In vivo perivascular implantation of encapsulated packaging cells for prolonged retroviral gene transfer

Long-term benefits of coronary angioplasty remain limited by the treatmentinduced renarrowing of arteries, termed restenosis. One of the mechanisms leading to restenosis is the proliferation of smooth muscle cells. Therefore, proliferating cells of the injured arterial wall, which can be selectively transduced by retroviruses, are potential targets for gene therapy strategies. A direct single-dose therapeutic application of retroviral vectors for inhibition of cell proliferation is normally limited by too low transduction efficiencies. Encapsulated retrovirus-producing cells release viral vectors from microcapsules, and may enhance the transduction efficiency by prolonged infection. Primary and immortal murine and porcine cells and murine retrovirusproducing cells were encapsulated in cellulose sulphate. Cell viability was monitored by analysing cell metabolism. Safety, stability, transfer efficiency and extent of restenosis using capsules were determined in a porcine restenosis model for local gene therapy using morphometry, histology, in situ betagalactosidase assay and PCR. Encapsulation of cells did not impair cell viability. Capsules containing retrovirus-producing cells expressing the beta-galactosidase reporter gene were implanted into periarterial tissue or a pig model of restenosis. Three weeks following implantation, beta-galactosidase activity was detected in the pericapsular tissue with a transduction efficiency of ~ 1 in 500 cells. Adventitial implantation of vector-producing encapsulated cells for gene therapy may, therefore, facilitate successful targeting of proliferating vascular smooth muscle cells, and allow stable integration of therapeutic genes into surrounding cells. The encapsulation of vector-producing cells could represent a novel and feasible way to optimize local retroviral gene therapy.     

Bowel Preparation Is Associated With Spillage of Bowel Contents in Colorectal Surgery

PURPOSE Infectious complications pose a significant cause of morbidity in colon and rectal surgery. This study was designed to assess the effect of bowel preparation on spillage of bowel contents into the peritoneal cavity during colorectal surgery, and its potential effect on the rate of postoperative infectious complications.METHODS The quality of bowel preparation and the incidence of spillage of bowel contents were prospectively assessed in patients undergoing elective colon and rectal resection. The patients were followed for 30 days for postoperative infectious and noninfectious complications.RESULTS A total of 333 patients were included in this study, of which 181 did not receive mechanical bowel preparation. Intraoperative spillage of bowel contents occurred in 48 patients (14 percent), whereas in 285 patients (86 percent), spillage did not occur. There was a trend toward a higher rate of overall surgical infectious and noninfectious complications in patients who had spillage of bowel contents compared with patients without spillage; however, this difference was not statistically significant (18.7 vs. 11 percent, and 29 vs. 19 percent, respectively). Preoperative mechanical bowel preparation and colocolonic or colorectal anastomosis was associated with a higher rate of bowel contents spillage, although this difference did not reach statistical significance. Liquid colonic contents caused significantly higher rates of spillage.CONCLUSIONS Spillage of bowel contents into the peritoneal cavity during colon and rectal surgery may increase the rate of postoperative infectious complications. In addition, inadequate mechanical bowel preparation, leading to liquid bowel contents, increases the rate of intraoperative spillage.Reprints are not available.     

Cloning of glycoprotein IIIa cDNA from human erythroleukemia cells and localization of the gene to chromosome 17

Platelet aggregation requires the binding of adhesive proteins such as fibrinogen to the heterodimer of membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa). Human erythroleukemia (HEL) cells synthesize both GPIIb and GPIIIa. Using poly(A+) RNA purified from HEL cells, we constructed a cDNA library in the lambda gt10 phage vector. This library was screened with a 38mer oligonucleotide derived from a platelet GPIIIa peptide, and three overlapping cDNAs were isolated. The three inserts encompassed 3.5 kilobases (kb), including the entire coding region of mature GPIIIa (2,286 basepairs, bp) and 1.3 kb of 3' untranslated sequence. All 222 residues determined directly from platelet GPIIIa tryptic peptides exactly matched the HEL cell-deduced amino acid sequence. The HEL cell sequence matched a previously reported endothelial cell cDNA sequence except for eight nucleotides. Five of these nucleotide differences were silent changes consistent with genetic polymorphisms. The other three differences resulted in changes in the deduced amino acid sequence of GPIIIa; reexamination of the endothelial cell cDNA sequence in these three areas revealed that it is actually identical to the HEL cell sequence. The virtual identity of the endothelial and HEL cell cDNA sequences provides direct evidence that GPIIIa is a subunit common to cell-adhesion receptors present in more than one cell type. We localized the gene for GPIIIa to chromosome 17, the same chromosome to which we had previously mapped the gene for GPIIb.     

Differential diagnosis of cardiac masses using contrast echocardiographic perfusion imaging

OBJECTIVES: We investigated the usefulness of echocardiographic contrast perfusion imaging in differentiating cardiac masses. BACKGROUND: Two-dimensional echocardiography is the primary diagnostic modality for cardiac masses. However, differentiation between the different types of cardiac masses may be difficult at times. We hypothesized that echocardiographic contrast perfusion imaging would differentiate the neo-vascularization of malignancies from the avascularity of thrombi and the sparse vascularity of stromal tumors. METHODS: Sixteen patients with cardiac masses underwent power-modulation imaging after echocardiographic intravenous contrast administration. Pixel intensities in the mass and an adjacent section of myocardium were analyzed visually and by dedicated software. All masses had a pathologic diagnosis or resolved after anticoagulation. In a subset of patients, video-intensity curves of contrast replenishment in the mass and myocardium over time were generated. The post-impulse steady-state pixel intensity (A) and initial rate of contrast replenishment after impulse (beta) were compared with an index of blood vessel area on pathology. RESULTS: In seven of 16 patients, contrast enhancement resulted in greater pixel intensity in the mass than in the adjacent myocardium. All of these masses were classified pathologically as malignant (n = 6) or benign and vascular (n = 1). Nine masses demonstrated decreased pixel intensity, compared with the myocardium, and were diagnosed pathologically as myxomas (n = 2) or thrombi (n = 5), or they resolved with anticoagulation (n = 2). For the subset of patients, beta correlated with the vessel area index (r = 0.60). CONCLUSIONS: Echocardiographic contrast perfusion imaging aids in the differentiation of cardiac masses. Compared with the adjacent myocardium, malignant and vascular tumors hyper-enhanced, whereas stromal tumors and thrombi hypo-enhanced.     

Effects of enteral arginine supplementation on the structural intestinal adaptation in a rat model of short bowel syndrome

The nitric oxide precursor L-arginine (ARG) has been shown to influence intestinal morphology and intestinal absorptive function. The purpose of the present study was to determine the effect of enteral ARG supplementation on structural intestinal adaptation, cell proliferation, and apoptosis in a rat model of short bowel syndrome (SBS). Thirty male Sprague-Dawley rats were divided into three experimental groups: Sham rats underwent bowel transection, SBS rats underwent 75% small bowel resection, and SBS-ARG rats underwent bowel resection and were treated with ARG given in the drinking water (2%). Parameters of intestinal adaptation, enterocyte proliferation and enterocyte apoptosis were determined on day 14 following operation. We have demonstrated that SBS-ARG animals had a lower jejunal and ileal mucosal weight, jejunal mucosal DNA and protein, ileal mucosal protein, jejunal villus height, jejunal and ileal crypt depth, and enterocyte proliferation index and a greater enterocyte apoptosis compared to SBS untreated animals. We conclude that in a rat model of SBS enteral L-arginine inhibits structural intestinal adaptation. Possible mechanism for this effect may be decreased cell proliferation and increased cell apoptosis.     

甲状腺术中喉返神经监测技术的标准化

目的在甲状腺手术中缺少术中神经监测(intra operative neuromonitoring,IONM)的标准化操作可导致结果变异性强,这些结果可产生错误信息并增加喉返神经损伤的危险性。因此有必要进行IONM操作的标准化。方法本研究共招募了289例进行过甲状腺切除术的患者(435根神经有危险),均由一位外科医师实施手术。每例患者均由同一位麻醉师使用EMG气管导管进行插管。每例患者均进行标准化IONM操作。该操作包括术前和术后对声带运动进行录像监测、保证电极在正确位置、喉返神经剥离前后刺激迷走神经并记录EMG信号,并摄像记录暴露的喉返神经。结果5例患者出现IONM波形异常,是由于电极错位所致,这一问题被立刻监测到。监测到1例患者在手术较早阶段出现非喉返神经损伤。甲状腺剥离时18例患者的神经失去了EMG信号,使用我们的标准化IONM操作后神经损伤的原因得以清楚阐明。结论标准化IONM操作不仅在消除错误的IONM结果方面有用且有帮助,而且有助于阐明喉返神经损伤的机制。在确定外科手术的缺陷并提高外科手术技巧后,本研究显著降低了神经麻痹的发生率。     

Hereditary hemochromatosis restores the virulence of plague vaccine strains

Nonpigmented Yersinia pestis (pgm) strains are defective in scavenging host iron and have been used in live-attenuated vaccines to combat plague epidemics. Recently, a Y. pestis pgm strain was isolated from a researcher with hereditary hemochromatosis who died from laboratory-acquired plague. We used hemojuvelin-knockout (Hjv(-/-)) mice to examine whether iron-storage disease restores the virulence defects of nonpigmented Y. pestis. Unlike wild-type mice, Hjv(-/-) mice developed lethal plague when challenged with Y. pestis pgm strains. Immunization of Hjv(-/-) mice with a subunit vaccine that blocks Y. pestis type III secretion generated protection against plague. Thus, individuals with hereditary hemochromatosis may be protected with subunit vaccines but should not be exposed to live-attenuated plague vaccines.     

Peroxisome proliferator-activated receptor alpha induction of uncoupling protein 2 protects against acetaminophen-induced liver toxicity

Acetaminophen (APAP) overdose causes acute liver failure in humans and rodents due in part to the destruction of mitochondria as a result of increased oxidative stress followed by hepatocellular necrosis. Activation of the peroxisome proliferator-activated receptor alpha (PPARα), a member of the nuclear receptor superfamily that controls the expression of genes encoding peroxisomal and mitochondrial fatty acid β-oxidation enzymes, with the experimental ligand Wy-14,643 or the clinically used fibrate drug fenofibrate, fully protects mice from APAP-induced hepatotoxicity. PPARα-humanized mice were also protected, whereas Ppara-null mice were not, thus indicating that the protection extends to human PPARα and is PPARα-dependent. This protection is due in part to induction of the PPARα target gene encoding mitochondrial uncoupling protein 2 (UCP2). Forced overexpression of UCP2 protected wildtype mice against APAP-induced hepatotoxicity in the absence of PPARα activation. Ucp2-null mice, however, were sensitive to APAP-induced hepatotoxicity despite activation of PPARα with Wy-14,643. Protection against hepatotoxicity by UCP2-induction through activation of PPARα is associated with decreased APAP-induced c-jun and c-fos expression, decreased phosphorylation of JNK and c-jun, lower mitochondrial H(2)O(2) levels, increased mitochondrial glutathione in liver, and decreased levels of circulating fatty acyl-carnitines. These studies indicate that the PPARα target gene UCP2 protects against elevated reactive oxygen species generated during drug-induced hepatotoxicity and suggest that induction of UCP2 may also be a general mechanism for protection of mitochondria during fatty acid β-oxidation.