。事实上，在该模型中，PET信号似乎能够预测糖尿病的发生。为了开发这项技术应用于临床介入试验，招募有T1 DM风险的人类，我们启动了几项研究，以评估11C-DTBZ-PET，使用来自人类身体捐赠者的胰岛以及我们可以使用的各种小鼠和非人类灵长类动物模型。 与技术承诺一致，我们发现： 1.人胰岛VMAT-2抗体与胰岛素抗体免疫染色结果基本一致，而其他胰岛内分泌细胞VMAT-2免疫染色均未见阳性反应。 2.可变的人胰岛数目与~3H-DTBZ混合产生的放射性计数与胰岛数目直接相关。 3.冷DTBZ可完全清除与人胰岛结合的~3H-DTBZ，提示其与特异性受体结合。 4.用~3H-DTBZ标记人胰岛，然后将标记的胰岛静脉注射到安乐死的小鼠体内，在动物的肺内产生与注射的胰岛数目一致的放射性计数。 5.非人灵长类动物注射~(11)C-DTBZ，然后用正电子发射计算机断层扫描(PET)进行研究，发现与动物胰腺的磁共振成像(MRI)相一致的明亮信号。 不幸的是，随着进一步的测试，我们发现： 1.在我们的小鼠模型中，天然的胰腺DTBZ-PET信号与预测的β细胞质量没有可靠的相关性。 2.小鼠和非人灵长类胰腺中抗VMAT2和抗胰岛素(或其他内分泌激素特异性抗体)的免疫染色与β细胞特异性VMAT2的表达不一致。为了检验β细胞VMAT2表达的明显缺失是否继发于物种差异(即产生了抗人VMAT2的染色抗体)，我们对其他已知表达VMAT2的灵长类组织(脑基底节和肾上腺髓质)进行了免疫染色，发现在那些阳性对照组织中有明显表达，而在适当的阴性对照组织中没有表达。 3.用流式细胞仪和逆转录-聚合酶链式反应(RT-PCR)对分离的非人灵长类胰岛单个细胞进行研究，未发现β细胞特异性VMAT2表达的证据。 4.11C-DTBZ既以正对映体的形式存在，又以镜像的负对映体的形式存在。只有阳性的11C-DTBZ对映体与VMAT2结合。我们在同一灵长类动物中使用这两种形式进行了正电子发射计算机断层扫描，发现两者产生了相似的正电子发射断层扫描信号。 综上所述(结合工作单元编号DK055110-01中报告的数据)，我们得出的结论是，尽管11C-DTBZ PET胰腺信号显示出相当大的前景，但对于胰腺β细胞来说，11C-DTBZ PET胰腺信号还不够特异，不能作为一种有用的技术在体内测量β细胞质量，我们已经将注意力转向其他技术。已经准备了一份描述这些结果的手稿，准备出版。 我们继续使用非人类灵长类动物模型来评估怀孕是否像在啮齿动物中所报道的那样促进β细胞的增殖。根据批准的动物使用方案，我们给怀孕的非人类灵长类动物注射了胸苷类似物，然后检查了动物的胰腺，以评估β细胞更新率。另一项这样的研究计划在接下来的几周内进行。初步数据表明，与在啮齿类动物中观察到的反应相反，成年灵长类β细胞即使在怀孕期间也表现出很少的增殖能力。

Much of the work described in this report emanated from observations made by Dr. Paul Harris at Columbia University who, using differential gene expression studies, noted that beta cells (to a much greater degree than other pancreatic cells) express the vesicular monoamine transporter-2 (VMAT-2). Dr. Harris and colleagues went on to perform studies using a VMAT-2 radiolabeled ligand named 11C-dihydrotetrabenazine (11C-DTBZ) as an imaging agent for positron-emission tomography (PET) studies in a rat model for T1DM with results suggesting that as beta cell mass fell during the autoimmune process, so did the 11C-DTBZ-PET pancreatic signal. Indeed, in that model the PET signal seemed able to predict diabetes onset. With an eye toward developing the technique for application in clinical interventional trials enrolling humans at risk for T1DM, we initiated several studies to assess 11C-DTBZ-PET using islets from human cadaveric donors and various mouse and non-human primate models at our disposal. Consistent with the techniques promise, we found that: 1. Human islets immunostained with anti-VMAT-2 and with anti-insulin revealed nearly coincident staining, while other islet endocrine cells did not stain for VMAT-2. 2. Mixing variable human islet numbers with 3H-DTBZ generated radioactive counts directly correlated with the islet number. 3. Cold DTBZ could complete away 3H-DTBZ bound to human islets suggesting binding to specific receptors. 4. Labeling human islets with 3H-DTBZ, then injecting the labeled islets intravenously into mice promptly euthanized after that injection, generated radioactivity counts in the animals lungs consistent with the islet number injected. 5. Non-human primates injected with 11C-DTBZ then studied using PET revealed bright signals that co-registered with magnetic-resonance imaging (MRI) of the animals pancreas. Unfortunately, with further testing we found: 1. In our mouse models, native pancreatic DTBZ-PET signals did not reliably correlate with predicted beta cell mass. 2. Immunostaining of both mouse and non-human primate pancreas with anti-VMAT2 and anti-insulin (or other endocrine hormone-specific antibodies) was not consistent with beta cell specific VMAT2 expression. To test whether the apparent absence of beta cell VMAT2 expression might be secondary to species differences (i.e. the staining antibody was raised against human VMAT2), we immunostained other primate tissue (brain basal ganglia and adrenal medulla) known to express VMAT2 and found clear expression in those positive control tissues, but absent staining in appropriate negative control tissues. 3. Individual cells from isolated non-human primate islets studied using both flow cytometry and reverse-transcriptase-polymerase chain reaction (RT-PCR) revealed no evidence for beta cell specific VMAT2 expression. 4. 11C-DTBZ exists as both a positive enantiomer, and a mirror image negative enantiomer. Only the positive 11C-DTBZ enantiomer binds to VMAT2. We performed PET scans using both forms in the same primate and found that both generated similar PET signals. Taken together (in conjunction with data reported in work unit number DK055110-01), we concluded that while it showed considerable promise, 11C-DTBZ PET pancreatic signals are not sufficiently specific for pancreatic beta cells to serve as a useful technique for measuring beta cell mass in vivo and we have turned our attention to other techniques. A manuscript describing these results has been prepared for publication. We have moved on to assess, using the non-human primate model, whether pregnancy promotes beta cell proliferation as is reported in rodents. Under an approved animal use protocol, we have administered thymidine analogs to a pregnant non-human primate, then examined the animal's pancreas to assess rates of beta cell turnover. Another such study is planned in the next few weeks. Preliminary data suggests that as opposed to responses observed in rodents, adult primate beta cells display little if any capacity for proliferation, even during pregancy.