Regulation of tissue ingrowth into proteolytically degradable hydrogels.

Regulation of the rate of cell ingrowth into and within a matrix is desirable for efficient tissue regeneration. Polyethylene glycol hydrogels crosslinked with matrix metalloproteinase (MMP) susceptible peptide sequences permit cell-controlled invasion. In this study, hydrogels of the same stiffness polymerised using different ratios of a readily degradable MMP peptide sequence (PAN-MMP) and a MMP peptide with a limited degradation capacity (MMP-9) were assessed both in vitro and in vivo for cellular invasion. The degree of invasion into the various hydrogels was found to be tightly linked to the relative proportion of each peptide both in vitro and in vivo. Furthermore a good correlation between in vitro and in vivo ingrowth was observed. These findings demonstrate a highly tunable model for regulating cellular invasion which is readily translatable to in vivo models. This finding may allow for further optimisation of aspects of regenerative scaffolds such as tissue invasion, growth factor release and cellular encapsulation. STATEMENT OF SIGNIFICANCE: Degradable hydrogels are used in a wide range of tissue regeneration approaches. A particularly advantageous variant of these hydrogels is where due to peptide based crosslinking of the polymeric hydrogels, cell invasion rate is dependent on cellular enzymatic activity. This present study demonstrates a further refinement whereby both cellular and tissue invasion rates are finely regulated through the polymerisation of a hydrogel with varying combinations of enzymatically degradable peptides. Importantly this allows for invasion rates to be controlled without altering the biomechanical properties of the hydrogel such as stiffness. The latter can further influence cellular behaviour thus potentially interfering with the desired outcome.

In vitro and in vivo characterisation of anti-murine IL-13 antibodies recognising distinct functional epitopes.

Interleukin-13 (IL-13) sequentially binds to IL-13Ralpha1 and IL-4Ralpha forming a high affinity signalling complex. This receptor complex is expressed on multiple cell types in the airway and signals through signal transducer and activator of transcription factor-6 (STAT-6) to stimulate the production of chemokines, cytokines and mucus. Antibodies have been generated, using the UCB Selected Lymphocyte Antibody Method (UCB SLAM), that block either binding of murine IL-13 (mIL-13) to mIL-13Ralpha1 and mIL-13Ralpha2, or block recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. Monoclonal antibody (mAb) A was shown to bind to mIL-13 with high affinity (K(D) 11 pM) and prevent binding of mIL-13 to mIL-13Ralpha1. MAb B, that also bound mIL-13 with high affinity (K(D) 8 pM), was shown to prevent recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. In vitro, mAbs A and B similarly neutralised mIL-13-stimulated STAT-6 activation and TF-1 cell proliferation. In vivo, mAbs A and B demonstrated equipotent, dose-dependent inhibition of eotaxin generation in mice stimulated by intraperitoneal administration of recombinant mIL-13. In an allergic lung inflammation model in mice, mAbs A and B equipotently inhibited muc5ac mucin mRNA upregulation in lung tissue measured two days after intranasal allergen challenge. These data support the design of therapeutics for the treatment of allergic airway disease that inhibits assembly of the high affinity IL-13 receptor signalling complex, by blocking the binding of IL-13 to IL-13Ralpha1 and IL-13Ralpha2, or the subsequent recruitment of IL-4Ralpha.

Roles for the interleukin-4 receptor and associated JAK/STAT proteins in human articular chondrocyte mechanotransduction.

OBJECTIVE: To identify functional interleukin-4 (IL4) receptor (IL4R) subtypes and associated Janus kinase/signal transducers and activators of transcription (JAK/STAT) molecules in human articular chondrocytes and assess the role of JAK/STAT proteins in chondrocyte mechanotransduction. METHODS: Expression of IL4R subunits and associated molecules was assessed by immunohistochemistry and western blotting. Functional IL4R were identified by chemical crosslinking of IL4-stimulated chondrocytes and western blotting. JAK and STAT phosphorylation was assessed by western blotting. RESULTS: Chondrocytes from normal and osteoarthritic (OA) cartilage express IL4Ralpha, gammac and IL13Ralpha1 subunits (components of the Type I and Type II IL4R). In the presence of IL4 only functional Type II IL4Rs were identified in normal or OA chondrocytes. With the exception of STAT2, no differences in JAK/STAT expression were detected between normal and OA cartilage. STAT2 was expressed in OA but not normal chondrocytes. Mechanical stimulation (MS) resulted in an IL4R-dependent increase in phosphorylated Tyk2 in normal chondrocytes, which could be abolished by IL1beta preincubation. No phosphorylation of STAT5 or STAT6 was detected in either normal or OA chondrocytes following mechanical stimulation (MS) IL4 stimulation resulted in a decrease in Tyk2 phosphorylation and an increase in phosphorylation of STAT6 in both normal and OA chondrocytes. CONCLUSION: Chondrocytes from normal and OA cartilage signal through a Type II IL4R. This signalling is via a STAT6-independent pathway. Differences in IL4 signalling are likely due to crosstalk between integrin and cytokine signalling pathways, and not differences in IL4R expression.

Reconstructed skin kits: reproducibility of cutaneous irritancy testing.

The aim of this study was to determine the reproducibility of data obtained from in vitro irritation testing using three industrial reconstructed human epidermis models, EpiDerm, Episkin and SkinEthic, and one in-house model developed at Wella/Cosmital. A common protocol was established based on the measurement of cytotoxicity in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and of extracellular release of proinflammatory mediators and cytosolic enzymes after a range of exposure times to sodium lauryl sulfate (SLS). This time course protocol was applied to 6 different batches of each skin model using triplicate tissue cultures per test condition. The parameters analyzed for intra- and inter-batch reproducibility were the cell viability determined as MTT reduction capacity and the ET-50 values in the 6 batches, as well as the release of the cytokine IL-1alpha and of the cellular enzymes LDH and GOT in 3 batches only. The MTT viability results showed that EpiDerm was the most resistant to the SLS treatment and at the same time the most reproducible model, SkinEthic was the most sensitive to SLS and the least reproducible, and Episkin and the Cosmital model were intermediate. Measurements of IL-1alpha release showed a relatively high intra- and inter-batch variability in all the skin models. It was not possible to detect the extracellular release of the enzymes LDH and GOT in the Episkin assay medium. With the 3 other models, the release of LDH and GOT varied in about the same range as that of IL-1alpha. For all the parameters in this study, the inter-batch variability was generally greater than the intra-batch variability. A possible reduction in the number of batches and replicates for future applications in routine irritancy testing is discussed on the basis of the results obtained using 6 batches in triplicate.

Predictive ability of reconstructed human epidermis equivalents for the assessment of skin irritation of cosmetics.

The aim of this study was to examine the concordance between human in vivo and in vitro skin irritation classifications of cosmetic products and to evaluate the correlations between the different parameters. For that purpose, 22 formulations from product development test series, covering the full range of in vivo scores and representing different cosmetic product classes, were tested in vivo (modified Frosch-Kligman Soap Chamber Patch Test with repetitive occlusive application) and in vitro using two epidermis equivalents commercially available as kits (EpiDerm and EPISKIN) and one in-house model (Cosmital). In vivo, skin reactions (erythema, dryness and fissures) were visually evaluated and, in addition, skin redness and transepidermal water loss (TEWL) were measured by means of technical instruments. The parameters measured in vitro were the percent cell viability in the MTT reduction assay, with ET(50) determination, and the extracellular release of the pro-inflammatory mediator IL-1alpha and of the cytosolic enzyme lactate dehydrogenase (LDH), into the culture medium collected after topical application of the products for different exposure times (time-course assay). In general, good Spearman rank correlations could be observed between the different in vivo parameters (with the exception of TEWL and dryness at day 2). Furthermore, high correlation coefficients were obtained by comparing the different in vitro parameters (except for LDH release) and different models, which allowed to conclude that the results obtained with the different reconstructed epidermis models were very similar. A comparison between in vivo and in vitro parameters resulted in the best rank correlation for ET(50), then in decreasing order, for the percent MTT viability at 16 h, the IL-1alpha release and finally, for LDH release, where the correlation was generally low. A direct comparison of the mean total scores (sum of erythema, dryness and fissures at day 5) of the 22 products with the best predictor, ET(50) obtained with the three reconstructed epidermis models, using simple linear regression analysis resulted in a coefficient of correlation R=0.94 for EpiDerm, R=0.90 for Cosmital and R=0.84 for EPISKIN. Multivariate descriptive statistics showed that the in vitro parameters, MTT viability evaluated after the 16-h exposure and ET(50), as well as the in vivo parameters, sum of visual scores at day 5 and chromameter value, were the best endpoints to discriminate between irritant and non-irritant products. Using the in vivo mean total scores at day 5 with a cut-off value at 2 and the in vitro percent MTT viability after the 16-h exposure with a cut-off value at 50% to classify the products, the same two-by-two contingency table was obtained for all the three reconstructed epidermis models with sensitivity=92%, specificity=100% and observed concordance=95% (kappa=0.91; 95% confidence interval 0.74-1.08). This classification system was a satisfactory and relevant approach to discriminate the "irritant" from the "non-irritant" cosmetic products in this study. In conclusion, this study demonstrated the usefulness of reconstructed human epidermis equivalents for the in vitro assessment of the irritation potential of a series of cosmetic products. These models allow the measurement of quantifiable and objective endpoints relevant to in vivo irritative phenomena.

Mitigation of bioprosthetic heart valve degeneration through biocompatibility: in vitro versus spontaneous endothelialization.

BACKGROUND: Glutaraldehyde-related cytotoxicity and transanastomotic ingrowth inhibition prevent the spontaneous endothelialization of bioprosthetic heart valves. In order to evaluate the ability of improved biocompatibility to reduce tissue degeneration, conventionally fixed aortic root prostheses were both glutaraldehyde-detoxified and in vitro endothelialized. METHODS: Entire aortic roots were fixed in 0.2% glutaraldehyde (GA) (control group) and either detoxified in acetic acid-buffered urazole (0.1 M) or detoxified and in vitro lined with cultured, autologousjugular vein endothelial cells. The valved roots were inserted in the distal aortic arch of 15 juvenile Merino sheep for a period of 12 weeks. Upon explant, leaflets, sinuses and aortic wall of the prostheses were analysed by SEM to assess the surface endothelium, histologically regarding tissue inflammation, and by atomic absorption spectrophotometry to determine the content of tissue calcium. RESULTS: There was no endothelium on control grafts, except for a short anastomotic pannus. The detoxified group showed an incomplete patchy endothelium on the aortic wall but hardly any on the leaflets, whereas, the in vitro lined group had aortic wall, sinuses and most of the leaflets confluently endothelialized. Tissue inflammation was prominent in the control group and least expressed in the endothelialized group (p < 0.05). Detoxification significantly reduced leaflet calcification. In the aortic wall, both detoxification and endothelial lining were required to significantly mitigate calcification. CONCLUSION: In the 12 week circulatory sheep model, the calcium mitigating effect of detoxification was more pronounced than that of in vitro endothelialization. Nevertheless, there was a distinct overall benefit if detoxification was combined with endothelialization.

The anticalcific effect of glutaraldehyde detoxification on bioprosthetic aortic wall tissue in the sheep model.

BACKGROUND: Increasing concentrations of glutaraldehyde (GA) lead to a decreased rather than increased calcification of bioprosthetic aortic wall tissue. This study determined to what extent the benefit of better cross-linking is masked by the intrinsic propensity of GA towards calcification. MATERIALS AND METHODS: Porcine aortic roots were immediately fixed at the abattoir at three different concentrations of GA (0.2%, 1.0%, and 3.0% for 1 week at 4 degrees C). Subsequently, roots underwent a GA extraction process using high volumes of Urazole solution (acetic acid buffer, pH 4.5, 37 degrees C, 1 week) followed by NaBH4 reduction (2 days, 37 degrees C). Roots were implanted in the distal aortic arch of young sheep for 6 weeks and 6 months. Calcium analysis was quantitatively done by atomic absorption spectrophotometry and qualitatively assessed by light microscopy on Von Kossa stains. RESULTS: There was a distinct anticalcification effect of GA detoxification after 6 weeks (56.8% to 97.9%; 95% confidence interval [CI]), which stabilized on a more moderate level after 6 months of implantation (19.1% to 31.6%; 95% CI). The most pronounced effect of GA extraction was seen in 0.2% fixed tissue, where aortic wall calcification was mitigated by 97% and 32% after 6 weeks and 6 months, respectively. Mitigation of aortic wall calcification was 71% (6 weeks) and 21% (6 months) in the 3.0% GA group. The combined effect of higher cross-link density and detoxification achieved an 82% (6 weeks) and 48% (6 months) reduction of calcium levels in the 3.0% GA group. In long-term implants (6 months), detoxification alone on top of standard 0.2% GA fixation was as effective (from 174.1 +/- 11.9 microg/mg without detoxification to 119.3 +/- 19.3 microg/mg with detoxification) as 3.0% fixation (114.8 +/- 10.0 microg/mg without detoxification to 91.3 +/- 11.5 microg/mg with detoxification). CONCLUSION: We were able to determine in the circulatory sheep model to what degree the intrinsic procalcific effect of GA counteracts the protective effect of higher cross-link density. Our study also established that the effect of detoxification is particularly pronounced in commercial low-grade fixation.

Matrix metalloproteinases and tissue valve degeneration.

Bioprosthetic heart valves have been used as replacements for diseased heart valves for over 30 years. More than 50% of bioprosthetic valves fail within 15 years because of structural deterioration. The role of proteolytic degradation, with particular reference to the matrix metalloproteinases (MMPs) in the degeneration of aortic bioprostheses, is appraised in this minireview. It is clear that both the intrinsic and host-derived proteolytic activities present in heart-valve bioprostheses may combine with mechanical stress to bring about valve failure.

Tumour necrosis factor levels during acute rejection and acute tubular necrosis in renal transplant recipients.

Plasma tumour necrosis factor levels were measured serially in 16 patients following renal transplantation, and in 10 patients on haemodialysis and in 12 patients on peritoneal dialysis. The patients on peritoneal dialysis had lower plasma TNF levels than the patients on haemodialysis. There was a decrease in TNF levels immediately following renal transplantation; this is probably related to the bolus doses of methylprednisolone administered intra-operatively. Patients with acute rejection had higher levels of TNF than non-rejecting patients. The increase in TNF levels in rejecting patients was observed 2 days before the clinical manifestation of acute rejection. There was a marked decrease in TNF levels in rejecting patients in response to treatment with steroids. Patients with delayed graft function had higher levels of TNF on the first post-operative day compared to patients with immediate function. These changes in plasma TNF levels following renal transplantation have important clinical and therapeutic implications.

Explaining trends in teenage childbearing in Sweden.

The teenage fertility rate fell precipitately in Sweden after 1966 and is now one of the lowest in Europe. This decline can be seen in the context of major reforms enacted in 1975 whereby the school sex-education curriculum was revised, contraceptive services were improved, and abortion was provided free and on demand. By means of microsimulation, the possible roles of contraception and induced abortion in causing teenage fertility to fall are examined. Before 1975, the decline appears to have been caused primarily by an increase in the number of induced abortions. After that date, however, an increase in the use of highly efficient methods of contraception led to a decline in the pregnancy rate in such a way that, even though the proportion of teenagers who sought abortion increased, the abortion rate declined. Parallels are drawn with the experience of other European countries, and contrasts with that of the United States, where no such developments have occurred, are noted.

PIP: This study examines the role of contraception and induced abortion in causing teenage fertility to fall in Sweden. The data used were drawn from the 1992 Swedish Family Survey, which sought event-history data from more than 3000 women born variously in 1949, 1954, 1956, 1964, and 1969. From these studies, it was found out that in the middle of 1970s, three interrelated changes occurred. First, the school sex education curriculum was revised in 1975; it no longer recommended abstinence among the youth, nor did it emphasize that sexual activity should take place only within marriage. Second, the abortion law was revised in 1975 to allow abortion on demand without charge. The pervasive fear was that relaxation of the law would lead to abortion's being used as a substitute for contraception. Third, the revision of the school sex-education curriculum, therefore, was accompanied by explicit contraceptive education, and special youth clinics were instituted to provide free contraceptives to young people. Due to the major reforms enacted in 1975, pregnancy rate declined primarily by an increase in the number of induced abortion. But after 1975, the decline was due to increased use of highly efficient contraceptive methods, which also caused the decline in the abortion rate.

Performance of the Neutral Red uptake assay in the COLIPA international validation study on alternatives to the rabbit eye irritation test.

The neutral red uptake (NRU) assay was included as part of the COLIPA international validation trial of in vitro alternatives to the Draize eye irritation test. In a blind trial, 55 substances were tested at four laboratories. Following testing, a prediction of the in vivo Draize modified maximum average score (MMAS) for each substance was made by each laboratory using a prediction model relating mean NR(50) value (concentration causing 50% reduction in NRU from that of untreated control cells) to MMAS. Following statistical analysis of the results and breaking of the code, assessment of the results and further analysis was carried out by the participating laboratories. This paper presents the conclusions with regard to the NRU assay. The initial trial analysis indicated that the interlaboratory reproducibility of results of the NRU assay was good. However, there was a poor correlation between observed and predicted MMAS (using the proposed prediction model) when all the test substances were analysed together (r=0.246). Data analysis of subsets of substances indicated that the best predictions were for pure surfactants only (r=0.843) although this data did not fit within the limits of the prediction model. The NRU assay therefore appears to have limited use as a complete Draize replacement. A further examination of the COLIPA trial data may identify combinations of assays which may be more useful than the individual assays which, like NRU, have been shown to be poor predictors of eye irritation.

COLIPA validation project on in vitro eye irritation tests for cosmetic ingredients and finished products (phase I): the red blood cell test for the estimation of acute eye irritation potentials. Present status.

The red blood cell test (RBC test) is part of the COLIPA Validation Project on Alternatives to Draize Eye Irritation. It shows good intra- and interlaboratory reproducibility (reliability) and represents one of the promising in vitro alternatives of this project with a good fit to prediction models (relevance) for the assessment of acute ocular irritancy caused by certain classes of chemicals (mainly surfactants) and formulations. Results obtained during the period of test development, prevalidation, and validation are summarized. The method is based on that of Pape et al. (1987), Pape and Hoppe (1990) and Lewis et al. (1993). The protocol has two endpoints: cellular lysis and changes in protein conformation which can be correlated with initial events in tissue injury inducing inflammatory responses as assessed by Draize eye irritation scoring. Both endpoints are detected by spectrophotometric changes in the haemoglobin absorption at 541nm. The protocol also includes a set of prediction models (PM). One PM is designed to predict three classes of irritancy (classification model) based on both endpoints and the three other PMs are designed to predict modified maximum average scores (MMAS) by algorithms based on data from cellular lysis only. These three PMs [with prediction intervals (PIs)] are: (i) for surfactant ingredients, (ii) for surfactant containing finished products, and (iii) for both groups together. The three PMs are based on a common algorithm derived from historic data. It is shown that PMs derived from historic data from several laboratories, by the same procedure, also produce a good fit with the presented data. Therefore, participating laboratories concluded that the protocol as used in this formal validation study can be considered to be validated for the estimation of acute eye irritation potential of surfactant-containing finished products.

The HET-CAM, a Useful In Vitro Assay for Assessing the Eye Irritation Properties of Cosmetic Formulations and Ingredients.

One of the most important biological properties of consumer products, and also of many raw materials, is the local compatibility to mucous membranes. Until now standardized in vivo tests are accepted by public health authorities as valid to estimate the irritation potential of chemicals and suitable for the risk assessment. Nevertheless, the controversial discussion on animal tests, and particularly on the Draize rabbit eye test, is increasing in the public and scientific domain. Efforts have been made to validate proper and suitable in vitro tests in international cosmetics industries during the last decade. One of the most important in vitro tests is the HET-CAM, the h en's e gg t est on the c horioa llantoic m embrane of fertilized chicken eggs. In this paper, the efforts to establish the HET-CAM protocol and the defined prediction model (PM) used in the COLIPA (The European Cosmetic, Toiletry and Perfumery Association) study on alternatives to the Draize rabbit eye test are described. Furthermore, the HET-CAM test results of the finalized phase I of the above-mentioned study are discussed in detail. Prior to the COLIPA validation study, the HET-CAM was prevalidated with about 100 test substances covering a broad spectrum of chemical structures and physical appearances and representing the range of chemicals in the cosmetics industry. This prevalidation was performed with a stringent in-house agreement in one company to test each chemical in the HET-CAM before any requested animal test was done. There was a high concordance of the HET-CAM results with in vivo data of the Draize test, especially for slightly irritating test articles. Based on these promising data, the HET-CAM protocol was taken as the final standard operating procedure (SOP) in the international COLIPA validation study, testing 55 coded chemicals in four different laboratories. The HET-CAM has been established and proven to be a robust test with a good prediction of irritation potential. According to strict associations of well-defined irritation categories (in vivo and in vitro), and with the concrete PM, the in vivo irritation potential of 29 out of 55 test articles (about 52%) were correctly predicted with the HET-CAM in at least three laboratories. This quality of prediction was of different success in the four categories of irritation severity. 90% of the slightly irritating chemicals but only 53% of the severely irritating articles were correctly predicted. The necessity to define a "gold standard" for validation purposes and the conflict with heterogeneous in vivo data were also pronounced this article. Here it is discussed, whether the evaluation of such heterogeneous responses and especially of persistent slight effects on the cornea can be done properly with additional data such as physicochemical data and biological information of the test substance.

High glutaraldehyde concentrations reduce rather than increase the calcification of aortic wall tissue.

BACKGROUND AND AIMS OF THE STUDY: This study was performed in order to: (i) determine whether a similar reduction of tissue calcification as seen after prolonged storage can be achieved through higher concentrations of glutaraldehyde (GA); and (ii) verify that well-preserved tissue integrity can suppress calcification. METHODS: Before fixation in 0.2% GA (PBS, 4 degrees C, seven days) porcine aortas were kept on ice for 48 h. Alternatively, tissue was immediately fixed at the abattoir in 0.2%, 1.0% or 3% glutaraldehyde (PBS, 4 degrees C, seven days). A second group of immediately fixed tissue (0.2%, 1.0%, 3.0% GA) (PBS, 4 degrees C, two days) had an interim step of L-lysine treatment (0.1M, 37 degrees C, acetic acid buffer, two days) in order to enhance cross-linking followed by warm-temperature fixation (PBS, 37 degrees C, five days). Two animal models were compared: subcutaneous implantation in rats (12 weeks) and vascular implantation in non-human primates, Chacma baboons (six weeks). RESULTS: In both animal models the highest level of calcification was found in the group with delayed fixation in 0.2% GA. In the rat model there was an inverse correlation between tissue calcification and the GA concentration used, with 3% GA-fixed tissue showing the lowest level of tissue calcium. Overall, increasing GA concentration had a significant benefit on calcification (p < 0.0001; two-factor analysis of variance). Enhancement of cross-linking with L-lysine further abrogated tissue calcium levels at all GA concentrations (p < 0.0001; two- factor analysis of variance). Although the short-term baboon model showed lower tissue calcium levels, the trend seen in the rat model was confirmed. CONCLUSIONS: Our results demonstrate the detrimental effect of delayed fixation and further suggest that, against previous beliefs, fixation at higher glutaraldehyde concentrations reduces the calcification tendency of cross-linked aortic tissue.

A summary report of the COLIPA international validation study on alternatives to the draize rabbit eye irritation test.

The principal goal of this study was to determine whether the results from a set of selected currently available alternative methods as used by cosmetics companies are valid for predicting the eye irritation potential of cosmetics formulations and ingredients and, as a consequence, could be valid replacements for the Draize eye irritation test. For the first time in a validation study, prediction models (PMs) that convert the in vitro data from an assay to a prediction of eye irritation were developed for each alternative method before the study began. The PM is an unequivocal description of the relationship between the in vitro and the in vivo data and allows an objective assessment of the reliability and relevance of the alternative methods. In this study, 10 alternative methods were evaluated using 55 test substances selected as representative of substances commonly used in the cosmetics industry (23 ingredients and 32 formulations). Twenty of the single ingredients were common to the European Commission/British Home Office (EC/HO) eye irritation validation study (Balls et al., 1995b). The test substances were coded and supplied to the participating laboratories. The results were collected centrally and analysed independently, using statistical methods that had been agreed before the testing phase began. Each alternative method was then evaluated for reliability and relevance in assessing eye irritation potential. Using the criteria of both reliability and relevance as defined in the study, the preliminary results indicate that none of the alternative methods evaluated could be confirmed as a valid replacement for the Draize eye irritation test across the full irritation scale. However, three alternative methods-the fluorescein leakage test, the red blood cell assay (classification model) and the tissue equivalent assay-each satisfied one criterion of reliability or relevance. Further investigation of the decoded data from this study to explore more fully the relationship between the in vitro data and the in vivo data is recommended. Such a review may allow the development of new prediction models to be tested in a subsequent validation study.

Cytoskeletal actin: the influence of major burns on neutrophil structure and function.

It has been noted that major trauma and burns patients who survive beyond 48 h most frequently succumb to sepsis and multiple organ failure. Furthermore, such patients are usually markedly hypermetabolic and in negative nitrogen balance at the time of their demise. Along with many other systemic and immune dysfunctions, the polymorphonuclear white blood cells in this setting become functionally impaired. Given that the motile white blood cells contain significant proportions of the contractile protein, actin, we speculated that the leucocyte dysfunction might in part be related to the overall systemic catabolism of actin stores. Accordingly, this hypothesis was explored by comparing the functions and cytoskeletal structure of neutrophilic leucocytes from normal control adults and victims of fresh, major thermal injuries. On days 1 and 7 after a burn of > 25 per cent of total body surface area, peripheral blood was drawn from 10 patients (mean age 33 years, mean burn area 44.2 per cent), and seven unburned controls (mean age 35.2 years). Neutrophils isolated from these specimens were tested for stimulated chemotactic rate, efficacy of intracellular killing as determined by superoxide production rate, and the levels of soluble and insoluble intracellular actin. In addition, both light microscopy and scanning electron microscopy were used to visualize the actin cytoskeleton. The results indicated that both chemotactic rate (12 mu/min vs. 38 mu/min--P < 0.05) and superoxide production rate (9 vs 43 mumol/ml10E6 cells--P < 0.05), were significantly reduced in the burn patients by day 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of flushing of the liver graft upon plasma calcium and magnesium concentrations.

Previous studies of total and ionized calcium in the plasma of liver transplant recipients have been conducted in patients with preexisting liver disease or who received blood transfusion. The intraoperative decline in plasma total and ionized calcium has been attributed to the effects of liver disease and/or the citrate in transfused blood. The present study was conducted in normal porcine recipients of liver stored either with EuroCollins or University of Wisconsin (UW) solution for 6 hr, compared with livers flushed with Ringer's lactate without storage. No blood transfusion was given. Mean total plasma calcium levels declined significantly after storage with UW solution to a nadir approximately 65-70% of preoperative levels. This decline persisted for two to five days. Mean levels of plasma ionized calcium declined lowest after flushing with UW solution but only to 82% of preoperative (NS). There was an increase in plasma total magnesium in the recipients of livers flushed with EuroCollins or UW solutions, which resolved within 30 min and which was probably related to magnesium content of the flushing solution. It is concluded that while the changes in plasma total and ionized calcium are moderate and of little clinical significance, they could be aggravated under clinical conditions by massive blood transfusion. Changes in plasma magnesium seemed to be directly attributable to the magnesium content of flushing solutions but the same relationship did not exist for changes in plasma calcium.