Predictors of mortality in adults with cystic fibrosis.

Assessment of prognostic indicators in patients with cystic fibrosis (CF) is important. The study's aim was to assess the relative contribution of gender, genetics and microbiology on survival in adults with CF. Adult patients were studied from 1995 to 2005 and data collected included FEV(1) (%predicted), body mass index (BMI), genetics, and microbiology. Data was available on 183 patients in 1995. Forty-five patients died in the subsequent 10 years. Patients who died during the study had lower mean (SD) FEV(1) %predicted in 1995 when compared to those remaining alive, 41.5 (15.2)% versus 69.8 (23.2)% predicted, respectively, P<0.001 and they had lower mean (SD) BMI in 1995, 19.2 (3.3) kg/m(2) in comparison to those remaining alive, 20.7 (3.4) kg/m(2), P=0.008. The proportion of patients infected with Pseudomonas aeruginosa and Burkholderia cepacia complex was higher in the group who died during the study compared to those remaining alive, odds ratio 20.9 P<0.0001 and 7.1 P<0.0001, respectively. The presence of the Delta F508 homozygous mutation did not alter survival, P=0.3. Patients infected with either P.aeruginosa or B.cepacia complex had reduced survival compared to those without infection, P=0.01 and P<0.0001, respectively. FEV(1)% (P<0.0001), infection with P.aeruginosa (P=0.005) or B.cepacia complex (P=0.03) were the only significant predictors of mortality. This study demonstrates adults who died were more likely to have worse lung function and be infected with either P.aeruginosa or B.cepacia complex. FEV(1)% and infection with P.aeruginosa or B.cepacia complex were the most significant predictors of survival in adults with CF.

A modification of the Wu and Hoak method for the determination of platelet aggregates and platelet adhesion.

The Wu and Hoak method for determining circulating platelet aggregates has poor reproducibility; problems have been reported with the composition of the buffer systems, haemolysis, the effects of blood collection technique and a divergence of the platelet aggregate ratio in blood for healthy donors from the theoretical value of 1. Our investigations suggest that the original technique is highly operator-dependent, especially the collection of blood and the method of counting platelets after centrifugation. We describe an improved modification of the Wu and Hoak technique; a new buffer system has been developed and the proportion of blood in the buffered EDTA and buffered EDTA-formalin solutions has been altered to obtain platelet rich plasma. The platelet aggregate ratio (PAR) by this modified method for healthy donors in two different studies was 0.97 +/- 0.02 and 0.98 +/- 0.01 respectively. Finally, the principle of Wu and Hoak was used to measure accurately platelet adhesion, without the role of platelet-platelet interactions (aggregation). Platelet adhesion and aggregation were then used to evaluate the thrombogenicity of various artificial surfaces, including silicone rubber and polytetrafluoroethylene (PTFE) vascular grafts.

Effect of dialyser membranes on extracellular and intracellular granulocyte and monocyte activation in ex vivo pyrogen-free conditions.

This study examined effects of blood-contacting materials on the monocyte reaction following the first contact of human blood with hollow fibre dialyser membranes under pyrogen-free conditions. Membrane materials were the unchanged regenerated cellulose, the synthetic polysulphone (PS), a positively charged diethylaminoethyl cellulose (DEAE-C), the negatively charged carboxymethyl cellulose (CMC) and acrylonitrile copolymer (AN). The experimental system involved perfusion with human fresh venous blood through different modules containing the materials in the form of hollow fibre membranes. Extracellular and intracellular aspects of blood reactions after the first contact with the materials were investigated in Ficoll-separated granulocytes and peripheral blood mononuclear cells. Investigations were done by release reactions of platelet activating factor (PAF), oxygen radical (O2-), leukotriene B4, prostaglandin E2 (PGE2) and cytokines (IL-1 beta, TNF-alpha, IL-6). The intracellular activation of peripheral blood mononuclear cells was done by mRNA transcription of IL-1 beta, TNF-alpha, IL-6, IL-8 and beta 2-microglobulin (beta 2-MG). From the set of parameters, release reactions were only measurable for PAF, PGE2 and O2- if a second stimulus (phorbol myristate acetate, lipopolysaccharide, zymosan and calcium ionophore) was used after blood-membrane interaction. Although the extent of the release reaction was weak, negatively charged membranes were, in general, more active. All dialysers exhibited the same increase in beta 2-MG mRNA transcription, suggesting that all blood-contacting membranes initiate the gene expression of beta 2-MG at the same level. TNF-alpha, IL-6, IL-1 beta and IL-8 mRNAs were demonstrated in the AN and CMC membranes rather than the other materials, which exhibit a lower transcription than the tubing set. As has been found, an enhanced generation of PGE2 for both CMC and AN membranes supports, therefore, the concept of an effect of the negative charges of the materials in the gene expression of cytokines. However, this initiation does not lead to the generation of cytokines, even after stimulation with pyrogens.

Screening of in vitro cytotoxicity by the adhesive film test.

A novel procedure, the adhesive film test, is proposed as a screening method to predict potential cytotoxicity of biomaterials. This in vitro test utilizes a sterile strip of acrylate-based medical adhesive as an anchorage substrate in cytotoxicity studies. The adhesive film allows direct fixation of test samples to the base of the petri dish, ensuring close contact between sample and cells. The test is based on the principle that toxic components present in the test material will readily leach out into the culture medium and adversely affect the local cell population. The main advantage of the adhesive film test is that a viable cell population can be added directly to the test plate and after an incubation period of 24 h, the cellular response can be recorded as either cytotoxic or cytocompatible. Microscopic examination can be followed by quantifying the results using a micrometer to measure cellular attachment areas, migration distances and zones of inhibition. In addition, the adhesive film used to attach the test samples is shown to support extensive fibroblast growth and attachment to its surface and hence can also function as a negative nontoxic control in cytotoxicity studies.

The attachment and growth of an established cell line on collagen, chemically modified collagen, and collagen composite surfaces.

The attachment and growth of an established cell line derived from mouse fibroblasts on collagen, chemically modified collagen, and collagen composite surfaces were compared. Tissue culture polystyrene dishes provided a suitable control. The substrates included native bovine dermal collagen, succinylated, acetylated and methylated collagen, and a series of composite materials formed from collagen and the glycosaminoglycans hyaluronic acid, chondroitin 4-sulphate and chondroitin 6-sulphate and the glycoprotein fibronectin. Attachment and growth of cells on each of these substrates were assessed by visual inspection under optical microscopy, by detachment of the cells using trypsinization and subsequent counting in a Coulter counter; and by 3H-thymidine incorporation studies. A very good correlation between the results was obtained by the three methods employed which showed that collagen, in comparison to polystyrene, is a relatively poor substrate for cellular attachment, growth and proliferation, but it may be improved by chemical modification and by incorporation of either fibronectin, chondroitin sulphate (5 and 10%), or low levels (less than 5%) of hyaluronic acid into the collagen matrix. Concentrations in excess of 5% hyaluronic acid into the collagen matrix, however, appeared to inhibit cellular attachment and growth and such materials provided a poorer substrate than native collagen.

Biomaterials for blood-contacting applications.

Consideration of biomaterials for blood-contacting applications should take into account blood-biomaterial interactions, factors influencing the blood response and evaluation procedures. Examination of blood-biomaterial interactions indicates that relevant features are protein adsorption, platelet reactions, intrinsic coagulation, fibrinolytic activity, erythrocytes, leucocytes and complement activation. Factors influencing the blood response to a biomaterial in clinical application are the biomaterial structure, the presence of an antithrombotic agent, the patient status as determined by the disease and drug therapy, and the nature of the application. Evaluation options for biomaterials are clinical, in vivo, ex vivo and in vitro, with ex vivo and in vitro procedures relevant for biomaterial development.

Heparin binding and release properties of DEAE cellulose membranes.

Two cellulose-based membranes, containing 5 and 20% N, N-diethyl-aminoethyl cellulose (DEAE) respectively, were investigated for ionic attachment of heparin and heparin release. Heparinization was achieved by recirculation of a heparin-containing glycine buffer over each membrane. Heparin was determined by chemical analysis or by 99mTc labelling. The heparin uptake was shown to be linearly dependent on the heparin content in the contacting fluid. Heparin release was studied by contacting heparinized membranes with saline, glycine buffer, phosphate buffer and plasma. Incubation with plasma brought about the release of 50% of the attached heparin. Crosslinking of the heparinized membrane with glutaraldehyde reduced the heparin release by one half. The release reaction is more critical in the case of increased heparin uptake and a more efficient immobilization of heparin appears necessary.

In vivo evaluation and comparison of collagen, acetylated collagen and collagen/glycosaminoglycan composite films and sponges as candidate biomaterials.

Native collagen, acetylated collagen, collagen/10% chondroitin sulphate, collagen/2.5% hyaluronic acid and collagen/20% hyaluronic acid were implanted both as film and as sponge into rat lumbar muscle for 7 and 14 d. After 7 d implantation, all materials elicited an acute inflammatory cell response characterized by numerous polymorphs and histocytes. The cell population after 14 d was principally mononuclear, i.e. leucocytes, neutrophils, macrophages, lymphocytes and fibroblasts. Both films and sponges followed a similar pattern. Native collagen elicited a subacute inflammatory response after 7 d. However, 14 d after implantation, a marked infiltration by neutrophils was apparent with subsequent degradation of existing collagen material. Acetylated collagen film evoked a much greater inflammatory cell response than native collagen. Both collagen/hyaluronic acid composites elicited a similar response. The collagen/10% chondroitin sulphate composite elicited the least inflammatory cell response at 7 d, whereas infiltration by host fibroblasts after 14 d implantation was clearly seen.

The preclinical evaluation of the water vapour transmission rate through burn wound dressings.

The control of evaporative water loss, following burn injury, is of major importance to the overall condition of the patient, whether this control is by natural eschar or by a dressing. It is therefore important to preclinically determine the water vapour transmission rate of these dressings, firstly to make comparisons between different materials and secondly to screen prototype materials, under controlled conditions. A preclinical (in vitro) technique is described and the results are given for several commercially available dressings which encompass foam, film and hydrogel material categories.

An in vitro assessment of wound dressing conformability.

An in vitro assessment technique has been developed to determine the conformability of wound dressings. The technique employed is based on an inflation technique which provides a measurement of the minimum radius of curvature which a specific dressing will adopt under pressure. A pressure of 40 mmHg was chosen as this had been shown to be the maximum tolerable pressure before the occurrence of tissue breakdown. This radius is then matched to the natural radii of the body surfaces and an assessment of conformability can be made. A series of commercially available dressings have been assessed with respect to their conformability, and to the enhancement of their conformability due to viscoelastic creep behaviour.

Biocompatibility assessment: application of fluorescent probe response (FPR) technique.

An in vitro test procedure capable of discriminating effectively between intact and membrane-damaged cells has been developed. This procedure utilizes fluorescein diacetate and ethidium bromide as fluorescent probes. The properties of the probes and the collapse in the selective cytoplasmic membrane permeability barrier of the damaged cells ensure the principal feature of the test procedure, that functional cells fluoresce bright green, but membrane-damaged cells fluoresce bright red. Investigations with natural rubber, silicone and acrylic polymers confirmed the suitability of the procedure to distinguish between materials on the basis of cytotoxicity.

Blood interactions with novel polyurethaneurea hydrogels.

The influence on blood of polyurethaneurea hydrogels in vitro was investigated based on poly(ethylene oxide). A hydrogel was compared with the regenerated cellulose membrane Cuprophan in terms of complement activation, as determined by measurement of C3a concentration. The hydrogel induced less complement activation and the presence of poly(ethylene oxide) is likely to be beneficial to platelet reactivity. The ability to vary the polymer composition and the solubility of the polymers in organic solvents makes the polyurethaneurea hydrogels strong candidates for composite biomaterials.

Principles of burn dressings.

Throughout history burn wounds have been treated by covering with dressings of many different materials. The successful application of a burn dressing remains an objective for biomaterial development. This paper examines how the burn wound differs from other skin injuries, the requirements of the ideal burn wound dressing, and reviews the type of dressings available. The dressings in common use in the treatment of burns are compared with the 'ideal' dressing, in so far as it can be defined.

Utilization of the platelet release reaction in the blood compatibility assessment of polymers.

The release reaction is directly associated with platelet adhesion and aggregation, which are primary events leading to thrombus formation following contact of blood with artificial surfaces. This investigation examined the release reaction from the alpha granules of platelets after blood-polymer interaction, and utilized the measurement of beta-thromboglobulin (BTG), a platelet-specific protein, in the assessment of the in vitro blood compatibility of polymers. A radioimmunoassay was used, to determine the release of BTG following contact of blood with tubes of siliconized glass and polypropylene and flat sheets of poly(vinyl chloride) and silicone rubber. Polypropylene tubes caused less release of BTG than those of siliconized glass and silicone rubber induced less BTG release than poly(vinyl chloride). The investigation indicates a role for BTG measurement in blood compatibility assessment.

In vitro investigation of the blood response to medical grade PVC and the effect of heparin on the blood response.

This paper reports the results of an investigation into the blood response of polymers in vitro, using non-anticoagulated and heparinised blood and plasma. The materials studied were regenerated cellulose, (Cuprophan), an acrylonitrile-allyl sulphonate copolymer (AN69S), and medical grade polyvinyl chloride plasticised with di-2-ethyl-hexyl-phthalate (PVC/DEHP). Blood-material or plasma-material contact was achieved using a parallel plate flow cell, and C3a generation and FXII-like activity measured. The results of the study with non-anticoagulated human blood show that PVC/DEHP is a high complement activator. C3a concentration in the blood was higher after contact with PVC/DEHP than after contact with regenerated cellulose. The introduction of heparin in the blood induced complex alterations in the blood response. C3a generation could be elevated, decreased, or remain the same, depending on the material. The FXII-like activity on the surface of the PVC/DEHP after contact with plasma was also higher than the other two polymers. The introduction of heparin could increase or decrease FXII-like activity, depending on material. The patterns of response obtained with non-anticoagulated blood in vitro for AN69S and Cuprophan bore a strong resemblance with patterns of response obtained in the clinic, whereas those obtained with heparinised blood in vitro did not.

Measurement of platelet loss in the blood compatibility assessment of biomaterials.

In the assessment of the in vitro blood compatibility of biomaterials, platelet loss is often attributed solely to platelet adhesion and consideration is not given to platelets lost in platelet aggregate formation. In order to distinguish between those platelets lost to adhesion and those lost to aggregate formation, the Wu and Hoak method for the quantification of circulating platelet aggregates in patients has been modified to establish a new test procedure. This procedure, which measures both platelet adhesion (PA) in the absence of platelets lost to aggregate formation and also the tendency of a material to induce aggregate formation, has been used to evaluate the influence of a range of polyamides and a hydrogel. The evaluation demonstrated the ability of polymers to induce readily platelet aggregates during in vitro blood-material contact. The sensitivity of the aggregate measurement was exemplified by the polyamides, where PA was similar for materials of different porosity but platelet aggregate formation increased significantly with porosity. The importance of considering platelets lost to aggregate formation was emphasized with the hydrogel, where PA was low.

In vitro contact phase activation with haemodialysis membranes: role of pharmaceutical agents.

Contact phase activation was investigated in vitro using flat sheet type of haemodialysis membranes, Cuprophan (Akzo, Faser, Germany) and AN69S (Hospal, France), and a negatively charged polyamide Ultipor NR 14225 membrane as a control. The investigation focussed on the determination of factor XII-like activity (FXIIA) as an indicator of contact phase activation in the supernatant phase and at the membrane surface after plasma-membrane contact using an incubation test cell. The findings were compared with the observations from a plasma-free system utilizing purified unactivated factor XII. The plasma FXIIA bound to the membrane surface was significantly different between the membranes, while the supernatant phase FXIIA exhibited no significant differences. In contrast, the plasma-free system exhibited significant differences in the supernatant FXIIA and membrane-bound FXIIA for all the materials used and the magnitude of the activity was significantly greater for negatively charged materials. This finding demonstrated the strong influence of the interaction of other plasma constituents on the membrane surface and as such the binding and subsequent activation of factor XII may be altered possibly due to competitive binding and steric hindrance. On the addition of anticoagulants such as heparin, low-molecular-weight heparin, citrate and hirudin, no significant differences were observed in plasma supernatant phase FXIIA. However, each anticoagulant appears to have a distinct influence on the magnitude of plasma membrane-bound FXIIA. On the addition of aprotinin (a kallikrein inhibitor), no significant differences were observed in the plasma supernatant FXIIA. In contrast, aprotinin appears to significantly reduce membrane-bound FXIIA on Cuprophan and polyamide NR, but significantly increase the magnitude of the membrane-bound FXIIA on AN69S.

Endotoxemia, complement, and white blood cell activation in cardiac surgery: a randomized trial of laxatives and pulsatile perfusion.

Endotoxin activates complement and white blood cells and all are implicated in the pathologic effects of cardiopulmonary bypass (CPB). We investigated if reduction in intestinal bacterial load with a laxative and/or pulsatile perfusion to improve bowel circulation during CPB reduced endotoxemia and complement and white blood cell activation. Sixty patients were randomized to four groups in a 2 x 2 factorial structure: group 1 (no laxative, nonpulsatile perfusion); group 2 (laxative, nonpulsatile perfusion); group 3 (no laxative, pulsatile perfusion); and group 4 (laxative, pulsatile perfusion). Plasma concentrations of endotoxin, C3a and C5a, and granulocyte elastase (GE) were measured before anesthesia, skin incision, and heparin administration; during CPB (1, 30, 60, 90, and 120 minutes and after protamine administration); and after CPB at 3, 6, 12, 24, and 48 hours and 7 days. In all groups there was a small increase in the concentration of endotoxin (overall from 6 ng/L before CPB to 11 ng/L at 90 to 120 minutes; p < 0.001) and significant increases in C3a, C5a, and GE levels but no significant differences among the groups. Endotoxin levels did not correlate with activation of complement or white blood cells. There was a weak correlation between duration of CPB and levels of C3a (r = 0.14; p < 0.03) and GE (r = 0.25; p = 0.001) but not endotoxin or C5a. There was a general correlation between levels of C3a and GE but not in individual patients. In conclusion, CPB results in statistically significant increases in endotoxin, C3a, C5a, and GE during CPB.(ABSTRACT TRUNCATED AT 250 WORDS)

Opg, Rank, and Rankl in tooth development: co-ordination of odontogenesis and osteogenesis.

Osteoprotegerin (OPG), receptor activator of nuclear factor-kappaB (RANK), and RANK ligand (RANKL) are mediators of various cellular interactions, including bone metabolism. We analyzed expression of these three genes during murine odontogenesis from epithelial thickening to cytodifferentiation stages. Opg showed expression in the thickening and bud epithelium. Expression of Opg and Rank was observed in both the internal and the external enamel epithelium as well as in the dental papilla mesenchyme. Although Rankl expression was not detected in tooth epithelium or mesenchyme, it was expressed in pre-osteogenic mesenchymal cells close to developing tooth germs. All three genes were detected in developing dentary bone at P0. The addition of exogenous OPG to explant cultures of tooth primordia produced a delay in tooth development that resulted in reduced mineralization. We propose that the spatiotemporal expression of these molecules in early tooth and bone primordia cells has a role in co-ordinating bone and tooth development.

Cytokines and inflammatory mediators in cystic fibrosis.

Airway disease in cystic fibrosis (CF) is characterised by a continuous cycle of chronic infection and inflammation dominated by a neutrophilic infiltrate. This inflammation is characterised by an increased production of pro-inflammatory cytokines in the lung. The relationship between the abnormal CFTR gene product and the development of inflammation and progression of lung disease in CF is not fully understood. This review article studied the mechanisms of pulmonary inflammation in CF, the profiles of cytokines and inflammatory mediators in the lung in CF, the mechanisms that predispose to chronic Pseudomonas aeruginosa infection, cytokine involvement in diseases other than CF and reviewed current therapeutic strategies for CF. Imbalances of cytokine secretion are now better understood due to recent advances in understanding CF at a molecular level and it is increasingly thought that the normal inflammatory process is deranged in CF early in the course of the disease and may occur in the absence of detectable infection. However, the relationship between this unbalanced cytokine production, the mutations in CFTR and its actual consequence for pathogenesis need further investigation.