The effects of use and simulated reuse on percutaneous transluminal coronary angioplasty balloons and catheters.

A series of studies was undertaken to determine the effects of single patient use and simulated reuse on percutaneous transluminal coronary angioplasty (PTCA) balloon catheters. Catheters were retrieved from Walter Reed Army Medical Center and were low-level disinfected and cleaned at the US Food and Drug Administration. They were then tested for balloon compliance, and the results were compared against the manufacturer's specifications. Selected groups of catheters were subjected to EO-resterilization and a simulated reuse protocol. The results demonstrated that the effects of use and EO-resterilization is model specific. Furthermore, some balloons demonstrated a time-dependent behavior while others recovered from the effects of simulated reuse by compliance testing at high pressure. Testing for the slipperiness of the catheters after repeated EO-resterilization also demonstrated that changes were model specific.

The effect of repeated ethylene oxide sterilization on the mechanical strength of synthetic absorbable sutures.

The purpose of this study was to determine the effect of repeated ethylene oxide sterilization using a standard clinical protocol on sutures, a type of medical device labeled for single use and reported to be reprocessed for use after being opened but not used. Four types of commonly used synthetic absorbable sutures were subjected to 1 and 2 ethylene oxide resterilization cycles. Knot tensile strength was determined for new sutures and for sutures that had been subjected to 1 and 2 ethylene oxide resterilization cycles. As has been found with other types of single-use devices, no general conclusions can be made for absorbable sutures. The strengths of different types of sutures increased, decreased, or stayed the same after repeated sterilization. In addition, the inner packages of some sutures were not intact after reprocessing, possibly exposing the sutures to increased humidity, which can produce degradation leading to loss of strength both immediately and after additional shelf aging and degraded performance after clinical use.

Effects of particulate and soluble cadmium species on biochemical and functional parameters in cultured murine macrophages.

Cultured murine macrophages (RAW 264.7) were used to evaluate the temporal relationships between cytotoxicity, phagocytosis, tumor necrosis factor-alpha (TNF-alpha), and nitric oxide (NO) production, and alterations in expression of stress proteins after exposure to cadmium oxide (CdO) or cadmium chloride (CdCl(2)), particulate and soluble forms of cadmium, respectively. Macrophages were exposed in vitro to CdO (25 or 50 microg) or CdCl(2) (30 or 40 microM) for 2 to 72 h. Cytotoxicity was not evident until 18 h when exposed to 30 microM CdCl(2) or 25 microg CdO, but occurred as early as 12 h after exposure to 40 microM CdCl(2) or 50 microg CdO. Relative to untreated controls, phagocytic activity decreased progressively from 2 to 24 h after exposure to both forms of cadmium. TNF-alpha levels increased to 2- to 3-fold after 4 h and remained elevated until 24 h after exposure to 25 and 50 microg CdO and 30 microM CdCl(2), but decreased by 18-24 h at 40 microM CdCl(2). CdCl(2) or CdO alone did not induce NO; however, both cadmium species reduced lipopolysaccharide (LPS)-stimulated NO production in a dose-dependent manner. Enhanced de novo synthesis of 70- and 90-kD heat shock, or stress, proteins was observed 2 to 8 h after exposure to both CdCl(2) and CdO; however, synthesis of these proteins returned to control levels by 24 h. Stress protein synthesis was enhanced by CdCl(2) or CdO prior to cytotoxicity, but coincided with a decrease in phagocytic capacity and an increase in TNF-a levels. The data suggest that cultured macrophages respond similarly in vitro to a particulate form and a soluble form of cadmium in a cell type that plays a pivotal role in inflammatory and immune responses.

Safety and cleaning of medical materials and devices.

A study was undertaken to evaluate different procedures to safely remove microorganisms, protein, and mammalian cells from materials and provide a suitable method for cleaning and assessing effectiveness of cleaning medical devices for reuse or for analysis of failure. Safety considerations for the personnel performing the cleaning or handling the device after cleaning are important issues. Polystyrene plates (96 well) were used to simulate device surfaces not amenable to manual scrubbing. Staphylococcus epidermidis, Candida albicans, Escherichia coli, Pseudomonas aeruginosa, and oral flora were grown in the plates. The plates were stained with crystal violet and the optical densities recorded. The results indicated that E. coli did not adhere well and Pseudomonas formed clumps that were easily detached from the surface of the plates. However, S. epi, C. albicans, and the oral organisms formed adherent biofilms that were difficult to remove from the plates. Detergents with enzymes and sodium hypochlorite (NaOCl) bleach were both effective in removing the biofilm. Other detergents and surfactants were not effective. The aldehyde agents did not remove the organisms and made further cleaning difficult. Allowing the biofilm to dry first made cleaning very difficult. Only the NaOCl bleach could subsequently remove the dried or aldehyde fixed organisms from the wells. The same 96-well polystyrene plate format was used to measure the amount of protein and cell adherence as well as the effectiveness of subsequent cleaning. Bradford reagent was used to detect protein as a measure of the cleaning efficacy. As with the bacteria, NaOCl bleach was effective at removing the protein and cells that had been dried or fixed by formalin or alcohol, whereas detergent with enzymes was not very effective. This study confirmed that used medical devices, contaminated with microorganisms, protein, and/or mammalian cells, should not be allowed to dry before cleaning and that a thorough cleaning procedure should precede sterilization or disinfection (with the exception of NaOCl bleach which also cleans).

Tissue colonization from implantable biomaterials with low numbers of bacteria.

This study was undertaken to evaluate the risk of infection (defined as the recovery of the relevant organism from the implant site) in a mouse model when low numbers of bacteria were present on an implanted biomaterial. Segments of different types of suture with adherent bacteria were implanted subcutaneously into mice. The infection risk with Staphylococcus aureus was greater than with Staphylococcus epidermidis RP62A or Candida albicans. The infection risk with the implantation of multifilament sutures was significantly greater than with monofilament sutures. When <10 colony forming units (cfu) of S. aureus were present on monofilament suture material, the infection rate was 3%. When <10 cfu of S. aureus were present on multifilament suture material, the infection rate was 7%. An infection rate of 15% occurred with <10 cfu of S. aureus on multifilament nylon sutures. When >10 but <20 cfu of S. aureus were present, the infection rates were 4 and 51%, respectively. These data confirm that the infection rate with multifilament sutures (or porous materials) is greater than with monofilament sutures (or solid materials) when the organisms are encountered at implantation (acute model) and indicate that a significant risk of infection may occur when only a few organisms are on a device at implantation.

Decontaminating particles exposed to bacterial endotoxin (LPS).

Lipopolysaccharide (LPS), which comes from the cell wall of gram-negative bacteria, can stimulate murine macrophage cells to produce nitric oxide (NO), cytokines, such as tumor necrosis factor-alpha, and interleukins, such as IL-6. When examining the biological effects of particles on macrophages, it is important to have no contaminating LPS associated with the particles and none with any cell culture media or supplies since even very low levels of LPS are stimulatory. The presence or absence of LPS was observed in two ways: (1) the amount of NO produced by RAW 264.7 murine macrophage cells, and (2) the Limulus amebocyte lysate (LAL) test. Treating particles with 70% ethanol at room temperature for 48 h, followed by washing the polymethylmethacrylate (PMMA) particles with endotoxin-free phosphate-buffered saline three times, decontaminated LPS and LPS-treated PMMA particles. When given LPS that had been treated with 70% ethanol for 48 h at room temperature or at 37 degrees C, cells did not produce NO above control levels. Negative LAL tests indicated the presence of extremely low levels or the complete absence of LPS in 70% ethanol-treated LPS.

Synergistic induction of tumor necrosis factor alpha by bacterial lipopolysaccharide and lipoteichoic acid in combination with polytetrafluoroethylene particles in a murine macrophage cell line RAW 264.7.

The induction of tumor necrosis factor alpha (TNF-alpha) by polytetrafluoroethylene (PTFE) particles (5-50 microns) and by bacterial lipopolysaccharide (LPS) and lipoteichoic acid (LTA) was examined in RAW cell cultures. Twenty-four-hour culture supernatants from the treated and control cells were assayed for TNF-alpha using a mouse L929 cell cytotoxicity assay. Untreated RAW cells produced low levels of endogenous TNF-alpha in the culture supernatants. Addition of 0.5 ng to 1 microgram/ mL LPS or 1 ng to 1 microgram/ml LTA increased the TNF-alpha production by 7-3570-fold and 2-815-fold, respectively. Addition of 1-5 mg PTFE increased the TNF-alpha production by 6-17-fold over the untreated control cell levels. The cells exposed to PTFE and 0.5 ng/mL LPS or 5 ng/mL LTA produced TNF-alpha levels that were significantly higher than those produced by any inducer alone. Thus, both LTA, a Gram-positive bacterial cell wall component and LPS, a Gram-negative bacterial cell wall component, can induce TNF-alpha production, which is further enhanced by PTFE particles in RAW cells.

Cytotoxic and mutagenic effects of ferric nitrilotriacetate on L5178Y mouse lymphoma cells.

An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces renal proximal tubular necrosis that leads to a high incidence of renal adenocarcinoma in rodents. Others have shown that Fe-NTA induces modified DNA base products both in vitro and in vivo. However, Fe-NTA is negative in the Ames Salmonella test with or without S9 activation. The goal of this project was to determine if Fe-NTA is cytotoxic and mutagenic using the L5178Y (TK +/-) mouse lymphoma assay. Our experiments showed a relationship between the concentration of Fe-NTA (0 to 1 mM) and the decrease in relative survival. An exposure-dependent increase in the number of mutations was observed with increasing concentrations of Fe-NTA. At 14% relative survival, there was about a 4-fold increase in mutations (trifluorothymidine resistance) over unexposed, control cells. Ferric nitrate or nitrilotriacetic acid alone induced a relatively low 1.5- or 1.1-fold increase in mutation, respectively. Our results establish that Fe-NTA is mutagenic in the L5178Y mouse lymphoma assay system.

Inhibition of herpes virus plaquing capacity in human diploid fibroblasts treated with gilvocarcin V plus near UV radiation.

The capacity of human fibroblasts to support plaque formation by Herpes simplex virus following treatment of the cells with gilvocarcin V, a polyaromatic C-glycoside, plus near ultraviolet radiation (UVA, 320-400 nm) was examined. Gilvocarcin V, plus UVA radiation, effectively inhibited host cell capacity at concentrations five orders of magnitude lower than that of 8-methyoxypsoralen required for capacity inhibition at similar levels of UVA radiation. This result extends the observation of unusual biological potency of UVA-activated gilvocarcins from bacterial cells to human cells.

Cytotoxicity and mutagenicity of ophthalmic solution preservatives and UVA radiation in L5178Y cells.

Four chemical preservatives commonly used in ophthalmic solutions were tested for their toxic and mutagenic potential in mouse lymphoma cells with and without exposure of the cells to ultraviolet A (UVA) radiation. The preservatives tested were benzalkonium chloride (BAK), chlorhexidine, thimerosal and ethylenediaminetetraacetic acid (EDTA). Cell survival and mutagenesis were measured using the L5178Y mouse lymphoma (TK +/-) system. Cells were exposed to varying amounts of preservatives for 1 h at 37 degrees C, and then aliquots were irradiated with UVA radiation (during the exposure to preservative). Cells were then assayed for survival, and for mutagenesis at the thymidine kinase (TK) locus. In concentrations commonly found in ophthalmic solutions, BAK, chlorhexidine, and thimerosal were toxic to cells, and thimerosal was slightly mutagenic. When cells were exposed to preservative and UVA radiation, chlorhexidine was mutagenic and the mutagenic activity of thimerosal was enhanced.

Induction of lambda prophage near the site of focused UV laser radiation.

DNA damage from photon scatter or beam spread during UV excimer laser irradiation was investigated using the induction of bacteriophage lambda in E. coli BR339. Prophage induction in these cells leads to the production of beta-galactosidase which can be detected colorimetrically by the application of appropriate substrates. An agar surface overlayed with BR339 cells was placed at various distances from the focal point of a converging lens and exposed to either 193 or 248 nm laser radiation. Energy densities ranging from approximately 5 mJ/cm2 to 30 J/cm2 were used. Ablation with 193 nm laser radiation produced an 800 microns wide clear 'trench' surrounded by a 500 microns zone of cells in which lambda had been induced. Following ablation with 248 nm laser radiation, the zone of induction was several millimeters wide. Exposures to 193 nm radiation at 170 mJ/cm2/pulse produced visible ablation of the agar surface at 1.7 J/cm2. Lambda induction was observed surrounding cleared ablation areas. The presence of induction in this system suggests that both 248 and 193 nm excimer laser radiation delivered at high energy densities has sufficient spread or scatter to damage DNA in cells surrounding areas of ablation.

Host cell reactivation by excision repair is error-free in human cells.

Do host cell repair processes affect the mutagenesis of UV-irradiated virus in human cells? The answer was obtained by investigating the mutagenesis of UV-irradiated herpes simplex virus after the irradiated virus was grown in human cells that possess normal repair capacity (normal) or lack excision repair (XPA) or post-replication repair (XP var). Evidence is presented which indicate that XPA cells express no host cell reactivation, while XP var cells express the normal level. Viral mutagenesis was measured as the fraction of the progeny of the surviving virus capable of plaque formation in the presence of iododeoxycytidine. In the normal and XPA cells mutagenesis of the irradiated virus increased linearly with UV exposure. The UV exposure needed to yield a given mutagenesis level for virus grown in XPA cells was much lower than that for virus grown in normal cells. However, when the mutation frequencies were compared at similar virus survival levels, the data from virus grown in normal cells and in XPA cells were indistinguishable. Mutagenesis in XP var cells increased as dose squared and was similar in magnitude to that in normal cells. Thus the excision repair of normal cells which provided host cell reactivation by removing lethal UV damage also removed mutagenic lesions from the virus with the same efficiency, while the repair deficiency of XP var cells had a minor role in host cell reactivation and in mutagenesis. This demonstrates that in human cells host cell reactivation by excision repair is primarily an error-free process.

Genetic regulation of aryl hydrocarbon hydroxylase activity in cultures of mouse fetal liver cells.

Primary cultures of fetal liver cells were established from six inbred strains of mice to determine whether the genes regulating the induction of aryl hydrocarbon hydroxylase (AHH) in adult livers in vivo also function in fetal liver cells maintained in culture. Fetuses were 19 days old and were derived from three inbred mouse strains (A/J, C3H/HeJ, C57BL/6J) which are classified genetically as aromatic hydrocarbon responsive and three strains (AKR/J, DBA/2J, SWR/J) which are classified as aromatic hydrocarbon non-responsive. Cells were induced with 3-methylcholanthrene (3-MC) and harvested for measurement of AHH activity, DNA content and amount of [3H]thymidine incorporated. The time course of induction of AHH activity by 3-MC was followed from around 60 h after cells were plated until 190 h when cells were at the end of exponential and at the start of stationary growth phase. Both basal and induced AHH activities generally rise to a peak value during exponential growth and then decline as cells reach stationary phase. During late exponential growth to early stationary growth phase, cells derived from responsive strains have higher induced enzyme activity than cells derived from non-responsive strains. Cells from aromatic hydrocarbon responsive mice attained maximal AHH activity with inducer concentrations of 0.05--0.10 micrograms 3-MC/ml. Cells from aromatic hydrocarbon non-responsive strains were maximally induced at 0.50 micrograms 3-MC/ml. The 5--10-fold difference between cells from responsive and non-responsive mice in concentration of 3-MC needed to induce AHH maximally is taken to indicate that genes controlling aromatic hydrocarbon responsiveness in adult mouse livers in vivo also function in fetal mouse liver cells maintained in culture.

Sequential metabolic events during encystment of Azobacter vinelandii.

Cells of Azotobacter vinelandii are specifically induced to encyst by beta-hydroxybutyrate (BHB). The process of differentiation, which occurs over a period of 36 h, was characterized by an ordered sequence of biochemical events. Upon initiation of encystment, nitrogen fixation and glucose-6-phosphate dehydrogenase activities decreased immediately to very low levels. This was followed by an increase in the specific activities of BHB dehydrogenase, isocitrate dehydrogenase, isocitrate lyase, and malate synthase first at 3 h and then again at 21 h. The peak activity of fructose 1,6-diphosphate aldolase occurred at 6 h, and the enzyme activity then decreased gradually. Fructose 1,6-diphosphatase had peak activities at 9 and 27 h. Deoxyribonucleic acid synthesis ceased just prior to the final cell division at 4 to 6 h, but ribonucleic acid synthesis continued until the 12th h. From labeling studies and the appearance of new enzyme activities, it appeared that protein synthesis continued throughout encystment.

Morphogenesis of cysts in Azotobacter vinelandii.

Cultures of Azotobacter vinelandii were induced to encystment with beta-hydroxybutyrate. The morphological events in the transition from cell to cyst were observed by electron microscopy of thin sections. Upon induction of encystment, cells became rounded and nonmotile. The exine coat developed by the continuous excretion of membranous components into the capsule surrounding the cell.