416

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

T. M. B. de Chalainl, M. Bracher’, W. Linley’, D. Gerneke’ and R. Hickman4 *Department of Plastic Surgery, Groote Schuur Hospital and University of Cape Town, ‘Surgical Research Laboratory, 3Electron Microscopy Unit and 4Division of Sureerv. University of Cape Town, South Africa

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 hypermetabo- lit and in negative nitrogen balance at the time of their demise. Along with many other systemic and immune dysfunctions, fhe polymor- phonuclear white blood cells in this setting become functionally impaired. Given that the motile white blood cells contain significanf proporfions of the contractile protein, actin, we speculated that the leucocyfe dysfunction might in part be relafed to the overall systemic catabolism of actin stores. Accordingly, this hypothesis was explored by comparing the functions ana’ cyfoskeletal structure of neutrophilic leucocytes from normal control adults ana’ victims of fresh, major thermal injuries. Ondays I and 7 after a burn of > 2~ 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 ana’ insoluble intracellular actin. In addition, both light microscopy and scanning electron microscopy were used to visualize the actin cytoskelefon. The results indicated that both chemotactic rate (12 p/min vs. 38plmin - P < 0.05) and superoxide production rate (9 us 43 ~mollmllOE6 cells - PC O.O5), were significantly reduced in the bum patients by day 7. Furthermore, the proportion of insoluble F-actin associated with the cytoskekton was also significantly reduced at 1 week postinjuy (324.7 vs. 769.8 - P < 0.07). while the total intracellular actin was also significantly less than control values at I weekpostinjuy (491.2 on day 7 vs. 13.25 for controls - P < 0.01). Specijic structural abnormalities of the cytoskel- etons of bum patients’ neutrophils could not be reliably characterized by either light or electron microscopy.

We therefore confirm that major thermal injury does indeed cause several forms of neutrophil dysfunction, the timing of which might be expected to contribute to the advent of sepsis. Furthermore, it appears that the numerically significant reduction in cyfoskeletal actin may be central to some of the observed neutrophil dysfunctions, although the mechanism by which this occurs is not yet entirely clear.

Bums (1994) 20, (5), 416-421

Introduction It has been noted that in patients who sustain major trauma, those surviving beyond 48 h most frequently die

0 1994 Butterworth-Heinemann Ltd 0305-4179/94/050416-06

of sepsis and multiple organ failure’. It has also been noted that such patients, especially those burned, become markedly hypermetabolic”. Despite aggressive nutritional support, protein synthesis is outstripped by breakdown and a state of net catabolism supervenes, with attendant loss of lean body mass3. Furthermore, along with numerous other systemic malfunctions, the polymor- phonuclear white cells in this clinical setting become functionally impaired, usually reaching a maximum in the second week after injury4. Given that the motile white blood cells contain a large proportion of the contractile protein actin, we speculated that the leucocyte dysfunction might be related to the catabolism of actin. Accordingly, we tested this hypothesis by comparing the cytoskeletal function and structure of neutrophilic leucocytes from normal volunteers and victims of major bums.

Materials and methods Clinical material Ten patients who were admitted to the Woodstock Hospital Bums Unit during the cohrse of 1992 were included in the study. Inclusion criteria included a fresh bum, greater than 25 per cent of total body surface area, and less than 12 hours old at the time of admission; age between I6 and 60 years; not pregnant, and no history or evidence of underlying disease such as neoplasia, diabetes mellitus or organ failure.

All patients were managed according to standard Bum Unit protocols by medical staff other than those carrying out the study. On days I and 7 postadmission, blood samples were taken for separation of neutrophilic leuco- cytes. These cells were then assessed in terms of structure and function, as described below.

For the purposes of valid comparison, a panel of healthy volunteers was used to obtain control data for all tested parameters. Full demographic data concerning the test and control groups are shown in Table 1.

Neutrophil superoxide production This test indirectly measures the ability of neutrophils to kill ingested bacteria. It assays the in vitro production, by neutrophils, of superoxide, a chemical intermediate in the formation of hydrogen peroxide, the agent which actually causes bacterial destruction. The test demands the isolation of neutrophils and their subsequent incubation with vari-

de Chalain et al.: Cytoskeletal actin 417

Table I. Clinical material

Age (vr) Sex %TBSA * Outcome

A. Patients 1. B.P. 2. F.W. 3. L.G. 4. O.M. 5. A.D. 6. A.N. 7. W.J. 8. M.W. 9. P.J.

10. P.M.

Means Range

B. Controls 1. M.B. 2. A.M. 3. H.B. 4. C.M. 5. SD. 6. A.J. 7. W.L.

Mean Range

32 M 40 26 M 50 28 F 60 28 M 30 48 M 29 38 F 28 34 M 50 52 17 30

33.3 17-52

44 41 32 32 28 29 41

35.2 28-44

M 45 M 45 M 65

2F/8M 44.2 28-65

2F/5M

Lived Lived Lived Died Lived Lived Lived Died Lived Lived

20 per cent Mortality

% TBSA, percentage total body surface area burned.

ous stimulants such as the tumour promoting agent phorbol myristate acetate (PMA), in the presence of cytochrome c. When superoxide is produced, cytochrome c is reduced, and this reduction can be followed and measured spectrophotometrically.

Cell preparation On days 1 and 7 postinjury and postadmission, 10ml of venous blood were drawn from each patient, directly into heparinized tubes. Cell fraction subsets in the whole blood sample were separated by centrifugation at 400g on a Ficoll-Isopaque density gradient for 3Omin at room temperature. The resultant mononuclear cell layer and plasma layer were aspirated and saved, leaving the polymorphonuclear cells plus red cells as a pellet. This pellet was then made up to 10 ml with phosphate-buffered saline (PBS). This suspension was then recentrifuged at 35Og for a further lOmin, and the supematant aspirated and discarded. The cell pellet was resuspended in I5 ml of PBS and the remaining red cells sedimented with 6 per cent Dextran. The supematant, containing neutrophils plus residual red cells, was aspir- ated, washed once and the cell pellet made up to 5 ml with PBS. To lyse the remaining red cells, 24 ml of water were added to the suspension and the tube mixed continuously for 30 s. Eight millilitres of 3.6 per cent saline were added and the cells washed once with PBS. The final cell pellet was resuspended in ice-cold PBS with glucose, at a concentration of 2 x lo6 cells/ml and the suspension kept on ice until used.

Measuring neutrophil superoxide production by dis- continuous assay For this assay, 12 by 100mm sili- conized glass tubes were required for each sample. The reagents required were:

?? Superoxide dismutase (SOD) (Sigma No. 2515; activi- ty = 3000 pg/mg protein) prepared as a 3 mg/ml solu-

tion in deionized water and kept at 4°C for a maximum of 5 days. Horse heart cytochrome c (Sigma Type VI C7752) prepared as a 3 1.4 mg/ml solution in PBS plus glucose, and kept at 4°C for a maximum of 5 days. Working PMA solution (Sigma P-8139: phorbol 12- myristate 13-acetate) prepared fresh daily. Fifteen microlitres of the stock solution (1 mg/ml ethanol) were added to 7.5 ml PBS plus glucose, and warmed to 37°C for each assay.

Assay procedure: for each patient, eight siliconized tubes (i.e. two rows of four tubes each) were labelled with the patient details plus the following information: Assay, Blank, 10min. Reagents were then added to the assay tubes as follows:

1. 500 cl1 of cell suspension: (1 X lo6 cells). 2. 50 ~1 cytochrome c. 3. 10 ~1 SOD (3 mg/ml) to rows 2 and 4. 4. 10 ~1 water to rows I and 3. The tubes were then mixed and incubated for 2 min at 32°C. 5. 500 ~1 PMA added to all tubes.

Once PMA had been added, the first row of four tubes was removed from the water-bath and placed on ice (t= Omin). The procedure was then repeated for the second row, at t = 10 min. At 10 min, all blank and IO-min tubes were centrifuged at 1500 rev./min for 20 min, at 4”C, to remove cells and particulate contaminants. The absorbances of the supematants were then measured at 550 nm using a double beam spectrophotometer (Hitachi U~OOO), with blank tubes as corresponding reagent blanks. At 550nm, the height A of the peak gives the amount of cytochrome c reduced during the incubation. The differ- ence between the amounts reduced in the presence and absence of SOD represents the amount of superoxide generated during the incubation.

Neutrophil chemotaxis The method employed was that of Addison and Babbage5 with minor modifications.

Neutrophil actin estimation

Cell preparation On days I and 7 post admission, fresh, heparinized blood samples were taken from the patients. The leucocytes were separated from the whole blood by the density gradient centrifugation method described above. After isolation, neutrophils were collected and washed in basic RPM1 medium containing 19 per cent human serum albumin (HSA). Thereafter, they were centri- fuged at 24Og, at room temperature, for IOmin, washed twice in basic medium with 0.2 per cent HSA and resuspended in 9.9 ml basic medium with 0.1 per cent HSA.

Determination 9.9 ml of the cell suspension were added to 0.1 ml of 500 mM phenylmethane sulphonyl fluoride (PMSF), in dimethyl sulphoxide (DMSO). This mixture was cooled on ice for 5 min and then centrifuged at 240g for 10min. The pellet was resuspended in 10ml basic medium with 0.1 per cent HSA. This centrifugation and resuspension was repeated and the final concentration of cells adjusted to 12 x lo6 cells/ml.

Previously prepared Eppendorf vials, each containing 10 ~(1 of 10mM EDTA, stored at - SO”C, were then removed from the freezer and each vial supplemented with

418 Burns (1994) Vol. to/No. 5

450 ~1 of cell suspension. These were then incubated at 37°C for 10 min. To test vials were added 10 ~1 of PMA in a final concentration of 0.01 PM, .while same-patient control vials received 10 ~1 DMSO, to a final concentra- tion of 0.4 per cent. Each vial was then supplemented with 30 ~1 of basic medium with 0.1 per cent HSA and the cell suspensions allowed to incubate for exactly timed intervals of 2,4,6,8, 10, 15, 20 or 30min. (Note: in perfecting the method, incubation times of 2-4 minutes were found optimal and these intervals were used for all the experi- mental work reported here.) Continuing reactions in each tube were stopped by cell lysis. This was done by adding 500 ~1 of a stock solution of ice-cold 2 per cent Triton-X- 100 to each vial. After cooling on ice for 10 min, the vials were allowed to equilibrate to room temperature, with occasional inversion mixing. Particulate debris was then separated by centrifugation in an Eppendorf microfuge, at 8008 for 4 min. From this, the supematant was drawn off and kept aside. The pellet was then resuspended in 10 ml of fresh Triton-X-100 stock solution, diluted 1: 1 with Hank’s medium. Again, the debris was separated by Eppendorf microcentrifugation at 8OOOg for 4min, and again the supematant was drawn off and added to the previously reserved supematant, to give a ‘combined supematant fraction’ sample. The vials were inverted to dry.

A sample buffer, comprising 10 per cent sodium dodecyl sulphate, 15 per cent glycerol, 62.5 mM Tris buffer (pH 6.8) and 0.001 per cent bromphenol blue was made up. One hundred microlitres of this buffer, supplemented with 0.2 ~1 of mercaptoethanol, was added to the dried pellet of debris in each vial, and these were incubated at 95°C with occasional vortex mixing, to aid dissolution of the pellet. This formed a second, ‘insoluble fraction’ sample.

Seventy microlitres of the combined supematant frac- tions were mixed with 35 l.tl of a 3 times concentration of the above sample buffer, and the mixture incubated at 95°C for 5 min.

Electrophoresis Ten per cent polyacrylamide gels were prepared as follows: Initially, four standard solutions were prepared. These were:

1. 2.

3. 4.

30 per cent acrylamide/O.S per cent bis-acrylamide. 0.75 M Tris-HCl buffer with 0.2 per cent SDS (sodium dodecyl sulphate). Adjust pH to 8.8. 20 mg/ml ammonium persulphate. 0.25 M Tris-HCl buffer with 0.2 per cent SDS. pH adjusted to 6.8.

Next, a mixture was made, incorporating 10 ml Solution I, 15 ml Solution II, 1 ml Solution III, 3.95 ml distilled water and 0.01 ml Temed. Solutions I, II and the water were mixed first, then Solution III was added and finally the Temed; immediately thereafter the gels were poured, covered with a little distilled water and left to set for 1 h.

Next, spacer gels were prepared by mixing 0.75 ml of Solution I with 3.75 ml of Solution IV and 2.75 ml of distilled water. 0.5 ml of Solution III was then added and mixed in well, and finally, 0.12 ml of Temed added, before a vigorous final mix. The spacer gel was poured immediately over the 10 per cent acrylamide gel and a 15 in ‘spacer comb inserted, before allowing 45 min for the gel to set. Thereafter, the comb was removed, the top of the gel apparatus loaded onto plates and the gels immersed in tank buffer.

Subsequently, standards as well as the soluble and insoluble fraction samples referred to above were run and

the gels stained with Coomassie Blue. For each sample 50 l.tl were loaded into each well and the apparatus run at 20 V per gel for 18 h before staining. This allowed isolation and densitometric quantitation of both soluble actin, the cytoplasmic G form, and insoluble, cytoskeleton- associated, F actin. Quantification was validated using pure samples to establish a standard curve.

Neutrophil cytoskeletal prepqation and scanning electron microscopy For purposes of scanning electron microscopy (SEM), neutrophils, isolated through density gradient centri- fugation as described above, were allowed to settle on, and adhere to carbon-stabilized, formvar-coated gold electron microscopy grids. Thereafter, the preparative technique followed that described by BelP.

Statistical methods All numerical data were analysed using the Wilcoxon Rank Sum test for small data sets. A value of PcO.05 was accepted as significant.

Results Superoxide production Reference to Figure I reveals that the patients’ neutrophilic production of superoxide was severely compromised. On

45

40

35

30

25

20

15

IO

5

0

Day 1

??Day7

Patients Controls

Figure 1. Neutrophil superoxide production is significantly impaired in patients, relative to unburned controls. This effect is manifest by day I postbum, and remains so through day 7.

Day 1

Day 7

Patients CWWOIS

Figure 2. Relative to controls, neutrophil chemotaxis is signifi- cantly faster on day 1 postinjury in burned patients. By day 7, however, the rate of neutrophil chemotaxis is significantly reduced in the burned patients.

de Chalain et al.: Cytoskeletal actin 419

day 1, the mean value was 11.55 (s.d. = 8.6), and on day 7, 9.76 (s.d. = 7.8), as compared with 43.65 (s.d. = 4.7) for the controls. There was no difference between the patient values at days 1 and 7, but on both these days patient values were significantly less than control values (P < 0.05).

Chemotaxis In comparing rates of chemotaxis between control cells and patients’ cells on days I and 7, Figure2 shows that, initially, chemotaxis in the patients’ cells was increased (day 1: 56.4 (s.d. = 21.0)), relative to the control value of 39.3 (s.d. = 13.7). By day 7, however, the chemotactic rate of patients’ neutrophils had declined to 12.3 (s.d. = 9.6). While the apparent increase in the chemotactic rate of patients’ neutrophils differed significantly from that of the controls (P < O.Ol), the subsequent decline in chemotactic rate seen by day 7 was also significantly different from both the rate on day I (P= 0.001) and that of the control group (P= 0.001).

Neutrophil actin content Reference to Table II and Figure 3 shows the values for all actin fractions in both bum patients’ and control neutro- phils. Actin F, the insoluble component believed to be associated with the cytoskeleton, was shown to differ significantly (I’= O.Ol), only between the patients’ neutro- phils on day 7, and controls. The patients’ values on day I,

Table II. Neutrophil actin levels

Day 1 Day 7 Controls

1. F actin 492.0 324.7 769.8 sd. 411 168 492 n a 6 7

2. G actin 164.1 166.5 555.64 s.d. 88.9 97.0 i 80.3 n a 6 7

4. Total actin 656.2 491.2 1325.5 s.d. 471 .l 252.1 1156.0 n a 6 7

Notes:s.d., standard deviation; n, number of individuals’ data included in the analysis.

, 5oo F Actin and Total Actin-D-7 vs. Controls: P~0.01 Total Actin

H GActin

??FActin + 1000 E cu 5 E 5 a 500

0

Patients D-l Patients D-7 Controls

Figure 3. By 1 week postinjury, both F-actin and total cytoskel- eta1 actin are significantly reduced in bum patients relative to controls. While this trend is manifest by day I, it becomes significant by day 7.

while apparently less than control values, were not significantly so (P= 0.09). By contrast, the values for Actin G, the soluble, cytoplasmic fraction, showed no significant differences between patients and controls.

When the individual values for the G and F fractions were summed to provide a ‘Total Actin Value’, it was found that only the day 7 values from patients’ neutrophils differed significantly from those of controls (P= 0.01).

Cytoskeletons Because neutrophils, once released from the bone marrow, have lifespans of only a few hours in the peripheral circulation, and 1-3 days in the tissues, an injury such as a bum will cause a change in the bone marrow progenitor cells with release of larger numbers of immature forms into the peripheral circulation, as well as attracting large numbers of inflammatory cells to the site of injury. Consequently, the neutrophils seen in a sample of periph- eral blood will be likely to represent a wide range of ages and states of activation. While this could be seen in the majority of micrographs, only two micrographs, represent- ing more or less ‘typical examples’ of patients’ and control neutrophils are presented here. In these it can be seen that there appears to be relatively less cytoskeletal protein remaining in the neutrophils from burned patients, as compared with those from uninjured controls.

Discussion

A major bum is one of the most catastrophic forms of injury. A bum involving as little as 25 per cent of total body surface area may be associated with significant morbidity and mortality, and this is exacerbated at the extremes of age, or where there is intercurrent illness or injury.

After a burn injury, there are a number of physiological derangements which contribute to the morbidity; in the initial, acute phase, there is circulatory shock with systemi- cally ‘leaky capillaries’ and massive fluid shifts. The large surface area of the wounds results in extensive losses of proteins and this is further complicated by both hormonal and inflammatory mediators, released in response to the insult, which promote a state of hypercatabolism in which protein degradation outstrips synthesis. The ensuing nega- tive nitrogen balance may be worsened by the advent of secondary sepsis.

Current research in wound healing has revealed a set programme of physiological responses to a bum. Early on, in the postbum period, within hours of the injury, a leucocytosis occurs and the wound sites become populated by neutrophils. These persist, as the primary defence, for some 48 h and are then superseded by activated cells of the monocyte/macrophage system. The neutrophils continue to play an important role in wound healing, however.

Various forms of trauma and sepsis have all been shown to affect neutrophil function adversely: both chemotaxis and chemiluminescence are impaired, but phagocytosis does not appear to be affected by burns’.

In the present study, the neutrophil superoxide levels (which are analogous to chemiluminescence data, as both reflect the efficacy of intracellular killing) were significantly depressed on both days I and 7 in the burned patients. Laboratory evidence suggests that this may be mediated by humoral factors4. Interestingly, at least one study has reported that impaired neutrophil bactericidal activity was seen in only one in six burned patients, and corrected itself

420 Bums (1994) Vol. 20/No. 5

Figure4 Scanning electron microscopy of neutrophil cytoskeletal proteins. a, Neutrophil cytoskeleton from uninjured control. ( x 370). b, Neutrophil cytoskeleton from burned patient, postbum day 7. The spherical structures at left and centre may represent undigested bacteria. ( x 370.)

by day 8’. Clearly, this was not the case in the present study, where all patients analysed showed similar impair- ment of neutrophil bactericidal activity. Furthermore, the mean superoxide levels fell further between days I and 7 in the burned patients, with only one patient showing any improvement in superoxide production.

While several authors have reported that neutrophil chemotaxis is depressed from as early as hours after trauma, bums and surgerys,9, it is interesting to note from the present data that no similar decline in functional motility was apparent at 24-36 h postinjury, the time when the day 1 blood samples were taken. By day 7, however, a marked suppression of chemotaxis was evident. Artursonl“, nevertheless reported that the loco- motion of polymorphonuclear white blood cells was increased during the first 1-3 days post-thermal injury, in all patients investigated. He ascribed this to an increase in heat-labile chemokinetic activity, and the subsequent decrease in locomotion, seen after 3 days, as being due to a deficiency of serum stimulator-y activity, and, in major bums, an increase in serum inhibitors.

In the present study, exactly why this delayed onset of chemotactic impairment occurred was not clear. While it is certainly possible that circulating inhibitors may have contributed, it is interesting to note that the data for F actin, the insoluble fraction associated with the cytoskel- eton, showed similar trends, with the day 7 values, but not those of day I, being significantly less than control values. Further, while the F actin decreased significantly from day I to day 7, the G actin, the soluble fraction associated with the neutrophilic cytosol, although also apparently reduced with time, was never significantly different from the control value. Significant differences between the total neutrophil act-in values on day 7 and control values are thus apparently, a function of the change in F actin. After the first week following a major bum injury, there would seem to. be significantly less actin associated with the cytoskeleton.

Given that the amount of cytoskeletal-associated actin can be influenced by a number of small peptide modulators, such as fMet-Leu-Phe, or PMA”, the changes in F actin seen in the bum patients are indeed likely to be a function of circulating modulators. Alternatively, this may consti- tute a further example of the autocannibalism more typically seen in the actin stores of skeletal muscle in

severely traumatized and septic patients; in this regard it should be noted that all our patients were in negative nitrogen balance at the time of the study.

It has been suggested by Nelson et a1.r2 that the observed post-thermal injury chemotactic dysfunction is a ‘global’ phenomenon, related, at least in part, to a non- receptor-mediated mechanism. They base their hypothesis on the observation that chemotaxis in relation to both non-specific attractants such as casein, as well as specific attractants like C5a in serum, or the synthetic tripep- tide N-formyl-methionyl-leucyl-phenylalanine, is impair- ed. They then go on to propose that there may be loss of random motility as the basis for impaired chemotaxis and present data suggesting that a possible mechanism may be auto-oxidation by excessively stimulated respir- atory chain enzymes and products. Their postulation is that thermally injured neutrophils will irreversibly polym- erize increased amounts of cytoplasmic actin, as a result of such overstimulation. Hinshaw13 has shown that cell-line fibroblasts exposed to hydrogen peroxidase will irrever- sibly polymerize cytoskeletal actin, and Nelson and col- leagues12 use this data to argue that auto-oxidative injury in the neutrophil leading to irreversible polymerization of the cellular actin interferes with the ready gelation and solvation of cellular actin, and hence to impaired motility. Our data refute the claim that the amount of F actin (i.e. the insoluble fraction associated with the cytoskeleton) under- goes irreversible polymerization, since we have shown clearly that levels of both F and total cellular actin fall significantly over the first week. Furthermore, it is signifi- cant that the chemotactic rate takes time to show impair- ment, being increased at 24-36 h postinjury, but reduced at 7 days. Not only does this corroborate earlier data suggesting that the period of maximal chemotactic dys- function is during the second week post injury”, coinci- dent with increased rates of bacterial infection’, but it also throws doubt on the validity of the auto-oxidation hypothesis of Nelson et al.rz. If overstimulation of neutrophils lead to auto-oxidation, with intracellular injury causing actin polymerization, then, since the superoxide data presented above suggest that the neutrophils’ respir- atory chain mechanism (which is implicated in intracellular killing) was already impaired by day 1 and remained so through day 7, it is difficult to explain why there should have been such differences in observed chemotactic rates

de Chalain et al.: Cytoskeletal actin 421

on days I and 7. It seems more likely that there may be another mechanism, probably unrelated to auto-oxidation, responsible for the observed changes in actin levels and chemotactic rates.

In considering the scanning electron microscopy data, marked differences in neutrophil cytoskeletal structure between days I and 7 could not be identified. At both times, careful analysis of the micrographs revealed hetero- geneity of cytoskeletal architecture, consistent with the suggestion that several subpopulations of neutrophils were present in each sample. It should therefore be noted that the numerical data presented probably represents means amongst these various subpopulations. Despite these caveats, however, the representative micrographs shown in Figwe suggest strongly that there is less neutrophil cytoskeletal protein to be seen in the burn patients than in the controls, an observation which correlates well with the numerical data.

References

Border JR, Hassett J, La Duca J et al. The gut origin septic state in blunt multiple trauma (ISS = 40) in the ICU. Ann Strrg 1987; 206: 427-430. Henley M. Feed that bum. Berms 1989; 15: 351-361. de Chalain TMB, Michell WL, O’Keefe SJ et al. The effect of fuel source on amino acid metabolism in critically ill patients.]Surg Res 1992; 52: 167-172. Stillwell M and Caplan ES. The septic multiple trauma patient. Crif Cure Chin 1988; 4: 345-354.

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Bell PB, Lindroth M, Fred&son B-A. Preparation of cells in culture for high resolution scanning and scanning- transmission electron microscopy. Scanning Micros 1988; 2:1647-1653. Alexander J, Hegg M, Altemeier W et al. Neutrophil function in selected surgical disorders. Ann Surg 1968; 168:447-450. Meakins J, Mclean A, Kelly R et al. Delayed hypersensitivity and neutrophil chemotaxis: effects of trauma. 1 Trutlma 1978;18:240-244. Heck E, Edgar M and Hunt J. A comparison of leucocyte function and bum mortality. 1 Tratrma 1980; 20: 75-79. Arturson G. Neutrophil granulocyte function in severely burned patients. Bums 1985; 11: 309-319. Sha’afi RI and Molski TFP. Signalling for increased cytoskel- eta1 actin in neutrophils. Biochem Biophys Res Commun 1987; 145: 934-936. Nelson PD, Hasslen SR, Ahrenholz DH et al. Polymorpho- nuclear leucocyte function following bum and mechanical injury: regulation and kinetics. In: Faist H, Ninnemann R and Green B, (eds) Immtlne Conseqtlences of Trauma, Shock and Sepsis. Berlin: Springer-Verlag, 1989; p 201. Hinshaw DB, Sklar LA, Bohl B et al. Cytoskeletal and morphologic impact of cellular oxidant injury. Am ] Puthol 1986; 123: 454-460.

Paper accepted 20 January 1994.

Addison IE and Babbage JW. A Raft technique for che- Correspondence should be addressed to: Dr T. M. B. de Chalain, motaxis: a versatile method suitable for clinical studies. ] Department of Plastic Surgery, The Emory Clinic, 1327 Clifton lmmunol Methods 1976;10:385-391. Road, N.E., Atlanta, GA 30322, USA.

“State of the Art Symposium” on

The Burn Wound

Response to Injury, Sepsis and Skin Replacement

17-18th October 1994

The Royal Society of Medicine, London

Ihe British Association of Plastic Surgeons in association with the European Burns Association are organizing the above symposium. Speakers will include;

Professor W. Boeckx Professor R. Hettich Professor C. Baxter Mr J.A. Clarke Professor I. Leigh Dr D. Heimbach Professor Donati Dr D. Ma&e Dr M. Robson Mr E. Freedlander Dr P. Wilson Dr D. Smith

For further information please contact: The British Association of Plastic Surgeons at The Royal College of Surgeons, 35-43 Lincoln’s Inn Fields, London WCZA 3PN. Tel: 071 831 5161/2 or Fax: 0718314041.