Behavior of cryopreserved endothelial cells in different phases: Their application in the seeding of vascular prostheses

Various methods of cryopreservation of human endothelial cells (EC) were studied to determine their viability and behavior when seeded onto vascular prostheses made of polytetrafluoroethylene (PTFE). Three different protocols were used: (1) cyropreservation of whole umbilical vein, (2) cyropreservation of freshly extracted umbilical EC in suspension, and (3) cryopreservation of EC derived from a first subculture. Fresh EC and EC from a first subculture were used as controls. The viability and growth of these cells in culture media were studied, and basal prostacyclin levels were determined. The cells were assessed morphologically after they were seeded onto PTFE discs. Our results showed that the cryopreservation method that maintained the greatest viability was that in which previously cultured EC were used. Basal prostacyclin levels were significantly different following cyropreservation. However, when these cells were seeded onto PTFE discs their behavior was similar to that of fresh EC.We thank W. L. Gore & Associates for supplying the prostheses used in this study.Supported by a grant from the Comision Interministerial de Cieucia y Tecnologia SAF 92/0875.     

Subarachnoid lumbar drains: A case series of fractured catheters and a near miss

PURPOSE: Lumbar subarachnoid catheters for cerebrospinal fluid (CSF) drainage (lumbar drains) are indicated for several medical and surgical conditions. A number of complications can occur from the placement of this type of catheter, including catheter breakage from excessive traction or shearing over the Tuohy needle. CLINICAL FEATURES: Five cases of lumbar subarachnoid catheter breakage/shearing and catheter fragment retention, as well as one near miss, were identified over a one-year period at a single institution. All (n = 6) patients were undergoing neurosurgical procedures. Four patients required surgical retrieval of the catheter fragments. No patient experienced log-term neurological sequelae. DISCUSSION: From these experiences, the following risks factors for catheter rupture are identified: 1) intentional or accidental retraction of the catheter through the needle during placement; 2) faulty use of the guidewire; or 3) use of excessive force during removal of the catheter. Methods to prevent such complications are suggested, including minimal use, or complete avoidance of a guidewire.     

Feasibility of near real-time image-guided sinus surgery using intraoperative fluoroscopic computed axial tomography.

OBJECTIVE: One of the main limitations of image-guided surgery is that navigation relies on the use of a CT scan obtained before surgery and is unable to be updated during the procedure. A software addition has been developed to allow reconstruction of CT-like images from a series of fluoroscopic scans and integrate these into an image-guided system (GE Healthcare Surgical Navigation, Lawrence, MA). We report our initial experience with a series of patients undergoing intraoperative fluoroscopic navigation in sinus surgery. STUDY DESIGN AND SETTINGS: After institutional review board clearance, we prospectively studied 14 consecutive patients undergoing image-guided sinus surgery with the use of intraoperative fluoroscopy. RESULTS: All patients had preoperative and postoperative fluoroscopic images reconstructed into CT-like images. By the conclusion of the study, images were adequate in quality and accurate navigation was achieved. CONCLUSION: Real-time image-guided sinus surgery using fluoroscopy is feasible. Future studies will need to focus on defining the procedures that could benefit, such as tumor resection, to enhance patient safety during these operations.     

Allele-Specific PCR Method Based on pncA and oxyR Sequences for Distinguishing Mycobacterium bovis from Mycobacterium tuberculosis: Intraspecific M. bovis pncA Sequence Polymorphism

An allele-specific amplification method based on two genetic polymorphisms to differentiate Mycobacterium tuberculosis from Mycobacterium bovis was tested. Based on the differences found at position 169 in the pncA genes from M. tuberculosis and M. bovis, a PCR system which was able to differentiate most of the 237 M. tuberculosis complex isolates tested in one of the two species was developed. All 121 M. tuberculosis strains showed the expected base (cytosine) at position 169. Most of the M. bovis isolates had a guanine at the cited position. Nevertheless, 18 of the 116 M. bovis isolates, all of them goat isolates, showed the pncA polymorphism specific to M. tuberculosis. These results suggest that goat M. bovis may be the nicotinamidase-missing link at the origin of the M. tuberculosis species. Based on the polymorphism found at position 285 in the oxyR gene, the same system was used to differentiate M. tuberculosis from M. bovis. In this case, DNAs from all 121 M. tuberculosis isolates had the expected base (guanine) at this position. In addition, all 116 M. bovis isolates, including those from goats, showed the identical polymorphism (adenine). The oxyR allele-specific amplification method can differentiate M. bovis from M. tuberculosis, is rapid (results can be obtained in less than 3 h), and is easy to perform.     

Epidemiology of extended-spectrum beta-lactamase-producing Enterobacter isolates in a Spanish hospital during a 12-year period

Fifteen Enterobacter clinical isolates (11 Enterobacter cloacae isolates, 3 Enterobacter aerogenes isolates, and 1 Enterobacter gergoviae isolate), representing 0.4% of all Enterobacter isolates recovered in our hospital from 1989 to 2000, were suspected of harboring an extended-spectrum beta-lactamase (ESBL). These isolates were recovered from 14 different patients. ESBLs were transferred by conjugation into an Escherichia coli recipient strain. Pulsed-field gel electrophoresis (PFGE) revealed a single clone of E. aerogenes and six different clones of E. cloacae. Four of these E. cloacae clonal types were represented by only one isolate each, but the other two were represented by three and four isolates, respectively. Isoelectric focusing, susceptibility phenotyping, PCR analysis, and sequencing demonstrated the presence of three different ESBLs. The most frequent was the recently characterized CTX-M-10 ESBL, which was found in the E. gergoviae isolate and in all but one of the E. cloacae isolates. The remaining E. cloacae isolate harbored a TEM-27 ESBL, and the three E. aerogenes isolates harbored a TEM-24 ESBL. PFGE revealed that our E. aerogenes strain was indistinguishable from the French TEM-24-producing E. aerogenes endemic clone. Although a low prevalence of ESBL-producing Enterobacter isolates was found in our institution over a 12-year period, a diversity of nonepidemic E. cloacae clones was detected, as was the persistence of the CTX-M-10 beta-lactamase. The presence of the TEM-24-producing E. aerogenes French clone in our institution also demonstrates the intercountry dissemination of ESBL-producing isolates.     

Listeriolysin O is essential for virulence of Listeria monocytogenes: direct evidence obtained by gene complementation.

The role of listeriolysin O in the intracellular multiplication of Listeria monocytogenes and, therefore, its pathogenicity was questioned through a genetic complementation study. A nonhemolytic mutant was generated by inserting a single copy of transposon Tn917 in the bacterial chromosome. This insertion was localized by DNA sequence analysis in hlyA, the gene coding for listeriolysin O. As was another mutant that we previously characterized, this mutant was avirulent in the mouse. It was transformed with a plasmid carrying only hlyA, able to replicate in L. monocytogenes, and stably maintained in vitro and in vivo. The complemented strain displayed a hemolytic phenotype identical to that of the wild-type strain and was fully virulent, therefore attributing a crucial role to listeriolysin O in virulence and excluding the hypothesis of a polar effect of the transposon insertion on genes adjacent to hlyA and possibly involved in virulence.     

Breakpoints for predicting Pseudomonas aeruginosa susceptibility to inhaled tobramycin in cystic fibrosis patients: use of high-range Etest strips

Inhaled administration of tobramycin assures high concentrations in cystic fibrotic lungs, improving the therapeutic ratio over that of parenteral tobramycin levels, particularly against Pseudomonas aeruginosa. Conventional Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards) breakpoints only consider parenteral levels and do not take into account these high antimicrobial concentrations. The Spanish Antibiogram Committee (The MENSURA Group) has tentatively defined specific breakpoint values for inhaled tobramycin when testing P. aeruginosa isolates from cystic fibrosis (CF) patients (susceptible, < or =64 microg/ml; resistant, > or =128 microg/ml). The antimicrobial susceptibilities of 206 prospectively collected CF P. aeruginosa isolates were determined by the reference agar dilution method. For tobramycin, the performance of high range tobramycin Etest strips (AB Biodisk, Solna, Sweden) and conventional tobramycin disks were assessed with the same collection. Applying MENSURA proposed breakpoints, 95.1% of the strains were categorized as susceptible to tobramycin, either using agar dilution or Etest high-range strips (99% categorical agreement between both methods). With CLSI breakpoints, susceptibility rates decreased to 79.1 and 81.1% for agar dilution and Etest strips, respectively (83.5% categorical agreement). Minor, major, and very major errors for Etest strips (CLSI criteria) were 13.6, 1.2, and 14.8%, respectively. Upon applying the new proposed criteria for inhaled tobramycin, only one major and one very major error were observed with Etest strips. Whenever inhaled tobramycin is considered for therapy, we suggest that P. aeruginosa strains from CF patients categorized as intermediate or resistant to tobramycin according to the CLSI criteria should be retested with high-range Etest strips and recategorized using MENSURA interpretive criteria. CLSI breakpoints should still be followed when intravenous tobramycin is used in CF patients, particularly during the course of exacerbations.     

Characterization of circulating idiotypes containing immune complexes and their presence in the glomerular mesangium in patients with IgA nephropathy.

The possible pathogenic role for idiotype-anti-idiotype interactions in kidney diseases has recently been suggested. Since patients with IgA nephropathy often present antibodies against alimentary antigens, like bovine serum albumin (BSA), we isolated an idiotypic antibody with BSA specificity from one of these patients. By means of a specific anti-idiotypic antibody raised in rabbits, we have studied the participation of these idiotypes in circulating and renal deposited immune complexes (IC) in patients with IgA nephropathy. On indirect immunofluorescence, the presence of cross-reactive idiotypes was detected in the glomeruli of 12 out of 42 (28%) patients with IgA nephropathy, but in none of 15 membranous or mesangiocapillary nephritis examined. The staining was located within mesangial and paramesangial areas, with a similar, but less intensive, pattern distribution than IgA. Previous adsorption of rabbit anti-idiotype antibodies on an idiotype-Sepharose column completely abolished that staining. A close relationship was found between the presence of cross-reactive idiotypes on mesangial immunoglobulins and the existence of increased levels of serum idiotypes and idiotype-containing IC. Serum analytical ultracentrifugation showed that circulating IC containing idiotypes have chiefly a large (greater than 19 S) and intermediate (13 S-19 S) size, while those containing anti-BSA antibodies were only between 7 S-13 S fractions, or absent. Our results suggest that in patients with IgA nephropathy, shared idiotypes participate in the formation of circulating and renal deposited IC. It is possible that the apposition of free anti-idiotype to idiotype already bound to glomeruli, and vice versa, could contribute to increasing the amount and size of mesangial immune deposits, and, therefore, facilitate or perpetuate tissue injury.