Noninvasive and quantitative monitoring of adult neuronal stem cell migration in mouse brain using bioluminescence imaging

It is now generally accepted that continuous neurogenesis occurs in the adult mammalian brain, including that of humans. Modulation of adult neurogenesis can provide therapeutic benefits for various brain disorders, including stroke and Parkinson's disease. The subventricular zone-olfactory bulb pathway is one of the preferred model systems by which to study neural stem cell proliferation, migration, and differentiation in adult rodent brain. Research on adult neurogenesis would greatly benefit from reliable methods for long-term noninvasive in vivo monitoring. We have used lentiviral vectors encoding firefly luciferase to stably mark endogenous neural stem cells in the mouse subventricular zone. We show that bioluminescence imaging (BLI) allows quantitative follow-up of the migration of adult neural stem cells into the olfactory bulb in time. Moreover, we propose a model to fit the kinetic data that allows estimation of migration and survival times of the neural stem cells using in vivo BLI. Long-term expression of brain-derived neurotrophic factor in the subventricular zone attenuated neurogenesis, as detected by histology and BLI. In vivo monitoring of the impact of drugs or genes on adult neurogenesis is now within reach.     

A bioluminescence technique for quantitative and structure-associated imaging of pyruvate

A novel bioluminescence assay has been developed for measuring pyruvate within sections of snap-frozen tissue in a quantitative manner as well as with a spatial resolution on a microscopical level. The assay was verified via HPLC and two independent photometric tests. The novel assay makes it possible to determine pyruvate concentrations in cryosections in the range of 0-5.0 micromol/g tissue (dry weight). Based on the analysis of samples of given pyruvate concentrations, the assay exhibits a recovery with a deviation < or =15%. The minimal detectable amount was 0.02 pmol based on a 20 microm thick tissue section with an area of 1 cm(2). Combination of the already established imaging bioluminescence techniques for ATP, glucose, and lactate with the novel pyruvate assay allows for a comprehensive characterization of the metabolic profile of individual tumors. As the redox state of cancer cells can be critical for the efficiency of irradiation and a number of chemotherapeutics, and as pyruvate and lactate are known to have radical scavenger functions, we hypothesize that the novel bioluminescence assay may be used for measuring the pretherapeutic lactate-to-pyruvate ratio which may predict the radiosensitivity of individual malignancies.     

Establishment and quantitative imaging of a 3D lung organotypic model of mammary tumor outgrowth

The lung is the second most common site of metastatic spread in breast cancer and experimental evidence has been provided in many systems for the importance of an organ-specific microenvironment in the development of metastasis. To better understand the interaction between tumor and host cells in this important secondary site, we have developed a 3D in vitro organotypic model of breast tumor metastatic growth in the lung. In our model, cells isolated from mouse lungs are placed in a collagen sponge to serve as a scaffold and co-cultured with a green fluorescent protein-labeled polyoma virus middle T antigen (PyVT) mammary tumor cell line. Analysis of the co-culture system was performed using flow cytometry to determine the relative constitution of the co-cultures over time. This analysis determined that the cultures consisted of viable lung and breast cancer cells over a 5-day period. Confocal microscopy was then used to perform live cell imaging of the co-cultures over time. Our studies determined that host lung cells influence the ability of tumor cells to grow, as the presence of lung parenchyma positively affected the proliferation of the mammary tumor cells in culture. In summary, we have developed a novel in vitro model of breast tumor cells in a common metastatic site that can be used to study tumor/host interactions in an important microenvironment.     

A porcine model of acute, haematogenous, localized osteomyelitis due to Staphylococcus aureus: a pathomorphological study

A porcine model of acute, haematogenous, localized osteomyelitis was established. Serial dilutions of Staphylococcus aureus [5-50-500-5000-50 000 CFU/kg body weight (BW) suspended in saline or saline alone] were inoculated into the right brachial artery of pigs (BW 15 kg) separated into six groups of two animals. During the infection, blood was collected for cultivation, and after the animals were killed from day 5 to 15, they were necropsied and tissues were sampled for histopathology. Animals receiving ≤500 CFU/kg BW were free of lesions. Pigs inoculated with 5000 and 50 000 CFU/kg BW only developed microabscesses in bones of the infected legs. In the centre of microabscesses, S. aureus was regularly demonstrated together with necrotic neutrophils. Often, bone lesions resulted in trabecular osteonecrosis. The present localized model of acute haematogenous osteomyelitis revealed a pattern of development and presence of lesions similar to the situation in children. Therefore, this model should be reliably applied in studies of this disease with respect to e.g. pathophysiology and pathomorphology. Moreover, because of the regional containment of the infection to a defined number of bones, the model should be applicable also for screening of new therapy strategies.     

Tomographic bioluminescence imaging reconstruction via a dynamically sparse regularized global method in mouse models

Generally, the performance of tomographic bioluminescence imaging is dependent on several factors, such as regularization parameters and initial guess of source distribution. In this paper, a global-inexact-Newton based reconstruction method, which is regularized by a dynamic sparse term, is presented for tomographic reconstruction. The proposed method can enhance higher imaging reliability and efficiency. In vivo mouse experimental reconstructions were performed to validate the proposed method. Reconstruction comparisons of the proposed method with other methods demonstrate the applicability on an entire region. Moreover, the reliable performance on a wide range of regularization parameters and initial unknown values were also investigated. Based on the in vivo experiment and a mouse atlas, the tolerance for optical property mismatch was evaluated with optical overestimation and underestimation. Additionally, the reconstruction efficiency was also investigated with different sizes of mouse grids. We showed that this method was reliable for tomographic bioluminescence imaging in practical mouse experimental applications.     

Rapid Staphylococcus aureus agr type determination by a novel multiplex real-time quantitative PCR assay

The accessory gene regulator (agr) is a crucial regulatory component of Staphylococcus aureus involved in the control of bacterial virulence factor expression. We developed a real-time multiplex quantitative PCR assay for the rapid determination of S. aureus agr type. This assay represents a rapid and affordable alternative to sequence-based strategies for assessing relevant epidemiological information.     

播散性鼠B细胞淋巴瘤动物模型的建立

目的 在具有免疫能力的BALB/c小鼠上构建播散性B淋巴瘤动物模型.方法 经尾静脉注射鼠源性B细胞淋巴瘤细胞株A20于同源BALB/c小鼠,0～3个月间观察动物成瘤情况;取动物脏器行石蜡包埋、病理切片、HE染色;流式细胞仪检测其CD19的表达.结果 尾静脉注射2×106、2×107细胞于14只BALB/c小鼠,成瘤时间分别为76.8±12.0天和26.1±7.9天;总体成瘤率分别为 71.4%(5/7)和100%(7/7);在肝脏、脾脏、淋巴结、肠、肠系膜、下肢、颈部、子宫和臀部等多脏器成瘤;流式检测瘤组织细胞mCD19表达强阳性.结论运用尾静脉注射方法构建了内脏器官广泛发生的播散性B淋巴瘤BALB/c小鼠动物模型,为进一步进行B淋巴瘤相关研究提供了实验依据.     

类人慢性粒细胞性白血病小鼠模型的建立

 目的：为了进一步阐明慢性粒细胞性白血病(chronic myeloid leukemia,CML) 的发病机制、寻找新的治疗靶点,有必要建立CML动物模型。方法：首先改进逆转录病毒包装技术,在相同实验条件下,改变以下条件之一,如质粒的质量、包装细胞的状态、转染当天包装细胞的密度等,进行病毒滴度的测定,优化实验条件后进行后续实验。实验组为BCR/ABL(含BCR/ABL融合基因的逆转录病毒载体)组小鼠。供体小鼠经5-氟尿嘧啶（5-FU）预处理后,骨髓细胞经p210 BCR/ABL逆转录病毒上清感染,感染的骨髓细胞经尾静脉注射给经致死剂量X光照射的受体小鼠。观察移植后BCR/ABL组小鼠的活动情况、发病鼠外周血和骨髓的细胞形态、肝脾病理改变等。同时观察MigR1(逆转录病毒空载体)组小鼠。结果：通过提高质粒的质量、改善包装细胞的状态、优化转染当天包装细胞的密度等,提高了病毒的滴度,用于小鼠移植模型实验。BCR/ABL组小鼠均于移植后19～25 d发病死亡。发病时外周血及骨髓均见中晚幼及成熟粒细胞大量增生,肝脾肿大伴正常结构破坏及大量粒细胞浸润。MigR1组小鼠均无发病。结论: 通过改进逆转录病毒包装技术,提高包装病毒的滴度,我们在体外将BCR/ABL转入小鼠骨髓细胞并移植入受体小鼠,成功建立了类人CML小鼠模型。     

A practical approach for quantitative estimates of voxel-by-voxel liver perfusion using DCE imaging and a compartmental model

Voxel-by-voxel estimation of liver perfusion using nonlinear least-squares fits of dynamic contrast enhanced computed tomography or magnetic resonance imaging data to a compartmental model is a computational expensive process. In this report, a "linear" least-squares method for estimation of liver perfusion is described. Simulated data and the data from an example case of a patient with intrahepatic cancer are presented. Compared to the nonlinear method, the new method can improve computational speed by a factor of approximately 400, which makes it practical for use in clinical trials.     

汉滩病毒感染神经元细胞模型的建立

目的 利用体外培养的小鼠胚胎脑皮腩神经元，感染汉潍病毒（Ｈａｎｔａａｎ ｖｉｒｕｓ．ＨＴ－ＮＶ），建立病毒感染的神经元细胞模型，供探讨病毒感染神经元后细胞的变化及损伤机制之用。方法 将孕１６￣１９ｄ的昆明种小鼠胚胎皮质神经元正常培养存活后，感染汉滩病毒Ａ９株造成细胞损伤模型。使用间接免疫荧光方法检测神经元荧光抗体阳性情况；利用ＭＴＴ比色试验检测细胞存活情况；通过免疫组化方法观察神经细胞生长的情况。     

Establishment of a diabetic mouse model with progressive diabetic nephropathy

Although diabetic animal models exist, no single animal model develops renal changes identical to those seen in humans. Here we show that transgenic mice that overexpress inducible cAMP early repressor (ICER Igamma) in pancreatic beta cells are a good model to study the pathogenesis of diabetic nephropathy. Although ICER Igamma transgenic mice exhibit extremely high blood glucose levels throughout their lives, they survive long enough to develop diabetic nephropathy. Using this model we followed the progress of diabetic renal changes compared to those seen in humans. By 8 weeks of age, the glomerular filtration rate (GFR) was already increased, and glomerular hypertrophy was prominent. At 20 weeks, GFR reached its peak, and urine albumin excretion rate was elevated. Finally, at 40 weeks, diffuse glomerular sclerotic lesions were prominently accompanied by increased expression of collagen type IV and laminin and reduced expression of matrix metalloproteinase-2. Nodular lesions were absent, but glomerular basement membrane thickening was prominent. At this point, GFR declined and urinary albumin excretion rate increased, causing a nephrotic state with lower serum albumin and higher serum total cholesterol. Thus, similar to human diabetic nephropathy, ICER Igamma transgenic mice exhibit a stable and progressive phenotype of diabetic kidney disease due solely to chronic hyperglycemia without other modulating factors.     

人源化SCID小鼠模型的建立及其鉴定

目的：探讨在SCID小鼠体内移植人免疫细胞，建立人源化SCID小鼠模型及其特性鉴定。方法：SCID小鼠腹腔注射环磷酰胺（CTX）抑制骨髓造血，连续4天后，通过腹腔注射移植人外周血单个核细胞（PBMC）。4、8和12周后分别取小鼠外周血、脾脏、肝脏。荧光显微镜下观察SCID小鼠外周血中人CD3^＋、CD19^＋细胞；流式细胞仪测定全血中人CD3^＋、CD19^＋细胞百分率；免疫组织化学分析SCID小鼠肝脏和脾脏中人CD3^＋、CD19^＋细胞；ELISA检测SCID小鼠血清中人免疫球蛋白含量。结果：（I）SCID小鼠移植人外周血单个核细胞4、8和12周后在小鼠外周血中通过荧光显微镜下可观察到人CD3^＋、CD19^＋细胞，4周后流式细胞仪测得小鼠外周血单个核细胞中人CD19^＋、CD3^＋细胞百分率分别为10．6％、31．7％；（2）免疫组织化学结果显示在小鼠脾脏中存在人CD3^＋、CD19^＋细胞；（3）移植人外周血单个核细胞4、8和12周后ELISA测得小鼠血清中人免疫球蛋白的含量分别为390、1100和1040μg／ml。结论：成功地在SCID小鼠体内建立了人免疫系统。     

小鼠听觉剥夺动物模型的建立

目的 探索一种建立稳定的小鼠听觉剥夺动物模型的方法.方法 选取出生2个月、听觉脑干诱发电位(ABR)正常的BALB/c小鼠40只,随机分为两组各20只.实验组采用耳后径路开放听泡,用微型钻在耳蜗表面钻孔损毁双侧耳蜗结构;对照组仅做耳后切口不破坏耳蜗结构.术后动物饲养4个月,通过比较手术前后ABR测试结果,进行客观听力学评估.结果 实验组小鼠术后耳廓反射消失,ABR测试未检测出反应波形.对照组听力正常.结论 耳后径路切除小鼠耳蜗,能够建立稳定可靠的听觉剥夺动物模型,对于听觉中枢可塑性的研究很有价值.     

实时荧光定量PCR测定胸腺近期输出功能方法的建立和应用

目的建立一种准确、快速的检测胸腺输出功能的方法。方法根据TCRδ基因序列,设计引物和探针,建立实时荧光定量PCR检测T细胞受体重排切除环(TRECs)的方法;并用于检测正常人及慢乙肝PBMCs中TRECs的含量。结果建立了测定TRECs含量的实时荧光定量PCR方法,产物与预计长度相符,序列正确;最低可扩增出5copies的模板;重复五次,Ct值的变异系数为1.06%。正常人21~45岁组的TRECs含量为(7767.4±2369.5)copies/106 PBMCs,16~20岁组为(28374.4±7820.4)copies/106 PBMCs,21~45岁慢乙肝组(6480.9±2031.2)copies/106 PBMCs,正常人21~45岁组分别与16~20岁组、同龄慢乙肝组比较差异均有统计学意义,P<0.05。结论实时荧光定量PCR检测TRECs的方法特异性强,灵敏度高,重复性好。正常人21~45岁组的胸腺输出功能低于16~21岁组,慢乙肝组低于同龄正常人。     

Transgenic reporter mouse for bioluminescence imaging of herpes simplex virus 1 infection in living mice

Bioluminescence imaging allows spatial and temporal progression of viral infection to be detected and quantified in living mice, thereby providing a new approach for studies of viral-host pathogenesis. It has been necessary to construct and validate recombinant reporter viruses that express firefly luciferase to investigate viral replication and spread with this imaging technology. This strategy greatly limits the ability to analyze multiple strains of virus and/or existing viral mutants, and reporter viruses also may be attenuated relative to the respective parental viruses. To facilitate bioluminescence imaging of herpes simplex virus type 1 (HSV-1), we developed a transgenic reporter mouse that uses the promoter from HSV-1 thymidine kinase to control expression of firefly luciferase. Infection with HSV-1 activated expression of firefly luciferase in corneal and flank models of infection, and amounts of bioluminescence increased in proportion to increasing input titers of virus. Imaging could detect infection with three different strains of HSV-1 with the following relative rank order of bioluminescence produced at the site of infection: McKrae > 17 > KOS. Corneal infection with as few as 1 x 10(3) pfu strain McKrae was detectable above background levels. By comparison, infection with vaccinia virus did not affect bioluminescence in the reporter mouse. Collectively, these data establish a new transgenic reporter mouse for infection with HSV-1, thereby enabling in vivo bioluminescence imaging studies of HSV-1 pathogenesis without constructing new reporter viruses.     

Dynamic imaging of arginine-rich heart-targeted vehicles in a mouse model

Efficacious delivery of drugs and genes to the heart is an important goal. Here, a radiolabeled peptide-targeted liposome was engineered to bind to the heart, and the biodistribution and pharmacokinetics were determined by dynamic positron emission tomography in the FVB mouse. Efficient targeting occurred only with an exposed ligand and a dense concentration of peptide (6000 peptides/particles). Liposomes targeted with CRPPR or other arginine-rich peptides with an exposed guanidine moiety bound within 100 s after intravenous injection and remained stably bound. With CRPPR-targeted particles, the radioisotope density in the heart averaged 44 +/- 9% injected dose/gram of tissue, more than 30-fold higher than in skeletal muscle. The rapid and efficient targeting of these particles can be exploited in drug and gene delivery systems and with dynamic positron emission tomography provides a model system to optimize targeting of engineered particles.     

Rapid detection of VHL exon deletions using real-time quantitative PCR

Various types of mutations exist that exert an effect on the normal function of a gene. Among these, exon/gene deletions often remain unnoticed in initial mutation screening. Until recently, no fast and efficient methods were available to detect this type of mutation. Molecular detection methods for gene copy number changes included Southern blot (SB) and fluorescence in situ hybridisation, both with their own intrinsic limitations. In this paper, we report the development and application of a fast, sensitive and high-resolution method for the detection of single exon or larger deletions in the VHL gene based on real-time quantitative PCR (Q-PCR). These deletions account for approximately one-fifth of all patients with the von Hippel-Lindau syndrome, a dominantly inherited highly penetrant familial cancer syndrome predisposing to specific malignancies including phaeochromocytomas and haemangioblastomas. Our VHL exon quantification strategy is based on SYBR Green I detection and normalisation using two reference genes with a normal copy number, that is, ZNF80 (3q13.31) and GPR15 (3q12.1). Choice of primer sequences and the use of two reference genes appears to be critical for accurate discrimination between 1 and 2 exon copies. In a blind Q-PCR study of 29 samples, all 14 deletions were detected, which is in perfect agreement with previously determined SB results. We propose Q-PCR as the method of choice for fast (within 3.5 h), accurate and sensitive (ng amount of input DNA) exon deletion screening in routine DNA diagnosis of VHL disease. Similar assays can be designed for deletion screening in other genetic disorders.     

Emergence of a clinical daptomycin-resistant Staphylococcus aureus isolate during treatment of methicillin-resistant Staphylococcus aureus bacteremia and osteomyelitis

The emergence of a clinically daptomycin-resistant Staphylococcus aureus isolate occurred during treatment of methicillin-resistant S. aureus bacteremia and probable vertebral osteomyelitis. The breakthrough isolate was indistinguishable from pretreatment daptomycin-susceptible isolates by pulsed-field gel electrophoresis. Daptomycin nonsusceptibility was confirmed by MIC and time-kill curve analyses.     

Quantification of MYCN, DDX1, and NAG gene copy number in neuroblastoma using a real-time quantitative PCR assay.

Amplification of the proto-oncogene MYCN is a strong adverse prognostic factor in neuroblastoma patients in all tumor stages. The status of the MYCN gene has become an important factor in clinical decision making and therapy stratification. Consequently, fast and accurate assessment of MYCN gene copy number is of the utmost importance and the use of two independent methods to determine MYCN status is recommended. For these reasons we have developed and evaluated a real-time quantitative PCR (Q-PCR) assay as an alternative for time-consuming Southern blot analysis (SB), and as a second independent technique in parallel with fluorescence in situ hybridization (FISH) analysis. Advantages of Q-PCR are a large dynamic range of quantification, no requirement for post-PCR sample handling and the need for very small amounts of starting material. The accuracy of the assay was illustrated by measurement of MYCN single gene copy changes in DNA samples of two patients with 2p deletion and duplication, respectively. Two different detection chemistries i.e., a sequence specific TaqMan probe and a generic DNA binding dye SYBR Green I were evaluated and shown to yield similar results. Also, two different calculation methods for copy number determination were used i.e., the kinetic method and the comparative C(T) method, and shown to be equivalent. In total, 175 neuroblastoma samples with known MYCN status, as determined by FISH and/or SB, were examined. Q-PCR data were highly concordant with FISH and SB data. In addition to MYCN copy number evaluation, DDX1 and NAG gene copy numbers were determined using a similar Q-PCR strategy. Survival analysis pointed out that DDX1 and/or NAG amplification has no additional adverse effect on prognosis.     

A modified quantitative precipitin test which is rapid, sensitive and reproducible.

A modification of the standard quantitative precipitin test is described. The new procedure, which utilizes polyethylene glycol (PEG) at both the 37 degrees C and 4 degrees C incubation stages, provides a convenient quantitative precipitin test assay which is more reliable and much faster than the standard assay. Moreover, the modified or PEG assay gives a quantitative measure of antibody concentration even when antisera of low titre are tested. The sensitivity of the capillary-tube test is also enhanced when the supernatant solutions contain PEG.