Staining:
PMLs surface antigens were labelled using monoclonal antibodies by indirect immunofluorescence technique. Briefly, for each antigen investigated a 100 ml of 1: 100 dilution of specific monoclonal antibody was added to 100 ml leukocytes and was incubated on ice for 30 minutes. After washing twice with PBS; a 100 ml of 1: 100 dilution of fluorescein isothiocyanate antibody (goat antimouse) conjugate (DAKO) was added, then was incubated for 30 minutes on ice. After a final wash, cells Were resuspended in 0.5 ml PBS. A negative control was prepared in the same manner ornitting the receptorspecific monoclonal antibody.

Flow cytometry:
The cells were analyzed using an EPICSPROFILE II (Coulter Electronics, FL, USA). The laser excitation wave length was 488 nm and standard filter setting were used. Lenk-ocytes subpopulations were differentiated by combined ineasurements of forward-angle and side-angle light scatter and the monoclonal antibody specific fluorescence gated on PMLs to separate lustograin. The PMLs surface receptors were expressed as the mean fluorescence of the PMLs population after staining with receptor specific monoclonal antibodies minus the negative control prepared from the sanie blood sample.

Candida albicans:

Preparation:
C.albicans was fixed by ethanol 70% for I hour, then suspended in PBS solution and adJusted to a count of 5 x l0⁷/ml.

Complement C3 opsonization:
For the opsonization of C.albicans with complement, Na₂-EDTA was added to pooled human serum (PHS) to bind divalent cations, before the PHS was observed twice with Na₂EDTA-washed C.albicans to remove antibodies against the fungi. The absorbed PHS was then reconstituted with Ca²⁺ and Mg²⁺ by addition of 0.1 ml 100 mol/L CaCl₂ and 100 mol/L MgCl₂ per milliters PHS. Then, the C.albicans (5 x 10⁷/ml) was rotated at 37'C for 45 minutes with PHS. The C3-opsonized C.albicans was washed twice, counted and resuspended in PBS to a final concentration of 5 x 10⁸ fungi/ml.

Immunoglobulin opsonization of C.albicans:
The PHS was heated at 56°C for 30 minutes to inactivate complement. Then, the C.albicans (5x 10⁷/ml) was rotated at 37°C for 45 minutes with heated PBS and the fungi washed, counted and ad usted in PBS to 5 x 10⁸/ml..

PHS opsonization:
The C.albicans (5 X10⁷/ml) was rotated with PHS at 37°C for 45 minutes & the fungi washed, counted and adjusted in PBS to 5 x 10⁸/ml.

Phagocytosis:
One hundred microliters of PML suspensions was mixed with 100 ml of preoponized C.albicans, before Hank's Balanced Salt Solution (HBSS) containing 0.5% Bovine Serum Albumin (BSA) was added to a final volume of 1 ml. This provided an initial fungus to PML ratio of 10:1. The mixtures were rotated at 37°C for 15 minutes. Then, the neutrophilic suspension was spreaded and stained by Leishman stain. Phagocytosis was measured cytomotphologically by determining the C.albicans phagocytic index according to Ballart et al. [23] using the following equation:                            

ICAM-1 assay:
Peripheral blood samples were collected on EDTA and centrifuged at 1000 g for 15 minutes within 2 hours. Plasma was stored at -70°C until analysis. Plasma concentrations were determined (at duplicate) by commercially available ELISA Kits (Biosource Europe S.A., Belgium). The minimum deteitable concentration was estimated to be 0.3 ng/ml.

RESULTS

Fig(1): Time course of neutrophil (PML) expression of Fcg RIII (A), CRI (B) and CRIII (C) in burn patients as determined by indirect immunofluorescence and FCM.The results are expressed as the percentage of the fluorescence obtained with PMLs from normal soggets in parallel measurements.The results are given as the mean ±SEM and the significance of difference between the patients and the controls is indicated as *p<0.05, **p<0.01. Fig. (2):Time course of neutrophil (PML) immunoglobuline-mediated phagocytosis (A),complement-mediated phagocytosis (B) and phagocytosis mediated by pooled human serum (PHS) (C) in burn patients as determined by phagocytic index.The results are expressed as the percentage of the number of candida albicans data were obtained from parallel measurements.The results are given as the mean ±SEM and the significance of difference between the patients and the controls is indicated as *p<0.05, **p<0.01.  Fig. (3):Time course of  ICAM-1 plasma levels in burn patients determined by ELISA.The results are expressed as the mean ±SEM (ng/ml).The control data are obtained from parallel measurements.The significance of difference between the patients and the controls is indicated as *p<0.05, **p<0.01.

PML expression of FcgRIII:
The patient PM1- expression of FcgRIII was decreased to 65.7% of control values at admission and remained low for the first 20 days (Fig. 1,a &Table 2).

PML expression of CRs:
The patient PMLs expression of CRI was increased to 162.9% of control values at 2 days, then gradually decreasing to control levels at 20 days (Fig. 1,b & Table 2). The expression of PMLs CR3 was increased by 199% at day 2 and remained high during the first 20 days (Fig. 1,b & Table 2). Both control and patient PMLs CRI-dependent and CR3-dependent fluorescence were shown to be monophaste throughout the investigation period.

Phagocytosis:
The PMLs lg-mediated phagocytosis of Calbicans was decreased to 84.7% of control values at day 2. The lowest lg-mediated phagocytosis was observed at day 10, with a reduction of 28.1% compared with controls (Fig. 2,a & Table 3).The patient PMLs complement-mediated phagocytosis of Calbicans was increased by about 54% of control level and remained high for the first 20 days (Fig. 2,b & Table 3).The patient PMLs phagocytosis of C.albicans opsonized with PHS (i.e. both IgG and complement factors being present) was increased by about 3 1 % of control values and remained higher than that of control for 20 days (Fig. 2, c & Table 3).

Plasma levels of sICAM-1:
The plasma sICAM-1 was increased by 31 % of control values at day 2 and remained high for the first 20 days with a maximum level at day 10 (Fig. 3 & Table 4).

Neutrophil activation by chemoattractants, enzymes and bacteriologically-derived peptides release PI linked FcRIII into plasma and in tissues with active inflammation [24]. PI-linked FcRIII mediates exocytosis of neutrophil granule proteins but does not mediate the initiation of the respiratory burst [25]. The present study, demonstrated that expression of PML Fe RIII following burn injury was decreased at day 2 and remained low for the first 20 days. This result is thus suggestive of a systemic activation of these cells that induce shedding of this receptor, a finding in agreement with Vindenes and Bjerknes [26].A marked and sustained increases in the expression of the complement receptors CRI and CR3 was documented in the present study. The increase in fluorescence that depends on complement receptors was always observed as a monophasic peak, indicating that all the cells had been activated by a stimulus that was systemic. Moore et al. [27] and Vindenes and Bjerknes [26] had reported similar results. The increased expression of CR3 is an important part of neutrophil priming and activation as this molecule is critically involved in adherence of PML to the endothelium. This is a necessary initial step in the emigration of PMLs from the circulation into the tissues. In addition, both CRI and CR3 are necessary for optimal phagocytosis of complement-coated bacteria and immune complexes [28].Previous studies have shown that a variety of mediators such as the complement split product C5a [27], TNF-a [28]; IL-8 [6]; granulocyte-macrophage colony - stimulating factors [5] and endotoxin [29], which might be locally produced at sites of infection are all capable of increasing complement receptor expression.There are intracellular pools for both the CR1and CR3, but the intracellular locations for these pools are distinct. The pool for C3 cosediments with specific granules, while the pool for CR1 does not [30]. The increased receptor expression occurs within minutes and represents translocation of presynthesized receptors from intracellular pools to the surface rather than new synthesis. So, increased expression of PML CRs following thermal injury strongly suggests PML degranulation. This is consistent with earlier reports of Alexander [31] and Davis et al.[32].This study revealed that plasma sICAM-1 levels were elevated during the first 20 days postburn with a maximum level at day 10. This finding could be referred to the activation of the inflammatory cytokines such as IL-1b, TNF-a and INF-a that induce or enhance the expression of both membrane and soluble ICAM-1 [15,33], Jaeschke et a]. [34] had reported an increased plasma sICAM-1 level endotoxin-challenged mice.In acute and chronic inflammatory processes, fibrin deposition and leukocyte accumulation are classic histopathologic hallmarks.Fibrin deposition on vascular endothelial cells (EC) can result in the upregulation of EC ICAM-1 which is an important ligand/receptor for CD1 lb/CD18 expressed on neutrophils. Fibrin stimulation of EC increased their adhesiveness for PMLs [35]. Circulating levels of soluble endothelial cell adhesion molecules may reflect the magnitude of expression of their membranebound counterparts [36]. Mulligan et a]. [37] have emphasized the role of ICAM-1 in the events that lead to neutrophil -mediated vascular injury of dermis and lung after thermal trauma to skin. These would cause cessation of neutrophil movement along the endothelial cells and transmigration.Systemic activation of PNILs may have harmful effects on the host. Increased PMLs CR3 expression as well as sICAM-1 plasma level may cause increased neutrophil adhesiveness and the formation of leukocyte microemboli which concentrate in the first capillary bed encountered, the lung [26,27]. Also there is depression of neutrophil chernotaxis [27]. Activated PNILs are known to degranulate and release lysosomal enzymes, as well as oxygen radicals toxic to the surrounding tissues [38]. The injury related PML activation can sustain and be further strengthened by subsequent superimposition of infectious foci (e.g. bum wound sepsis) [29] or by transintestinal transport of LPS or micro-organisms [39,40,41]. This recruitment and activation of PMLs in the course of thermal injury may exceed physiologic needs and induce PML tissue infiltration and destruction. Therefore we recommend that apparent source of neutrophil activating substances, the burn wound, should be excised as early as clinically feasible to free the patient of the burden of systemetically activated neutrophils [29] and early initiation of enteral feeding [42]. The use of anti-proinflammatory cytokines and/or antiICAM-1 needs further investigation [37].

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