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Cultured keratinocytes and antibody problems
68
Burns (1989) 15,(I). 68-70 Pn'ntdin Greaf Britain
Abstracts
CLINICAL STUDIES
leukotrienes from human polymorphonuclear granulocytes severely burned patients. 1. Truuma 28, (6), 733-740.
of
Chemical injuries A detailed survey of the chemical injuries observed in the US Army bum unit over a IT-year period showed an incidence of 2.1per cent (8714212). Th e mortality rate was 13.8per cent (12187). White phosphorus, the most common cause of chemical injury of the skin, affected 49 patients. The injury was caused by acid in 18 patients, alkalies in 10 patients and organic solvents in five patients. Systemic toxicity arose from contact with phenol, nitrates and formates. Acute tubular necrosis followed overzealous treatment of white phosphorus injury with copper sulphate. Prompt lavage of the injury with copious volumes of water is mandatory if the severity of the injuries is to be minimized. Mozingo D. W., Smith A. A., McManus W. F. et al. (1988) Chemical bums. 1. Trmma 28,(5), 642-647.
Diarrhoea and tube feeds In an attempt to specify which components in the tube feed given to burned children induced diarrhoea, 50 patients with a wide range of burned areas were studied; 16 of the children developed diarrhoea. Stool cultures were negative for pathogenic microorganisms. Significant risk factors associated with diarrhoea were antibiotic administration, dietary lipid content and a low vitamin A intake. In contrast, implementation of tube feeding within 48 h of bum injury was associated with a markedly decreasedincidence of diarrhoea. Factors not associated with diarrhoea were tube feed osmolality, drugs used to prevent stress ulcers and hypoalbuminaemia. Gottschlich M. M., Warden G. D., Michel M. et al. (1988) Diarrhoea in tube fed bum patients. Incidence, etiology, nutritional impact and prevention. J. Parent. Entr. N&r. 12, (4,
338-345. Granulocyte
contamination
of lymphocyte
preparations
When isolating lymphocytes from the blood of burned patients using Ficoll-Hypaque techniques it is obligatory to make differential counts on the samples of obtained cells since they may be extensively contaminated with granulocytes. Serial studies in 15 patients with a wide range of burned areas showed that this contamination reached about 40 per cent at 24 h postburn and was still about 25 per cent at 4 weeks postbum. Such contamination was not a reflection of a granulocytosis with normal cells but the formation of a substantial number of granulocytes with a lower density than normal: such cells remain at the FicollHypaque interface following centrifugation. Calvano S. E., Greenlee P. G., Reid A. M. M. et al. (1988) Granulocyte contamination of Ficoll-Hypaque preparations of mononuclear cells following thermal injury may lead to substantial overestimation of lymphocyte recovery. 1. Truuma 28,
(3), 353-361.
Granulocyte
function and leukotrienes
Opioids and immune function Based on the evidence of raised beta endorphin levels in the plasma from severely burned patients, and that this opioid may alter immune function, in vitro studies tested the effects of beta endorphin, met enkephalin, naloxone and morphine on normal neutrophil and lymphocyte function. Morphine markedly decreased neutrophil chemotaxis and increased bactericidal activity for Staph. uurew and resting and zymosan-stimulated oxygen consumption. In contrast, the other opioids had no direct effect on neutrophil chemotaxis or bactericidal activity. Both naloxone and met enkephalin increased neutrophil oxygen consumption in a dose-dependent manner; beta endorphin reduced neutrophil oxygen consumption. None of the opioids altered resting lyrnphocyte blastogenesis. Only beta endorphin reduced the ability of normal lymphocytes to respond to mitogen stimulation at physiologically relevant doses. Deitch E. A., Xu D. Z. and Bridges R. M. (1988) Opioids modulate human neutrophil and lymphocyte function; thermal injury alters plasma beta endorphin levels. Srrrgery 104, (I),
41-48. Studies were made of the release of leukotrienes (LTC, and LTB,) from the polymorphonuclear granulocytes from six severely burned patients. The release of these materials fell to nil soon after burning and then slowly recovered to normal values by 40 days postinjury. In patients with fatal bums the leukotriene release was always markedly subnormal. An enhanced LTC, production coincided with an increase in the proportion of eosinophils in the granulocyte fraction. A reduced LTB, content seemed to be the result of metabolism to less active products exceeding synthesis. These changes in leukotriene production were associated with the onset of invasive microbial growth as determined by quantitative bacteriological analysis of full skin thickness biopsy specimens. Kiiller M., Konig W., Brom J. et al. (1988) Generation of
Cultured keratinocytes
and antibody problems
Sera from 10 patients with bums who had been grafted with cultured keratinocytes were analysed for the presence of antibodies derived from the fetal bovine serum or mouse fibroblasts used during the in vitro multiplication of the keratinocytes. All patients had detectable amounts of antibody to the fetal bovine serum (mainly to the albumin moiety) and no detectable antibody to the mouse fibroblasts. The titres of the bovine serum antibody varied between patients and with the time after graft application. Generally the titres were moderate by 30 days, then declined but showed a second rise 5-6 months after exposure to
69
Abstracts
the antigens. Whether these antibodies will pose clinical probIems of future graft rejection, anaphylaxis or serum sickness remains to be determined. Meyer A., Manktelow A., Johnson M. et al. (1988) Antibody response to xenogeneic proteins in burned patients receiving cultured keratinocyte grafts. J. Trauma 28, (7), 1054-1059.
muscle of animals seeded with Staph. aureus and E. cloacae was less in animals treated with CDSS than with SS. Snelling C. F. T. and Roberts F. 1. (1988) Comparison of 1% silver sulfadiazine with and without 1% chlorhexidine digluconate for topical antibacterial effect in the burnt infected rat. J. Burn Cure Rehabil. 9, (I), 35-40.
Wound healing and metabolism
Age, burns and cardiac function
Retrospective analyses of sequential metabolic studies in nine severely burned patients indicated that the reduction in the hypermetabolic response was correlated with the number of days postbum, the percentage of the wound still unhealed, the average body temperature and evaporative heat loss. However, decrements in metabolic rate between sequential studies correlated only with the changes in percentage open wound and evaporative heat loss. While changes in heat loss are not capable of predicting all of the variation in metabolic rate, burned patients show an increase in heat production (related to altered central thermoregulation) which is largely secondary to the inability to conserve heat effectively in the periphery. Cone J. B., Wallace 8. H. and Caldwell F. T. (1988) The effect of staged bum wound closure on the rates of heat production and heat loss of burned children and young adults. J. Truuma 28, (7), 968-972.
The contractile responses of young and aged hearts from burned and non-burned animals were assessed. In the absence of injury, aged control hearts showed significantly lower left ventricular performances than young hearts at the same left ventricular enddiastolic pressures. A full skin thickness bum covering about 40 per cent of the total body surface area reduced the left ventricular performance of both the young and the aged hearts significantly. Contractile defects in the aged hearts were not related to altered beating frequency or to maximally effective increases in diastolic stretch. Although varying extracellular calcium concentrations modified the performance of both the young and aged hearts from burned animals, the age difference persisted. Within the group of aged hearts from burned animals the peak left ventricular performance and the rate of relaxation were uniformly decreased compared with control aged hearts. Horton J. W., Baxter C. R. and White J. (1988) The effects of aging on the cardiac contractile response to unresuscitated thermal injury. J. Bum Cure Rehabil. 9, (I), 41-51.
Effects of HSL resuscitation Hypertonic sodium lactate (HSL) was used to resuscitate 14 patients with bums covering between 20 and 60 per cent of the body surface area. From admission onwards for 7 days sequential measurements were made of the plasma concentrations of atria1 natriuretic peptide, arginine vasopressin, angiotensin II, adrenaline and noradrenaline. Serum sodium concentrations and osmolalities were lowest on admission and were persistently elevated following HSL resuscitation. Plasma arginine vasopressin levels were highest on admission and correlated with the size of the bum. Between days 4 and 5 plasma atria1 natriuretic peptide levels rose while the plasma levels of arginine vasopressin returned to normal; neither of these levels correlated with serum osmolality or serum sodium concentrations on admission or after HSL resuscitation. The plasma levels of angiotensin II, adrenaline and noradrenaline were elevated throughout the 7day period of study and were unrelated to the size of the bum. Crum R., Bobrow B., Shackford S. et al. (1988) The neurohumoral response to bum injury in patients resuscitated with hypertonic saline. 1. Trauma 28. (8), 1181-1187.
DMSO and inhalation injury Sheep with a cotton smoke inhalation injury received either dimethyl sulphoxide (DMSO) alone or DMSO plus heparin or heparin alone or no treatment. All animals had ventilatory support to maintain their PuO, above 60mmHg and their P&O, below 45 mmHg. By 72 h after injury all the seven control animals were dead, two of the six heparin-treated animals had died, four of the six DMSO-alone animals had died whereas all the DMSO plus heparin-treated animals were still alive. Lung lymph flow was not as high in the DMSO plus heparin group as in the heparin only group. Whether this beneficial effect of DMSO plus heparin can be used to alleviate smoke inhalation injury in man remains to be determined. Brown M., Desai M., Traber L. D. et al. (1988) Dimethylsulfoxide with heparin in the treatment of smoke inhalation injury. 1. Burn Cure Rehabil. 9, (I), 22-25.
Influx of leucocytes to infected scalds ANIMAL
STUDIES
Benefits of chlorhexidine
added to silver sulphadiazine
The benefits of adding 1 per cent chlorhexidine to I per cent silver sulphadiazine cream were assessed in rats having deep bums covering 20 per cent of the body surface area. The bums of groups of these injured animals were seeded with 10’ organisms of Staph. aurepw or Ps. aeruginosa or Enterobacfer cloacae or Strep. fuecalis, all species having been isolated from human bum wounds. Daily topical application to the bum wound of either silver sulphadiazine alone (SS) or a mixture of SS with chlorhexidine (CDSS) showed the superiority of CDSS, since the mean concentration of all four organisms recovered after 1 week from biopsy specimens of full skin thickness eschar was less in the CDSS animals than in the SS-treated animals. Microbial invasion into subadjacent muscle was less frequent in animals seeded with Strep. foecalis. The mean concentration of bacteria recovered from
Rats previously sensitized to keyhole limpet haemocyanin (KLH) were given a scald covering 30 per cent of the body surface area or a sham scald. Three days later the animals received an intradermal inoculation of KLH, 10’ Staph. aurew organisms and an iv. injection of indium-labelled leucocytes. In the sham scald rats the peak influx of labelled leucocytes to the site of the delayed type hypersensitivity (DTH) area was at 2-4 h and into the staphylococcal abscess, biphasic peaks occurred at 3 and 6-8 h. In contrast, in the scalded rats there was a markedly increased influx of leucocytes at 2 h in both the DTH and abscess sites, followed by a significantly lower than normal leucocyte influx at 6-24 h postinjury. The marked early leucocyte influx following scalds coincided with and was directly related to a reduced DTH reaction and an increase in the extent of the Staph. aureus abscess. Tchervenkov J. I., Latter D. A., Psychogios J. et al. (1988) Altered leukocyte delivery to specific and non-specific inflammatory skin lesions following bum injury. J. Trauma 28, (5), 582-588.
Burns (1989) 15,(I). 68-70 Pn'ntdin Greaf Britain
Abstracts
CLINICAL STUDIES
leukotrienes from human polymorphonuclear granulocytes severely burned patients. 1. Truuma 28, (6), 733-740.
of
Chemical injuries A detailed survey of the chemical injuries observed in the US Army bum unit over a IT-year period showed an incidence of 2.1per cent (8714212). Th e mortality rate was 13.8per cent (12187). White phosphorus, the most common cause of chemical injury of the skin, affected 49 patients. The injury was caused by acid in 18 patients, alkalies in 10 patients and organic solvents in five patients. Systemic toxicity arose from contact with phenol, nitrates and formates. Acute tubular necrosis followed overzealous treatment of white phosphorus injury with copper sulphate. Prompt lavage of the injury with copious volumes of water is mandatory if the severity of the injuries is to be minimized. Mozingo D. W., Smith A. A., McManus W. F. et al. (1988) Chemical bums. 1. Trmma 28,(5), 642-647.
Diarrhoea and tube feeds In an attempt to specify which components in the tube feed given to burned children induced diarrhoea, 50 patients with a wide range of burned areas were studied; 16 of the children developed diarrhoea. Stool cultures were negative for pathogenic microorganisms. Significant risk factors associated with diarrhoea were antibiotic administration, dietary lipid content and a low vitamin A intake. In contrast, implementation of tube feeding within 48 h of bum injury was associated with a markedly decreasedincidence of diarrhoea. Factors not associated with diarrhoea were tube feed osmolality, drugs used to prevent stress ulcers and hypoalbuminaemia. Gottschlich M. M., Warden G. D., Michel M. et al. (1988) Diarrhoea in tube fed bum patients. Incidence, etiology, nutritional impact and prevention. J. Parent. Entr. N&r. 12, (4,
338-345. Granulocyte
contamination
of lymphocyte
preparations
When isolating lymphocytes from the blood of burned patients using Ficoll-Hypaque techniques it is obligatory to make differential counts on the samples of obtained cells since they may be extensively contaminated with granulocytes. Serial studies in 15 patients with a wide range of burned areas showed that this contamination reached about 40 per cent at 24 h postburn and was still about 25 per cent at 4 weeks postbum. Such contamination was not a reflection of a granulocytosis with normal cells but the formation of a substantial number of granulocytes with a lower density than normal: such cells remain at the FicollHypaque interface following centrifugation. Calvano S. E., Greenlee P. G., Reid A. M. M. et al. (1988) Granulocyte contamination of Ficoll-Hypaque preparations of mononuclear cells following thermal injury may lead to substantial overestimation of lymphocyte recovery. 1. Truuma 28,
(3), 353-361.
Granulocyte
function and leukotrienes
Opioids and immune function Based on the evidence of raised beta endorphin levels in the plasma from severely burned patients, and that this opioid may alter immune function, in vitro studies tested the effects of beta endorphin, met enkephalin, naloxone and morphine on normal neutrophil and lymphocyte function. Morphine markedly decreased neutrophil chemotaxis and increased bactericidal activity for Staph. uurew and resting and zymosan-stimulated oxygen consumption. In contrast, the other opioids had no direct effect on neutrophil chemotaxis or bactericidal activity. Both naloxone and met enkephalin increased neutrophil oxygen consumption in a dose-dependent manner; beta endorphin reduced neutrophil oxygen consumption. None of the opioids altered resting lyrnphocyte blastogenesis. Only beta endorphin reduced the ability of normal lymphocytes to respond to mitogen stimulation at physiologically relevant doses. Deitch E. A., Xu D. Z. and Bridges R. M. (1988) Opioids modulate human neutrophil and lymphocyte function; thermal injury alters plasma beta endorphin levels. Srrrgery 104, (I),
41-48. Studies were made of the release of leukotrienes (LTC, and LTB,) from the polymorphonuclear granulocytes from six severely burned patients. The release of these materials fell to nil soon after burning and then slowly recovered to normal values by 40 days postinjury. In patients with fatal bums the leukotriene release was always markedly subnormal. An enhanced LTC, production coincided with an increase in the proportion of eosinophils in the granulocyte fraction. A reduced LTB, content seemed to be the result of metabolism to less active products exceeding synthesis. These changes in leukotriene production were associated with the onset of invasive microbial growth as determined by quantitative bacteriological analysis of full skin thickness biopsy specimens. Kiiller M., Konig W., Brom J. et al. (1988) Generation of
Cultured keratinocytes
and antibody problems
Sera from 10 patients with bums who had been grafted with cultured keratinocytes were analysed for the presence of antibodies derived from the fetal bovine serum or mouse fibroblasts used during the in vitro multiplication of the keratinocytes. All patients had detectable amounts of antibody to the fetal bovine serum (mainly to the albumin moiety) and no detectable antibody to the mouse fibroblasts. The titres of the bovine serum antibody varied between patients and with the time after graft application. Generally the titres were moderate by 30 days, then declined but showed a second rise 5-6 months after exposure to
69
Abstracts
the antigens. Whether these antibodies will pose clinical probIems of future graft rejection, anaphylaxis or serum sickness remains to be determined. Meyer A., Manktelow A., Johnson M. et al. (1988) Antibody response to xenogeneic proteins in burned patients receiving cultured keratinocyte grafts. J. Trauma 28, (7), 1054-1059.
muscle of animals seeded with Staph. aureus and E. cloacae was less in animals treated with CDSS than with SS. Snelling C. F. T. and Roberts F. 1. (1988) Comparison of 1% silver sulfadiazine with and without 1% chlorhexidine digluconate for topical antibacterial effect in the burnt infected rat. J. Burn Cure Rehabil. 9, (I), 35-40.
Wound healing and metabolism
Age, burns and cardiac function
Retrospective analyses of sequential metabolic studies in nine severely burned patients indicated that the reduction in the hypermetabolic response was correlated with the number of days postbum, the percentage of the wound still unhealed, the average body temperature and evaporative heat loss. However, decrements in metabolic rate between sequential studies correlated only with the changes in percentage open wound and evaporative heat loss. While changes in heat loss are not capable of predicting all of the variation in metabolic rate, burned patients show an increase in heat production (related to altered central thermoregulation) which is largely secondary to the inability to conserve heat effectively in the periphery. Cone J. B., Wallace 8. H. and Caldwell F. T. (1988) The effect of staged bum wound closure on the rates of heat production and heat loss of burned children and young adults. J. Truuma 28, (7), 968-972.
The contractile responses of young and aged hearts from burned and non-burned animals were assessed. In the absence of injury, aged control hearts showed significantly lower left ventricular performances than young hearts at the same left ventricular enddiastolic pressures. A full skin thickness bum covering about 40 per cent of the total body surface area reduced the left ventricular performance of both the young and the aged hearts significantly. Contractile defects in the aged hearts were not related to altered beating frequency or to maximally effective increases in diastolic stretch. Although varying extracellular calcium concentrations modified the performance of both the young and aged hearts from burned animals, the age difference persisted. Within the group of aged hearts from burned animals the peak left ventricular performance and the rate of relaxation were uniformly decreased compared with control aged hearts. Horton J. W., Baxter C. R. and White J. (1988) The effects of aging on the cardiac contractile response to unresuscitated thermal injury. J. Bum Cure Rehabil. 9, (I), 41-51.
Effects of HSL resuscitation Hypertonic sodium lactate (HSL) was used to resuscitate 14 patients with bums covering between 20 and 60 per cent of the body surface area. From admission onwards for 7 days sequential measurements were made of the plasma concentrations of atria1 natriuretic peptide, arginine vasopressin, angiotensin II, adrenaline and noradrenaline. Serum sodium concentrations and osmolalities were lowest on admission and were persistently elevated following HSL resuscitation. Plasma arginine vasopressin levels were highest on admission and correlated with the size of the bum. Between days 4 and 5 plasma atria1 natriuretic peptide levels rose while the plasma levels of arginine vasopressin returned to normal; neither of these levels correlated with serum osmolality or serum sodium concentrations on admission or after HSL resuscitation. The plasma levels of angiotensin II, adrenaline and noradrenaline were elevated throughout the 7day period of study and were unrelated to the size of the bum. Crum R., Bobrow B., Shackford S. et al. (1988) The neurohumoral response to bum injury in patients resuscitated with hypertonic saline. 1. Trauma 28. (8), 1181-1187.
DMSO and inhalation injury Sheep with a cotton smoke inhalation injury received either dimethyl sulphoxide (DMSO) alone or DMSO plus heparin or heparin alone or no treatment. All animals had ventilatory support to maintain their PuO, above 60mmHg and their P&O, below 45 mmHg. By 72 h after injury all the seven control animals were dead, two of the six heparin-treated animals had died, four of the six DMSO-alone animals had died whereas all the DMSO plus heparin-treated animals were still alive. Lung lymph flow was not as high in the DMSO plus heparin group as in the heparin only group. Whether this beneficial effect of DMSO plus heparin can be used to alleviate smoke inhalation injury in man remains to be determined. Brown M., Desai M., Traber L. D. et al. (1988) Dimethylsulfoxide with heparin in the treatment of smoke inhalation injury. 1. Burn Cure Rehabil. 9, (I), 22-25.
Influx of leucocytes to infected scalds ANIMAL
STUDIES
Benefits of chlorhexidine
added to silver sulphadiazine
The benefits of adding 1 per cent chlorhexidine to I per cent silver sulphadiazine cream were assessed in rats having deep bums covering 20 per cent of the body surface area. The bums of groups of these injured animals were seeded with 10’ organisms of Staph. aurepw or Ps. aeruginosa or Enterobacfer cloacae or Strep. fuecalis, all species having been isolated from human bum wounds. Daily topical application to the bum wound of either silver sulphadiazine alone (SS) or a mixture of SS with chlorhexidine (CDSS) showed the superiority of CDSS, since the mean concentration of all four organisms recovered after 1 week from biopsy specimens of full skin thickness eschar was less in the CDSS animals than in the SS-treated animals. Microbial invasion into subadjacent muscle was less frequent in animals seeded with Strep. foecalis. The mean concentration of bacteria recovered from
Rats previously sensitized to keyhole limpet haemocyanin (KLH) were given a scald covering 30 per cent of the body surface area or a sham scald. Three days later the animals received an intradermal inoculation of KLH, 10’ Staph. aurew organisms and an iv. injection of indium-labelled leucocytes. In the sham scald rats the peak influx of labelled leucocytes to the site of the delayed type hypersensitivity (DTH) area was at 2-4 h and into the staphylococcal abscess, biphasic peaks occurred at 3 and 6-8 h. In contrast, in the scalded rats there was a markedly increased influx of leucocytes at 2 h in both the DTH and abscess sites, followed by a significantly lower than normal leucocyte influx at 6-24 h postinjury. The marked early leucocyte influx following scalds coincided with and was directly related to a reduced DTH reaction and an increase in the extent of the Staph. aureus abscess. Tchervenkov J. I., Latter D. A., Psychogios J. et al. (1988) Altered leukocyte delivery to specific and non-specific inflammatory skin lesions following bum injury. J. Trauma 28, (5), 582-588.