An experimental and theoretical evaluation of the influence of pretargeting antibody on the tumor accumulation of effector

In treating tumors by pretargeting, the antitumor antibody and the cytotoxic effector (e.g., toxins and radioactivity) are separately administered. Therefore, pretargeting is more complicated with many variables. We are conducting studies to understand the influence of each variable using a novel recognition pair of mutually complementary phosphorodiamidate morpholino oligomers (MORF/cMORF). Earlier we developed a semi-empirical model capable of accurately predicting the behavior of a radiolabeled cMORF effector with variations in dosages and timing. We have now extended the model to predict the effector behavior, in particular, its maximum percent tumor accumulation (MPTA) in mice pretargeted with three different MORF-conjugated antibodies (MN14, B72.3, and CC49). The MN14 and the CC49 target different antigens in the same tumor, whereas the CC49 and the B72.3 target the same antigen but with very different tumor accumulation. By comparing the pretargeting results of these three antibodies with our prediction, we confirmed that the MPTA of the radiolabeled cMORF effector in the LS174T tumor is independent of the antibodies. In conclusion, the MPTA cannot be improved through the use of different pretargeting antibodies, although different antibodies may improve the maximum absolute tumor accumulation, the heterogeneity, and/or the tumor-to-normal tissue ratios of the effector. This conclusion will apply equally well to effectors carrying a fluorescent probe, an anticancer agent, or a radioactive imaging agent.     

Replacing 99mTc with 111In Improves MORF/cMORF Pretargeting by Reducing Intestinal Accumulation

Purpose  To reduce accumulation in the abdomen by MORF/cMORF pretargeting, ¹¹¹In was compared to ^99mTc as the radiolabel. Procedures  After receiving either ^99mTc (MAG3)-cMORF or ¹¹¹In (DTPA)-cMORF, normal mice were imaged and killed for pharmacokinetics. Thereafter, tumored mice were pretargeted with MORF-antibody, 48 h later were given an injection of ^99mTc- or ¹¹¹In-cMORF, and finally were imaged repeatedly. Results  The cMORF biodistribution in both normal and pretargeted tumored mice was influenced by its radiolabel. While excretion of both ^99mTc-cMORF and ¹¹¹In-cMORF was rapid and mainly through the kidneys, about 2% of ^99mTc accumulated in the intestines compared to essentially no intestinal accumulation for ¹¹¹In at any time. Tumor accumulation was unchanged. Conclusion  In applications of MORF/cMORF pretargeting intended to image organs deep within the abdomen such as the pancreas, radiolabeling with ¹¹¹In may be superior to ^99mTc.     

In Vivo Near-Infrared Fluorescence Imaging of Integrin αvβ3 in an Orthotopic Glioblastoma Model

Purpose Expression of cell adhesion molecule integrin α_vβ₃ is significantly up-regulated during tumor growth, and sprouting of tumor vessels and correlates well with tumor aggressiveness. The purpose of this study was to visualize tumor integrin α_vβ₃ expression in vivo by using near-infrared fluorescence (NIRF) imaging of Cy5.5-linked cyclic arginine–glycine–aspartic acid (RGD) peptide in an orthotopic brain tumor model.Procedures U87MG glioma cells transfected with the firefly luciferase gene were stereotactically injected into nude mice in the right frontal lobe. Bioluminescence imaging (BLI) using d-luciferin substrate and small animal magnetic resonance imaging (MRI) using gadolinium contrast enhancement were conducted weekly after tumor cell inoculation to monitor intracranial tumor growth. Integrin α_vβ₃ expression was assessed by using a three-dimensional optical imaging system (IVIS 200) 0–24 hours after administration of 1.5 nmol monomeric Cy5.5-RGD via the tail vein. Animals were injected intravenously with both Texas Red–tomato lectin and Cy5.5-RGD prior to sacrifice to visualize peptide localization to tumor vasculature using histology.Results Fluorescence microscopy demonstrated specific Cy5.5-RGD binding to both U87MG tumor vessels and tumor cells with no normal tissue binding. NIRF imaging showed highest tumor uptake and tumor to normal brain tissue ratio two hours postinjection (2.64 ± 0.20). Tumor uptake of Cy5.5-RGD was effectively blocked by using unlabeled c(RGDyK), and injection of Cy5.5 dye alone showed nonspecific binding.Conclusions Optical imaging via BLI and NIRF offer a simple, effective, and rapid technique for noninvasive in vivo monitoring and semiquantitative analysis of intracranial tumor growth and integrin α_vβ₃ expression. This study suggests that NIRF via fluorescently labeled RGD peptides may provide enhanced surveillance of tumor angiogenesis and anti-integrin treatment efficacy in orthotopic brain tumor models.     

Performance of a new fluorescence‐labeled MMP inhibitor to image tumor MMP activity in vivo in comparison to an MMP‐activatable probe

Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade structural components of the extracellular matrix and participate in pathologies such as cancer and inflammatory disorders. The development of novel contrast agents for optical imaging of MMP activity in vivo is of great interest. The commonly used near‐infrared fluorescence‐compatible agents are dye‐quenched probes that do not emit fluorescence until they interact with MMPs. In contrast, fluorescent synthetic low‐molecular‐weight MMP inhibitors have not been systematically employed. The aim of this study was to evaluate the performance of our recently developed Cy5.5‐labeled MMP inhibitor to image MMP activity in tumors in vivo compared with activatable fluorescent MMP‐sensing probes. The dynamic uptake of Cy5.5‐AF489 into four different tumor entities was analyzed in xenografted mice by intravenous injection and subsequent fluorescence reflectance imaging. Tumors were characterized in regard to their MMP‐2 and ?9 mRNA expressions (qRT‐PCR analysis), proteins (immunohistochemistry) and gelatinase/collagenase activities (in situ zymography). Cy5.5‐AF489 was compared with MMPSense^TM 680 and MMPSense^TM 750 FAST, two commercially available MMP‐activatable probes. Cy5.5‐AF489 showed a specific tumor uptake, which was blocked by pre‐injection of the unlabeled MMPI, and discriminated between tumors with high or low MMP‐2/‐9 expressions. Our optical probe facilitated faster visualization of MMP‐active tumors accompanied by excellent tumor‐to‐background ratios when compared with activatable probes. The MMP inhibitor Cy5.5‐AF489 permits fast in vivo imaging of MMP expression/activity in tumors. Given its small molecular weight and non‐peptidic structure, translational imaging from a preclinical application to a diagnostic tool for MMP‐related diseases seems feasible. Copyright © 2012 John Wiley & Sons, Ltd.     

细胞凋亡近红外线荧光显像在评价肿瘤放疗早期疗效中的作用

目的：研究近红外线荧光（NIRF）标记的annexinV是否能显示小鼠体内细胞凋亡的情况。方法：合成了Cy5．5-annexinV，并对其体外与磷脂酰丝氨酸（PS）的结合特性进行研究。建立小鼠乳腺癌（MCA29）动物模型，将其分为放射治疗组与非放射治疗组，于放射治疗后6h静脉注射Cy5．5-annexJnV，24小时后进行显像，比较两组浓聚Cy5．5-annexinV的差异。结果：Cy5．5-annexinV与PS结合的结合力（Kd）为2．51×10^-9，未标记的annexinV与Ps的Kd为1．54×10^-9，两者间无显著差异，表明经Cy5．5标记后并未改变annexinV与PS的Kd。采用流式细胞检测仪及共聚焦激光扫描显微镜研究表明Cy5．5-annexinV与PS结合的特性与FITC—annexinV相似，说明Cy5．5-annexinV在体外具有鉴别凋亡细胞与正常细胞的能力。小鼠MCA29肿瘤细胞动物模型显像表明经放射治疗后的肿瘤细胞浓聚Cy5．5-annexinV高于未治疗的肿瘤细胞。结论：Cy5．5-annexinV可以用于肿瘤放射治疗的早期疗效评价。     

In vivo tumor imaging in mice with near-infrared labeled endostatin

Endostatin is a potent inhibitor of angiogenesis currently in phase I clinical trials. Imaging technologies that use near-infrared fluorescent probes are well suited to the laboratory setting. The goal of this study was to determine whether endostatin labeled with a near-infrared probe (Cy5.5) could be detected in an animal and whether it would selectively localize to a tumor. Endostatin was conjugated to Cy5.5 monofunctional dye and injected into mice bearing Lewis lung carcinoma tumors (350 mm2). Mice were imaged at various time points while under sedation using a lightproof box affixed to a fluorescent microscope mounted with a filter in the near-infrared bandwidth consistent with Cy5.5 fluorescence. After i.p. injection, endostatin-Cy5.5 was absorbed producing a near-infrared fluorescent image within the tumors at 18 h reaching a maximum at 42 h after injection. No signal was emitted from mice injected with unlabeled endostatin or Cy5.5 dye alone or those that received no injection. Further results show that a dose response exists with injection of endostatin-Cy5.5. Mimicking the clinical route of administration, an i.v. injection had a peak signal emission at 3 h but also persisted to 72 h. Finally, to determine the intratumoral binding site for endostatin, we performed immunofluorescence on tumor specimens and demonstrated that endostatin binds to tumor vasculature and colocalizes with platelet/endothelial cell adhesion molecule 1 expression. This study demonstrates that endostatin covalently bound to Cy5.5 will migrate from a distant i.p. injection site to a tumor. These data indicate that endostatin-Cy5.5 is appropriate for selectively imaging tumors in uninjured experimental animals.     

Dual-Modality Micro-Positron Emission Tomography/Computed Tomography and Near-Infrared Fluorescence Imaging of EphB4 in Orthotopic Glioblastoma Xenograft Models

Purpose

In glioblastoma, EphB4 receptors, a member of the largest family of receptor tyrosine kinases, are overexpressed in both tumor cells and angiogenic blood vessels. The purpose of this study was to examine whether the EphB4-binding peptide TNYL-RAW labeled with both ⁶⁴Cu and near-infrared fluorescence dye Cy5.5 could be used as a molecular imaging agent for dual-modality positron emission tomography/computed tomography [PET/CT] and optical imaging of human glioblastoma in orthotopic brain tumor models.

Materials and Methods

TNYL-RAW was conjugated to Cy5.5 and the radiometal chelator 1,4,7,10-tetraazadodecane-N,N′,N″,N?-tetraacetic acid. The conjugate was then labeled with ⁶⁴Cu for in vitro binding and in vivo dual μPET/CT and optical imaging studies in nude mice implanted with EphB4-expressing U251 and EphB4-negative U87 human glioblastoma cells. Tumors and brains were removed at the end of the imaging sessions for immunohistochemical staining and fluorescence microscopic examinations.

Results

μPET/CT and near-infrared optical imaging clearly showed specific uptake of the dual-labeled TNYL-RAW peptide in both U251 and U87 tumors in the brains of the nude mice after intravenous injection of the peptide. In U251 tumors, the Cy5.5-labeled peptide colocalized with both tumor blood vessels and tumor cells; in U87 tumors, the tracer colocalized only with tumor blood vessels, not with tumor cells.

Conclusions

Dual-labeled EphB4-specific peptide could be used as a noninvasive molecular imaging agent for PET/CT and optical imaging of glioblastoma owing to its ability to bind to both EphB4-expressing angiogenic blood vessels and EphB4-expressing tumor cells.     

Highly selective in-vivo imaging of tumor as an inflammation site by ROS detection using hydrocyanine-conjugated, functional nano-carriers

Previously, the optical imaging of chitosan-functionalized, Pluronic-based nano-carriers by Cy5.5 conjugation revealed a good tumor targeting characteristic of the nano-carriers in vivo [J. Control. Release, 147 (2010) 109–117]. However, in spite of the relatively strong signal from tumor site, they also showed strong fluorescence signals from other organs, especially liver. Thus, for the detection of pathological sites, the direct use of the Cy5.5-conjugated nano-carriers is limited due to significant background signals associated with non-specific delivery of the probes. To overcome this limitation, in this study, we prepared hydrocyanine-conjugated and chitosan-functionalized Pluronic-based nano-carriers (Hydrocyanine-NC) that can detect ROS in pathological sites. The reduction of cyanine to hydrocyanine of the nano-carriers resulted in complete disappearance of fluorescence emission, and the fluorescence could be recovered by ROS-induced re-oxidization. Hydrocyanine-NC could detect various ROS including superoxide anion (O₂⁻) and hydroxyl radical (OH⁻) in a dose-dependent manner. Hydrocyanine-NC was also stable in serum-containing media and did not show acute cytotoxicity. Hydrocyanine-NC developed strong fluorescence by the intracellular ROS formation in LPS-stimulated macrophage cells in vitro. As an in-vivo inflammation site imaging, SCC7 tumor-bearing mice were optically monitored after the i.v. injection of the dye-conjugated nano-carriers. When non-reduced, cyanine-conjugated and chitosan-functionalized Pluronic-based nano-carriers (Cyanine-NC) were injected, strong fluorescence emission was observed from the abdominal area as well as from the tumor site, and it remained over 2 days. In contrast, in the case of Hydrocyanine-NC, the initially very weak fluorescence emission from the abdominal area disappeared over time whereas the fluorescence emission from the tumor site was similar to that of Cyanine-NC. Therefore, the re-oxidation of Hydrocyanine-NC by ROS in vivo specifically eliminated the background signals from non-specific delivery of the probes, but it produced fluorescence emission strong enough to monitor the target inflammation site selectively.     

Radioisotopic Localization of 90Yttrium–Ibritumomab Tiuxetan in Patients with CD20+ Non-Hodgkin’s Lymphoma

Purpose   ⁹⁰Yttrium-ibritumomab-tiuxetan (Zevalin) is an effective treatment for relapsed or refractory low-grade, follicular, or transformed B-cell NHL. The purpose of this study is to assess whether tissue and cellular localization of ⁹⁰Y-ibritumomab–tiuxetan determined by autoradiography and radioactivity localized to tumor tissue might enhance our understanding of the mechanism of action of radioimmunotherapy. Methods  Eight eligible patients had CD20+ NHL, a bulky peripheral lymph node, and were scheduled for ⁹⁰Y-ibritumomab–tiuxetan treatment. 2-Deoxy-2-[F-18]fluoro-d-glucose-positron emission tomography/computed tomography (FDG-PET/CT) was performed prior to treatment and at 12 weeks after therapy for assessment of response. Bone marrow, lymph node, and blood samples were collected 114 ± 3 h after 14.8 MBq/kg ⁹⁰Y-ibritumomab-tiuxetan and processed for histology, scintillation counting, and microscopic autoradiography. Results  Pericellular membrane localization of ⁹⁰Y-ibritumomab–tiuxetan to lymphoma cells was observed by autoradiography in the involved areas of lymph node with absence of significant localization in histologically normal sections of bone marrow. Pericellular radioactivity and the highest quantitative radioactivity were observed in lymph node samples of responding patients. Conclusions   ⁹⁰Y-ibritumomab-tiuxetan localizes to the surface membrane of CD20+ lymphoma cells in affected lymph nodes. The patients with the highest quantitative concentration of radioactivity to the lymph node as determined by scintillation counting were observed to have a clinical and FDG-PET/CT response.     

Continuous cerebral autoregulation monitoring by improved cross-correlation analysis: comparison with the cuff deflation test

Objectives To improve the cross-correlation method for noninvasive, continuous monitoring of cerebral autoregulation, to evaluate this method in humans with intact and impaired autoregulatory capacity, and to compare it to the cuff deflation test. Design and setting Prospective study in the intensive care unit of a university hospital. Patients and participants Fourteen patients with severe head injury, six patients with subarachnoid hemorrhage, and nine healthy volunteers. Interventions and measurements Middle cerebral artery flow velocities and arterial blood pressure were monitored continuously. Aaslid's thigh cuff tests were performed and results were scored using Tiecks' model for autoregulation index. Data were then collected without any patient manipulation. The mean time delay between slow spontaneous oscillations of blood pressure and middle cerebral artery flow velocity was calculated by cross-correlation analysis. Data are expressed as median (lower/upper quartile). Results Healthy subjects had a higher autoregulation index than patients, 5.0 (5.0/5.5) vs. 3.3 (2.0/4.5). Slow oscillations of blood pressure and middle cerebral artery flow velocity showed a time delay of –2.0 s (–2.7/–1.7) in healthy subjects but were almost synchronal in patients, –0.07 s (–0.5/0.45). Inter-method agreement in diagnosing an intact or impaired cerebral autoregulation was obtained in 108 of 147 examinations of autoregulation (73.5%) and was considered moderate. Conclusions Cross-correlation analysis may serve as a simple, noninvasive, and continuous measure of cerebral autoregulation. The time delay of –2.0 s in healthy subjects is in good agreement with other studies. Short-term autoregulation tests and monitoring techniques based on slow spontaneous oscillations should not be used interchangeably. Electronic supplementary material Electronic supplementary material to this contribution can be obtained by using the Springer Link server located at . Financial support for this study was provided by Kuratorium ZNS, Hannelore Kohl Stiftung, Bonn, Germany (no. 2001011).     

Signal processors in duplex sonography: in vitro comparison between analog and digital methods

Using a new flow-test phantom, which respects the acoustic properties of real blood as well as the proximal and distal impedances of body circulation, we assessed the performance of two duplex sonography signal processors on blood-flow measurements. With both the analog and the dynamic signal processor (Fast Fourier Transform), the correlation between duplex sonography and quantitative flow measurements was high (0.96–0.99) for different dynamic conditions (steady or pulsatile blood flow, varying heart rate, blood pressure, and hematocrit) and for different mechanical conditions (silicon tube or animal vessel). The real blood flow was overestimated by duplex sonography the over-estimation was more pronounced with the analog processor (factor 1.87–4.20) than with the digital processor (factor 1.22–1.64,P<0.05). Applied to the study of asymmetric stenoses, the digital processor was not superior to the analog processors described in the literature.     

人骨髓瘤细胞株基因表达谱经沙利度胺诱导的变化

本研究通过检测沙利度胺(thalidomide,TLD)作用于多发性骨髓瘤细胞株RPMI-8226后基因表达谱的改变,探索TLD抗骨髓瘤作用的分子机制。通过逆转录PCR,将TLD处理前后的人骨髓瘤细胞株RPMI-8226的mRNA分别制备成2种探针,应用Cy3和Cy5荧光染料分别标记,随后与包含1 152条待研究的人类基因表达谱基因芯片杂交,通过扫描和计算机软件分析,寻找经TLD作用后表达有差异的基因,并分析这些差异表达基因的功能。结果表明,100μmol/L TLD作用于RPMI8226细胞72小时后,筛选出22个差异表达基因,其中4个基因下调,分别为rpl32、scya3、mmp1和igbp1基因,18个基因表达上调,其中主要为wars、tubb4q、ube1l、txnrd1和fyb基因等5个基因表达明显上调。研究显示:①TLD能使编码色氨酸-tRNA合成酶的wars基因表达上调,同时使编码基质金属蛋白酶1的mmp1表达下调,这2个基因的变化可能与TLD抑制血管生成的作用有关;②TLD能使编码巨噬细胞炎症反应蛋白1a(MIP-1a)的scya3和编码免疫球蛋白结合蛋白1的igbp1表达下调,其表达下降可能参与了TLD抑制细胞增殖的过程;③TLD能诱导编码微管蛋白β4的tubb4q、编码泛素激活酶E1相关蛋白的ube1l和编码硫氧还蛋白还原酶1(TR1)的txnrd1表达上调,这些基因的表达变化与TLD的促凋亡作用有关;④TLD能使编码酪氨酸激酶Fyn结合蛋白(Fyb)的fyb表达上调,该基因的表达上调与TLD杀伤骨髓瘤细胞的作用有关。结论:22个差异表达基因通过不同的作用环节和途径发挥抗骨髓瘤功能,它们分别涉及到蛋白质的合成和降解、细胞信号转导、细胞骨架运动、免疫调节、细胞代谢、抑癌基因的调控、细胞凋亡等方面,直接或间接与TLD的分子作用机制有关。     

近红外线光学成像诊断微小肝癌的初步研究

目的探讨近红外线光学成像在动物活体的皮下HepG2移植瘤的成像表现。方法取HepG2皮下荷瘤鼠10只,应用小动物分子成像仪和非特异性探针Cy5.5对裸鼠移植瘤行近红外线光学成像(NearInfra-redOpticalImaging),以得到早期肿瘤的大小和荧光强度信息。结果10只HepG2皮下荷瘤裸鼠中有8只肿瘤清晰成像,成像敏感度为80%,成像瘤体的平均径线为3.717mm×2.612mm,平均体积为15.768mm3。其余2只受背景荧光影响而肿瘤边界显示不清。结论近红外线光学成像具有敏感性高的优点,应用非特异性探针即可在较早期阶段发现微小肿瘤。而背景荧光干扰和成像深度有限是影响实验结果的瓶颈。     

Persistently low plasma thioredoxin is associated with meningococcal septic shock in children

Objective To compare plasma levels of thioredoxin (Trx), TNF-α and IL-1β in children during the acute phase of meningococcal septic shock (MSS) and in convalescence. Design and setting Retrospective, observational study in the paediatric intensive care unit of a postgraduate teaching hospital. Patients Thirty-five children requiring intensive care for meningococcal sepsis; paired convalescent samples from 30 survivors (median interval between samples 62 days); 25 healthy control children. Measurements and results Plasma Trx levels were significantly lower in the children with MSS, both during the acute illness (5.5 ng/ml, IQR 1.4–11.4) and in convalescence (2.5 ng/ml, IQR 0.4–6.9) than controls (18.8 ng/ml, IQR 7.9–25.0). Levels of IL-1β and TNF-α were higher in patients with acute MSS (30.3 pg/ml, IQR 3.6–63.6, and 145.9 pg/ml, IQR 31.8–278.1 respectively) than controls (3.7 pg/ml, IQR 0–36.9, and 23.8 pg/ml, IQR 0–124.3, respectively). Levels fell in convalescence (3.7 pg/ml, IQR 0–25.5, 3.7 pg/ml, IQR 0–304.8, respectively). Plasma Trx was higher in non-survivors, albeit a small group (n = 5), than in survivors (n = 30). Trx, IL-1β, and TNF-α levels were not correlated with predicted mortality as assessed by the paediatric risk of mortality (PRISM) score. Conclusions Children with MSS exhibit persistently low plasma levels of Trx during acute illness and in convalescence.     

Near-Infrared Fluorescence Imaging of Tumor Integrin αvβ3 Expression with Cy7-Labeled RGD Multimers

Purpose Cell adhesion molecule integrin α_vβ₃ is an excellent target for tumor interventions because of its unique expression on the surface of several types of solid tumor cells and on almost all sprouting tumor vasculatures. Here, we describe the development of near-infrared (NIR) fluorochrome Cy7-labeled RGD peptides for tumor integrin targeting.Procedures Mono-, di-, and tetrameric RGD peptides were synthesized and conjugated with Cy7. The integrin specificity of these fluorescent probes was tested in vitro for receptor binding assay and fluorescence microscopy and in vivo for subcutaneous U87MG tumor targeting.Results The tetrameric RGD peptide probe with the highest integrin affinity showed the highest tumor activity accumulation and strongest tumor-to-normal tissue contrast. This uptake is integrin-specific as the signal accumulated in the tumor can be effectively blocked by unconjugated RGD peptide antagonist of integrin α_vβ₃.Conclusions Noninvasive NIR fluorescence imaging is able to detect and semiquantify tumor integrin expression based upon the highly potent tetrameric RGD peptide probe.     

E‐selectin targeting to visualize tumorsin vivo

Generally angiogenic factors induce the expression of E‐selectin in vascular endothelial cells in the tumors. In this study, we employed an anti‐E‐selectin monoclonal antibody to target tumors in vivo and evaluated an optical imaging reagent to visualize tumor regions. The anti‐E‐selectin antibody was conjugated on the surface of liposomes, which encapsulated the near‐infrared fluorescent substances Cy3 or Cy5.5. The liposomes efficiently recognized human umbilical vein endothelial cells only when E‐selectin was induced by angiogenic factors such as TNF‐α in vitro. Cy5.5 encapsulated into liposomes that were conjugated with an anti‐E‐selectin antibody successfully visualized Ehrlich ascites tumor cells when transplanted into mice. Thus, E‐selectin targeting with liposomes containing a near‐infrared fluorescent dye was found effective in visualizing tumors in vivo. This strategy should be extremely useful as a method to identify sentinel lymphatic nodes and angiogenic tumors as well as use for drug delivery to tumor cells. Copyright © 2010 John Wiley & Sons, Ltd.     

A Non-Peptidic S100A9 Specific Ligand for Optical Imaging of Phagocyte Activity <Emphasis Type="Italic">In Vivo</Emphasis>

Purpose

Non-invasive assessment of inflammatory activity in the course of various diseases is a largely unmet clinical challenge. An early feature of inflammation is local secretion of the alarmin S100A8/A9 by activated phagocytes. We here evaluate a novel S100A9-targeted small molecule tracer Cy5.5-CES271 for in vivo optical imaging of inflammatory activity in exemplary disease models.

Procedures

Dynamics of Cy5.5-CES271 was characterized in a model of irritant contact dermatitis by sequential fluorescence reflectance imaging (FRI) up to 24 h postinjection (p.i.). Specificity of Cy5.5-CES271 binding to S100A9 in vivo was examined by blocking studies and by employing S100A9^?/? mice. Finally, S100A9 secretion in acute lung inflammation was assessed by Cy5.5-CES271 and FRI of explanted lungs.

Results

In ear inflammation, we were able to non-invasively follow the time course of S100A9 expression using Cy5.5-CES271 and FRI over 24 h p.i. (peak activity at 3 h p.i.). Specificity of imaging could be shown by a significant signal reduction after predosing and using S100A9^?/? mice. In acute lung injury, local and systemic S100A8/A9 levels increased over time and correlated significantly with FRI signal levels in explanted lungs.

Conclusions

Cy5.5-CES271 shows significant accumulation in models of inflammatory diseases and specific binding to S100A9 in vivo. This study, for the first time, demonstrates the potential of a small molecule non-peptidic tracer enabling imaging of S100A9 as a marker of local phagocyte activity in inflammatory scenarios suggesting this compound class for translational attempts.

     

预定位技术用于超声分子成像的体外实验

目的 探讨预定位技术提高超声造影剂靶向肿瘤细胞的能力。方法 采用两步法预定位技术靶向SKOV-3卵巢癌细胞：第一步加入生物素化的CEA抗体,第二步加入结合链霉亲和素的造影剂。结果 制备的结合链霉亲和素的PLGA造影剂平均粒径为（346.20±74.49）nm。链霉亲和素与造影剂的连接效率为（95.02±0.62）%。预定位靶向组细胞结合的造影剂明显多于其余各组。结论 预定位技术能提高造影剂的靶向性,可提高超声分子成像的敏感性。     

Low density lipoprotein cholesterol, statins and cardiovascular events: a meta–analysis

Summary A recent meta–analysis of the Cholesterol Treatment Trialists’ (CTT) Collaboration comes to the clear conclusion that a reduction in LDL–C using statins of 1 mmol/l (39 mg/dl) leads to a decrease in overall mortality by 12%, in coronary mortality by 19% and in the incidence of strokes by 17%, independent of the LDL–C level prior to the start of treatment. We conducted a systematic review retrieving 18 studies with a total of 97 861 participants. Differences in average LDL–C reductions between the intervention and control groups during the follow–up and relative risks according to different clinical endpoints were extracted from the original publications. Metaregression analyses showed that reduction in LDL–C accounted for more than 75% of the variance in risk reductions for overall mortality and cardiovascular endpoints. On the basis of our estimates, a reduction in LDL–C of 1 mmol/l (39 mg/dl) leads to reductions in overall mortality, coronary mortality, incidence of non–fatal myocardial infarction, the combination of coronary mortality and non–fatal myocardial infarction, stroke and any vascular event by 15% (95% CI: 11–20%), 24% (95% CI: 20–28%), 27% (95% CI: 20–32%), 25% (95% CI: 22–29%), 24% (18–29%) and 22% (95% CI: 19–26%), respectively. We conclude that the extent to which statins lower LDL–C is strongly related to the improvement of clinical outcomes achieved by this class of drugs. An erratum to this article is available at .     

Detection of miRNA cancer biomarkers using light activated Molecular Beacons

Early detection of cancer biomarkers can reduce cancer mortality rate. miRNAs are small non-coding RNAs whose expression changes upon the onset of various types of cancer. Biosensors that specifically detect such biomarkers can be engineered and integrated into point-of-care devices (POC) using label-free detection, high sensibility and compactness. In this paper, a new engineered Molecular Beacon (MB) construct used to detect miRNAs is presented. Such a construct is immobilized onto biosensor surfaces in a covalent and spatially oriented way using the photonic technology Light Assisted Molecular Immobilization (LAMI). The construct consists of a Cy3 labelled MB covalently attached to a light-switchable peptide. One MB construct contains a poly-A sequence in its loop region while the other contains a sequence complementary to the cancer biomarker miRNA-21. The constructs have been characterized by UV-Vis spectroscopy, mass spectrometry and HPLC. LAMI led to the successful immobilization of the engineered constructs onto thiol functionalized optically flat quartz slides and Silicon on Insulator (SOI) sensor surfaces. The immobilized Cy3 labelled MB construct has been imaged using confocal fluorescence microscopy (CFM). The bioavailability of the immobilized engineered MB biosensors was confirmed through specific hybridization with the Cy5 labelled complementary sequence and imaged by CFM and FRET. Hybridization kinetics have been monitored using steady state fluorescence spectroscopy. The label-free detection of miRNA-21 was also achieved by using integrated photonic sensing structures. The engineered light sensitive constructs can be immobilized onto thiol reactive surfaces and are currently being integrated in a POC device for the detection of cancer biomarkers.

Photonic based detection strategies of cancer miRNA biomarkers after Light Assisted Molecular Immobilization (LAMI) of peptide-MB biosensor constructs.