The effect of a disinfectant/coolant irrigant on microbes isolated from dental unit water lines.

The purpose of this study was to assess water samples from a hospital dental clinic to determine whether a disinfectant/coolant irrigant containing chlorhexidine (Lines, Micrylium Laboratories) affects the presence of microbial organisms in dental unit waterlines. Water samples from three hospital dental operatories were collected at baseline and after overnight treatment with a disinfectant-containing irrigant followed by sterile water irrigation. Saliva of treated patients and sterile water rinse specimens were collected from the waterlines of these operatories for three consecutive days, then weekly for eight weeks after treatment. Specimens were cultured to identify total heterotrophic plate counts as well as presence of Pseudomonas aeruginosa and Candida species. Baseline organism counts varied from 10(3) to 10(5) colony-forming units per milliliter. After treatment, no organisms were detected in waterline discharge. Decontamination of dental unit waterlines is possible using a disinfectant/irrigant followed by sterile water irrigation. The potential for contamination of the lines from patients' saliva may have been reduced due to use of anti-retraction valves and the disinfectant/sterile water irrigation, as conducted in this study.

Hypothetical ratings of coronary angiography appropriateness: are they associated with actual angiographic findings, mortality, and revascularisation rate? The ACRE study.

OBJECTIVE: To determine whether ratings of coronary angiography appropriateness derived by an expert panel on hypothetical patients are associated with actual angiographic findings, mortality, and subsequent revascularisation in the ACRE (appropriateness of coronary revascularisation) study. DESIGN: Population based, prospective study. The ACRE expert panel rated hypothetical clinical indications as inappropriate, uncertain, or appropriate before recruitment of a cohort of real patients. SETTING: Royal Hospitals Trust, London, UK. PARTICIPANTS: 3631 consecutive patients undergoing coronary angiography (no exclusion criteria). MAIN OUTCOME MEASURES: Angiographic findings, mortality (n = 226 deaths), and revascularisation (n = 1556 procedures) over 2.5 years of follow up. RESULTS: The indications for coronary angiography were rated appropriate in 2253 (62%) patients. 166 (5%) coronary angiograms were performed for indications rated inappropriate, largely for asymptomatic or atypical chest pain presentations. The remaining 1212 (33%) angiograms were rated uncertain, of which 47% were in patients with mild angina and no exercise ECG or in patients with unstable angina controlled by inpatient management. Three vessel disease was more likely among appropriate cases and normal coronaries were more likely among inappropriate cases (p < 0.001). Mortality and revascularisation rates were highest among patients with an appropriate indication, intermediate in those with an uncertain indication, and lowest in the inappropriate group (log rank p = 0.018 and p < 0.0001, respectively). CONCLUSION: The ACRE ratings of appropriateness for angiography predicted angiographic findings, mortality, and revascularisation rates. These findings support the clinical usefulness of expert panel methods in defining criteria for performing coronary angiography.

Underuse of coronary revascularization procedures in patients considered appropriate candidates for revascularization.

BACKGROUND: Ratings by an expert panel of the appropriateness of treatments may offer better guidance for clinical practice than the variable decisions of individual clinicians, yet there have been no prospective studies of clinical outcomes. We compared the clinical outcomes of patients treated medically after angiography with those of patients who underwent revascularization, within groups defined by ratings of the degree of appropriateness of revascularization in varying clinical circumstances. METHODS: This was a prospective study of consecutive patients undergoing coronary angiography at three London hospitals. Before patients were recruited, a nine-member expert panel rated the appropriateness of percutaneous transluminal coronary angioplasty (PTCA) and coronary-artery bypass grafting (CABG) on a nine-point scale (with 1 denoting highly inappropriate and 9 denoting highly appropriate) for specific clinical indications. These ratings were then applied to a population of patients with coronary artery disease. However, the patients were treated without regard to the ratings. A total of 2552 patients were followed for a median of 30 months after angiography. RESULTS: Of 908 patients with indications for which PTCA was rated appropriate (score, 7 to 9), 34 percent were treated medically; these patients were more likely to have angina at follow-up than those who underwent PTCA (odds ratio, 1.97; 95 percent confidence interval, 1.29 to 3.00). Of 1353 patients with indications for which CABG was considered appropriate, 26 percent were treated medically; they were more likely than those who underwent CABG to die or have a nonfatal myocardial infarction--the composite primary outcome (hazard ratio, 4.08; 95 percent confidence interval, 2.82 to 5.93)--and to have angina (odds ratio, 3.03; 95 percent confidence interval, 2.08 to 4.42). Furthermore, there was a graded relation between rating and outcome over the entire scale of appropriateness (P for linear trend=0.002). CONCLUSIONS: On the basis of the ratings of the expert panel, we identified substantial underuse of coronary revascularization among patients who were considered appropriate candidates for these procedures. Underuse was associated with adverse clinical outcomes.

Correlation of human CD56+ cell cytotoxicity and IFN-gamma production.

The use of an IFN-gamma ELISPOT assay to evaluate cellular immune responses has gained increasing popularity, especially as a surrogate measure for cytotoxic T lymphocyte (CTL) responses. We have compared the IFN-gamma ELISPOT assay and the traditional(51)Cr release assay for detection of human natural killer (NK) cell activity. The cell populations used for evaluation of these assays included freshly isolated and IL-2-activated peripheral blood mononuclear cells (PBMC). CD56-positive cells were demonstrated to be the primary source of the IFN-gamma signal when PBMC were evaluated with NK-sensitive targets in the IFN-gamma ELISPOT assay. IFN-gamma ELISPOT and(51)Cr release assays showed excellent correlation suggesting that NK activity can be reliably evaluated with methods other than the traditional(51)Cr release assays.

Magnitude and consequences of error in coronary angiography interpretation (the ACRE study).

In the routine reporting of coronary angiograms, there are no contemporary estimates of the magnitude and consequences of interobserver variability. We therefore measured the agreement beyond chance between (1) the number of narrowed arteries on an angiographic report extracted from case notes and independent assessments by 2 cardiologists, and (2) actual patient management over an 18-month follow-up period and each cardiologist's hypothetical management proposal based on abstracted clinical details. Two hundred nine angiograms were randomly selected from 4,121 patients in a prospective study (Appropriateness of Coronary Revascularisation [ACRE study]). The number of narrowed arteries was defined using Coronary Artery Surgery Study (CASS) criteria. For the number of narrowed arteries, cardiologists A and B agreed with the angiographic report in 126 patients (60%, weighted kappa = 0.64) and 124 patients (59%, weighted kappa = 0.63), respectively. In a subset of 92 patients (44%) there was unanimous agreement on the number of narrowed arteries (both cardiologists agreed with the angiographic report). Comparing actual management (34 percutaneous transluminal coronary angioplasty and 39 coronary artery bypass grafting procedures on follow-up) with each of the cardiologist's management recommendations showed agreement in 150 patients (72%, kappa = 0.46) and 154 patients (74%, kappa = 0.48) for cardiologists A and B, respectively. These agreements on management improved (p = 0.05) for cardiologist B (but not A) when analysis was confined to the subset of 92 patients, showing agreement in 73 patients (79%, kappa = 0.60). Thus, in routine clinical practice, the agreement beyond chance in interpretation of the number of narrowed arteries was good. Disagreements on subsequent patient management arose as a result of, and independent of, errors in angiographic interpretation.

Waiting for coronary angiography: is there a clinically ordered queue?

Among over 3000 patients undergoing coronary angiography in the absence of a formal queue-management system, we found that a-priori urgency scores were strongly associated with waiting times, prevalence of coronary-artery disease, rate of revascularisation, and mortality. These data challenge the widely held assumption that such waiting lists are not clinically ordered; however, the wide variation in waiting times within urgency categories suggests the need for further improvements in clinical queueing.

Natural killer cell-endothelial cell interactions in xenotransplantation.

Interest in xenotransplantation derives from the documented need for more organs and tissues than can be expected from living or cadaveric donors. Although the barriers to xenotransplantation are formidable, the scientific rewards in addressing these problems have been significant. The first and most potent barrier to xenotransplantation is hyperacute rejection mediated by xenoreactive natural antibodies and serum complement. The majority of the xenoreactive antibodies appear to be directed at terminal galactose epitopes, especially gal alpha1-3 gal. Significant progress has been made in surmounting hyperacute rejection, and this has led to an examination of underlying mechanisms of delayed xenograft rejection. One of these delayed mechanisms concerns the potential role of graft recipient, natural killer (NK) cells. NK cells can cause variable, low-level cytotoxicity of xenogeneic endothelial cells in vitro that may be enhanced in the presence of xenoreactive IgG. The specificity of NK cell-mediated cytotoxicity appears to overlap with a major subset of xenoreactive natural antibodies. These cytotoxic interactions can be regulated by "humanizing" the endothelial cells through expression of the appropriate human MHC class I genes. More important, NK cells induce endothelial cell activation, which results in changing the nature of the endothelial cell surface from an anticoagulant surface to a procoagulant surface. These findings parallel those observed in allogeneic NK cell-endothelial cell interactions and suggest these important observations may be extended to NK cell-endothelial cell interactions in general.

Getting residents involved in research: a challenge in the era of managed care.

The changing climate of health care, with the associated increasing financial constraints, challenges faculty and residents to find time for mentoring and pursuing research. Departments and institutions can encourage residents to conduct research by assuring them that they have a supportive infrastructure. Only when the institution and faculty value research will residents do the same. More than half of academic surgeons spend less than 20% of their effort in research; how can residency programs expect residents to enthusiastically pursue research when members of the teaching faculty are themselves not writing and publishing? If the ACGME is serious about asking residents to devote their time and energy to becoming more active in research activities, we must be prepared to decrease service commitments, and we must provide clinically active mentors and visible career models.

Rating the appropriateness of coronary angiography, coronary angioplasty and coronary artery bypass grafting: the ACRE study. Appropriateness of Coronary Revascularisation study.

BACKGROUND: Previous studies investigating the appropriateness of invasive management of coronary disease had not reported the internal consistency of their ratings and may now be out of date. The aim of this study was to measure the influence of clinical factors on contemporary ratings of the appropriateness of coronary angiography, percutaneous transluminal coronary angioplasty (PTCA) and coronary artery bypass graft (CABG) in the Appropriateness of Coronary Revascularisation (ACRE) study. METHODS: The Delphi-RAND technique was used, in which an expert panel (four cardiologists, three cardiothoracic surgeons, a general physician and a general practitioner), meeting in 1995, rated mutually exclusive indications (n = 2178 for angiography, n = 995 for PTCA and n = 984 for CABG). The main outcome measures were the appropriateness category (inappropriate, uncertain or appropriate) for each of the three procedures and treatment preference. RESULTS: For revascularization, the strongest determinant of inappropriateness was coronary anatomy. The odds ratio (OR) for inappropriate PTCA was 10.6 (95 per cent confidence interval (CI) 4.8-23.5) for the effect of left main stem or three-vessel disease versus single-vessel disease, and for CABG it was 0.06 (95 per cent CI 0.03-0.15). The number of diseased vessels was strongly related to preference for medical, PTCA or CABG treatment (p for linear trend <0.001). Mild versus severe anginal symptoms were associated with inappropriate angiography (OR 2.0 (95 per cent CI 0.9-9.8), although this effect was stronger when only the cardiologists' ratings were considered (OR 10.1 (95 per cent CI 2.4-42.6)). CONCLUSION: These are the first UK ratings of appropriateness covering all three procedures. The associations with clinical factors provide evidence of the internal consistency of these ratings. Prospective validation of these ratings against clinical outcomes is under way in the ACRE study.

The role of calnexin in NK-target cell interaction.

In this study, a relationship between target cell sensitivity to natural killing and target cell expression of the molecular chaperone++ calnexin was assessed. The NK-resistant cell line NKR was originally derived from the NK-sensitive, human T-cell line CEM and does not synthesize calnexin protein or mRNA. The cell lines CEM, NKR and 1B9 (NKR transfected with a calnexin cDNA) were compared in a number of assays. All the lines but CEM were resistant to NK in conventional 4 h cytotoxicity assay, but were highly sensitive to IL-2 activated NK. Incubation of NK cells with CEM but not with the other two lines led to increased expression of the NK cell activation marker CD69. Treatment of effector cells with PGE2 and TGF-beta resulted in an inhibition of NK activity and CD69 expression. The calnexin transfected clone 1B9 clone had intermediate ability to block cytotoxicity in cold target inhibition assay compared to CEM and NKR. Expression of the adhesion molecules CD44 and LFA-1alpha was significantly higher on both calnexin positive cell lines compared to NKR. These data suggest that calnexin controls the expression of some, but not all, target structures that are necessary for binding and activation of NK cells.

Conformation dependence of MHC class I in the modulation of target cell sensitivity to natural killing.

C1R.Aw68 delta 242 is a human B cell line expressing a mutant class I molecule that is defective in assembly and transport at 37 degrees C but is stably expressed at room temperature. This cell line has been utilized to study the conformation dependence of MHC class I in the modulation of target cell sensitivity to natural killing. Surface expression of MHC class I molecules was monitored by the antibodies W6/32 (detecting a pan-class I specificity that is beta 2-microglobulin and conformation dependent) and HC.10 (detecting free HLA-B heavy chain and a subset of HLA-A heavy chains). C1R.Aw68 delta 242 was cultured at reduced temperature to induce cell surface expression of class I molecules, and then the temperature was shifted to 37 degrees C. During the first 2 h at 37 degrees C, C1R.Aw68 delta 242 displayed a higher level of HC.10 reactivity than W6/32. Conjugation of C1R.Aw68 delta 242 to NK cells correlated inversely with W6/32 expression, but not with HC.10 reactivity as revealed by flow cytometry. The sensitivity of the C1R.Aw68 delta 242 cells to NK-mediated lysis was also examined as a function of temperature, and the level of C1R.Aw68 delta 242 cytolysis correlated inversely with W6/32 expression but not HC.10. The fact that both the conjugation rate and target cell cytolysis increased with decreased reactivity with the conformation-dependent antibody W6/32 and not with HC.10, is consistent with the hypothesis that NK cell inhibitory receptors (KIR) detect a conformation-dependent epitope(s).

Induction of procoagulant function in porcine endothelial cells by human natural killer cells.

NK cells may mediate effector functions other than target cell cytotoxicity. To explore such noncytotoxic effector mechanisms, we tested whether human PBL and purified NK (CD56+) cells might induce expression of tissue factor by cultured porcine aortic endothelial cells. Tissue factor is the major coagulation factor that binds to factor VIIa and initiates coagulation. The addition of freshly isolated NK cells but not T cells to endothelial cells resulted in the induction of tissue factor activity. NK-depleted (CD56-) effector cells did not induce tissue factor activity; however, the combination of CD56+ cells and NK-depleted cells induced tissue factor activity to the same extent as unseparated cells. PBL induced tissue factor mRNA in porcine endothelial cells and NK depletion resulted in a significant decrease of the induction. Induction of tissue factor activity in porcine endothelial cells by human NK cells required direct cell-to-cell contact, as transfer of supernatants from NK-endothelial cell cultures to secondary cultures did not induce tissue factor activity, and anti-LFA-1alpha Abs inhibited the induction of tissue factor activity. Induction of tissue factor activity in endothelial cells by NK cells may represent one of a variety of ways in which NK cells mediate noncytotoxic effects.

Differential expression of natural killer cell markers: human versus baboon.

The use of baboons as a model for the study of allo- and xenotransplantation has become increasingly important, but there are few studies on the basic immunological responses in baboons that might be relevant for a rejection reaction. In present study, the cell-surface phenotype, cytokine-induced activation and growth, and cytotoxicity of baboon and human natural killer (NK) and lymphokine-activated killer (LAK) cells were compared. A panel of murine monoclonal antibodies specific for human cell-surface markers expressed on lymphocytes was used to compare relevant baboon and human peripheral blood lymphocytes (PBL). Baboon PBL were 52.1+/-2.9% CD8+, 18.5+/-2.2% CD16+, 3.0+/-0.5% CD25+, and 5.5+/-1.8% CD69+. The corresponding proportions in humans were 23.8+/-7.1%, 12.8+/-3.2%, 4.5+/-1.0%, and 2.3+/-1.1%. In contrast to human PBL, less than 1% of baboon lymphocytes expressed CD56, CD57, and CD122 (interleukin [IL]-2Rbeta). Baboon lymphocytes showed NK cytotoxic activity against the human K562 and CEM cell lines, which was comparable to human NK activity. Depletion of baboon CD16+ or CD8+ cells led to dramatic decreases in NK cytotoxicity, and removal of both subsets completely abrogated NK activity. Incubation of baboon lymphocytes with human recombinant IL-2 for 1 week led to the appearance of CD56+ cells (11.3+/-2.8%). Most of the baboon CD56+ cells induced in culture were in S and G2 phases of cell cycle. Both baboon and human IL-2-activated lymphocytes were highly cytotoxic against the human LAK-sensitive cell line Daudi. Depletion of baboon CD8+ but not CD56+ cells significantly decreased LAK activity. These studies revealed differences in the NK system of humans and baboons that should be taken into consideration when analyzing immune responses to allo- and xenotransplantation in baboons.

Platelet surface activation antigen expression at baseline and during elective angioplasty in patients with mild to moderate coronary artery disease.

Platelet activation is an important pre-thrombotic event. The elucidation of its pathophysiology could contribute to a reduction in the mortality associated with coronary artery disease-the foremost cause of death in the UK. We examined the platelets of 27 patients with angiographically documented coronary artery disease. All patients had stable angina and were taking their regular medication-including aspirin. We demonstrated significantly increased expression of GP53 and activated GPIIb/IIIa on the platelet surface using a sensitive flow cytometric method of detection. Comparison was made with a control group of 35 patients. Seventeen of the patients had coronary angioplasty carried out. Serial studies of these patients demonstrate an immediate and sustained increase in platelet activation and this has important implications for prevention of restenosis after angioplasty.

Class I-like CD1A-C do not protect target cells from NK-mediated cytolysis.

Previous studies have validated the capacity of classical (HLA-A,B,C) and nonclassical (HLA-G) MHC class I antigens expressed by target cells to influence natural killer (NK) cell-mediated cytolysis. Generally, elevated expression of these HLA class I molecules is correlated with enhanced resistance to lysis by NK cells. In this study, we have evaluated the effect of transfected class I-like CD1A, CD1B, and CD1C molecules on C1R (HLA-A,B null) target cell sensitivity to natural killing. We report that while each of these molecules was expressed at physiologically relevant levels on the cell surface of clonal transfectants, no change was observed in target cell susceptibility to fresh or lymphokine-activated NK-mediated lysis.

Human natural killer cells induce morphologic changes in porcine endothelial cell monolayers.

In this study, we have investigated the early in vitro effects of natural killer (NK) cells on porcine aortic endothelial cell (PAEC) monolayers. Incubation of effector cells containing about 70% CD56+ cells on PAEC monolayer led to time-dependent changes in PAEC monolayer morphology. As little as 20 min of incubation resulted in changes in PAEC shape and in the appearance of gaps between the cells. These effects have been observed for up to 6 hr, but not before 20 min or after 6 hr. When NK-depleted effector cells were used, no morphological changes were observed in comparison with the same effectors before depletion; if CD56+ cells were added back, the effects were comparable with those on nondepleted effector cells. There was no detectable NK cell-mediated cytolytic activity during the 1-6 hr of incubation of peripheral blood lymphocytes with PAEC monolayers. These data indicate that NK cells may participate in endothelial cell changes leading to xenograft rejection.

Heat treatment of leukemic cell lines can increase their sensitivity to NK lysis.

The effect of stress on target cell susceptibility to human natural killer cell-mediated lysis was examined. Targets were incubated at 37, 42, or 45 degrees C for 1 hr and then tested for NK sensitivity in chromium-release assays. The T cell target JURKAT displayed minor increases in susceptibility to NK lysis with 42 degrees C pretreatment (20-50% increases) and dramatic increases in lysability with 45 degrees C pretreatment (100-300% increase) compared to control. In contrast, lysis of the NK prototypic target K562 is not increased after 42 or 45 degrees C pretreatment. Kinetic studies indicated an optimal NK sensitivity enhancement time of 1 hr at 45 degrees C for JURKAT. Inhibition of target cell protein synthesis by emetine pretreatment does not produce an increase in susceptibility to NK lysis. JURKAT cells pretreated with sodium arsenite exhibited a comparable increase in NK sensitivity to the heat treatments. Cold target inhibition assays suggest that the increase in sensitivity after heat treatment is at a postbinding stage. This was exemplified by the increased sensitivity of JURKAT, but not K562, to lysis mediated by isolated rat NK granules. These results indicate that a heat-sensitive, de novo protein synthesis-independent defense mechanism against lysis may exist in some tumors, altering their susceptibility to lysis by NK cells.

MHC class I expression and transport in a calnexin-deficient cell line.

The human leukemic cell line, CEM, and an NK-resistant variant of CEM, called CEM-NKR, were analyzed for protein differences by two-dimensional gel electrophoresis. One protein was found to be completely absent in CEM-NKR. This protein has been identified as calnexin. CEM-NKR also completely lacks calnexin RNA. Calnexin is thought to act as a molecular chaperone by assisting in the assembly and/or retention of MHC class I and many other membrane and secreted proteins. The surface expression of class I molecules on CEM-NKR was compared with CEM by several Abs. There was no significant class I expression differences between CEM and CEM-NKR using w6/32 (a conformational and beta 2-microglobulin-dependent mAb), HC-10 (a conformational and beta 2-microglobulin-independent mAb), and an anti-class I antiserum that reacts with native and denatured class I. The transport rate of class I in both cell lines was examined by pulse-chase experiments, immunoprecipitating class I with w6/32 and anti-class I antiserum. The results show that class I molecules in the calnexin-deficient cell line and its parent cell line are transported at similar rates. These results indicate that calnexin is not absolutely required for the viability of CEM or the transport and surface expression of human MHC class I molecules.

Clustering of fibronectin adhesins toward Treponema denticola tips upon contact with immobilized fibronectin.

Treponema denticola has been shown to bind to immobilized fibronectin (Fn) by its tips. Yet labeling of cells in suspension with an Fn-gold conjugate to localize the Fn adhesins shows that they are distributed in patches along the entire cell length. Subsequent experiments have shown that the number and proportion of tip-oriented cells increase with time, suggesting that Fn contact stimulates T. denticola to rearrange adhesins toward its tips. To test this hypothesis, T. denticola cells were allowed to migrate in a 2% methylcellulose column toward nitrocellulose filters coated with Fn, laminin, bovine serum albumin, or phosphate-buffered saline. Cells close to and distant from the filters were collected, labeled with Fn-gold probes, and examined by transmission electron microscopy. The number of gold particles on each of 20 cells was counted, dividing each cell into thirds along its length: the end third with the most label (end 1), the middle third, and the end with the least label (end 2). The mean number (+/- standard deviation) of gold probes per third was calculated. Fn-gold probes clustered toward one end of T. denticola cells when in contact with Fn-coated nitrocellulose, with > 55% of probes in end 1. In contrast, no clustering toward T. denticola ends occurred with laminin-, bovine serum albumin, or phosphate-buffered saline-coated filters or in the absence of a filter. Blocking access of the T. denticola cells to the Fn-coated nitrocellulose filter by placing an uncoated filter between them prevented clustering of Fn-gold. Removal of T. denticola cells from direct contact with the Fn-coated filter did not promote redistribution of clustered probes. These data suggest that T. denticola is stimulated to cluster Fn adhesins irreversibly toward its tips when it migrates into contact with immobilized Fn. This might be significant for establishing multiple adhesive interactions with host cells and ligands.