Concise Communications
248
13.
14.
15.
16.
17.
18.
surement of the sensitivity of herpes simplex viruses to antiviral agents. Antiviral Res 1983;3:223-234 Martin JL, Ellis MN, Keller PM, Biron KK, Lehrman SN, Barry OW, Furman PA. Plaque autoradiography assay for the detection and quantitation of thymidine kinase-deficient and thymidine kinase-altered mutants of herpes simplex virus in clinical isolates. Antimicrob Agents Chemother 1985;28:181-187 Parker AC, Craig JIO, Collins P, Oliver N, Smith 1. Acyclovir-resistant herpes simplex virus infection due to altered DNA polymerase [letter]. Lancet 1987;2:1461 Ellis MN, Keller PM, Fyfe JA, Martin JL, Rooney JF, Straus SE, Nusinoff Lehrman 5, Barry Ow. Clinical isolate of herpes simplex virus type 2 that induces a thymidine kinase with altered substrate specificity. Antimicrob Agents Chemother 1987;31:1117-1125 Field H, McMillan A, Darby G. The sensitivity of acyclovir-resistant mutants of herpes simplex virus to other antiviral drugs. J Infect Dis 1981;143:281-285 Whitley RJ, Spruance S, Hayden FG, Overall J, Alford CA Jr, Gwaltney JM Jr, Soong SJ, the NIAID Collaborative Antiviral Study Group. Vidarabine therapy for mucocutaneous herpes simplex virus infections in the immunocompromised host. J Infect Dis 1984;149:1-8 Chatis PA, Miller CH, Schrager LE, Crumpacker CS. Successful treatment with Foscarnet of an acyclovir-resistant mucocutaneous infection with herpes simplex virus in a patient with acquired immunodeficiency syndrome. N Engl J Med 1989;320:297-300
C4B Deficiency: A Risk Factor for Bacteremia with Encapsulated Organisms Nancy A. Bishof, Thomas R. Welch, and Linda S. Beischel
From the Division of Nephrology, Children's Hospital Medical Center, Cincinnati, Ohio
The fourth component of complement (C4) is crucial to the activation of the classical complement pathway, a key defense against invading microorganisms. The two isotypes of C4, C4A and C4B, have very different in vitro activities. An increased incidence of total C4B deficiency was found in white patients with Streptococcus pneumoniae, Haemophilus influenzae, or Neisseria meningitidis infection (14% of bacteremic children vs. 2% of race-matched controls, P;= .02). In black patients, however,there was no difference in incidence of C4B deficiency between bacteremic patients and race-matched controls (7% and 5%, respectively, P> .5). These data suggest that, at least in whites, total C4B deficiency is a risk factor for invasive disease with these three encapsulated organisms.
Activation of the complement system, a central event in host defense, produces chemoattractants and anaphylatoxins, prepares microorganisms for opsonization by depositing C3b on their surfaces, and causes direct lysis of some organisms [1].
Received 25 September 1989; revised 22 January 1990. The study was approved by the Institutional Review Board of Children's Hospital Medical Center. Reprints and correspondence: Dr. Thomas R. Welch, Children's Hospital Medical Center, Division of Nephrology, EIland & Bethesda Avenues, Cincinnati, OH 45229-2899. The journal of Infectious Diseases 1990;162:248-250 © 1990 by The University of Chicago. All rights reserved. 0022-1899/90/6201-0039$01.00
C4 plays a key role in antibody-mediated complement activation by forming part of the classical pathway C3 convertase [1]. Most individuals have two C4 isotypes, C4A and C4B. These isotypes are the products of two distinct genetic loci on chromosome 6. Both are recognized by polyclonal C4 antisera, sothey are not distinguishable in measurements of serum complement levels. Both types bind to surfaces after activation to C4b reveals a reactive thiolester group. C4A preferentially attaches to amino-rich surfaces through an amide linkage, while C4B more avidly forms an ester linkage with hydroxyl-containing carbohydrate surfaces [2]. Since only bound C4b is capable of forming an efficient C3 convertase, this attachment is crucial for further complement activation [1].
Downloaded from http://jid.oxfordjournals.org/ at University of Regina on September 7, 2015
5. Crumpacker CS, Schnipper LE, Marlowe 51, Kowalsky PN, Hershey BJ, Levin MJ. Resistance to antiviral drugs of herpes simplex virus isolated from a patient treated with acyclovir. N Engl J Med 1982; 306:343-346 6. Wade JC, McLaren C, Meyers JD. Frequency and significance of acyclovirresistant herpes simplex virus isolated from marrow transplant patients receiving multiple courses of treatment with acyclovir. J Infect Dis 1983;148:1077-1082 7. Erlich KS, Mills J, Chatis P, Mertz GJ, Busch OF, Follansbee SE, Grant RM, Crumpacker CS. Acyclovir-resistant herpes simplex virus infections in patients with the acquired immunodeficiency syndrome. N Engl J Med 1989;320:293-296 8. Ambinder RF, Bums WH, Lietman PS, Saral R. Prophylaxis: a strategy to minimise antiviral resistance. Lancet 1984;1:1154-1155 9. Sibrack CD, Gutman LT, Wilfert CM, McLaren C, St Clair MH, Keller PM, Barry Ow. Pathogenicity of acyclovir-resistant herpes simplex virus type 1 from an immunodeficient child. J Infect Dis 1982;146: 673-682 10. Field HJ, Darby G. Pathogenicity in mice of strains of herpes simplex virus which are resistant to acyclovir in vitro and in vivo. Antimicrob Agents Chemother 1980;17:209-216 11. Ellis MN, Waters R, Hill EL, Lobe DC, Selleseth OW, Barry Ow. Orofacial infection of athymic mice with defined mixtures of acyclovirsusceptible and acyclovir-resistant herpes simplex virus type 1. Antimicrob Agents Chemother 1989;33:304-310 12. McLaren C, Ellis MN, Hunter GA. A colorimetric assay for the mea-
JID 1990;162 (July)
1ID 1990;162 (July)
Concise Communications
Isolated C4B deficiency has been noted in rv2 % of whites [3]. We reasoned that such deficiency would impair the immune response to infection with carbohydrate-encapsulated bacteria and tested this hypothesis in a group of children undergoing investigation for bacteremia.
Materials and Methods Patients.
Results The incidence of homozygous C4B deficiency in whites who were not bacteremic (control group) was 2 %, similar to previous studies in healthy populations [3]. Similarly, the blacks with negative blood cultures (control group) had an incidence of homozygous C4B deficiency of 7 %, as in a previous population-based study [6]. Of the 50 patients (29 white and 21 black).with blood cultures positive for either S. pneumoniae, H. infiuenzae, or N. meningitidis, five (10%) were homozygous C4B-deficient. Because the frequency of C4B
Table 1. Incidence of C4B deficiency in bacteremic and nonbacteremic children. Patients
Nonbacteremic
Bacteremic
P
2 % (2/100) 7% (7/100)
14% (4/29) 5% (1121)
>.5
White Black
.02
Odds ratio
7.8 0.7
deficiency differed in white and black controls, data were analyzed separately for each group (table 1). Fourteen percent of whites bacteremic with encapsulated organisms (bacteremic group) were C4B-deficientcompared with 2 % of those without bacteremia (P = .02, odds ratio = 7.8). In blacks, however, C4B deficiency was not increased in frequency in bacteremic patients (5% bacteremic, 7% nonbacteremic) . The distribution of organisms causing bacteremia was similar in C4B-deficient and C4B-competent individuals (S. pneumoniae [n = 34], 3 C4B-deficient, 31 C4B-competent; H. infiuenzae [n = 12], 1 C4B-deficient, 11 C4B-competent; N. meningitidis [n = 4], 1 C4B-deficient, 3 C4B-competent). There were no deaths among the infected children. Bacteremic children with C4B deficiency were no more likely than bacteremic C4B-competent children to be hospitalized for their current illness (60 % of C4B-competent vs. 80 % of C4B-deficient, P = .36). Of those children admitted, the length of stay did not differ between the two groups (8.5 days for C4B-competent, 7.8 days for C4B-deficient). The diagnoses of those admitted did not differ significantly between the C4B-competent and -deficient groups. Of the 27 C4Bcompetent children, 13 (48 %) had meningitis, 4 (15%) had periorbital cellulitis, 3 (11 %) had septicemia, 2 (7 %) had epiglottitis, 3 (11 %) had pneumonia, 1 (4 %) had peritonitis, and 1 (4 %) had bilateral otitis media. Of the C4B-deficient patients admitted, 1 each had meningitis, sinusitis, epiglottitis, and septicemia.
Discussion C4, a polymorphic protein, is encoded by two tandem genes linked to the major histocompatibility complex on chromosome 6. Most individuals express four gene products, although various combinations of deletions and duplications exist [7]. Complete C4A or C4B deficiency occurs in rv3% of whites; a single gene product is absent in 20 %- 25 % [3]. C4 is necessary for antibody-mediated complement activation and host defense against invading microorganisms. IgM and IgG antibodies directed against bacterial components lead to complement deposition on the bacterium via the classical pathway. In organisms without a polysaccharide capsule, C3b is deposited directly on the cell wall, and efficient opsonization, as well as formation of the membrane attack complex, occurs. In organisms with a polysaccharide capsule, antibodies directed at the cell wall still direct deposition of C3b onto
Downloaded from http://jid.oxfordjournals.org/ at University of Regina on September 7, 2015
All children undergoing blood culture as part of the emergency room evaluation of fever were eligible for study. Plasma was obtained from 50 consecutive children bacteremic with Streptococcuspneumoniae, Haemophilusinfiuenzae, or Neisseriameningitidis (bacteremic group). In addition, plasma was available from 100 black and 100 white children similarly evaluated whose blood cultures were negative for these three pathogens (control group). Children with known immunodeficiencies were excluded from study. All individuals were children less than 21 years of age. C4 protein typing. C4 typing was performed by the method of Awdeh and Alper [3]. Briefly, plasma samples, which had been stored at -70°C, were incubated with neuraminidase from Clostridium perfringens (type VI; Sigma, S1. Louis) at a concentration of 8 munits/ILl of plasma for 15 h at 4°C with continuous dialysis against 0.1 M phosphate and 5 mM Na 2EDTA, pH 6.8. Desialated plasma samples were then subjected to electrophoresis in 0.75% agarose (Seakem LE; FMC Bio Products, Rockland, ME) using a Tris-glycine-barbital discontinuous buffer system, pH 8.6. Immunofixation was performed by applying 1 ml of goat anti-human C4 (provided by Dr. Robert Wyatt, Memphis) to the gel. After 1 h of incubation, gels were washed in 0.9% NaCI, dried, and stained with 0.25% Coomassie brilliant blue. Homozygous C4B deficiency was confirmed by absence of lysis in a C4 functional overlay [3]. Samples were treated with carboxypeptidase B (type I; Sigma) [4] at 0.5 units/ul of plasma for 30 min at 25°C, then incubated with neuraminidase as above before electrophoresis. The overlay consisted of sheep erythrocytes sensitized with rabbit antibody in 0.6% agarose (Seaplaque; FMC Bio Products) containing veronal buffered saline, 150 ILM Ca'", 5 mM Mg?", and C4-deficient guinea pig serum. Hemolysis was allowed to develop at 37°C for 1-3 h. Statistics. Before.data collection began, the hypothesis to be tested was that a significantly greater frequency of C4B deficiency would be found in children with positive blood cultures than in controls. Data were analyzed by Fisher's exact test [5], and odds ratios were calculated as cross-product ratios.
249
250
Concise Communications
have been found to have an increased frequency of homozygous C4B deficiency [13]. Our data demonstrate yet another specific disease association, that of increased risk of invasive infection with encapsulated organisms in individuals who are C4B-deficient. Acknowledgment We acknowledge the skillful secretarial assistance of Barbara Pieper and the cooperation of the Hematology Laboratory personnel.
References 1. Frank MM. Complement in the pathophysiology of human disease. N Engl J Moo 1987;316:1525-1530 2. Isenman DE, Young JR. Covalent binding properties of the C4A and C4B isotypes of the fourth component of human complement on several Cl-bearing cell surfaces. J Immunol 1986;136:2542-2550 3. Awdeh ZL, Alper CA. Inherited structural polymorphism of the fourth component of human complement. Proc Natl Acad Sci USA 1980; 77:3576-3580 4. Sim E, Cross SJ. Phenotyping of human complement component C4, a class-III HLA antigen. Biochem J 1986;239:763-767 5. Gustafson TL. EPISTAT software, 1984 6. Wilson WA, Perez MC, Armatis PE. Partial C4A deficiency is associated with susceptibility to systemic lupus erythematosus in black Americans. Arthritis Rheum 1988;31:1171-1175 7. Yu CY, Campbell RD. Definitive RFLPs to distinguish between the human complement C4A/C4B isotypes and the major Rodgers/Chido determinants: application to the study of C4 null alleles. Immunogenetics 1987;25:383-390 8. Joiner KA, Brown EJ, Frank MM. Complement and bacteria: chemistry and biology in host defense. Annu Rev ImmunoI1984;2:461-491 9. Rowe PC, McLean RH, Wood RA, Leggiadro RI, Winkel stein JA. Association of homozygous C4B deficiency with bacterial meningitis. J Infect Dis 1989;160:448-451 10. Ross SC, Densen P. Complement deficiency states and infection: epidemiology, pathogenesis and consequences of neisserial and other infections in an immune deficiency. Medicine 1984;63:243-273 11. Hauptmann G, Tappeiner G, Schifferli JA. Isherited deficiency of the fourth component of human complement. Immunodeficiency Rev 1988;1:3-22 12. Welch TR, Beischel LS, Balakrishnan K, Quillian M, West CD. Major histocompatibility complex extended haplotypes in systemic lupu: erythematosus. Dis Markers 1988;6:247-255 13. Welch TR, Berry A, Beischel LS. C4 isotype deficiency in IgA nephrop athy. Pediatr Nephrol 1987;1:136-139
Downloaded from http://jid.oxfordjournals.org/ at University of Regina on September 7, 2015
the cell wall through the porous capsule; however, these C3b particles are relatively "hidden" from phagocytes and thus are not efficient opsonins. Therefore, in encapsulated organisms, antibodies directed against capsular determinants provide the most efficient means of opsonization leading to phagocytosis [8]. It is known that activated C4A more efficiently binds to amino groups, while C4B more avidly binds to hydroxyl groups [2]. As all bacterial capsules are composed of polysaccharides rich in hydroxyl groups, it can be hypothesized that C4B would be more important than C4A during assembly of the classical pathway C3 convertase on such surfaces. If this were true, it could be predicted that individuals lacking the C4B isotype would be suboptimally equipped for dealing with encapsulated organisms. Consistent with this prediction is a study by Rowe et al. [9], who examined 46 children with bacterial meningitis for complement deficiencies and found an increased frequency of C4B deficiency. We chose three common encapsulated childhood pathogens and studied the association between C4B deficiency and bacteremia with these organisms. In whites, the hypothesis was confirmed in that the odds ratio for bacteria in children with C4B deficiency was significantly >1 (1.1-51.0).This observation was not made in blacks. Quite possibly, other factors predisposing to bacteremia in the black population (e.g., hemoglobinopathies) were more important, thus diluting any contribution from C4 dysfunction. Various complement deficiencies have been associated with an increased risk of infection [10]. For instance, individuals with deficiencies of any of the terminal complement pathway components or properdin have an increased incidence of neisserial infection, while those with C3 deficiency have an increased incidence of bacterial infections. Complete C4 deficiency is an exceedinglyrare condition, with rv20 reported cases [11], and is associated with a lupus-like syndrome and an increased risk of infection. With the advent of C4 protein typing and the discovery that isotypic (C4A or C4B) deficiencies are fairly common, further specific disease associations became apparent. An increased frequency of C4A null genes in systemic lupus erythematosus has been reported by several investigators [12]; patients with the immune glomerulopathy IgA nephropathy
JID 1990;162 (July)
248
13.
14.
15.
16.
17.
18.
surement of the sensitivity of herpes simplex viruses to antiviral agents. Antiviral Res 1983;3:223-234 Martin JL, Ellis MN, Keller PM, Biron KK, Lehrman SN, Barry OW, Furman PA. Plaque autoradiography assay for the detection and quantitation of thymidine kinase-deficient and thymidine kinase-altered mutants of herpes simplex virus in clinical isolates. Antimicrob Agents Chemother 1985;28:181-187 Parker AC, Craig JIO, Collins P, Oliver N, Smith 1. Acyclovir-resistant herpes simplex virus infection due to altered DNA polymerase [letter]. Lancet 1987;2:1461 Ellis MN, Keller PM, Fyfe JA, Martin JL, Rooney JF, Straus SE, Nusinoff Lehrman 5, Barry Ow. Clinical isolate of herpes simplex virus type 2 that induces a thymidine kinase with altered substrate specificity. Antimicrob Agents Chemother 1987;31:1117-1125 Field H, McMillan A, Darby G. The sensitivity of acyclovir-resistant mutants of herpes simplex virus to other antiviral drugs. J Infect Dis 1981;143:281-285 Whitley RJ, Spruance S, Hayden FG, Overall J, Alford CA Jr, Gwaltney JM Jr, Soong SJ, the NIAID Collaborative Antiviral Study Group. Vidarabine therapy for mucocutaneous herpes simplex virus infections in the immunocompromised host. J Infect Dis 1984;149:1-8 Chatis PA, Miller CH, Schrager LE, Crumpacker CS. Successful treatment with Foscarnet of an acyclovir-resistant mucocutaneous infection with herpes simplex virus in a patient with acquired immunodeficiency syndrome. N Engl J Med 1989;320:297-300
C4B Deficiency: A Risk Factor for Bacteremia with Encapsulated Organisms Nancy A. Bishof, Thomas R. Welch, and Linda S. Beischel
From the Division of Nephrology, Children's Hospital Medical Center, Cincinnati, Ohio
The fourth component of complement (C4) is crucial to the activation of the classical complement pathway, a key defense against invading microorganisms. The two isotypes of C4, C4A and C4B, have very different in vitro activities. An increased incidence of total C4B deficiency was found in white patients with Streptococcus pneumoniae, Haemophilus influenzae, or Neisseria meningitidis infection (14% of bacteremic children vs. 2% of race-matched controls, P;= .02). In black patients, however,there was no difference in incidence of C4B deficiency between bacteremic patients and race-matched controls (7% and 5%, respectively, P> .5). These data suggest that, at least in whites, total C4B deficiency is a risk factor for invasive disease with these three encapsulated organisms.
Activation of the complement system, a central event in host defense, produces chemoattractants and anaphylatoxins, prepares microorganisms for opsonization by depositing C3b on their surfaces, and causes direct lysis of some organisms [1].
Received 25 September 1989; revised 22 January 1990. The study was approved by the Institutional Review Board of Children's Hospital Medical Center. Reprints and correspondence: Dr. Thomas R. Welch, Children's Hospital Medical Center, Division of Nephrology, EIland & Bethesda Avenues, Cincinnati, OH 45229-2899. The journal of Infectious Diseases 1990;162:248-250 © 1990 by The University of Chicago. All rights reserved. 0022-1899/90/6201-0039$01.00
C4 plays a key role in antibody-mediated complement activation by forming part of the classical pathway C3 convertase [1]. Most individuals have two C4 isotypes, C4A and C4B. These isotypes are the products of two distinct genetic loci on chromosome 6. Both are recognized by polyclonal C4 antisera, sothey are not distinguishable in measurements of serum complement levels. Both types bind to surfaces after activation to C4b reveals a reactive thiolester group. C4A preferentially attaches to amino-rich surfaces through an amide linkage, while C4B more avidly forms an ester linkage with hydroxyl-containing carbohydrate surfaces [2]. Since only bound C4b is capable of forming an efficient C3 convertase, this attachment is crucial for further complement activation [1].
Downloaded from http://jid.oxfordjournals.org/ at University of Regina on September 7, 2015
5. Crumpacker CS, Schnipper LE, Marlowe 51, Kowalsky PN, Hershey BJ, Levin MJ. Resistance to antiviral drugs of herpes simplex virus isolated from a patient treated with acyclovir. N Engl J Med 1982; 306:343-346 6. Wade JC, McLaren C, Meyers JD. Frequency and significance of acyclovirresistant herpes simplex virus isolated from marrow transplant patients receiving multiple courses of treatment with acyclovir. J Infect Dis 1983;148:1077-1082 7. Erlich KS, Mills J, Chatis P, Mertz GJ, Busch OF, Follansbee SE, Grant RM, Crumpacker CS. Acyclovir-resistant herpes simplex virus infections in patients with the acquired immunodeficiency syndrome. N Engl J Med 1989;320:293-296 8. Ambinder RF, Bums WH, Lietman PS, Saral R. Prophylaxis: a strategy to minimise antiviral resistance. Lancet 1984;1:1154-1155 9. Sibrack CD, Gutman LT, Wilfert CM, McLaren C, St Clair MH, Keller PM, Barry Ow. Pathogenicity of acyclovir-resistant herpes simplex virus type 1 from an immunodeficient child. J Infect Dis 1982;146: 673-682 10. Field HJ, Darby G. Pathogenicity in mice of strains of herpes simplex virus which are resistant to acyclovir in vitro and in vivo. Antimicrob Agents Chemother 1980;17:209-216 11. Ellis MN, Waters R, Hill EL, Lobe DC, Selleseth OW, Barry Ow. Orofacial infection of athymic mice with defined mixtures of acyclovirsusceptible and acyclovir-resistant herpes simplex virus type 1. Antimicrob Agents Chemother 1989;33:304-310 12. McLaren C, Ellis MN, Hunter GA. A colorimetric assay for the mea-
JID 1990;162 (July)
1ID 1990;162 (July)
Concise Communications
Isolated C4B deficiency has been noted in rv2 % of whites [3]. We reasoned that such deficiency would impair the immune response to infection with carbohydrate-encapsulated bacteria and tested this hypothesis in a group of children undergoing investigation for bacteremia.
Materials and Methods Patients.
Results The incidence of homozygous C4B deficiency in whites who were not bacteremic (control group) was 2 %, similar to previous studies in healthy populations [3]. Similarly, the blacks with negative blood cultures (control group) had an incidence of homozygous C4B deficiency of 7 %, as in a previous population-based study [6]. Of the 50 patients (29 white and 21 black).with blood cultures positive for either S. pneumoniae, H. infiuenzae, or N. meningitidis, five (10%) were homozygous C4B-deficient. Because the frequency of C4B
Table 1. Incidence of C4B deficiency in bacteremic and nonbacteremic children. Patients
Nonbacteremic
Bacteremic
P
2 % (2/100) 7% (7/100)
14% (4/29) 5% (1121)
>.5
White Black
.02
Odds ratio
7.8 0.7
deficiency differed in white and black controls, data were analyzed separately for each group (table 1). Fourteen percent of whites bacteremic with encapsulated organisms (bacteremic group) were C4B-deficientcompared with 2 % of those without bacteremia (P = .02, odds ratio = 7.8). In blacks, however, C4B deficiency was not increased in frequency in bacteremic patients (5% bacteremic, 7% nonbacteremic) . The distribution of organisms causing bacteremia was similar in C4B-deficient and C4B-competent individuals (S. pneumoniae [n = 34], 3 C4B-deficient, 31 C4B-competent; H. infiuenzae [n = 12], 1 C4B-deficient, 11 C4B-competent; N. meningitidis [n = 4], 1 C4B-deficient, 3 C4B-competent). There were no deaths among the infected children. Bacteremic children with C4B deficiency were no more likely than bacteremic C4B-competent children to be hospitalized for their current illness (60 % of C4B-competent vs. 80 % of C4B-deficient, P = .36). Of those children admitted, the length of stay did not differ between the two groups (8.5 days for C4B-competent, 7.8 days for C4B-deficient). The diagnoses of those admitted did not differ significantly between the C4B-competent and -deficient groups. Of the 27 C4Bcompetent children, 13 (48 %) had meningitis, 4 (15%) had periorbital cellulitis, 3 (11 %) had septicemia, 2 (7 %) had epiglottitis, 3 (11 %) had pneumonia, 1 (4 %) had peritonitis, and 1 (4 %) had bilateral otitis media. Of the C4B-deficient patients admitted, 1 each had meningitis, sinusitis, epiglottitis, and septicemia.
Discussion C4, a polymorphic protein, is encoded by two tandem genes linked to the major histocompatibility complex on chromosome 6. Most individuals express four gene products, although various combinations of deletions and duplications exist [7]. Complete C4A or C4B deficiency occurs in rv3% of whites; a single gene product is absent in 20 %- 25 % [3]. C4 is necessary for antibody-mediated complement activation and host defense against invading microorganisms. IgM and IgG antibodies directed against bacterial components lead to complement deposition on the bacterium via the classical pathway. In organisms without a polysaccharide capsule, C3b is deposited directly on the cell wall, and efficient opsonization, as well as formation of the membrane attack complex, occurs. In organisms with a polysaccharide capsule, antibodies directed at the cell wall still direct deposition of C3b onto
Downloaded from http://jid.oxfordjournals.org/ at University of Regina on September 7, 2015
All children undergoing blood culture as part of the emergency room evaluation of fever were eligible for study. Plasma was obtained from 50 consecutive children bacteremic with Streptococcuspneumoniae, Haemophilusinfiuenzae, or Neisseriameningitidis (bacteremic group). In addition, plasma was available from 100 black and 100 white children similarly evaluated whose blood cultures were negative for these three pathogens (control group). Children with known immunodeficiencies were excluded from study. All individuals were children less than 21 years of age. C4 protein typing. C4 typing was performed by the method of Awdeh and Alper [3]. Briefly, plasma samples, which had been stored at -70°C, were incubated with neuraminidase from Clostridium perfringens (type VI; Sigma, S1. Louis) at a concentration of 8 munits/ILl of plasma for 15 h at 4°C with continuous dialysis against 0.1 M phosphate and 5 mM Na 2EDTA, pH 6.8. Desialated plasma samples were then subjected to electrophoresis in 0.75% agarose (Seakem LE; FMC Bio Products, Rockland, ME) using a Tris-glycine-barbital discontinuous buffer system, pH 8.6. Immunofixation was performed by applying 1 ml of goat anti-human C4 (provided by Dr. Robert Wyatt, Memphis) to the gel. After 1 h of incubation, gels were washed in 0.9% NaCI, dried, and stained with 0.25% Coomassie brilliant blue. Homozygous C4B deficiency was confirmed by absence of lysis in a C4 functional overlay [3]. Samples were treated with carboxypeptidase B (type I; Sigma) [4] at 0.5 units/ul of plasma for 30 min at 25°C, then incubated with neuraminidase as above before electrophoresis. The overlay consisted of sheep erythrocytes sensitized with rabbit antibody in 0.6% agarose (Seaplaque; FMC Bio Products) containing veronal buffered saline, 150 ILM Ca'", 5 mM Mg?", and C4-deficient guinea pig serum. Hemolysis was allowed to develop at 37°C for 1-3 h. Statistics. Before.data collection began, the hypothesis to be tested was that a significantly greater frequency of C4B deficiency would be found in children with positive blood cultures than in controls. Data were analyzed by Fisher's exact test [5], and odds ratios were calculated as cross-product ratios.
249
250
Concise Communications
have been found to have an increased frequency of homozygous C4B deficiency [13]. Our data demonstrate yet another specific disease association, that of increased risk of invasive infection with encapsulated organisms in individuals who are C4B-deficient. Acknowledgment We acknowledge the skillful secretarial assistance of Barbara Pieper and the cooperation of the Hematology Laboratory personnel.
References 1. Frank MM. Complement in the pathophysiology of human disease. N Engl J Moo 1987;316:1525-1530 2. Isenman DE, Young JR. Covalent binding properties of the C4A and C4B isotypes of the fourth component of human complement on several Cl-bearing cell surfaces. J Immunol 1986;136:2542-2550 3. Awdeh ZL, Alper CA. Inherited structural polymorphism of the fourth component of human complement. Proc Natl Acad Sci USA 1980; 77:3576-3580 4. Sim E, Cross SJ. Phenotyping of human complement component C4, a class-III HLA antigen. Biochem J 1986;239:763-767 5. Gustafson TL. EPISTAT software, 1984 6. Wilson WA, Perez MC, Armatis PE. Partial C4A deficiency is associated with susceptibility to systemic lupus erythematosus in black Americans. Arthritis Rheum 1988;31:1171-1175 7. Yu CY, Campbell RD. Definitive RFLPs to distinguish between the human complement C4A/C4B isotypes and the major Rodgers/Chido determinants: application to the study of C4 null alleles. Immunogenetics 1987;25:383-390 8. Joiner KA, Brown EJ, Frank MM. Complement and bacteria: chemistry and biology in host defense. Annu Rev ImmunoI1984;2:461-491 9. Rowe PC, McLean RH, Wood RA, Leggiadro RI, Winkel stein JA. Association of homozygous C4B deficiency with bacterial meningitis. J Infect Dis 1989;160:448-451 10. Ross SC, Densen P. Complement deficiency states and infection: epidemiology, pathogenesis and consequences of neisserial and other infections in an immune deficiency. Medicine 1984;63:243-273 11. Hauptmann G, Tappeiner G, Schifferli JA. Isherited deficiency of the fourth component of human complement. Immunodeficiency Rev 1988;1:3-22 12. Welch TR, Beischel LS, Balakrishnan K, Quillian M, West CD. Major histocompatibility complex extended haplotypes in systemic lupu: erythematosus. Dis Markers 1988;6:247-255 13. Welch TR, Berry A, Beischel LS. C4 isotype deficiency in IgA nephrop athy. Pediatr Nephrol 1987;1:136-139
Downloaded from http://jid.oxfordjournals.org/ at University of Regina on September 7, 2015
the cell wall through the porous capsule; however, these C3b particles are relatively "hidden" from phagocytes and thus are not efficient opsonins. Therefore, in encapsulated organisms, antibodies directed against capsular determinants provide the most efficient means of opsonization leading to phagocytosis [8]. It is known that activated C4A more efficiently binds to amino groups, while C4B more avidly binds to hydroxyl groups [2]. As all bacterial capsules are composed of polysaccharides rich in hydroxyl groups, it can be hypothesized that C4B would be more important than C4A during assembly of the classical pathway C3 convertase on such surfaces. If this were true, it could be predicted that individuals lacking the C4B isotype would be suboptimally equipped for dealing with encapsulated organisms. Consistent with this prediction is a study by Rowe et al. [9], who examined 46 children with bacterial meningitis for complement deficiencies and found an increased frequency of C4B deficiency. We chose three common encapsulated childhood pathogens and studied the association between C4B deficiency and bacteremia with these organisms. In whites, the hypothesis was confirmed in that the odds ratio for bacteria in children with C4B deficiency was significantly >1 (1.1-51.0).This observation was not made in blacks. Quite possibly, other factors predisposing to bacteremia in the black population (e.g., hemoglobinopathies) were more important, thus diluting any contribution from C4 dysfunction. Various complement deficiencies have been associated with an increased risk of infection [10]. For instance, individuals with deficiencies of any of the terminal complement pathway components or properdin have an increased incidence of neisserial infection, while those with C3 deficiency have an increased incidence of bacterial infections. Complete C4 deficiency is an exceedinglyrare condition, with rv20 reported cases [11], and is associated with a lupus-like syndrome and an increased risk of infection. With the advent of C4 protein typing and the discovery that isotypic (C4A or C4B) deficiencies are fairly common, further specific disease associations became apparent. An increased frequency of C4A null genes in systemic lupus erythematosus has been reported by several investigators [12]; patients with the immune glomerulopathy IgA nephropathy
JID 1990;162 (July)
