Incomplete rescue of cystic fibrosis transmembrane conductance regulator deficient mice by the human CFTR cDNA

We have used a mouse model to study the ability of human CFTR to correct the defect in mice deficient of the endogenous protein. In this model, expression of the endogenous Cftr gene was disrupted and replaced with a human CFTR cDNA by a gene targeted 'knock-in' event. Animals homozygous for the gene replacement failed to show neither improved intestinal pathology nor survival when compared to mice completely lacking CFTR. RNA analyses showed that the human CFTR sequence was transcribed from the targeted allele in the respiratory and intestinal epithelial cells. Furthermore, in vivo potential difference measurements showed that basal CFTR chloride channel activity was present in the apical membranes of both nasal and rectal epithelial cells in all homozygous knock-in animals examined. Ussing chamber studies showed, however, that the cAMP-mediated chloride channel function was impaired in the intestinal tract among the majority of homozygous knock-in animals. Hence, failure to correct the intestinal pathology associated with loss of endogenous CFTR was related to inefficient functional expression of the human protein in mice. These results emphasize the need to understand the tissue- specific expression and regulation of CFTR function when animal models are used in gene therapy studies.      

Human MSH2 binds to trinucleotide repeat DNA structures associated with neurodegenerative diseases

The expansion of trinucleotide repeat sequences is associated with several neurodegenerative diseases. The mechanism of this expansion is unknown but may involve slipped-strand structures where adjacent rather than perfect complementary sequences of a trinucleotide repeat become paired. Here, we have studied the interaction of the human mismatch repair protein MSH2 with slipped-strand structures formed from a triplet repeat sequence in order to address the possible role of MSH2 in trinucleotide expansion. Genomic clones of the myotonic dystrophy locus containing disease-relevant lengths of (CTG)n x (CAG)n triplet repeats were examined. We have constructed two types of slipped-strand structures by annealing complementary strands of DNA containing: (i) equal numbers of trinucleotide repeats (homoduplex slipped structures or S-DNA) or (ii) different numbers of repeats (heteroduplex slipped intermediates or SI-DNA). SI-DNAs having an excess of either CTG or CAG repeats were structurally distinct and could be separated electrophoretically and studied individually. Using a band-shift assay, the MSH2 was shown to bind to both S-DNA and SI-DNA in a structure- specific manner. The affinity of MSH2 increased with the length of the repeat sequence. Furthermore, MSH2 bound preferentially to looped-out CAG repeat sequences, implicating a strand asymmetry in MSH2 recognition. Our results are consistent with the idea that MSH2 may participate in trinucleotide repeat expansion via its role in repair and/or recombination.      

Cone-beam computed tomography with a flat-panel imager: initial performance characterization

The development and performance of a system for x-ray cone-beam computed tomography (CBCT) using an indirect-detection flat-panel imager (FPI) is presented. Developed as a bench-top prototype for initial investigation of FPI-based CBCT for bone and soft-tissue localization in radiotherapy, the system provides fully three-dimensional volumetric image data from projections acquired during a single rotation. The system employs a 512 x 512 active matrix of a-Si:H thin-film transistors and photodiodes in combination with a luminescent phosphor. Tomographic imaging performance is quantified in terms of response uniformity, response linearity, voxel noise, noise-power spectrum (NPS), and modulation transfer function (MTF), each in comparison to the performance measured on a conventional CT scanner. For the geometry employed and the objects considered, response is uniform to within 2% and linear within 1%. Voxel noise, at a level of approximately 20 HU, is comparable to the conventional CT scanner. NPS and MTF results highlight the frequency-dependent transfer characteristics, confirming that the CBCT system can provide high spatial resolution and does not suffer greatly from additive noise levels. For larger objects and/or low exposures, additive noise levels must be reduced to maintain high performance. Imaging studies of a low-contrast phantom and a small animal (a euthanized rat) qualitatively demonstrate excellent soft-tissue visibility and high spatial resolution. Image quality appears comparable or superior to that of the conventional scanner. These quantitative and qualitative results clearly demonstrate the potential of CBCT systems based upon flat-panel imagers. Advances in FPI technology (e.g., improved x-ray converters and enhanced electronics) are anticipated to allow high-performance FPI-based CBCT for medical imaging. General and specific requirements of kilovoltage CBCT systems are discussed, and the applicability of FPI-based CBCT systems to tomographic localization and image-guidance for radiotherapy is considered.     

Cone-beam computed tomography with a flat-panel imager: effects of image lag

A system for cone-beam computed tomography (CBCT) has been developed based upon the technology of active matrix flat-panel imagers (FPIs), and the system has demonstrated the potential for fully three-dimensional volumetric imaging with high spatial and contrast resolution. This paper investigates the effects of image lag (arising from charge trapping and release in the FPI pixels) upon CBCT reconstructions. Hypotheses were derived based upon a simple, geometrical/physical model, suggesting that image lag in the projection data results primarily in two artifacts: a spatial blurring artifact in the direction opposite to the direction of rotation (called a "comet") and a line artifact along the direction of the first few projections (called a "streak"). The hypotheses were tested by means of computer simulations and experimental measurements that yielded CBCT images of a simple cylindrical water phantom containing an attenuating rod of varying size and composition. The computer simulations generated projection images based upon analysis of the system geometry and a simple model of the FPI that allowed free adjustment of the image lag. Experimental measurements involved CBCT scans of the phantom under various conditions and modes of acquisition followed by examination of the resulting CBCT axial slices for lag artifacts. Measurements were performed as a function of exposure level, position and contrast of the rod, and for three modes of acquisition designed to isolate and/or minimize the two hypothesized artifacts. The results clearly illustrate the comet and streak artifacts, particularly in relation to high-contrast objects imaged at high exposure levels. The significance of such artifacts under clinical conditions is expected to be small, considering the magnitude of the effect relative to the morphology and composition of typical anatomy. The artifacts may become appreciable, however, in the presence of high-contrast objects, such as marker BBs, dental fillings, and metal prosthetics. A procedural method of reducing lag artifacts is demonstrated.     

Multiple cerebral metastases: detectability with Gd-DTPA-enhanced MR imaging

Sixteen patients with suspected cerebral metastases were studied with magnetic resonance (MR) imaging before and after the intravenous administration of 0.1 mmol/kg of gadolinium diethylenetriaminepenta-acetic acid. The images were interpreted blindly by two neuroradiologists; all clinical, radiologic (computed tomographic and MR imaging), and pathologic data were reviewed to arrive at a final "best diagnosis," which was then compared with the prior blinded interpretations. Of seven patients found to have multiple metastases, six (86%) had at least one tumor nodule depicted by postinfusion MR imaging that was missed by one or both observers on review of preinfusion images alone. Lesions missed on preinfusion studies were usually small nodules hidden by or not detected next to regions of high-signal edema thought to be related to the adjacent tumor nodule. The authors believe that contrast enhancement improves detection of metastatic foci with MR imaging and that the findings indicate broader implications for the detection of multiple lesions from other causes.     

Osteopetrosis: MR characteristics at 1.5 T

Four patients with proved osteopetrosis (three with the infantile malignant form and one with the benign form) were examined with magnetic resonance imaging at 1.5 T. All patients were studied in the coronal and sagittal planes using both short and long repetition time/echo time sequences. The infantile malignant form was characterized by a complete lack of signal from the marrow alternating with a signal intensity equivalent to that of the intervertebral disks, resulting in a "stepladder" appearance. In the benign form or after successful marrow transplantation in the infantile malignant form, intermediate or high signal intensity in the vertebrae was noted, suggesting the presence of some marrow elements.     

Accuracy of finite element model-based multi-organ deformable image registration

As more pretreatment imaging becomes integrated into the treatment planning process and full three-dimensional image-guidance becomes part of the treatment delivery the need for a deformable image registration technique becomes more apparent. A novel finite element model-based multiorgan deformable image registration method, MORFEUS, has been developed. The basis of this method is twofold: first, individual organ deformation can be accurately modeled by deforming the surface of the organ at one instance into the surface of the organ at another instance and assigning the material properties that allow the internal structures to be accurately deformed into the secondary position and second, multi-organ deformable alignment can be achieved by explicitly defining the deformation of a subset of organs and assigning surface interfaces between organs. The feasibility and accuracy of the method was tested on MR thoracic and abdominal images of healthy volunteers at inhale and exhale. For the thoracic cases, the lungs and external surface were explicitly deformed and the breasts were implicitly deformed based on its relation to the lung and external surface. For the abdominal cases, the liver, spleen, and external surface were explicitly deformed and the stomach and kidneys were implicitly deformed. The average accuracy (average absolute error) of the lung and liver deformation, determined by tracking visible bifurcations, was 0.19 (s.d.: 0.09), 0.28 (s.d.: 0.12) and 0.17 (s.d.: 0.07) cm, in the LR, AP, and IS directions, respectively. The average accuracy of implicitly deformed organs was 0.11 (s.d.: 0.11), 0.13 (s.d.: 0.12), and 0.08 (s.d.: 0.09) cm, in the LR, AP, and IS directions, respectively. The average vector magnitude of the accuracy was 0.44 (s.d.: 0.20) cm for the lung and liver deformation and 0.24 (s.d.: 0.18) cm for the implicitly deformed organs. The two main processes, explicit deformation of the selected organs and finite element analysis calculations, require less than 120 and 495 s, respectively. This platform can facilitate the integration of deformable image registration into online image guidance procedures, dose calculations, and tissue response monitoring as well as performing multi-modality image registration for purposes of treatment planning.     

Ewing’s肉瘤细胞X-射线照射后TNF-α和TGF-β mRNA表达的研究

 目的　研究Ewing’s肉瘤细胞系 (RM 82 )X 射线外照射后肿瘤坏死因子 (TNF α)和转化生长因子 (TGF β)mRNA表达水平的变化 ,探讨X 射线诱导内源性TNF α和TGF β产生的可能性及意义。 方法　应用实时荧光RT PCR ,检测接受不同剂量X 线照射 (2Gy ,5Gy ,10Gy ,2 0Gy ,30Gy ,4 0Gy)和受照后不同时间 (1h ,3h ,6h ,12h ,2 4h ,4 8h ,72h)。TNF α和TGF βmRNA表达水平的变化。 结果　RM 82细胞TNF αmRNA表达水平较外照射前显著升高。一方面受照后TNF αmRNA表达逐渐升高 ,照射剂量达 4 0Gy时TNF αmRNA表达水平达高峰 ,为正常对照组的 10 8倍 ;另一方面 ,照射后 3h后TNF αmRNA表达逐渐升高 ,6h达高峰 ,为正常对照组的 18倍。相反 ,TGF βmRNA表达水平X 射线照射前后无显著变化。结论　Ewing’s肉瘤细胞系 (RM 82 )接受X 线照射后TNF αmRNA表达明显升高 ,且呈现时间、剂量依赖性。放射治疗可诱导Ewing’s肉瘤细胞系 (RM 82...     

Pancreatic elastase activates pulmonary nuclear factor kappa B and inhibitory kappa B, mimicking pancreatitis-associated adult respiratory distress syndrome

BACKGROUND: Select pancreatic enzymes, primarily elastase, precipitate pulmonary injury similar to pancreatitis-associated adult respiratory distress syndrome and stimulate leukocyte cytokine production in vitro via nuclear factor kappa B (NF-kappaB) activation. This study explores the effect of systemic pancreatic enzymes on pulmonary NF-kappaB and inhibitory kappa B (IkappaB) proteins and their role in enzyme-induced pulmonary injury. METHODS: Mice received pancreatic elastase, amylase, lipase, or trypsin intraperitoneally. Bronchoalveolar lavage IkappaBalpha/IkappaBbeta proteins were measured by immunoblot. Pulmonary NF-kappaB activation, tumor necrosis factor (TNF) gene expression, and neutrophil infiltration (myeloperoxidase) were determined and myeloperoxidase experiments repeated in p55 TNF receptor-deficient (TNF KO) animals. Additional animals received pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB activation, and TNF protein and pulmonary microvascular permeability were measured after elastase administration. RESULTS: Pancreatic elastase induced pulmonary IkappaBalpha/IkappaBbeta degradation (30 minutes), NF-kappaB activation (60 minutes), and TNF gene expression (60 minutes) with subsequent neutrophilic inflammation (4 hours) and microvascular leakage (24 hours), whereas amylase, lipase, and trypsin did not. Furthermore, lung injury was markedly reduced in TNF KO animals and PDTC significantly attenuated TNF production and pulmonary microvascular leakage. CONCLUSIONS: Pancreatic elastase induces cytokine-mediated lung injury and this pathway involves the NF-kappaB second messenger system, further supporting elastase as a factor linking pancreatic inflammation to systemic illness during severe acute pancreatitis.     

Fat gram target to achieve high energy intake in cystic fibrosis

Higher fat and energy intakes confer a survival advantage in cystic fibrosis (CF). There is a need to develop effective nutrition programmes that ensure optimal energy intake in CF.

Methodology:

A cross-sectional measurement of clinical characteristics and energy and fat intakes in patients attending the CF outpatients clinic of the John Hunter Hospital, Newcastle was undertaken. Twenty-nine subjects, mean age 12 years (range 4.3–20.2), completed weighed food records to determine the contribution of fat to the percentage of the recommended energy intake obtained and to document use of pancreatic enzyme replacement therapy.

Results:

Diets with a high percentage of energy derived from fat did not guarantee that individuals with CF met their energy requirements. Subjects with total fat intakes of 100 g per day or greater, however, achieved in excess of 110% recommended daily intake (RDI) for energy. Up to 47% of subjects consumed more pancreatic enzyme replacement capsules than shown to give maximum effectiveness.

Conclusion:

Setting a 100 g daily fat target is a realistic way of ensuring high energy intakes in CF. Fat ready reckoners would identify the fat content of food and prescribe specific numbers of pancreatic enzyme replacement capsules to be consumed with each meal or food item.