Pre-operative vessel mapping and early post-operative surveillance duplex scanning of arteriovenous fistulae.

PURPOSE: To increase the proportion of dialysis patients using native arteriovenous fistulae (AVF), improved selection of the most appropriate procedure must be coupled with early access surveillance to determine which access would likely mature and when intervention might lead to access salvage. This study was aimed at auditing pre-operative vessel mapping and post-operative access surveillance against the primary outcome measure of AVF maturation. METHODS: Between January 2006 and August 2007, 113 AVF created in 101 patients were studied. Data on pre-operative vessel mapping, type of AVF, post-operative surveillance scans were analyzed against the outcome AVF. RESULTS: Pre-operative mapping and post-operative scanning were carried out in 86% and 91%, respectively. A maturing fistula on post-operative scan highly correlated with a satisfactory outcome (p<0.001). The sensitivity and specificity of the post-operative scan were 100% and 85%, respectively. There were 79 brachiocephalic and 34 radiocephalic fistulae with a primary failure rate of 23% and 47%, respectively, giving an overall failure rate of 30%. Nine fistulae had further intervention (angioplasty or thrombolysis) and five (56%) were salvaged. Seventy-two AVF matured satisfactorily giving a primary cumulative patency of 71% (72/102). CONCLUSION: This study shows that preoperative vessel mapping provides useful information regarding the choice of AVF. Access surveillance duplex scanning at 6-8 weeks post-operatively is viable and has a high sensitivity and specificity for final outcome of fistula. Identifying AVF with potential problems early means that further intervention or surgery can be planned earlier, which will have a positive impact on patients.

Tissue oxygen saturation, measured by near-infrared spectroscopy, and its relationship to surgical-site infections.

BACKGROUND: Surgical-site infections (SSIs) are common after major abdominal and groin bypass surgery. Tissue oxygen tension has been shown to predict these infections accurately. This study assessed whether a non-invasive measurement of tissue oxygenation, tissue oxygen saturation as measured by spectrophotometry, was as accurate. METHODS: Fifty-nine patients having major abdominal or groin bypass surgery had tissue oxygen saturation measured by near-infrared spectrophotometry at the incision site and in the arm before operation, and at 12, 24 and 48 h after surgery. Masked outcome assessments for SSI were made at 7 and 30 days after operation. RESULTS: In this retrospective analysis, 17 patients (29 per cent) developed an SSI. At 12 h after operation there was a significant difference in tissue oxygen saturation at the surgical site between patients who developed an SSI and those who did not (mean(s.d.) 43.4(18.1) versus 55.8(22.0) per cent; P = 0.032). These oxygen saturation readings were found to be more specific and sensitive in predicting SSIs than the National Nosocomial Infection Surveillance system. DISCUSSION: There is a difference in postoperative surgical-site oxygen saturation between patients who subsequently develop SSIs and those who do not. Prediction of SSIs provides opportunities for intervention and prevention.

The regulatory C proteins from different restriction-modification systems can cross-complement.

The BamHI restriction-modification system contains a third gene, bamHIC, which positively regulates bamHIR. Similar small genes from other systems were tested in vivo for their ability to cross-complement. C.BamHI protein was identified, purified, and used to raise polyclonal antibodies. Attempts to detect other C proteins in cell extracts by cross-reactivity with C.BamHI antibodies proved unsuccessful.

Purification and characterization of C.BamHI, a regulator of the BamHI restriction-modification system.

The bamHIC gene, controlling the BamHI restriction-modification (R-M) system can functionally be replaced by providing pvuIIC or smaIC in trans. C.BamHI, the protein product encoded by bamHIC, has been purified and shown to bind a 345-bp DNA fragment within the BamHI R-M system.

Regulation of the BamHI restriction-modification system by a small intergenic open reading frame, bamHIC, in both Escherichia coli and Bacillus subtilis.

BamHI, from Bacillus amyloliquefaciens H, is a type II restriction-modification system recognizing and cleaving the sequence G--GATCC. The BamHI restriction-modification system contains divergently transcribed endonuclease and methylase genes along with a small open reading frame oriented in the direction of the endonuclease gene. The small open reading frame has been designated bamHIC (for BamHI controlling element). It acts as both a positive activator of endonuclease expression and a negative repressor of methylase expression of BamHI clones in Escherichia coli. Methylase activity increased 15-fold and endonuclease activity decreased 100-fold when bamHIC was inactivated. The normal levels of activity for both methylase and endonuclease were restored by supplying bamHIC in trans. The BamHI restriction-modification system was transferred into Bacillus subtilis, where bamHIC also regulated endonuclease expression when present on multicopy plasmid vectors or integrated into the chromosome. In B. subtilis, disruption of bamHIC caused at least a 1,000-fold decrease in endonuclease activity; activity was partially restored by supplying bamHIC in trans.

A limited number of Bacillus subtilis strains carry a tetracycline-resistance determinant at a site close to the origin of replication.

Several strains of Bacillus subtilis, e.g., 168 derivatives and R, were found to carry a single copy of a tetracycline-resistance (TcR) determinant (named tetBS908) at a site close to the origin of replication on the chromosome. This gene is highly homologous (80% identical) to the TcR determinant of plasmids widely dispersed among aerobic spore-forming bacilli. B. subtilis RM125 (168 strain) transformants which carry a varying number of tetBS908 sequences in a tandem array on the chromosome were constructed and examined for their TcR level. A nearly proportional relationship between the TcR level and copy number of tetBS908 existed.

Characterization of the cloned BamHI restriction modification system: its nucleotide sequence, properties of the methylase, and expression in heterologous hosts.

The BamHI restriction modification system was previously cloned into E. coli and maintained with an extra copy of the methylase gene on a high copy vector (Brooks et al., (1989) Nucl. Acids Res. 17, 979-997). The nucleotide sequence of a 3014 bp region containing the endonuclease (R) and methylase (M) genes has now been determined. The sequence predicts a methylase protein of 423 amino acids, Mr 49,527, and an endonuclease protein of 213 amino acids, Mr 24,570. Between the two genes is a small open reading frame capable of encoding a 102 amino acid protein, Mr 13,351. The M. BamHI enzyme has been purified from a high expression clone, its amino terminal sequence determined, and the nature of its substrate modification studied. The BamHI methylase modifies the internal C within its recognition sequence at the N4 position. Comparisons of the deduced amino acid sequence of M. BamHI have been made with those available for other DNA methylases: among them, several contain five distinct regions, 12 to 22 amino acids in length, of pronounced sequence similarity. Finally, stability and expression of the BamHI system in both E. coli and B. subtilis have been studied. The results suggest R and M expression are carefully regulated in a 'natural' host like B. subtilis.

Characterization of chromosomal DNA amplifications with associated tetracycline resistance in Bacillus subtilis.

Endogenous chromosomal DNA amplifications with associated tetracycline resistance (Tcr) in Bacillus subtilis were first described by C. R. Wilson and A. E. Morgan (J. Bacteriol. 163:445-453, 1985). We have confirmed and extended their results, and we show that fusion of protoplasts from Tcs B. subtilis 168 trpC2 with polyethylene glycol and regeneration on medium containing 20 micrograms of tetracycline per ml induces Tcr regenerants that contain amplified DNA. This phenomenon appeared to be recE dependent and requires the addition of polyethylene glycol. Along with three regenerants kindly provided by Wilson and Morgan (RAD1, RAD6, and RAD7), we characterized three strains (CLI20, CLI22, CLI30) isolated in this laboratory. All six contain an amplified region of DNA which was independently cloned on plasmid pCIS7. Integration of pCIS7 into the wild-type (Tcs) B. subtilis chromosome and amplification of the plasmid sequences generated a Tcr phenotype, even though the DNA on pCIS7 was cloned from Tcs B. subtilis KS162 (Ives and Bott, J. Bacteriol. 171:1801-1810, 1989). The amplified DNA also showed homology (through hybridization analysis) with pAM alpha 1 delta 1, a gram-positive Tcr plasmid, indicating that B. subtilis normally contains a silent integrated copy of the gene whose amplification confers Tcr. The amplifications were determined to lie between purA and gyrB on the B. subtilis chromosome, and the endpoints were mapped. RAD6 and CLI30 may share the same left-hand endpoint, but the other endpoints are different in each isolate. The amplified DNAs of RAD1, RAD6, CLI20, and CLI30 end near known DNA membrane binding sites. The number of amplified units of DNA was determined through dot blot analysis to do approximately 80 to 100 copies per cell, with corresponding increases in transcription of RAD1, RAD6, CLI20, CLI22, and CLI30.

Analysis of the tet gene of plasmid pCIS7 isolated from Bacillus subtilis.

We have previously shown that plasmid pCIS7, which contains 11.5 kb of Bacillus subtilis DNA isolated from a tetracycline-sensitive (TcS) strain, confers Tc resistance when integrated and amplified in the chromosome of TcS B. subtilis 168trpC2 [Ives and Bott, J. Bacteriol. 171 (1989) 1801-1810]. Here, we report that the number of integrated plasmid sequences required to confer Tc resistance is greater than the 20 copies seen with increasing chloramphenicol selection and, by dot-blot analysis, exceeds 100 copies per cell. The amplification is accompanied by a corresponding increase in mRNA encoding the tet gene. The tet gene sequence of pCIS7 has been compared to B. subtilis tetGSY908 [Sakaguchi et al., Biochim. Biophys. Acta. 94 (1988) 49-57] and other Gram-positive tet genes. The tet gene of pCIS7 is a member of the class L TcR determinants, and probably confers Tc resistance by increasing the efflux of Tc from the bacterial cell.

Cloned Bacillus subtilis chromosomal DNA mediates tetracycline resistance when present in multiple copies.

Plasmid pCIS7, containing 11.5 kilobases (kb) of Bacillus subtilis DNA, was isolated from a Tn917 transposon insertion in tetracycline-sensitive B. subtilis KS162. When integrated into the chromosome of B. subtilis 168, this plasmid conferred tetracycline resistance upon reiteration of the plasmid DNA sequences in the chromosome. Deletions and subclones of pCIS7 were constructed and introduced into an Escherichia coli in vitro transcription-translation system. A 72-kilodalton protein was localized to a 3.1-kb PstI-EcoRI fragment of the plasmid. Amplification of the 3.1-kb PstI-EcoRI fragment was required for expression of tetracycline resistance in B. subtilis 168. By hybridization to previously characterized clones, the 11.5-kb fragment was localized to the origin region of the chromosome. Through contour-clamped homogeneous electric field electrophoresis, this cluster of clones was shown to reside on a 200-kb NotI fragment bridging SfiI fragments of 150 and 250 kb and was oriented with respect to the purA and guaA loci, developing an accurate physical map of the region surrounding the origin of replication.

Mechanical effects on endothelial cell morphology: in vitro assessment.

Endothelial cells are subjected to fluid mechanical forces which accompany blood flow. These cells become elongated and orient their long axes parallel to the direction of shear stress when the cultured cells are subjected to flow in an in vitro circulatory system. When the substrate is compliant and cyclically deformed, to simulate effects of pressure in the vasculature, the cells elongate an orient perpendicular to the axis of deformation. Cell shape changes are reflected in the alignment of microtubule networks. The systems described provide tools for assessing the individual roles of shear stress, pressure, and mechanical strain on vascular cell structure and function.

Seeding of arteriovenous prostheses with homologous endothelium. A preliminary report.

Endothelial cell seeding of expanded polytetrafluoroethylene (e-PTFE) arteriovenous prostheses was performed to compare seeding with homologous vs. autologous cells and to study the effect of homologous seeding with a larger vs. a smaller number of cells. Sixteen dogs were randomly assigned to four equal groups: I, control; II, light homologous seeded; III, heavy homologous seeded; and IV, autologous seeded. Bilateral femoral arteriovenous loop grafts were inserted in all. Efficiency of seeding was assessed by cell counts of instilled vs. retained cells. All grafts remained patent and were harvested approximately 8 weeks after implantation. Samples were evaluated grossly by scanning electron microscopy and light microscopy. There was a direct correlation between number of cells instilled and number retained for each graft; group III received and retained the largest number (p less than 0.001 and p less than 0.05, respectively). The amount of thrombus deposition on the lumen of all grafts was grossly the same. Endothelium was demonstrated in samples obtained from the midgraft of groups III and IV; in contrast no endothelium was seen in groups I and II. The percentage of endothelialized surface was not determined. No immunologic cellular reaction was detected in any of the samples. We conclude that in the animal laboratory it is possible to seed e-PTFE arteriovenous prostheses successfully with homologous cells and to improve the efficiency of seeding by implanting a larger number of cells obtained from an endothelial cell bank. The potential applications of this technique to the clinical field are discussed herein.

Flow effects on prostacyclin production by cultured human endothelial cells.

Endothelial cell functions, such as arachidonic acid metabolism, may be modulated by membrane stresses induced by blood flow. The production of prostacyclin by primary human endothelial cell cultures subjected to pulsatile and steady flow shear stress was measured. The onset of flow led to a sudden increase in prostacyclin production, which decreased to a steady rate within several minutes. The steady-state production rate of cells subjected to pulsatile shear stress was more than twice that of cells exposed to steady shear stress and 16 times greater than that of cells in stationary culture.

Response of cultured endothelial cells to steady flow.

A system has been developed for subjecting endothelial cell monolayers to prolonged steady flow while maintaining normal culture conditions. Cloned bovine endothelial cells were grown to confluence on one wall of a square glass tube which was then incorporated in the flow circuit. Flow rates of 19-21 ml/min were sustained for periods of 6-45 hr, subjecting the cells along the center line of the wall of the tube to a maximum shear stress of 34 dyn/cm2. The cells in all the experiments remained attached and viable when subjected to this shear stress. Photographic data from experimental runs were qualitatively assessed for changes in cell morphology, confluence, and orientation and were compared to data from matched stationary controls. Five experiments were chosen for quantitative morphometric analysis. In three experiments, the cells showed elongation with their long axes aligned with the direction of flow in 6.5, 21, and 22 hr. In the other experiments, either the cells formed a swirling pattern or no change in morphology was apparent. Although cell shape (form) changed in response to shear stress, cell area remained unaffected by exposure to flow.