Targeted transgastric drainage of isolated pancreatic duct segments to cure persistent pancreaticocutaneous fistulas from pancreatitis.

Chronic pancreaticocutaneous fistulas can be difficult to treat. This article presents a snare-target technique for draining a nondilated pancreatic duct into the stomach, diverting pancreatic fluid away from the pancreaticocutaneous fistula to allow it to heal. Internal or internal/external transgastric pancreatic duct or fistula drains were placed in six patients. After an average of 4 months of drainage, all six patients experienced resolution of the cutaneous fistula. Two patients developed a pseudocyst but no recurrent fistula after drain removal, and the other four patients had no pseudocyst or fistula after an average 27-month follow-up (range, 6-74 mo).

Overexpression of GTP cyclohydrolase 1 feedback regulatory protein is protective in a murine model of septic shock.

Overproduction of nitric oxide (NO) by inducible NO synthase contributes toward refractory hypotension, impaired microvascular perfusion, and end-organ damage in septic shock patients. Tetrahydrobiopterin (BH4) is an essential NOS cofactor. GTP cyclohydrolase 1 (GCH1) is the rate-limiting enzyme for BH4 biosynthesis. Under inflammatory conditions, GCH1 activity and hence BH4 levels are increased, supporting pathological NOS activity. GCH1 activity can be controlled through allosteric interactions with GCH1 feedback regulatory protein (GFRP). We investigated whether overexpression of GFRP can regulate BH4 and NO production and attenuate cardiovascular dysfunction in sepsis. Sepsis was induced in mice conditionally overexpressing GFRP and wild-type littermates by cecal ligation and puncture. Blood pressure was monitored by radiotelemetry, and mesenteric blood flow was quantified by laser speckle contrast imaging. Blood biochemistry data were obtained using an iSTAT analyzer, and BH4 levels were measured in plasma and tissues by high-performance liquid chromatography. Increased BH4 and NO production and hypotension were observed in all mice, but the extents of these pathophysiological changes were attenuated in GFRP OE mice. Perturbations in blood biochemistry were similarly attenuated in GFRP OE compared with wild-type controls. These results suggest that GFRP overexpression regulates GCH1 activity during septic shock, which in turn limits BH4 bioavailability for iNOS. We conclude that the GCH1-GFRP axis is a critical regulator of BH4 and NO production and the cardiovascular derangements that occur in septic shock.

Drainage of collections associated with hernia mesh: is it worthwhile?

PURPOSE: Synthetic mesh has revolutionized abdominal wall hernia repair. However, mesh infections present a clinical problem because the standard practice of surgical excision is fraught with increased morbidity. Here, single-institutional outcomes in managing mesh-related collections via percutaneous drainage are retrospectively reviewed to assess its effectiveness. MATERIALS AND METHODS: A total of 21 patients underwent drainage of perimesh collections. Three types of mesh were employed: polytetrafluoroethylene (PTFE; n = 5), polypropylene (n = 14), and porcine dermal collagen (n = 3). One patient received both polypropylene and PTFE. Drainage was performed with ultrasound guidance (n = 19) or surgical drain exchange (n = 2). Mesh type, culture results, fluid collection size, and location were analyzed with respect to need for mesh excision. RESULTS: Sixteen of 21 patients (76%) were successfully treated with drainage. One required additional surgical capsulectomy; the mesh was salvaged. Four required mesh excision because of recurrent infection (n = 2) or lack of improvement of clinical course (n = 2). Recurrent infection occurred in six patients, with mesh salvage via conservative management or new drainage in four. Fluid cultures were positive in 68% of patients (n = 13), with Staphylococcus aureus the most common organism. Cultures did not predict mesh excision (P = .26). The PTFE excision rate trended higher compared with polypropylene (40% vs 14%; P = .27). No porcine dermal collagen mesh was excised. Neither fluid collection size nor location predicted mesh excision. Mean follow-up was 319 days (range, 6-1,406 d). CONCLUSIONS: Percutaneous drainage of suspected mesh-related abscess is effective. The use of PTFE mesh trended toward a higher excision rate.

CT-guided tube pericardiostomy: a safe and effective technique in the management of postsurgical pericardial effusion.

OBJECTIVE: The purpose of this study was to analyze the efficacy and examine the competitive cost of CT-guided tube pericardiostomy in the management of symptomatic postsurgical pericardial effusion. MATERIALS AND METHODS: Over a 4-year period, 36 patients with symptomatic pericardial effusion were treated with CT-guided percutaneous placement of an indwelling pericardial catheter, for a total of 39 CT-guided tube pericardiostomy procedures. Thirty-three patients (92%) had undergone major cardiothoracic surgery, and three patients (8%) had undergone minimally invasive procedures. The medical records were retrospectively reviewed for clinical presentation, surgical history, imaging studies performed, procedural details, fluid characterization, and outcome. Charge comparison was performed with the American Medical Association Current Procedural Terminology codes and information acquired from the billing department at our facility. RESULTS: All 39 CT-guided tube pericardiostomy procedures were performed successfully without clinically significant complications. After 33 of the 39 procedures (85%), symptoms did not recur after the catheter was removed. Three of 36 patients (8%) had a recurrence of pericardial effusion. Comparison of procedure charges showed an 89% saving over intraoperative pericardial window procedures and no significant difference compared with ultrasound-guided tube pericardiostomy. Eight patients (21% of procedures) needed pleural drainage procedures, all of which were performed in the CT suite immediately after the tube pericardiostomy procedure. CONCLUSION: CT-guided tube pericardiostomy is a safe and effective alternative to surgical drainage in the care of patients with clinically significant pericardial effusion after cardiothoracic surgery and has the additional benefit of substantial cost savings.

Cell-based assays using primary endothelial cells to study multiple steps in inflammation.

Cell-based assays are powerful tools for drug discovery and provide insight into complex signal transduction pathways in higher eukaryotic cells. Information gleaned from assays that monitor a cellular phenotype can be used to elucidate the details of a single pathway and to establish patterns of cross talk between pathways. By selecting the appropriate cell model, cell-based assays can be used to understand the function of a specific cell type in a complex disease process such as inflammation. We have used human umbilical vein endothelial cells to establish three cell-based, phenotypic assays that query different stages of a major signaling pathway activated in inflammation. One assay analyzes the tumor necrosis factor alpha (TNFalpha)-induced translocation of the transcription factor NF-kappaB from the cytoplasm into the nucleus 20 min after stimulation with TNFalpha. Two more assays monitor the expression of E-selectin and VCAM-1, 4 and 24 h after stimulation with TNFalpha. Indirect immunofluorescence and high-throughput automated microscopy were used to analyze cells. Imaging was performed with the IN Cell Analyzer 3000. All assays proved to be highly robust. Z' values between 0.7 and 0.8 make each of the three assays well suited for use in high-throughput screening for drug or probe discovery.

The zebrafish gene map defines ancestral vertebrate chromosomes.

Genetic screens in zebrafish (Danio rerio) have identified mutations that define the roles of hundreds of essential vertebrate genes. Genetic maps can link mutant phenotype with gene sequence by providing candidate genes for mutations and polymorphic genetic markers useful in positional cloning projects. Here we report a zebrafish genetic map comprising 4073 polymorphic markers, with more than twice the number of coding sequences localized in previously reported zebrafish genetic maps. We use this map in comparative studies to identify numerous regions of synteny conserved among the genomes of zebrafish, Tetraodon, and human. In addition, we use our map to analyze gene duplication in the zebrafish and Tetraodon genomes. Current evidence suggests that a whole-genome duplication occurred in the teleost lineage after it split from the tetrapod lineage, and that only a subset of the duplicates have been retained in modern teleost genomes. It has been proposed that differential retention of duplicate genes may have facilitated the isolation of nascent species formed during the vast radiation of teleosts. We find that different duplicated genes have been retained in zebrafish and Tetraodon, although similar numbers of duplicates remain in both genomes. Finally, we use comparative mapping data to address the proposal that the common ancestor of vertebrates had a genome consisting of 12 chromosomes. In a three-way comparison between the genomes of zebrafish, Tetraodon, and human, our analysis delineates the gene content for 11 of these 12 proposed ancestral chromosomes.

Sequence characteristics of functional siRNAs.

RNA interference in mammalian cells is actively used to conduct genetic screens, to identify and to validate targets, and to elucidate regulators and modifiers of cellular pathways. To ensure the specificity and efficacy of the active 21mer siRNA molecules, it is pertinent to develop a strategy for their rational design. Here we show that most functional siRNAs have characteristic sequence features. We tested 601 siRNAs targeting one exogenous and three endogenous genes. The efficacy of the siRNAs was determined at the protein level. Using a decision tree algorithm in combination with information analysis, our analyses revealed four sets of rules with a mean knockdown efficacy ranging from 60% to 73%. (To distinguish between percentages used to describe the quality of an siRNA and the percentages used to describe parts of data sets we underlined the former throughout this paper.) The best rule comprises an A/U at positions 10 and 19, a G/C at position 1, and more than three A/Us between positions 13 and 19, in the sense strand of the siRNA sequence. Using these rules, there is a 99.9% chance of designing an effective siRNA in a set of three with more than 50% knockdown efficiency in a biological readout.

Rapid mapping of zebrafish mutations with SNPs and oligonucleotide microarrays.

Large-scale genetic screens in zebrafish have identified thousands of mutations in hundreds of essential genes. The genetic mapping of these mutations is necessary to link DNA sequences to the gene functions defined by mutant phenotypes. Here, we report two advances that will accelerate the mapping of zebrafish mutations: (1) The construction of a first generation single nucleotide polymorphism (SNP) map of the zebrafish genome comprising 2035 SNPs and 178 small insertions/deletions, and (2) the development of a method for mapping mutations in which hundreds of SNPs can be scored in parallel with an oligonucleotide microarray. We have demonstrated the utility of the microarray technique in crosses with haploid and diploid embryos by mapping two known mutations to their previously identified locations. We have also used this approach to localize four previously unmapped mutations. We expect that mapping with SNPs and oligonucleotide microarrays will accelerate the molecular analysis of zebrafish mutations.