The Journal of International Medical Research 1991; 19: 451 - 456
Effect of Oral Administration of Bacterial Extracts on the Bactericidal Capacity of Polymorphonuclear Leucocytes in Children with Recurrent Respiratory Infections A. Kantar', N. Oggiano', G.G. RomagnonF and P.L. GiorgP 'Paediatric Clinic, University ofAncona, Ancona, Italy; 2Medical Direction, Simes SpA, Bresso-Milan, Italy
The effect oforally administered hacterial extracts given intermittently over 16 weeks on the bactericidal capacity of polymorphonuclear leucocytes (PMNs) in children with recurrent respiratory infections was investigated using a luminol-amplified chemiluminescence assay. Chemiluminescence of PMNs stimulated with zymosan or N.formylmethionyl-Ieucyl-phenylalanine (fMLP) before and after treatment with bacterial extracts or intramuscular benzanthine penicillin was evaluated. Chemiluminescenceinduced by opsonizedzymosanincreased significantly (P < 0.05) after treatment with bacterial extracts, whereas no significant changes were observed in the fMLP-stimulated PMNs. Long-acting penicillin treatment did not significantly affect zymosanor fMLP-stimulated chemiluminescence. The data suggest that orally administered bacterial extracts can increase the opsonic capacity of serum and thus the bactericidal capacity of PMNs in subjects with recurrent respiratory infections. KEY WORDS: Bacterial extracts; respiratory infections; polymorphonuclear leucocytes; bactericidal capacity; chemiluminescence.
INTRODUCTION
R
ecurrent respiratory infections in early childhood are common clinical
Received for publication 27 August 1991;accepted 10 September 1991. Address for correspondence: Dr A. Kantar, Paediatric Clinic, University of Ancona, Via Corridoni 11,1-60213 Ancona, Italy.
© Copyright 1991 by Cambridge Medical Publications Ltd
problems that face paediatricians during the winter months.' The majority of children with recurrent infections have no defects in host defence or detectable abnormalities predisposing to infection and several environmental factors are believed to increase the susceptibility to infection including exposure to cigarette smoking in the household, chemical pollutants, or 451
A. Kantar, N. Oggiano, G.G. Romagnoni et at.
continuous contact with infection in the home or the daycare setting. I - 3 Chemoprophylaxis, immunoprophylaxis and modification of certain environmental factors are widely used to prevent recurrent respiratory infections.':" Recent studies have demonstrated the efficacy of various bacterial extracts that can stimulate the immune system in preventing these recurrent infections.v Lln the present study, a luminol-amplified chemiluminescence assay of polymorphonuclear leucocytes (PMNs) was employed to investigate the effect of an orally administered bacterial extract on the bactericidal capacity of PMNs in children with recurrent respiratory infections. The preparation used was a bacterial derivative, Immucytal" tablets, resulting from the association of membrane glycoprotein fractions extracted from Klebsiella pneumoniae and four ribosomal extracts from the bacteria most commonly responsible for respiratory infections (K. pneumoniae, Diplococcus pneumoniae, Haemophilus influenzae and Streptococcus pyogenes group A).8.9 PATIENTS AND METHODS
Patients In total, 37 children with recurrent respiratory infections were initially included in the study after appropriate informed consent had been obtained; however, three children were subsequently dropped because of poor compliance. There was no evidence of any congenital respiratory or immunological abnormalities in the children based on laboratory studies that evaluated humoral, cellular and mucosal defences, sweat electrolyte concentrations and serum (Xl-antitrypsin concentrations. Treatment The children were assigned to active or Immucytal" is a registered tradename of Simes SpA. Italy.
452
control treatment: 10 males and six females (mean age 3.7 ± 0.7 years, range 2.4 - 5.2 years) received bacterial extracts; 12 males and six females (mean age 3.8 ± 0.7 years, range 2.6 - 5.2 years) received control treatment. The active treatment group received tablets of the bacterial extracts according to the following schedule: month 1, bacterial extracts for 14 days, no treatment for 7 days and extracts for 7 days; months 2, 3 and 4, extracts for 14 days and no treatment for 14 days. The control group was treated with 600 000 IV benzathine penicillin injected intramuscularly once every 14 days for 16 weeks. Parents were instructed to record all the episodes of respiratory tract infections and any drugs received during the study period.
Laboratory assays In the absence of any signs of acute infection and after overnight fasting, prior to treatment erythrocyte sedimentation rate, differential white cell count in whole blood, immunoglobins (Ig) A, M and G, and the complement factors C3 and C4 were measured. A 3.5 ml sample of heparinized blood was used for chemiluminescence measurements. The PMNs were isolated using a Mono-Poly Resol ving Medium (ICN Biomedicals, UK) as previously described 10 and cells were resuspended in Krebs - Ringer phosphate solution containing 1 mg/ml glucose. A reaction mixture of 104 cells and 50 nmol/ml lumino1 (Sigma, USA) in 5 ml Krebs - Ringer phosphate plus glucose was prepared and the basal chemiluminescence was measured immediately in a LKB RackBeta 1211 beta counter (LKB, Wallac, Finland) as previously described." The PMNs were activated by the addition of 10 IlM N-formyl-methionyl-leucyl-phenylalanine (fMLP, Sigma, USA) or 3.0 mg/ml zymosan (Sigma, USA). The increase in chemiluminescence was monitored and the peak value was determined as previously described; 11,12 results were expressed as counts/min.
Bacterial extracts and bactericidal capacity
The evaluations were repeated following 16 weeks of treatment with oral bacterial extracts or long-acting penicillin in the active and control groups, respectively.
Statistical analysis The significance of the values obtained was calculated using the Mann-Whitney V-test and Student's t-test. RESULTS A decrease in the frequency of respiratory tract infections in the children treated with bacterial extracts with respect to the control group was observed during the period of treatment: four to six episodes in control group compared with zero to three in bacterial extracts-treated group. The use of antibiotics (ampicillin, cefaclor, cephazolin, erythromycin, or co-trimoxazole) in the treatment of these episodes was higher in
the control group: three to five antibiotics used in control group compared with zero to three in bacterial extracts-treated group. The serum concentrations of IgM, C3 and C4 in both treatment groups showed no marked alterations from the normal ranges. In the children treated with the bacterial extracts, serum concentrations of IgG (900 ± 165 mg/dl before compared with 1600 ± 250 mg/dl after therapy) and IgA (120 ± 47 mg/dl before compared with 150 ± 27 mg/dl after therapy) were significantly (P < 0.05) increased. Prior to treatment, there was no significant difference in the chemiluminescence of PMNs stimulated with either zymosan or fMLP between the two treatment groups. A typical luminal-amplified chemiluminescence of PMNs stimulated with zymosan is shown in Fig. 1. Figure 2 demonstrates peak chemiluminescence values of PMNs
3000 ?
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v: a;
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..c
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0
0
30
60
90
Time (min)
Fig. 1. Luminol- (50 nmol/ml) amplified chemiluminescence of 104 polymorphonuclear leucocytes stimulated with 3.0 mg/ml zymosan (the arrow indicates activation).
453
A. Kantar, N. Oggiano, G.G. Romagnoni et al.
•
Basal
E;j Zymosan-stimulated
D
3000
tMLP-stimulated
=
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Fig. 2. Mean (± SD) basal and peak 3 mg/ml zymosan- and 10 11M N.formyl-methionyl-Ieucylphenylalanine- (fMLP·) stimulated 50 nmol/ml luminol-amplified chemiluminescence of polymorphonuclear leucocytes from children with recurrent respiratory infections before and after treatment with oral bacterial extracts for 16 weeks.
3000
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Basal
E;j Zymosan-stimulated
=
D
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tMLP-stimulated
;::l
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b
2000
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Q)
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After
Fig. 3. Mean (± SD) basal and peak 3 mg/ml zymosan- and 10 11M N-formyl.methionyl-Ieucylphenylalanine- (fMLP-) stimulated 50 nmol/ml luminol-amplified chemiluminescence of polymorphonuclear leucocytes from children with recurrent respiratory infections before and after intramuscular treatment with 600000 IV benzathine penicillin every 14 days for 16 weeks.
454
Bacterial extracts and bactericidal capacity
stimulated with zymosan or tMLP in patients before and after treatment with bacterial extracts. A significant (P < 0.05) increase in chemiluminescence of PMNs stimulated with zymosan was observed after treatment: peak values were (1475 ± 202) x 103 counts/min before treatment compared with (2550 ± 220) x 103 counts/min after 16 weeks' treatment. Figure 3 shows chemiluminescence of PMNs stimulated with either zymosan or tMLP in the children treated with the long-acting penicillin; no significant changes in peak chemiluminescence values were observed. DISCUSSION
Antimicrobial mechanisms of PMNs are now classified as either oxygenindependent or oxygen-dependent. The oxygen-dependent mechanisms are expressed when PMNs undergo the respiratory burst that results from the activation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) - oxidase system." This enzyme system is responsible for generating superoxide anions (02-) at the expense of NADPW 3, I4 and 02- formed dismutes rapidly to produce hydrogen peroxide, the 02- and hydrogen peroxide being starting materials for the production of powerful microbicidal oxidants of phagocytosis." - 15 These include hypochlorite produced by the myeloperoxidase - hydrogen peroxide - halide reaction, chloramines and hydroxyl radicals. The importance of oxygen-dependent antimicrobial activities is illustrated by the greatly increased susceptibility to infections in patients with chronic granulomatous disease, a genetic defect in the oxidase system. 16 A remarkably wide variety of physiological substances are capable of activating the respiratory burst, among which are the Fe region ofIgG, C3b fragment and chemotactic tMLP, as well as various inflammatory mediators such as leucotrienes and platelet activating factor. 10.15.17
An important recognition-conferring mechanism is the ability of serum to coat objects with specific proteins, i.e. opsonization. The best characterized opsonization systems of serum are immunoglobulins and the complement system, which work by binding the bacteria so that they can be subsequently bound and ingested by phagocytes. The practice of incubating zymosan with fresh serum produces opsonized zymosan which is coated with properdin, C3 fragments and serum immunoglobulins. These particles, therefore, display ligands for PMNs receptors and activate the respiratory burst. 19The respiratory burst of PMNs can be monitored by measuring oxygen consumption, superoxide dismutase-inhibitable ferricytochrome c reduction, oxidation of radiolabelled glucose, or chemiluminescence assay.":" In order to demonstrate chemiluminescence, a reaction has to yield a product in an electronically excited state that on returning to ground state produces light. Luminol is one of the most commonly used chemiluminescence reagents" and phagocytosis-induced luminol-amplified chemiluminescence is dependent upon a myeloperoxidase-mediated reaction." The results of the present study demonstrated that chemiluminescence in PMN s induced by opsonized zymosan increased significantly in children with recurrent respiratory infections treated with the bacterial extracts, but no significant changes were observed in tMLP-activated PMN s after treatment with bacterial extracts or long-acting penicillin compared with before treatment. These data suggest an increase in the opsonic capacity of serum with no changes in the tMLP-induced activation ofthe respiratory burst. It seems likely that the increase in the opsonic capacity is one of the mechanisms responsible for the efficacy of bacterial extracts in the prevention of recurrent respiratory infections in children. 455
A. Kantar, N. Oggiano, G.G. Romagnoni et al. REFERENCES 1. Rubin BK: The evaluation of the child with recurrent chest infections. Pediatr Infect Dis J 1985; 4: 88 - 98. 2. Chern Y,Li W, YuS:Influenceofpassivesmoking for respiratory illness in early childhood. Br Med J 1986; 293: 303 - 306. 3. De Martino M, Vierucci A, Appendino C, et aL: II bambino con infezioni respiratorie ricorrenti. ImmunoL Pediatr 1981; 1: 76 -79. 4. Marcy SM: Prevention of respiratory infections. Pediatr Infect Dis J 1985; 4: 442 - 446. 5. Fiocchi A, Arancio R, Cinquepalmi P, et aL: Recurrent respiratory infections in childhood: experience with a bacterial extract plus bacterial ribosomes (Immucytal'"). J Int Med Res 1990; 18: 50-60. 6. Puigdollers JM, Rodesserna G, Hernandez Del Rey I, et aL: Immunoglobulin production in man stimulated by orally administered bacterial lysate (Broncho-Vaxom). Respiration 1980; 40: 142145. 7. Bousquet J, Dussourd d'Hinteriand L: Prevention de I'infection bacterienne chronique des voies respiratoires par un vaccin ribosomal. Mid Hyg 1982; 40: 146 - 157. 8. Dussourd d'Hinterland L, Serre H, Normier G: Preparation de vaccins a base de fractions ribosomiales antigeniques. Brevet Pierre Fabre SA, depose Ie 10.12.1973; no. 73.43957. 9. Dussourd d'Hinteriand L, Normier G, Pinel AM, et al: Preparation de vaccins a base d' acides nucleiques associes a des polysaccarides extraits de germes microbiens. Brevet Pierre Fabre SA, depose Ie 2.4.1975; no. 75.10252. 10. Kantar A, WilkinsG, Swoboda B, etal: Alterations of the respiratory burst of polymorphonuclear leukocytes from diabetic children. Acta Paediatr Scand 1990; 79: 535 - 541. II. Fiorini R, Curatola G, Bertoli E, et al: Changes of fluorescence anisotropy in plasma membrane of human polymorphonuclear leukocytes during the respiratory burst phenomenon. FEBS Lett 1990; 273: 122 - 126.
456
12. Kantar A, Giorgi PL, Bertoli E, et al: Luminolchemiluminescence assay for the evaluation of the effect of antibiotics on the microbicidal activities of polymorphonuclear leukocytes. Pharmacol (Life Sci Adv) 1990; 9: 483 - 488. 13. Babior BM: The respiratory burst of phagocytes. J Clin Invest 1984; 73: 599 - 601. 14. Rossi F: The O 2- forming NADPH oxidase of the phagocytes: nature, mechanisms of activation and function. Biochim Biophys Acta 1986; 853: 65 89. 15. Baggiolini M, Wymann MP: Turning on the respiratory burst. Trends Biochem Sci 1990; 15: 69 -72. 16. Smith RM, Curnutte JT: Molecular basis of chronic granulomatous disease. Blood 1991; 77: 673 686. 17. Babior BM: The respiratory burst oxidase. Trends Biochem Sci 1987; 12: 241 - 243. 18. Cohen HJ, Newburger PE, Chovaniec ME, et al: Opsonized zymosan-stimulated granulocytes activation and activity of the superoxidegenerating system and membrane potential changes. Blood 1981; 58: 975 - 982. 19. Goldstein 1M, Roos D, Kaplan HB, et al: Complement and immunoglobulins stimulate superoxide production by human leukocytes independently of phagocytosis. J Clin Invest 1975; 56: 1155 - 1163. 20. Babior BM, Cohen HJ: Measurement of neutrophil function: phagocytosis, degranulation, the respiratory burst and bacterial killing. In: Leukocyte Function (Cline MJ, ed). New York: Churchill Livingstone, 1981; pp I - 38. 21. Allen RC, Stjernholm RL, Steel RH: Evidence for the generation of an electronic excitation state(s) in human polymorphonuclear leukocytes and its participation in bactericidal activity. Biochem Biophys Res Commun 1972; 47: 679 - 684. 22. Dahlgren C, Stendahl 0: Role of myeloperoxidase in luminol-dependent chemiluminescence of polymorphonuclear leukocytes. Infect Immun 1983; 39: 736 -741.
Effect of Oral Administration of Bacterial Extracts on the Bactericidal Capacity of Polymorphonuclear Leucocytes in Children with Recurrent Respiratory Infections A. Kantar', N. Oggiano', G.G. RomagnonF and P.L. GiorgP 'Paediatric Clinic, University ofAncona, Ancona, Italy; 2Medical Direction, Simes SpA, Bresso-Milan, Italy
The effect oforally administered hacterial extracts given intermittently over 16 weeks on the bactericidal capacity of polymorphonuclear leucocytes (PMNs) in children with recurrent respiratory infections was investigated using a luminol-amplified chemiluminescence assay. Chemiluminescence of PMNs stimulated with zymosan or N.formylmethionyl-Ieucyl-phenylalanine (fMLP) before and after treatment with bacterial extracts or intramuscular benzanthine penicillin was evaluated. Chemiluminescenceinduced by opsonizedzymosanincreased significantly (P < 0.05) after treatment with bacterial extracts, whereas no significant changes were observed in the fMLP-stimulated PMNs. Long-acting penicillin treatment did not significantly affect zymosanor fMLP-stimulated chemiluminescence. The data suggest that orally administered bacterial extracts can increase the opsonic capacity of serum and thus the bactericidal capacity of PMNs in subjects with recurrent respiratory infections. KEY WORDS: Bacterial extracts; respiratory infections; polymorphonuclear leucocytes; bactericidal capacity; chemiluminescence.
INTRODUCTION
R
ecurrent respiratory infections in early childhood are common clinical
Received for publication 27 August 1991;accepted 10 September 1991. Address for correspondence: Dr A. Kantar, Paediatric Clinic, University of Ancona, Via Corridoni 11,1-60213 Ancona, Italy.
© Copyright 1991 by Cambridge Medical Publications Ltd
problems that face paediatricians during the winter months.' The majority of children with recurrent infections have no defects in host defence or detectable abnormalities predisposing to infection and several environmental factors are believed to increase the susceptibility to infection including exposure to cigarette smoking in the household, chemical pollutants, or 451
A. Kantar, N. Oggiano, G.G. Romagnoni et at.
continuous contact with infection in the home or the daycare setting. I - 3 Chemoprophylaxis, immunoprophylaxis and modification of certain environmental factors are widely used to prevent recurrent respiratory infections.':" Recent studies have demonstrated the efficacy of various bacterial extracts that can stimulate the immune system in preventing these recurrent infections.v Lln the present study, a luminol-amplified chemiluminescence assay of polymorphonuclear leucocytes (PMNs) was employed to investigate the effect of an orally administered bacterial extract on the bactericidal capacity of PMNs in children with recurrent respiratory infections. The preparation used was a bacterial derivative, Immucytal" tablets, resulting from the association of membrane glycoprotein fractions extracted from Klebsiella pneumoniae and four ribosomal extracts from the bacteria most commonly responsible for respiratory infections (K. pneumoniae, Diplococcus pneumoniae, Haemophilus influenzae and Streptococcus pyogenes group A).8.9 PATIENTS AND METHODS
Patients In total, 37 children with recurrent respiratory infections were initially included in the study after appropriate informed consent had been obtained; however, three children were subsequently dropped because of poor compliance. There was no evidence of any congenital respiratory or immunological abnormalities in the children based on laboratory studies that evaluated humoral, cellular and mucosal defences, sweat electrolyte concentrations and serum (Xl-antitrypsin concentrations. Treatment The children were assigned to active or Immucytal" is a registered tradename of Simes SpA. Italy.
452
control treatment: 10 males and six females (mean age 3.7 ± 0.7 years, range 2.4 - 5.2 years) received bacterial extracts; 12 males and six females (mean age 3.8 ± 0.7 years, range 2.6 - 5.2 years) received control treatment. The active treatment group received tablets of the bacterial extracts according to the following schedule: month 1, bacterial extracts for 14 days, no treatment for 7 days and extracts for 7 days; months 2, 3 and 4, extracts for 14 days and no treatment for 14 days. The control group was treated with 600 000 IV benzathine penicillin injected intramuscularly once every 14 days for 16 weeks. Parents were instructed to record all the episodes of respiratory tract infections and any drugs received during the study period.
Laboratory assays In the absence of any signs of acute infection and after overnight fasting, prior to treatment erythrocyte sedimentation rate, differential white cell count in whole blood, immunoglobins (Ig) A, M and G, and the complement factors C3 and C4 were measured. A 3.5 ml sample of heparinized blood was used for chemiluminescence measurements. The PMNs were isolated using a Mono-Poly Resol ving Medium (ICN Biomedicals, UK) as previously described 10 and cells were resuspended in Krebs - Ringer phosphate solution containing 1 mg/ml glucose. A reaction mixture of 104 cells and 50 nmol/ml lumino1 (Sigma, USA) in 5 ml Krebs - Ringer phosphate plus glucose was prepared and the basal chemiluminescence was measured immediately in a LKB RackBeta 1211 beta counter (LKB, Wallac, Finland) as previously described." The PMNs were activated by the addition of 10 IlM N-formyl-methionyl-leucyl-phenylalanine (fMLP, Sigma, USA) or 3.0 mg/ml zymosan (Sigma, USA). The increase in chemiluminescence was monitored and the peak value was determined as previously described; 11,12 results were expressed as counts/min.
Bacterial extracts and bactericidal capacity
The evaluations were repeated following 16 weeks of treatment with oral bacterial extracts or long-acting penicillin in the active and control groups, respectively.
Statistical analysis The significance of the values obtained was calculated using the Mann-Whitney V-test and Student's t-test. RESULTS A decrease in the frequency of respiratory tract infections in the children treated with bacterial extracts with respect to the control group was observed during the period of treatment: four to six episodes in control group compared with zero to three in bacterial extracts-treated group. The use of antibiotics (ampicillin, cefaclor, cephazolin, erythromycin, or co-trimoxazole) in the treatment of these episodes was higher in
the control group: three to five antibiotics used in control group compared with zero to three in bacterial extracts-treated group. The serum concentrations of IgM, C3 and C4 in both treatment groups showed no marked alterations from the normal ranges. In the children treated with the bacterial extracts, serum concentrations of IgG (900 ± 165 mg/dl before compared with 1600 ± 250 mg/dl after therapy) and IgA (120 ± 47 mg/dl before compared with 150 ± 27 mg/dl after therapy) were significantly (P < 0.05) increased. Prior to treatment, there was no significant difference in the chemiluminescence of PMNs stimulated with either zymosan or fMLP between the two treatment groups. A typical luminal-amplified chemiluminescence of PMNs stimulated with zymosan is shown in Fig. 1. Figure 2 demonstrates peak chemiluminescence values of PMNs
3000 ?
]v:
e:
= 0 u
2000
'"0
>(
a;
u
c
a; U
v: a;
c
.§ ..=: .§
1000
a;
..c
U
0
0
30
60
90
Time (min)
Fig. 1. Luminol- (50 nmol/ml) amplified chemiluminescence of 104 polymorphonuclear leucocytes stimulated with 3.0 mg/ml zymosan (the arrow indicates activation).
453
A. Kantar, N. Oggiano, G.G. Romagnoni et al.
•
Basal
E;j Zymosan-stimulated
D
3000
tMLP-stimulated
=
~~
;::l
0
u M
-
2000
b
>< Q)
u
cQ)
u
Vl Q)
c
.§
1000
..=
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0
Before
After
Fig. 2. Mean (± SD) basal and peak 3 mg/ml zymosan- and 10 11M N.formyl-methionyl-Ieucylphenylalanine- (fMLP·) stimulated 50 nmol/ml luminol-amplified chemiluminescence of polymorphonuclear leucocytes from children with recurrent respiratory infections before and after treatment with oral bacterial extracts for 16 weeks.
3000
•
Basal
E;j Zymosan-stimulated
=
D
~c
tMLP-stimulated
;::l
0
u M
b
2000
>< Q)
u
C
Q)
u
Vl Q)
c .§ 1000
..=
'§ Q) ..c U
0
Before
After
Fig. 3. Mean (± SD) basal and peak 3 mg/ml zymosan- and 10 11M N-formyl.methionyl-Ieucylphenylalanine- (fMLP-) stimulated 50 nmol/ml luminol-amplified chemiluminescence of polymorphonuclear leucocytes from children with recurrent respiratory infections before and after intramuscular treatment with 600000 IV benzathine penicillin every 14 days for 16 weeks.
454
Bacterial extracts and bactericidal capacity
stimulated with zymosan or tMLP in patients before and after treatment with bacterial extracts. A significant (P < 0.05) increase in chemiluminescence of PMNs stimulated with zymosan was observed after treatment: peak values were (1475 ± 202) x 103 counts/min before treatment compared with (2550 ± 220) x 103 counts/min after 16 weeks' treatment. Figure 3 shows chemiluminescence of PMNs stimulated with either zymosan or tMLP in the children treated with the long-acting penicillin; no significant changes in peak chemiluminescence values were observed. DISCUSSION
Antimicrobial mechanisms of PMNs are now classified as either oxygenindependent or oxygen-dependent. The oxygen-dependent mechanisms are expressed when PMNs undergo the respiratory burst that results from the activation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) - oxidase system." This enzyme system is responsible for generating superoxide anions (02-) at the expense of NADPW 3, I4 and 02- formed dismutes rapidly to produce hydrogen peroxide, the 02- and hydrogen peroxide being starting materials for the production of powerful microbicidal oxidants of phagocytosis." - 15 These include hypochlorite produced by the myeloperoxidase - hydrogen peroxide - halide reaction, chloramines and hydroxyl radicals. The importance of oxygen-dependent antimicrobial activities is illustrated by the greatly increased susceptibility to infections in patients with chronic granulomatous disease, a genetic defect in the oxidase system. 16 A remarkably wide variety of physiological substances are capable of activating the respiratory burst, among which are the Fe region ofIgG, C3b fragment and chemotactic tMLP, as well as various inflammatory mediators such as leucotrienes and platelet activating factor. 10.15.17
An important recognition-conferring mechanism is the ability of serum to coat objects with specific proteins, i.e. opsonization. The best characterized opsonization systems of serum are immunoglobulins and the complement system, which work by binding the bacteria so that they can be subsequently bound and ingested by phagocytes. The practice of incubating zymosan with fresh serum produces opsonized zymosan which is coated with properdin, C3 fragments and serum immunoglobulins. These particles, therefore, display ligands for PMNs receptors and activate the respiratory burst. 19The respiratory burst of PMNs can be monitored by measuring oxygen consumption, superoxide dismutase-inhibitable ferricytochrome c reduction, oxidation of radiolabelled glucose, or chemiluminescence assay.":" In order to demonstrate chemiluminescence, a reaction has to yield a product in an electronically excited state that on returning to ground state produces light. Luminol is one of the most commonly used chemiluminescence reagents" and phagocytosis-induced luminol-amplified chemiluminescence is dependent upon a myeloperoxidase-mediated reaction." The results of the present study demonstrated that chemiluminescence in PMN s induced by opsonized zymosan increased significantly in children with recurrent respiratory infections treated with the bacterial extracts, but no significant changes were observed in tMLP-activated PMN s after treatment with bacterial extracts or long-acting penicillin compared with before treatment. These data suggest an increase in the opsonic capacity of serum with no changes in the tMLP-induced activation ofthe respiratory burst. It seems likely that the increase in the opsonic capacity is one of the mechanisms responsible for the efficacy of bacterial extracts in the prevention of recurrent respiratory infections in children. 455
A. Kantar, N. Oggiano, G.G. Romagnoni et al. REFERENCES 1. Rubin BK: The evaluation of the child with recurrent chest infections. Pediatr Infect Dis J 1985; 4: 88 - 98. 2. Chern Y,Li W, YuS:Influenceofpassivesmoking for respiratory illness in early childhood. Br Med J 1986; 293: 303 - 306. 3. De Martino M, Vierucci A, Appendino C, et aL: II bambino con infezioni respiratorie ricorrenti. ImmunoL Pediatr 1981; 1: 76 -79. 4. Marcy SM: Prevention of respiratory infections. Pediatr Infect Dis J 1985; 4: 442 - 446. 5. Fiocchi A, Arancio R, Cinquepalmi P, et aL: Recurrent respiratory infections in childhood: experience with a bacterial extract plus bacterial ribosomes (Immucytal'"). J Int Med Res 1990; 18: 50-60. 6. Puigdollers JM, Rodesserna G, Hernandez Del Rey I, et aL: Immunoglobulin production in man stimulated by orally administered bacterial lysate (Broncho-Vaxom). Respiration 1980; 40: 142145. 7. Bousquet J, Dussourd d'Hinteriand L: Prevention de I'infection bacterienne chronique des voies respiratoires par un vaccin ribosomal. Mid Hyg 1982; 40: 146 - 157. 8. Dussourd d'Hinterland L, Serre H, Normier G: Preparation de vaccins a base de fractions ribosomiales antigeniques. Brevet Pierre Fabre SA, depose Ie 10.12.1973; no. 73.43957. 9. Dussourd d'Hinteriand L, Normier G, Pinel AM, et al: Preparation de vaccins a base d' acides nucleiques associes a des polysaccarides extraits de germes microbiens. Brevet Pierre Fabre SA, depose Ie 2.4.1975; no. 75.10252. 10. Kantar A, WilkinsG, Swoboda B, etal: Alterations of the respiratory burst of polymorphonuclear leukocytes from diabetic children. Acta Paediatr Scand 1990; 79: 535 - 541. II. Fiorini R, Curatola G, Bertoli E, et al: Changes of fluorescence anisotropy in plasma membrane of human polymorphonuclear leukocytes during the respiratory burst phenomenon. FEBS Lett 1990; 273: 122 - 126.
456
12. Kantar A, Giorgi PL, Bertoli E, et al: Luminolchemiluminescence assay for the evaluation of the effect of antibiotics on the microbicidal activities of polymorphonuclear leukocytes. Pharmacol (Life Sci Adv) 1990; 9: 483 - 488. 13. Babior BM: The respiratory burst of phagocytes. J Clin Invest 1984; 73: 599 - 601. 14. Rossi F: The O 2- forming NADPH oxidase of the phagocytes: nature, mechanisms of activation and function. Biochim Biophys Acta 1986; 853: 65 89. 15. Baggiolini M, Wymann MP: Turning on the respiratory burst. Trends Biochem Sci 1990; 15: 69 -72. 16. Smith RM, Curnutte JT: Molecular basis of chronic granulomatous disease. Blood 1991; 77: 673 686. 17. Babior BM: The respiratory burst oxidase. Trends Biochem Sci 1987; 12: 241 - 243. 18. Cohen HJ, Newburger PE, Chovaniec ME, et al: Opsonized zymosan-stimulated granulocytes activation and activity of the superoxidegenerating system and membrane potential changes. Blood 1981; 58: 975 - 982. 19. Goldstein 1M, Roos D, Kaplan HB, et al: Complement and immunoglobulins stimulate superoxide production by human leukocytes independently of phagocytosis. J Clin Invest 1975; 56: 1155 - 1163. 20. Babior BM, Cohen HJ: Measurement of neutrophil function: phagocytosis, degranulation, the respiratory burst and bacterial killing. In: Leukocyte Function (Cline MJ, ed). New York: Churchill Livingstone, 1981; pp I - 38. 21. Allen RC, Stjernholm RL, Steel RH: Evidence for the generation of an electronic excitation state(s) in human polymorphonuclear leukocytes and its participation in bactericidal activity. Biochem Biophys Res Commun 1972; 47: 679 - 684. 22. Dahlgren C, Stendahl 0: Role of myeloperoxidase in luminol-dependent chemiluminescence of polymorphonuclear leukocytes. Infect Immun 1983; 39: 736 -741.
