LIST OF ABBREVIATIONS
ABP actin binding protein ADCC antibody-dependent cellular cytotoxicity BPI bactericidal permeability increasing protein C3 complement component 3 CAMP cyclic adenosine monophosphate cfu colony forming unit CGD chronic granulomatous disease CM I cell mediated immunity CR1 complement receptor type 1 CR2 complement receptor type 2 CR3 complement receptor type 3 CR4 complement receptor type 4 CRP C-reactive protein CSF colony stimulating factor CV coefficient of variation DAC diacylglycerol EBV Epstein-Barr virus EGTA ethylene glycol-bis(3-aminoethyl ether)N,N,N',N'-tetraacetic acid E-LAM endothelium leucocyte adhesion molecule ELISA enzyme-1 inked immunosorbent assay FcocR receptor for the Fc portion of IgA FMLP N-formyl-L-methionyl-L-leucyl-L-phenylalanine GABHS group A p-haemolytic streptococcus GBS Group B streptococcus G-PROTEIN guanine nucleotide binding protein G-CSF granulocyte colony stimulating factor GM-CSF granulocyte-monocyte colony stimulating fa c to r HMS hexose monophosphate shunt HNP human neutrophil peptide IC internal control IFNy interferon gamma IgA immunoglobulin A igG immunoglobulin G IL3 interleukin 3 inositol 1,4-5 trisphosphate l5a leucocyte adhesion deficiency LPS 1ipopolysaccharide LTB4 leukotriene B4 mAb monoclonal antibody MBP mannose binding p ro te in min minute MPO myeloperoxidase NA neutrophil antigen NADG N-acetyl-D-glucosamine NADPH nicotinamide adenine dinucleotide phosphate NAP neutrophil activating peptide PA p ro te in A PBS phosphate buffered saline PBSAT phosphate buffered saline-azide-tween PG peptidoglycan PIP- phosphoinositol 4,5-bisphosphate PKC protein kinase C PLC phospholipase C PMN polymorphonuclear leucocyte PNH paroxysmal nocturnal haemogloninuria RBC erythrocyte RCA regulator of complement activation RGD Arg i n i ne-Glyc i ne-Aspartate SAP serum amyloid protein STSS staph ylococcal to x ic shock syndrome TA teichoic acid TNF tumour necrosis factor TNF-oc tumour necrosis factor-alpha TSLS toxic shock-like syndrome WCH Westminster Children's Hospital 3A STATISTICAL ANALYSIS
3A.1 Staphylococcal Inhibition assay
3A.1.1 Reference Range
3A.1.2 Intra-assay variation
3A.1.3 In te r-a ssa y and b io lo g ic a l v a ria tio n
3A.1.4 Analysis of cross-over studies
3A.2 Staphylococcal inhibition assay in the presence of heat inactivated plasma
3A.3 Streptococcal inhibition assay
3A.4 Anti-staphylococcal ELISA
3A.5 Statistical analysis of patients
3A.5.1 Patients with staphylococcal infections
3A.5.1.1 Clinical characteristics of patients with staphylococcal infections
3A .5.1.2 The 27 p a tie n ts w ith S.aureus infections but normal opsonophagocytosis
3A .5.1.3 A comparison of Fc-mediated phagocytosis in patients with S.aureus infections, patients with other infections and normal volunteers
3A .5.1.4 A comparison of anti- S.aureus IgGl and IgG2 an tib o d ie s in p a tie n ts w ith S.aureus infections, patients with other in fe c tio n s and healthy volunteers
3A .5.1.5 Correlation between Fc-mediated phagocytosis and a n t i-S.aureus IgGl and IgG2 subclass an tib o d ie s LIST OF TABLES IN CHAPTER 3A
3A.1 Results showing the intra-assay variation of the staphylococcal inhibition assay 3
3A.2 Results of 13 consecutive tests of the S .aureus inhibition assay using PMNs and plasma from a single healthy volunteer 4
3A.3 A comparison of staphylococcal inhibition using patient plasma in the presence of p a tie n t (PcPp) and co n tro l PMN (NcPp) 7
3A.4 Variation in % inhibition when heat inactivated control plasma was incubated with PMN from 3 donors 8
3A.5 Serial dilution of 4 different broth cultures of S.pyogenes 10
3A.6 Fc-mediated opsonophagocytosis using plasma from patients with S.aureus infections, other infections and healthy normal controls 19
3A.7 A n t i- 5 .aureus to ta l IgG, IgGl and IgG2 le v e ls in patients with S.aureus infections, other p a tie n ts and normal volu n teers 21
LIST OF FIGURES IN CHAPTER 3A
3A.1 Frequency histogram of the results of S .aureus inhibition tests using PMN and plasma from 29 healthy volunteers 2
3A.2 Graph showing the relationship between broth b a c te ria l con cen tration and absorbance of S.pyogenes at 650nm 10
3A.3 Phagocytosis of 2 clinical isolates of S.pyogenes (JB & FS) using PMNs from 3 donors (Ncl-3) with plasma from 10 different healthy volunteers (A-J) 11
3A.4 Comparison of IgGl (A) and IgG2 (B) a n t i- S.aureus subclass antibodies in patient and reference plasma samples 14
3A.5 Frequency histogram of the S.aureus inhibition results of patients with significant staphylococcal infections 15
3A.6 Frequency distribution of patients' ages 16
3A.7 Frequency distribution of the ages of patients with non-staphylococcal in fe c tio n s 19 CHAPTER 3A: STATISTICAL ANALYSIS
3A.1 STAPHYLOCOCCAL INHIBITION ASSAY
3A.1.1 REFERENCE RANGE
The staphylococcal inhibition assay (see 3.9, page 67) was used to measure staphylococcal opsonization by patient
and control plasma and phagocytosis of this organism by
p a tie n t and c o n tro l PMNs. The r e s u lt s were expressed as %
inhibition. PMNs in autologous plasma from 29 healthy adult
volu n teers were tested by t h is method and produced a range
of 88-99% in h ib it io n w ith a median o f 95.7%. A p lo t o f the
frequency distribution of these results suggested a skewed
distribution (Fig 3A.1). This reference range agrees well
with that of Foroozanfar who defined the lower lim it of her
normal range as 88% {Foroozanfar,1976}.
A separate reference range for children was not
established because of the difficulty in obtaining adequate
quantities of blood from healthy children. Sporadic sibling
studies and measurement of staphylococcal phagocytosis by
PMNs obtained from 21 children with non-staphylococcal
infections fell within the adult reference range, thereby
suggesting that this reference range is also valid for
phagocytic studies with children.
1 FIGURE 3A.1. Frequency histogram o f the re s u lts o f S.aureus
inhibition tests using PMNs and plasma from 29 healthy volunteers.
2 3A.1.2. INTRA-ASSAY VARIATION
The intra-assay variation of the staphylococcal
inhibition test was determined by testing one sample of PMNs and plasma from a s in g le healthy vo lu n teer. The re s u lts set out in Table 3A.1 are the averages obtained from triplicate determinations. The mean was found to be 98.15% with a standard error of the mean (SEM) of 0.3.
TABLE 3A.1. Results showing the intra-assay variation of the
staphylococcal inhibition assay.
% S.aureus TEST NO. INHIBITION 1. 95.0 2 . 97.3 3 . 97.7 4 . 98.0 5 . 98.1 6 . 98.3 7 . 98.4 8 . 98.5 9 . 98.5 10. 98.6 11. 98.7 12. 98.7 13. 98.8 14. 98.9 15. 99.1
3 3A.1.3. INTER-ASSAY AND BIOLOGICAL VARIATION
The results of 13 consecutive tests performed over 2 years using freshly prepared PMNs and plasma from a single
healthy volunteer are shown in Table 3A.2. The mean was found to be 95.9% with a SEM of 0.5.
TABLE 3A.2 Results of 13 consecutive tests of the S. aureus
in h ib itio n assay using PMNs and plasma from a single healthy volunteer
% S.aureus TEST NO. INHIBITION 1 95.0 2 96.4 3 93.0 4 96.4 5 93.0 6 95.0 7 99.0 8 97.7 9 96.0 10 96.4 11 95.0 1 2 96.8 13 96.6
4 It is difficult to assess separately the inter-assay and biological variations in the staphylococcal inhibition assay because two components, the PMNs and the S. aureus organisms, were always freshly prepared before use. However, the contribution of plasma storage to inter-assay variability was assessed by comparing staphylococcal phagocytosis in the presence of stored and freshly separated plasma from a single individual. The % in h ib itio n obtained using fresh plasma and samples stored fo r 4 months and 18 months were 98%, 97% and 95%, resp ectively. Therefore, plasma storage does not appear to contibute s ig n ific a n tly to
inter-assay variation. Nevertheless, most patient plasma are fre s h ly obtained and in the few cases where stored plasma were used, these were always tested w ithin a month of receiving them.
The reproducibi lty of an assay is dependent on the effects of intra-assay, inter-assay and biological variations. In the staphylococcal inhibition assay, the
intra-assay v a ria b ilty was low (CV 1.04%) and factors such as plasma storage contributed minimally to the inter-assay variation. Thus, the range of 7% obtained when fresh samples from a single individual were tested (Table 3A.2) was probably due to biological variation. Some factors which could have contributed to biological variation in the
staphylococcal inhibition assay include:
1) Variation in the physiology of the PMN.
2) Activation of the PMN by the separation procedure.
5 3) Variation in the quantity and quality of humoral factors.
4) Variation in the growth characteristics of S,aureus micro-organisms which are phagocytosed.
3A.1.4.ANALYSIS OF CROSS-OVER STUDIES
Staphylococcal inhibition tests were usually set up to d istin g u ish humoral and c e llu la r defects by using four combinations of control PMNs with control (NcNp) and patient plasma (NcPp), as well as patient neutrophils with patient (PcPp) and control plasma (PcNp). The mixture of normal c e lls with patient plasma (NcPp) was used to assess the opsonic capacity of the patient plasma. Combinations of patient cells in the presence of autologous (PcPp) and control plasma (PcNp) were used to test the ability of the patient's neutrophils to phagocytose S. aureus and the reaction mixture of control cells in control plasma served as an internal control.
The opsonic properties of a p a tie n t's plasma were o ccasio n ally measured in the absence of autologous c e lls .
Since the reference range is based on results obtained using control c e lls in autologous plasma (NcNp), it was important to compare the results of staphylococcal inhibition obtained with combinations of patient plasma with control PMN (NcPp) and autologous cells (PcPp). The results from 25 patients, without significant staphylococcal disease, who were referred for neutrophil function testing are listed in Table
3A.3. These results show no significant statistical
6 difference between opsonophagocytosis in the presence of control and patient PMNs (t=-0.7,p=0.5). Therefore, it is also valid to use the reference range for plasma samples tested in the absence of autologous PMNs.
TABLE 3A.3. A comparison of staphylococcal in h ib itio n using patient plasma in the presence of patient (PcPp) and control
PMN (NcPp).
PcPp PATIENT NO. NcPp % INHIBITION 1 94 98 2 99 99 3 97 99 4 99 98 5 96 97 6 99 99 7 99 99 8 93 98 9 92 95 10 99 99 11 94 93 12 95 96 13 98 98 14 99 97 15 97 96 16 98 97 17 96 95 18 91 92 19 98 95 20 99 98 21 99 99 22 90 94 23 98 98 24 93 96 25 94 93
PcPp = patient cells with patient plasma NcPp = control cells with patient plasma
7 3A.2.STAPHYLOCOCCAL INHIBITION ASSAY IN THE PRESENCE OF HEAT
INACTIVATED PLASMA
In this series of experiments, Fc-mediated
phagocytosis was measured using patient and control plasma which had been stored at -20°C for up to 3 years. A ll plasma were decomplemented by heat in a ctiva tio n at 56°C fo r 35 minutes before being used in a modified form of the
staphylococcal inhibition assay (see 3.9.2, page 68).
Control neutrophils were obtained from 10 different healthy
volunteers. A ll patient plasma were tested sim ultaneously with control samples that had been separated out on the same
day. However, since many volunteers were used frequently as
controls for different patients, it followed that the stored control plasma would have been tested against different
normal PMNs. I t was th e re fo re n e c e ssa ry to t e s t decomplemented control plasma against d iffe re n t normal,
ABO-compatible PMNs. The results obtained by incubating plasma from a single control with PMNs from three donors on a sin g le day are tabulated in Table 3A.4. These re su lts showed that a difference of up to 17% inhibition was obtained by using different normal cells.
TABLE 3A.4 Variation in % in h ib ition when heat inactivated control plasma was incubated with PMN from 3 donors
CONTROL PLASMA PMN SOURCE % inhibition ( + /- SEM) NcA 50 + /- 3% NcB 37 + /- 1.3% NcC 49.4 + /- 3%
Nc A,B,C = Control PMN from 3 donors
8 After decomplentation, samples with either high or low opsonic capacity remained so despite storage. Thus a patient's plasma that had been stored for 3 years retained a high Fc-mediated phagocytosis (68%) while the control of the day retained a low level of decomplemented phagocytosis
(0%). Therefore, the storage of plasma at -20°C did not adversely affect phagocytosis in the presence of decomplemented plasma.
3A.3.STREPTOCOCCAL INHIBITION ASSAY
The phagocytosis of 3 clinical isolates of S.pyogenes obtained from patients with severe streptococcal infection was measured by a streptococcal inhibition assay. It was not possible to establish a normal range for th is assay due to a limited availability of these virulent organisms. Moreover, in order to maintain the organismsms in their initial virulent state it was necessary to minimise further subculture. Thus, w h ilst on any given sample experiments were performed in trip lic a te , such testing at times was not repeated. For example, the effect of heat-inactivated plasma on streptococcal phagocytosis (Fig 3.6, page 73) and the comparison of the phagocytosis of one of these virulent organisms with an untyped strain (Fig 5.3, page 124) were performed only once using c e lls and autologous plasma from 2 healthy volunteers.
For a comparison of the phagocytosis of the three clinical isolates of S.pyogenes (FS,JB,MB), equal quantities
9 Absorbance at 650 nm 650nm FIG.3A.2. Graph showing the re la tio n sh ip between broth broth between ip sh n tio la re the showing Graph FIG.3A.2. b a cte ria l concentration and absorbance of of absorbance and concentration l ria cte a b TABLE 3A.5 Serial d ilu tio n of 4 differen t broth cultures of of cultures broth t differen 4 of n tio ilu d Serial 3A.5 TABLE S. pyogenes utrs o gvn bobne 05 t 5 nm. 650 at 0.5 absorbance, given a to cultures standardise the quantity of organisms by d ilu tin g a ll broth broth ll a g tin ilu d by organisms of quantity the 3A.2 standardise (Fig absorbance to related n tio rly a ria e a v lin were wide showed concentrations lly a ic p y t lts su re The determined. prxmae al entaton o ec i ate e t la o is each of n tio tra n ce n o c l the ia and r e t c a plates lly b ia agar r e s onto ate placed were assay: approxim each cultures in broth used were diluted organisms three the of (T a b le 3 A .5 ). S in c e the i n i t i a l b ro th b a c t e r i a l l a i r e t c a b th ro b l a i t i n i the e c in S ). .5 A 3 le b a (T is representative of this relationship) it was possible to to possible was it relationship) this of representative is CU cln frig nt SM sadr err f h mean the of error standard SEM= units forming colony *CFU= * id tan f S.pyogenes of strain wild ** S.(UNKNOWN)** S.(JB) S.(MB) S.(FS) S.PYOGENES tetcca cnetain 1 mlin u/ l) /m fu c million (10 concentration Streptococcal AVEARAGE CFU/ml CFU/ml AVEARAGE ) 1 ( 0 0 1 3 (5) 33 (3) 61 (8) 85 ( - SEM)* /- + S.pyogenes.
at at 10 The measurement of streptococcal phagocytosis in the presence of neutrophils from 13 volunteers is discussed at page 125 (Fig. 5.4). These experiments were preceded by experiments using equal concentrations of the clinical isolates of S.pyogenes with cells from 3 donors in the presence of 10 non-autologous, ABO compatible plasma
(Fig .3A .4). Here too, the phagocytosis of S.(JB) (median
39%, range 1-68.4%) was found to be less than that of S.(FS)
(median 62%, range 37-88.9%) though this difference was more marked when a larger group of normal PMNs were tested.
FIG.3A.3. Phagocytosis of 2 clinical isolates of 5.pyogenes
(JB & FS) using PMNs from 3 donors (Ncl-3) with plasma from
10 different healthy volunteers (A-J).
100 T 90 S.pyogenes (JB) 80 -- C 70 -- rh E 60 - - D rh G 50 F nh 40 -- rh 30 -- * 20 -- 10 100 Nc1 C Nc2 Nc3 90 -- B ® S.pyogenes (FS) 80 -- 70-- I °i 60 - - A I 50 -- 40-- 30 as 20 10 0 1 I Nc1 Nc2 Nc3
11 The results (Fig.3A.3 & 5.4) and their interpretation
could nevertheless have been affected by spontaneous changes
in the relative virulence of these bacteria during in vitro
culture and the small number of clinical isolates studied, respectively.
3A.4.ANTI-STAPHYLOCOCCAL ELISA
The inter-assay coefficients of variation for total
a n ti-5 .awreus IgG (see section 3.13.2, page 74) and IgGl and
IgG2 subclass anti-S.aureus antibody levels (see section
3.13.3, page 77) were found to be 13, 34 and 25%, respectively. Since interassay precision of <15% is considered acceptable for clinical immunoassays (Hamilton,
1987), those obtained from s p e c ific subclass measurement
(34, 25%) were high. Some factors which could have contributed to these high values include:-
(i) Variation in the coating of the ELISA plates. For example, approximately 250 ug/ml of staphylococcal
suspension was added to each well but 500 ug/ml was used in the to ta l IgG assay. However, an attempt was made to minimise this effect by using Nunc maxi-sorp plates which have a limited intra-well coating variation of < 5%.
(ii) Non-specific binding as a result of the low
16-fold plasma d ilu tio n used in these assays.
(iii) The lack of a high positive control as the reference plasma in the subclass ELISA. Thus, Persson et al
(1985) found that the reproducibity of their assays was
improved from 17-58% to 8-26% i f a high positive reference
12 serum was used in the assay instead of a standard myeloma plate. In the present assay although the reference serum was not a particularly high positive standard, it did enable a comparison to be made between high antibody levels from patients and normal plasma (see Fig.3A.4)
The CV for the anti- S.aureus IgGl and IgG2 subclass
levels (34% & 25%) were similar to the results of Persson
(1985) who used bacterial antigens such as purified
Salmonella B 0-antigen and Streptococcus pneumoniae polysaccharide type 6A in separate ELISA systems.
13 ?
0.D.405nm O.D. at 405nm FIG.3A.4. Comparison of of Comparison FIG.3A.4. ucas niois n ain ad eeec pam samples plasma reference and patient in antibodies subclass I q GI A ad q2 B ant-5 ti- n a (B) IqG2 and (A) .aureus 14
3A.5 STATISTICAL ANALYSIS OF PATIENTS
3A.5.1. PATIENTS WITH STAPHYLOCOCCAL INFECTIONS
In these studies 31 patients with significant S.aureus
infections were grouped according to whether their
neutrophils produced either abnormal or normal
staphylococcal inhibition results. As seen from Fig.3A.5,
four patients produced markedly abnormal staphylococcal
inhibition results (49-79%) which differed significantly
from the reference range (z=-3.18,p=.0015). These 4 patients
have therefore been considered to have defective
opsonophagocytosis of S.aureus and are discussed separately.
The other 27 patients with normal staphylococcal
opsonization and phagocytosis gave results which were within
the reference range (>88%).
FIGURE 3A.5. Frequency histogram of the S.aureus inhibition
results of patients with significant staphylococcal
infections
x o c CD Z5 CT CD
% staphylococcal inhibition 15 A511 CIIA CAATRSIS F AINS WITH PATIENTS OF CHARACTERISTICS CLINICAL 3A.5.1.1. years. recurrent abscesses being the presenting complaint in 24 of of 24 in complaint presenting the being abscesses recurrent STAPHYLOCOCCAL INFECTIONSSTAPHYLOCOCCAL I.A6 Feuny srbuton o ptet' ages patients' of n tio u istrib d Frequency FIG.3A.6. h 3 ptet. oe hn 0 o tee ains had patients these of 80% than More patients. 31 the predominance of males in th is group with a male: female female male: a with 1. group 1.8: of is th tio ra in either a males patients of suggests predominance of .6 -) the .3A ajority (Fig m of the with n tio n u presentation tio u trib is d istrib d at bimodal ages frequency A ts' n tie a years. p 16 of median a ains, h ee 2 ains ad oe ( ptet. The patient). (1 bones and of of patients) s duration (2 ite s eye the Other patients), abscesses. of crops more or 10 had had ufrd t es 3 psds f bis bt (0) f them of (20%) 5 but "boils" of episodes 3 least at suffered freq ue n cy a was There old. years 25 than more or years 5 than less with years 61 to year 1 of ages the between ranged infection nf ton icue te lo ( paint) te oi s (3 ts in jo the ts), atien p (3 blood the included n ctio fe in The 31 p a tie n ts w ith s ig n ific a n t stap h ylo co ccal ccal co ylo h stap t n a ific n ig s ith w ts n tie a p 31 The The skin was the most common s ite of in fe ctio n with with n ctio fe in of ite common s most the was skin The S.aureus netos agd rm mnh t 30 to months 6 from ranged infections 3A5.1.2 THE 27 PATIENTS WITH S.AUREUS INFECTIONS BUT NORMAL
OPSONOPHAGOCYTOSIS.
Neutrophil mobility studies (chemotaxis and random migration) as well as the Candida killing assay were performed on the cells from the 27 patients with clinically significant 5. aureus in fectio n but normal opsonophagocytosis, to rule out other possible causes of an increased susceptibility to bacterial infections.
Using the technique of Evans (1989), chemotaxis to zymosan-activated plasma was measured and was found to be low in 8 patients, 6 of whom were children who were also characterised using the adult reference range (Table
4 .7 ,page 98). The Candida k illin g assay (Lehrer 1969) which assesses the total killing capacity of the neutrophil especially the oxygen-dependent killing mechanisms, was found to produce markedly decreased results when PMNs from 3
CGD patients were studied. However, of the 5.aureus patients studied, all the results were within the normal range (>25%)
(Table 4.7, page 98).
Plasma causes of immunodeficiency such as agammaglobulinaemia, hypocomplementaemia and IgG subclass deficiencies were ruled out by measuring total
immunoglobulin class and subclass antibodies and complement concentrations. The results of these investigations are summarised in tables 4.5 and 4.6, pages 96-97.
17 3A5.1.3. A COMPARISON OF Fc-MEDIATED PHAGOCYTOSIS IN
PATIENTS WITH S.AUREUS INFECTIONS. PATIENTS WITH OTHER
INFECTIONS AND NORMAL VOLUNTEERS.
A benign non-encapsulated strain of S.aureus (Oxford strain) was used in the inhibition tests. However, in vivo more virulent, and possibly encapsulated organisms are present which are frequently only phagocytosed in the presence of high levels of specific opsonic antibodies. The presence of such e ffic ie n t opsonins in patients' plasma was detected by measuring staphylococcal phagocytosis in decomplemented plasma (Fc-mediated phagocytosis).
The results of Fc-mediated phagocytosis with plasma from the 27 patients were compared with those obtained from two control populations namely, normal adult laboratory workers (69 samples) and 20 patients with non-staphylococcal infections (Table 3A.6). These patients had been referred for neutrophil function tests and suffered with a variety of conditions ranging from recurrent v ira l infections, benign neutropenia, recurrent aphthous ulceration and bacterial infections caused by S.pyogenes, S.pneumoniae and
H. influenzae. Their ages ranged between 8 months to 46 years with a bimodal distribution (Fig.3A.7) also seen with the staphylococcal patients.
18 Fiq.3A.7. Frequency distribution of the ages of patients
with non-staphvlococcal infections.
(age in years)
TABLE 3A.6 Fc-mediated opsonophaqocvtosis using plasma from
patients with S.aureus infections, other infections and
healthy normal controls
PARAMETER S.AUREUS PTS. OTHER PTS CONTROLS
No.of samples 27 20 69
Median (% inhibition) 54.5 44 18.6
Range (% inhibition) 0-86 0-69 0-75
p value* p=.0002 p=.03
p value ** p=.04 p value* = significant difference when compared with controls p value** = significant difference when compared with the other patient group
19 When non-parametric s ta tis tic a l analysis (Mann-Whitney
U test) was applied to these data, the S.aureus patients were found to have significantly increased Fc-mediated
phagocytosis compared with the healthy volunteers (p=0.0002)
and with the other patients (p=0.04).
3A5.1.4. A COMPARISON OF ANTI-5.AUREUS IqGl AND IoG2
ANTIBODIES IN PATIENTS WITH S.AUREUS INFECTIONS. PATIENTS
WITH OTHER INFECTIONS AND HEALTHY VOLUNTEERS.
Since staphylococcal patients had the highest median
level of Fc-mediated phagocytosis, an attempt was made to
correlate these findings with specific anti-5 .aureus IgG
subclass levels. Table 3A.7 contains a summary of the total
IgG, IgGl and IgG2 levels found in the staphylococcal
patients, the other patient group and in healthy controls.
IgGl concentrations in both patient groups differed markedly from the control population (p= 0.001 for the S.aureus patients and p=0.05 for the other patients). Additionally, the IgGl levels in the staphylococcal patients were
significantly higher than those in the other patients (p=0.02).
The comparison between the results obtained with the staphylococcal patients and the healthy volunteers is further discussed in section 5.2, page 113.
20 TABLE 3A.7. Anti-S.aureus total IqG. I qGI and IqG2 levels in patients with S.aureus infections, other patients and normal volunteers a) Total IqG
PARAMETER STAPH PTS. OTHER PTS. CONTROL PTS.
No.of samples 27 20 65 median (ELISA INDEX) 0.82 0.6 0.81 range (ELISA INDEX) 0.22-1.16 0.17-1.14 0.11-1.29
n.s. n.s.
b) IoGl
PARAMETER STAPH PTS OTHER PTS CONTROLS
No.of samples 26 13 51
Median (mg/l) 1.79 1.3 1.00
Range (mg/l) 0 .9 8 -3 .9 0 .6 4 -3 .1 6 0 .4 -2 .6 5
p value* p=.001 p=.05 o CM n a p value** •
21 c) IqG2
PARAMETER STAPH PTS OTHER PTS CONTROLS
No.of samples 26 13 51
Median (mg/l) 45 43.5 46.9
Range (mg/l) 10.7 -7 1 .3 8.45-59.7 7.3-74
n.s. n.s. n.s.= no significant difference * = compared with controls ** = compared with other patients
3A5.1.5. CORRELATION BETWEEN Fc-MEDIATED PHAGOCYTOSIS AND
mi-S.AUREUS IqGl AND IqG2 SUBCLASS ANTIBODIES.
Fc-mediated phagocytosis was found not to be linearly
dependent on specific IgGl and IgG2 concentrations by
regressional analysis. Thus, there was poor correlation
between Fc-mediated phagocytosis and either IgGl
concentration, (r= 0.330, p = 0.124) or Ig G 2
concentration,(r=0.07, p=0.75), respectively. Whilst it is
not e n tire ly clear why there was such poor correlation the
following factors cannot be overlooked:-
(i) Two different strains of S.aureus were used in the
phagocytosis and ELISA assays, the latter requiring the use
of a protein A deficient strain in order to minimise
non-specific binding of IgG.
22 (ii) The Fc-mediated phagocytosis may not be linearly related to specific IgGl or IgG2 levels owing to the complexity of opsonophagocytosis. For example, different plasma factors, some as yet unidentified, interact with different membrane receptors to cause the formation of pseudopodia and ultimately a phagosome. Furthermore, the removal of complement may c a ll into play the e ffe cts of other opsonins like CRP, mannose binding protein or adhesive proteins as fibrinogen which could act synergistically with specific IgG.
In conclusion, there appears to be an association between specific IgGl levels and higher Fc-mediated phagocytosis in patients with S.aureus infections. The two parameters are however not linearly correlated.
23 this thesis is dedicated with much love to my husband, Rene and our three daughters, Noele, Dominique and Danielle COCCAL INFECTIONS DUE TO OPSONIC DEFECTS
By
Michele Anne Monteil BSc (Hons), MB.BS (Hons)
A thesis submitted to the University of London for the degree of Doctor of Philosophy in the Faculty of Medicine
Department of Immunology Charing Cross and Westminster Medical School LONDON SW1P 2AR June, 1990
1 ABSTRACT OF THESIS
Staphylococcal and streptococcal infections are a common cause of sig n ifica n t morbidity and m ortality in the general population. Indeed, patients with recurrent, chronic or deep-seated staphylococcal infections account for about
40% of all patients referred for investigation of neutrophil function at the Department of Immunology,
Westminster Hospital. Though cellular defects of phagocytes can predispose to recurrent or severe infections, these are rare. However, since humoral defects were shown to be more common, at least in a single large study, the possibility of subtle opsonophagocytic defects predisposing to significant coccal disease, was explored.
The phagocytosis of Staphylococcus aureus was measured with a functional assay in patients with staphylococcal infections and in healthy controls. A specific functional defect in opsonization was identified in 4 patients and in 2 of them, purified IgG therapy was found to be effective in decreasing the frequency and severity of their lesions. The measurement of plasma levels of specific anti-S.aureus IgG class and subclass antibodies, by enzyme linked immunosorbent assay (ELISA), in a ll patients studied were similar to levels in healthy controls.
Thus, a deficiency of specific antibodies contributing to the frequency and severity of infections in these patients, was not identified in this study.
2 The phagocytic assay was adapted to measure the opsonophagocytosis of Streptococcus pyogenes and used in a study of the interaction of normal neutrophils with virulent isolates of S.pyogenes M1/T1/0F-, obtained from patients with septic scarlet fever. These organisms were found to be markedly resistant to phagocytosis and this resistance may p a rtly explain th e ir capacity to overwhelm normal host immune defences.
3 ACKNOWLEDGEMENTS
I am greatly indebted to Professor J.R. Hobbs for allowing me the opportunity and privilege of continuing my postgraduate education as a member of his department and for his generosity and support.
My special thanks to the following very special people,
Mrs. Ann Kaniuk for her unswerving support and encouragement and without whom, this thesis would never have been finished.
Drs. Anne Evans and David Chambers, who introduced me to the n eutrophil and who have both shown me of what good scientists are made.
Dr. Pamela Riches for her constructive criticism and advice.
Dr. Jennifer Lortan for many stimulating conversations.
Miss Jo Sheldon and Dr. Robert Beetham, who always extended a helping hand whenever it was needed and a ll my other colleagues at the department of Immunology, for th e ir kind encouragement.
To my mother, Mrs.Merle Bain, my grandmother, Mrs.Ivy Attale and aunt Mrs.Joan Wharton-Lake, I would lik e to say thanks for a lifetim e of love and support. Last but ce rta in ly not least, my mother-in-law, Mrs.Titta Monteil, without whose help I would not have been able to continue my career away from my children.
4 TABLE OF CONTENTS
PAGE
List of abbreviations front pocket
T itle page 1
Abstract 2
Acknowledgements 4
Table of contents 5
List of tables 13
List of figures 15
1 REVIEW OF NEUTROPHIL FUNCTION 17
1.1 Introduction 17
1.1.1 Neutrophil morphology, production and development 17
1.1.2 Neutrophils in the blood and tissues 18
1.2 Neutrophil functions 19
1.2.1 Adherence 19
1.2.2 Movement 20
1.2.3 Degranulation 21
1.2.4 Microbicidal mechanisms 22
1.2.4.1 Oxygen dependent mechanisms 22
1.2.4.2 Oxygen independent bactericidal mechanisms 23
1.2.5 Digestion 24
1.3 Opsonisation & phagocytosis 24
1.3.1 Historical perspectives 24
1.3.2 D efinition of terms 25
1.4 Opsonins 26
1.4.1 Immunoglobulin G 26
1.4.2 Immunoglobulins A,M,D,E 27
1.4.3 Complement derived opsonins 27
1.4.3.1 C3 opsonins 28
5 1.4.3.2 C4 opsonins 28
1.4.4 C-reactive protein, fibronectin and mannose binding protein 29
1.4.5 Non opsonic phagocytic enhancers 30
1.5 Neutrophil phagocytic receptors 30
1.5.1 Fc receptors 30
1.5.1.1 Human FcyRI 31
1.5.1.2 Human FcyRII 31
1.5.1.3 Human FcyRIII 31
1.5.1.4 Human FcaR 33
1.5.2 Complement receptors 33
1.5.2.1 Complement receptor type 1 33
1.5.2.2 Complement receptor type 2 34
1.5.2.3 Complement receptor type 3 35
1.5.2.4 Complement receptor type 4 37
1.6 Mechanisms of phagocytosis 37
1.6.1 The differential effects of IgG and com plement derived opsonins on phagocytosis 41
1.6.2 Surface phagocytosis 42
1.7 Tests of phagocytosis and opsonization 43
1.8 Defects of opsonization and phagocytosis 45
1.8.1 The clinical presentation of patients with phagocytic defects 45
1.8.2 The investigation of patients with phagocytic defects 48
2 THE COCCI 50
2.1 Staphylococcus aureus 50
2.1.1 Bacterial structure 50
2.1.2 Virulence factors 51
2.1.2.1 The capsule 51
2.1.2.2 Enzymes and toxins 52
2.1.3 Staphylococcal infections 53
6 2.1.4 Host resistance to staphylococcal infections 54
2.2 Group A beta-haemolytic streptococci (5treptococcus pyogenes) 56
2.2.1 Structure 56
2.2.2 Virulence factors 57
2.2.2.1 Surface components 57
2.2.2.2 Enzymes and toxins 57
2.2.3 Streptococcal diseases 58
2.2.4 Host resistance to streptococcal infection 59
2.3 Aims of the project 60
3 MATERIALS AND METHODS 61
3.1 Subjects 61
3.2 Buffers and media 61
3.2.1 Medium 199 61
3.2.2 Phosphate buffered saline (PBS) /PBS-Azide-Tween 62
3.3 Separation of the polymorphonuclear leucocytes 62
3.4 Plasmas and sera 63
3.4.1 Separation and storage 63
3.4.2 Heat inactivation of plasma 63
3.5 Bacteria 63
3.5.1 Staphylococcus aureus 63
3.5.2 Streptococcus pyogenes 64
3.6 Bacterial growth media 64
3.6.1 Tryptic soy and Todd-Hewitt broths 64
3.6.2 Agar slopes and agar plates 65
3.7 Methods of assessing bacterial numbers 65
3.7.1 Colony counting method 65
3.7.2 Spectrophotometric measurements 66
7 3.8 Preparation of bacteria for use in the phagocytic assays 66
3.9 The staphylococcal inhibition assay 67
3.9.1 The principle 67
3.9.2 The method 67
3.10 Optimisation of the staphylococcal inhibition assay 68
3.10.1 Effects of different plasma concen trations 68
3.10.2 Effects of different staphylococci: neutrophil ratios 69
3.10.3 Effects of shaker speeds on the staphylococcal inhibition assay 70
3.10.4 Time course of staphylococcal phagocytosis 70
3.10.5 Assessment of the opsonic contribution of complement in the staphylococcal inhibition assay 71
3.11 Measurement of blocking activity in patients' plasmas 73
3.12 Streptococcal inhibition assay 73
3.13 Enzyme-1 inked immunosorbent assays 74
3.13.1 Measurement of a n ti-S.pyogenes IgG levels 74
3.13.2 Measurement of total IgG levels sp e cific for S.aureus 74
3.13.2.1 Optimisation of the total IgG assay 75
3.13.2.2 Determination of assay specificity 76
3.13.3 Measurement of IgG subclass levels of a n ti-5.aureus antibodies 77
3.13.3.1 Optimisation of specific subclass assay 77
3.14 Equal potency method 79
3.15 Measurement of immunoglobulin and complement concentrations 81
3.16 Measurement of the effect of 4 drugs on S.aureus phagocytosis 81
3.17 Statistical analysis 82
3A STATISTICAL ANALYSIS in front pocket 4 DETAILS OF PATIENTS STUDIED 83
4.1 Overview 83
4.2 Patients with abnormal staphylococcal inhibition tests 83
4.2.1 Patient 1: L.C. 83
4.2.2 Patient 2: I.E. 85
4.2.3 Patient 3: R.J-S. 86
4.2.4 Patient 4: R.T. 88
4.3 Patients with recurrent staphylococcal infections and normal staphylococcal inhibition tests 89
4.3.1 Clinical summaries of patients with normal S.aureus in h ib itio n but s ig n if i cant staphylococcal infections 89
4.3.1.1 Patient 1: G.B. 89
4.3.1.2 Patient 2: R.B. 89
4.3.1.3 Patients J.B., N.B., V.B., Y.B. 90
4.3.1.4 Patient R.B. 90
4.3.1.5 Patient M.C. 91
4.3.1.6 Patient D.C. 91
4.3.1.7 Patient D.H. 91
4.3.1.8 Patient L.J. 91
4.3.1.9 Patient D.M. 92
4.3.1.10 Patients A.N., C.N., R.N. 92
4.3.1.11 Patient S.P. 92
4.3.1.12 Patient D.P. 93
4.3.1.13 Patient S.R. 93
4.3.1.14 Patient J.R. 93
4.3.1.15 Patient M.S. 94
4.3.1.16 Patient G.S. 94
4.3.1.17 Patient J.S. 94
4.3.1.18 Patient F.C. 94
9 4.3.1.19 Patient R.C. 95
4.3.1.20 Patient P.D. 95
4.3.1.21 Patient E.F. 95
4.3.1.22 Patient K.K. 95
4.4 The patients with septic scarlet fever 99
4.4.1 Patient 1: M.B. 99
4.4.2 Patient 2: J.B. 99
4.4.3 Patient 3: F.S. 100
5 RESULTS AND DISCUSSION 103
5.1 Patients with abnormal staphylococcal 103 inhibition tests
5.1.1 In vitro correction of the defect 104
5.1.2 In vivo correction of the defect 105
5.1.3 A n ti-5.aureus IgG class and subclass 106 antibodies in patients with defective staphylococcal inhibition
5.1.4 Blocking a c tiv ity in the plasmas of 107
patients with defective staphylococcal phagocytosis
5.1.5 Evidence for a specific staphylococcal 108
opsonin deficiency
5.2 Patients with normal staphylococcal 113 inhibition tests but with recurrent or severe staphylococcal infections
5.2.1 Specific anti-5.aureus IgG antibodies 117
5.2.2 A n ti-5.aureus IgG2 subclass antibodies 117
5.2.3 A n ti-5 .aureus IgG3 & IgG4 subclass 118 antibodies
5.2.4 A n ti-5.aureus IgGl subclass antibodies 119
5.2.5 A n ti-5.aureus subclass antibodies in 119 children and adults
5.2.6 A n ti-5 .aureus IgGl levels and opsono- 122 phagocytosis in patients with recurrent or deep S.aureus infections
10 5.2.7 Other possible defects causing recurrent 122 or deep S.aureus infections
5.3 Phagocytosis of clinical isolates of 123 S.pyogenes type M1/T1/0F- obtained from three patients with septic scarlet fever
5.3.1 The re-emergence of strains of invasive 123 S.pyogenes which cause severe infection and a toxic shock-like syndrome
5.3.2 Difference in the phagocytosis of 3 iso- 123 lates of invasive S.pyogenes
5.3.3 Phagocytosis of S.pyogenes MI/TI/OF- 126 from different c lin ic a l situations
5.3.4 hnti-S.pyogenes IgG antibodies 127
5.3.5 S.pyogenes MI/TI/OF- virulence factors 128
5.3.6 Defective phagocytosis in septic scarlet 130 fever
5.3.7 Viral suppression of neutrophil function 131 and invasive S.pyogenes disease
6 GENERAL DISCUSSION AND CONCLUSIONS 132
6.1 The abscess 132
6.2 Defective opsonophagocytosis and failure 132 of bacterial containment
6.3 Bacterial hypersensitivity and abscess 133 formation
6.4 Opsonic defects and recurrent infections 133
6.5 Prophylaxis against recurrent S,aureus 134 infections
6.6 Treatment of patients with recurrent 135 S.aureus infections
6.7 Treatment of patients with sp e cific 136 opsonic defects
6.8 Defective phagocytosis of virulent 137 S.pyogenes in the pathogenesis of streptococcal toxic shock-like syndrome
6.9 Management of streptococcal toxic 138 shock-like syndrome
11 6.10 Conclusions 141
6.10.1 Aims of Project 141
6.10.2 The future 142
LIST OF REFERENCES 144-162
APPENDIX - Normal ranges 163-164
1. Immunoglobulin G, A, M
2. Immunoglobulin G subclasses
3. White Blood C ells, Neutrophils, Lymphocytes
Photocopies of Publications:
1. Monteil,M.A., Hobbs,J.R., Citron,K. (1987)
2. Shaunak,S., Wendon,J., M onteil,M .A., Gordon,A.M. (1988)
3. Riches,P.G., Hancock,M., Jurges,E., Kurobane,I., Gray,C., Montei1,M.A., Hobbs,J.R. (1989)
12 LIST OF TABLES PAGE 1.1 Methods for measuring opsonization & phagocytosis 44
1.2 C e llu la r causes of defective phagocytosis 46
1.3 Diseases associated with defective opsonization 47
3.1 Duplicate colony counts of 5.aureus from 3 consecutive overnight broth cultures 66
4.1 Summary of haematological and immunological results of patient L.C. 84
4.2 Summary of haematological and immunological results of patient I.E. 86
4.3 Results of haematological and immunological investigations of patient R.J-S. 87
4.4 Results of investigations of patient R.T. 88
4.5 Immunoglobulin and complement concentrations in 27 patients with significant staphylo coccal infections 96
4.6 Immunoglobulin G subclass levels in 27 patients with staphylococcal infections 97
4.7 Results of phagocyte function tests in 27 patients with significant S.aureus infections 98
4.8 Immunological investigations of patients with septic scarlet fever 101
4.9 Immunoglobulin G subclass levels in acute and convalescent sera from patients with septic scarlet fever 102
5.1 Summary of staphylococcal inhibition results in 4 patients with defective S.aureus opsonization 103
5.2 In vitro correction of staphylococcal phagocytosis by the addition of purified immunoglobulin G 104
5.3 Effect of IgG therapy on staphylococcal inhibition in patient LC 105
5.4 A n ti-S.aureus IgG class and subclass antibodies in patients with defective staphylococcal inhibition 106
5.5 Results of experiment to detect blocking a c tiv ity in patients' plasmas 108 13 5.6 Differential diagnosis of recurrent staphylococcal infection 112
5.7 Clinical details and specific anti-5 .aureus IgGl and IgG2 concentrations of the 27 patients with staphylococcal infections 114
5.8 Median concentrations and ranges of a n ti-5 .aureus IgG class and subclasses in patients and controls 115
5.9 Median values and ranges of a n ti-5.aureus IgG2 antibodies in children and adults 120
5.10 Median values and ranges of a n ti-S.aureus IgGl antibodies in children and adults 121
5.11 A n ti-S.aureus IgG class and subclass concentrations in healthy controls and patients aged 5 years and under 121
5.12 Medians and ranges of phagocytosis of 3 clinical isolates of S.pyogenes by PMNs from 13 healthy controls 125
5.13 Median values and ranges of the opsono- phagocytosis of S(septicaemia)f S(abd.pus) and S(pneumonia) by control PMNs in 4 d ifferen t normal plasmas 126
5.14 Median values and ranges of anti-S.pyogenes IgG binding to 6 clinical isolates of S.pyogenes M1/T1/0F- measured by ELISA 127
5.15 Phagocytosis of S(JB) by normal PMNs in the presence of normal plasmas and convalescent FS serum 131
14 LIST OF FIGURES PAGE
2.1 Overnight growth of Staphylococcus aureus on blood agar 51
2.2 Overnight growth of Streptococcus pyogenes on blood agar 56
3.1 The effect of increasing concentrations of normal plasma on the phagocytosis of 5 .aureus 69
3.2 Effect of increasing S.aureusiPM ratios on staphylococcal phagocytosis 69
3.3 The effects of shaker speed on S.aureus phagocytosis 70
3.4 % staphylococcal inhibition after 5, 15, 30 minutes incubation with plasmas from 10 patients and 10 healthy controls 71
3.5(a) Staphylococcal phagocytosis in the presence of normal and Mg-EGTA treated plasmas 72
3.5(b) Staphylococcal phagocytosis in the presence of normal and heat-inactivated plasmas 72
3.6 Phagocytosis of S. pyogenes in the presence of normal and heat-inactivated plasmas 73
3.7 Chequerboard analysis of increasing dilu tio n s of S.aureus suspension and increasing d ilu tio n s of normal plasmas 75
3.8 Anti-5. aureus IgG levels in doubling d ilu tio n s of normal plasma and plasma absorbed against S.aureus Wood 46 strain 76
3.9 A n ti-S.aureus IgG subclass binding to doubling dilutions of S.aureus suspension after the addition of normal plasma (1/16 dilution) 78
3.10 A n ti-S.aureus IgG subclass binding to S.aureus antigen (1/400 dilution) after the addition of doubling dilutions of normal plasma 78
3.11 Standard curves of absorbance (405 nm) against SPS-01 d ilu tio n s (a) IgGl (b) IgG2 80
5.1 IgGl a n ti-5 .aureus antibody concentrations in patients and controls 115
5.2 Fc-dependent phagocytosis of S.aureus in patients and healthy controls 116
15 5.3 Phagocytosis of S(MB) M1/T1/0F- and an untyped clinical isolate of S.pyogenes 124
5.4 Phagocytosis of three clinical isolates of S.pyogenes M1/T1/0F- from cases of septic scarlet fever by PMNs from healthy controls in autologous plasmas 125
6.1 Effects of preincubation of control PMNs with increasing concentrations of rifampicin, clindamycin, erythromycin and ampicillin on the phagocytosis of S.aureus 139
16 CHAPTER 1: REVIEW OF NEUTROPHIL FUNCTION
1.1 INTRODUCTION
Phagocytosis is the process by which c e lls engulf par
ticulate matter and it is the way in which specialised
leucocytes, the PHAGOCYTES, remove foreign invaders and ef
fete tissue. The phagocytes are classified as the
MONONUCLEAR PHAGOCYTES which include c e lls of the monocyte-
macrophage series and the POLYMORPHONUCLEAR PHAGOCYTES con
sisting of neutrophils and eosinophils. Of these, the
neutrophils provide the body's first method of defence
against bacteria and fungi so that quantitative and qualita
tive abnormalities of these cells are associated with an in
creased risk of severe infection {Segal, 1980}.
1.1.1 NEUTROPHIL MORPHOLOGY. PRODUCTION AND DEVELOPMENT
The mature polymorphonuclear leucocyte (PMN) is a
granular cell of 12-15K.ni in diameter with a coarse multi-
lobed nucleus and abundant cytoplasm which contains a com
plex cytoskeleton and a variety of granules {Klebanoff,
1978}. The primary granules which account for a third of all
granules are lysosomes and contain an array of m icrobicidal
enzymes including myeloperoxidase. The more abundant
specific granules stain deeply for glycoprotein and contain
lactoferrin, vitamin B12 binding protein, lysozyme and
several membrane receptors. In addition, the recently iden
tified tertiary granules carry gelatinase and possibly
complement receptors type 3 (CR3) {Boxer, 1988}. The
17 neutrophil is enclosed by a plasma membrane in abundance in which several enzyme systems and receptors fo r a variety of chemoattractants and opsonins are found.
Mature PMNs are produced in the bone marrow from stem c e lls by a process known as GRANULOCYTOPOIESIS. This takes approximately 14 days {Williams, 1983} and the daily produc tio n rate in a normal adult is about 100 b illio n c e lls
{Segal, 1981}. The proliferation, maturation and release of
PMNs from the marrow into the circulation are regulated by
glycoprotein COLONY STIMULATING FACTORS (CSF) e sp ecially IN
TERLEUKIN 3 (IL3 ), GRANULOCYTE-MONOCYTE CSF (GM-CSF) and
GRANULOCYTE-CSF (G-CSF) {reviewed by Metcalf, 1989}.
1.1.2 NEUTROPHILS IN THE BLOOD AND TISSUES
Granulocytes in the blood either circu late fre e ly or adhere
randomly to the endothelium of small vessels to produce CIR
CULATING and MARGINATING pools of approximately equal size.
However, strenuous exercise or an in je ctio n of adrenaline
can augment the circulating pool by displacing marginated
c e lls from the endothelium {Athens, 1961}. The PMNs with a
mean h a lf - lif e in the blood of only 6.7 hours leave the c i r
culation randomly {Williams, 1983}.
The emigration of neutrophils from the blood into in
flammatory lesions is one of the major hallmarks of the
acute inflammatory response and occurs predominantly from
the post-capillary venules {Movat, 1987}. First, the cells
marginate to the walls of the inflamed vessel (PAVEMENTING)
and then squeeze through spaces between the endothelial
18 cells (DIAPEDESIS) into the tissues. The egress of these cells from the circulation is hastened by the presence of chemotaxins which alter endothelial cell stickiness and stimulate PMN chemotaxis {Colditz, 1985}.
L it t le is known of the a c t iv it ie s of PMNs in normal tissu es where they are thought to survive fo r about 2-3 days. Moreover, although they are known to migrate into various tissues their fate remains uncertain {Williams,
1983}. However, in inflammed tissues, PMNs appear to undergo apoptotic changes and are removed by tissues histiocytes.
Savill {1989} has observed that recognition of aged PMNs oc curs via vitronectin receptors on the surface of the tissue macrophages which he believes may be an important mechanism for removing neutrophils during the resolution of inflam matory lesions.
1.2 NEUTROPHIL FUNCTIONS
1.2.1 ADHERENCE
Leucocyte adherence to the endothelium of normal and inflamed vessels has long been known but the relevance of neutrophil adhesion to the inflammatory response was only appreciated after the development of assays which measured
PMN adhesion to nylon fib re {Cates, 1981}. More recen tly, studies on PMN adherence to cultured endothelia have defined neutrophil-dependent and endothelial-dependent mechanisms of adhesion {Dobrina, 1989}. The former involve direct stimulation of the neutrophils by activating agents which upregulate the surface expression of adhesive
19 molecules like the LEUCOCYTE INTEGRINS. These molecules promote cell-endothelial adhesion by binding to their ligands present on the endothelial cell surface {Dobrina,
1989; Tonnesen, 1989}. A ltern atively, activating agents can enhance neutrophil stickiness to the endothelium by inducing the synthesis of adhesive molecules such as the ENDOTHELIUM
LEUCOCYTE ADHESION MOLECULES (E-LAM) which bind to their ligands on the polymorphs {Dobrina, 1989}.
1.2.2 MOVEMENT
The movement of PMNs along a concentration gradient
(CHEMOTAXIS) ensures that the neutrophils accumulate at an inflammatory focus in adequate numbers. However, the PMN is also capable of other forms of movement such as RANDOM
MIGRATION and CHEMOKINESIS {reviewed by Smith, 1983}.
CHEMOATTRACTANTS, derived from many sources, are substances which induce a chemotactic response and those thought to be of physiolo gical importance are complement degradation product C5a {Yancey, 1988}, b a cte ria l m etabolites such as the N-formylmethionyl peptides (eg.FMLP) {Allen, 1988}, leukotriene B4 (LTB4) {Payan, 1984} and neutrophil a ctiv a t ing peptide (NAP/IL8) {Thelen, 1988}. The neutrophil detects chemotactic stimuli by specific receptors at its surface
{Becker, 1980} and receptors for FMLP, C5a, LTB4 {Snyderman,
1984} and IL8 {Samanta, 1989} have already been charac terised.
After FMLP-receptor interaction the ligand-receptor complex combines with and activates a guanine nucleotide
20 binding protein (G-protein) which activates phospholipase C
(PLC) present in the plasma membrane. PLC hydrolyses phos- phoinositol 4,5-bisphosphate (PIP2) to diacylglycerol (DAC) and inositol 1,4-5 trisphosphate (IP3), the latter releasing calcium ions from internal stores while the former activates protein kinase C (PKC) and translocates it to the plasma membrane. Consequently, there is an increased expression of chemotactic receptors and adhesion molecules, enhanced secretion of chemotactic factors and changes in the cytos- keletal organization which lead to PMN movement {Allen,
1988}. Increased free cytoplasmic calcium ion levels are thought to be responsible for both the localised changes in cytoskeletal consistency and the actin-myosin contraction which causes the directionality of PMN locomotion {Becker,
1980}. However, the mechanism by which signal transduction ultimately results in cell locomotion is still not fully un derstood {Allen, 1988}. The amount of intracellular cyclic adenosine monophosphate (cAMP) is also increased following receptor-ligand binding and appears to have an inhibitory influence on the activation of neutrophil functions {Boxer,
1987}.
1.2.3 DEGRANULATION
Degranulation is the process by which the cytoplasmic granules discharge their contents into the phagocytic vacuole or to the exterior of the neutrophil (SECRETION).
The mechanisms by which granules coalesce with e ith e r the vacuolar membrane or the plasma membrane have been likened
21 to the adrenal chromaffin granule system where a protein, synexin, in association with calcium, promotes the aggrega tion and fusion of these granules to membranes {Boxer, 1987;
Boxer, 1988}. The primary granules bring about microbial destruction within the phagosomes {Segal, 1981} whilst the specific granules discharge their contents extracellularly to cause complement activation and replenish plasma membrane receptors {Boxer, 1987}.
1.2.4 MICROBICIDAL MECHANISMS
The microbicidal activities of phagocytic cells arise either from OXYGEN DEPENDENT or OXYGEN INDEPENDENT systems : the former may be either myeloperoxidase (MPO) mediated or
MPO independent {Klebanoff, 1978} and the latter consist of granular proteins and peptides which are designed for microbial destruction and dissolution {Lehrer, 1988}.
1.2.4.1 OXYGEN DEPENDENT MECHANISMS
P e rtu rb a tio n of the n e u tro p h il membrane during phagocytosis causes the RESPIRATORY BURST which is charac terised by an oxygen consumption of over ten times the re st ing respiratory rate, a markedly increased consumption and oxidation of glucose via the hexose monophosphate shunt
(HMP) and an increased production of superoxide anion (02_)
{reviewed by Segal, 1985; Wardle, 1986}. NICOTINAMIDE
ADENINE DINUCLEOTIDE PHOSPHATE (NADPH) OXIDASE in the PMN plasma membrane contains a unique cytochrome, CYTOCHROME
B-245 which reduces the molecular oxygen to produce large
22 quantities of the superoxide {Wardle, 1986}. This nucleophile undergoes a number of chemical reactions to gen erate several reactive oxygen intermediates such as hydrogen peroxide, hydroxyl ion and singlet oxygen which act alone or in combination with granule constituents such as
MYELOPEROXIDASE or LACTOFERRIN {Gabay, 1988} to destroy bac te ria .
MPO is a large tetramer situated within the primary granules of PMNs and monocytes and accounts fo r greater than
5% of the dry weight of the cell. The green colour of pus is due to this enzyme. Following fusion of the granules with the phagosome, MPO is discharged into the phagocytic vacuole where it combines with H202 and halide ions to gen erate hypohalite agents which damage bacteria {Klebanoff,
1978}.
1.2.4.2 OXYGEN INDEPENDENT BACTERICIDAL MECHANISMS
In addition to the production of reactive oxygen in termediates the PMN also contains an array of microbicidal proteins and peptides in its cytoplasmic granules especially w ithin the membranes of the a zu ro p h ilic granules {Gabay,
1986}. Of these, CATHEPSIN G, BACTERICIDAL PERMEABILITY IN
CREASING PROTEIN (BPI) and the DEFENSINS have been best characterised. Cathepsin G is active against gram-positive and gram-negative bacteria and the yeast form of Candida
species. It impairs the respiration and active transport of molecules across bacterial membranes and so decreases the
synthesis of nucleic acids and proteins within these or
23 ganisms {Lehrer, 1988}. BPI binds prim arily to components of the b a cte ria l 1ipopolysaccharide (LPS) and increases the permeability of the outer membrane to hydrophobic molecules.
It is selectively active against Gram-negative organisms
{Gabay, 1988}. Human defensins, HUMAN NEUTROPHIL PEPTIDES 1,
2, 3 (HNPs) are small cationic proteins (MWt 4000-8000) which make up 5% of the total protein content of the cell
{Lehrer, 1988} and display antimicrobial activity against bacteria, viruses, fungi and cells in tissue culture {Ganz,
1985; Gabay, 1988}. They a lte r the permeability of the bac terial membranes and reduce the synthesis of proteins and nucleic acids leading to loss of microbial viability.
1.2.5 DIGESTION
After ingestion, microorganisms undergo extensive degradation of their lipids, proteins and nucleic acids to form soluble products. The presence of these osmotically ac tive degradative products attracts water into the vacuole causing it to swell, thereby making contact and fusing with the plasma membrane {Segal, 1981}. Thereafter, the phagocytic contents are extruded into the extracellular medium.
1.3 0PS0NISATI0N & PHAGOCYTOSIS
1.3.1 HISTORICAL PERSPECTIVES
By the latter part of the 19th century, it was generally thought that microbes actively invaded host
leucocytes and used these cells as a means of dispersal
24 throughout the body. The white blood cells were thought to
have no role in the removal and destruction of invading o r ganisms. However, in 1882 Metchnikoff discovered that mesodermal amoeboid c e lls , which he called phagocytes, par
ticip ated in the defence of the organism against foreign in
vaders. He concluded that "diapedesis and accumulation of white corpuscles in inflammatory diseases must be regarded
as modes of defence of the organism against micro
organisms." {Metchnikoff, 1905}.
This theory was not widely accepted and the anti
bacterial properties of serum were preferred to explain the
host resistance to microbial infection. For example, von Be
hring and Kitasato demonstrated the protective function of
antitoxins in normal animals when challenged with diphtheria
or tetanus toxins. Also, Ehrlich proposed that preformed
cellular receptors and side chains gave rise to these an
titoxins and to antibodies which contributed to humoral im
munity {Metchnikoff, 1905; Bellanti, 1985}. This conflict
was reconciled by Wright and Douglas who showed that
microorganisms were p re fe re n tia lly ingested by phagocytes
after being coated with serum factors which they called
"opsonins" {Wright & Douglas, 1903}.
1.3.2 DEFINITION OF TERMS
HEAT STABLE opsonins i.e. immunoglobulins or HEAT
LABILE opsonins which are components of complement that lose
th e ir opsonic a c t iv it y when normal serum is heated to 56°C
for 30 minutes, generally interact to prepare particles for
25 ingestion. Opsonization is the process by which particles
and complexes are coated with specific serum proteins to render them more readily ingestible by phagocytic cells.
Phagocytosis is the process by which phagocytes internalize
opsonized or unopsonized particles within a vacuole known as
the PHAGOSOME.
1.4 OPSONINS
1.4.1 IMMUNOGLOBULIN G
Early researchers were aware that "immune serum" con
tained heat stable opsonins which were sp e cific fo r the im
munizing strain and effective at high dilutions even in the
absence of heat-labile factors. In contrast, the opsonic
a c tiv ity of normal serum was removed or markedly reduced by
heating the sample at 56°C fo r h a lf an hour {reviewed by
Klebanoff, 1978}. Later, specific antibodies were
demonstrated for a range of bacteria including Streptococcus
pneumoniae, Staphylococcus aureus and Streptococcus pyogenes
and in a ll cases the main immunoglobulin opsonin present was
shown to be of Immunoglobulin G (IgG) isotype {Nickerson,
1969; Humphreys, 1974; Laxdal, 1968; Williams, 1969}. It is
now known that the concentration of specific antibody
produced against these organisms is p artly determined by the
site of infection, high concentrations being common in bac
terial endocarditis {Laxdal, 1968} but chronic osteomyelitis
is associated with a lack of heat stable gamma-globulin
{W illiam s, 1969}. Moreover, work with the four IgG sub
classes has shown that PMNs respond differently to stimula
26 tion with these subclasses and the order of effective
stim ulation is IgG3 > IgGl > IgG2 > IgG4 {Leffnel, 1975}.
1.4.2 IMMUNOGLOBULINS A.M.D.E
Immunoglobulin A (IgA) also exhibits opsonic a c tiv ity
as shown by Henson {Henson, 1972} who found that aggregated
IgAl and IgA2 adhered to and were ingested by neutrophils
and moreover high levels of opsonic anti-yeast mannan IgA
were found in the sera of two patients with liver disease
{Yeaman, 1987}. The other immunoglobulin classes IgM, IgD
and IgE do not have any sig n ifica n t opsonic a c tiv ity though
IgE-coated cells readily bind with eosinophils {Walsh,
1986}. IgG also shows non-specific opsonic a c t iv it y by
hydrophobic binding to particles which is thought to be the
operative mechanism in the phagocytosis of polystyrene beads
{Klebanoff, 1978}.
1.4.3 COMPLEMENT DERIVED OPSONINS
The heat labile opsonins are formed by the activation
of either the classical or alternate pathways of complement.
The p rin cip a l opsonic fragments, formed from fixe d C3 and
C4, have been designated C3b, iC3b, C4b and iC4b and of
these iC3b is thought to be the most important {Ross, 1986}.
The classical pathway is activated in vivo by the binding
of Cl to immune complexes or aggregates containing IgG or
IgM. This results in the sequential activation and binding
of components C4 and C2 generating a C3 convertase enzyme
(C4b2a) complex which cleaves C3 into fragments C3a, an
27 anaphylatoxin and C3b. C3b can act as an opsonin, become in corporated into the C4b2a complex to form the C5 convertase
(C4b2a3b) or react with Factor B to form the alternate path way convertase (C3bBb) {Law, 1988}. In contrast, the a lt e r native pathway can be directly activated by microbial cell walls and capsular components without the involvement of any specific antibody, thereby providing a way in which non- immune sera can e ffe ctiv e ly opsonize microbes {Brown, 1981}.
1.4.3.1 C3 OPSONINS
The C3 molecule consists of a- and b-polypeptide chains held together by interchain disulphide bonds.
Cleavage of the alpha chain by C3 convertase produces the fragments C3a and C3b and exposes an internal thioester linkage in the latter. This reactive group allows the C3b moiety to become covalently linked to a nearby cell surface but this can be rapidly converted to iC3b by Factor I {Law,
1988}. The extent of conversion of C3b to iC3b is variable
(20 - 80%) and depends on the a b ilit y of the p a rticle sur face to protect C3b from the action of Factor H. Both C3b and iC3b can be further degraded to C3dg or C3d which are poor opsonins {Ross, 1986; Ehlenberger, 1977}.
1.4.3.2 C4 OPSONINS
The importance of the C4 derived opsonins in the phagocytic process remains obscure. Fixed C4b has been shown to opsonize sheep erythrocytes (RBCs) in a plasma free sys tem but because it is rapidly broken down by Factor I to
28 iC4b which is a poor opsonin, it has been held to have limited opsonic activity in vivo. However, C4b opsonization may play a major part in patients with genetic deficiency of
C3 {Ross, 1986}.
1.4.4 C-REACTIVE PROTEIN. FIBRONECTIN AND MANNOSE BINDING
PROTEIN
C-reactive protein (CRP) is an acute phase protein which is synthesised in the liver and binds to numerous ligands including the peptido-polysaccharides of microbial surfaces. It has opsonic a c tiv ity both in vivo and in vitro
{Nakayama, 1982; Horowitz, 1987; Shephard, 1986; Holzer,
1984} and has been shown to be an effective opsonin of some serotypes of S.pneumoniae {Holzer, 1984}. Fibronectin is a large glycoprotein (Mr 440kD) which is widely distributed in plasma and tissues where i t plays an important ro le in promoting cell-cell and cell-substratum interactions {La
Fleur, 1987}. It is secreted from a variety of cells and even though its role as an opsonin has been disputed
{Lanser, 1981; Verbrugh, 1981; Wan de Water, 1983}, i t is known to stimulate monocyte and PMN phagocytosis {Pommier,
1984}. Mannose binding protein (MBP), produced by the liver, is an acute phase reactant which has been shown to bind to mannose rich glycans {Kuhlman, 1989}. It augments the phagocytosis of mannose rich strains of S.montevideo and is thought to interact with the Clq receptor on surfaces of phagocytes {Kuhlman, 1989}.
29 1.4.5 NON OPSONIC PHAGOCYTIC ENHANCERS
Several substances are known to enhance phagocytosis without acting as opsonins. These include the tetrapeptide
TUFTSIN {Fridkin, 1981}, Substance P, a neuropeptide with a tuftsin-like N-terminal residue {Bar-Shavit, 1980},
Neurotensin {Goldman, 1983}, the cytokines interferon gamma
(IFN-c) and tumour necrosis factor (TNF) {Shalaby, 1985} and hyaluronic acid {Hakansson, 1980}.
1.5 NEUTROPHIL PHAGOCYTIC RECEPTORS
1.5.1 Fc RECEPTORS
Fc receptors consist of "a group of surface membrane molecules that s p e c ific a lly recognize and bind homologous immunoglobulin via the Fc portion, and which putatively mediate b io lo g ic functions" {Unkeless, 1988}. Three d i f ferent Fee receptors which have antigenic, biochemical and functional differences {Fleit, 1988} and which are desig nated huFccRI, huFccRII and huFccRIII have been id e n tifie d on human leucocytes. Cloning studies have shown that the
Fee receptors are members of the immunoglobulin superfamily which are thought to have evolved from a common primordial gene {W illiam s, 1988}. Of these, FccRII and FccRIII are normally expressed on the neutrophil though IFN-c can induce the expression of the FcRI on PMNs {Unkeless, 1988}. Reset tin g experiments {Wong, 1975; Henson, 1969} with antibody coated RBCs were in itially used to demonstrate the presence of Fc receptors on the neutrophil surface but have been re placed by the use of sp ecific monoclonal antibodies (mAbs)
30 which enable these receptor molecules to be identified and
characterized.
1.5.1.1 HUMAN FccRI
HuFccRI (CD64) is a monovalent 72 kD sialoglycoprotein
present on monocytes and macrophages which can bind
monomeric IgG with a high a f f in it y (Ka = 10“8 to 10“9 M)
{O'Grady, 1986}. It is thought to mediate antibody-
dependent cellular cytotoxicity (ADCC) and its expression on
these cells can be increased by stimulation with IFN-c and
C5a {Unkeless, 1988}.
1.5.1.2 HUMAN FccRII
The second receptor FccRII (CDw32), encoded on
chromosome 1 {Peltz, 1989}, is a 40kD protein with low a f
f in it y for IgG and which has been identified on monocytes,
p la te le ts , neutrophils and eosinophils {Looney, 1986}.
Neutrophils express about 10,000 - 20,000 of these molecules
per cell and IgG binding to these receptors has been shown
to be essential for IgG induced activation of the
respiratory burst and IgG-dependent phagocytosis {Huizinga,
1989}.
1.5.1.3 HUMAN FccRIII
The third receptor, Fc RIII (CD16) is also widely dis
tributed on NK cells, eosinophils, neutrophils and tissue
macrophages though not on monocytes {Unkeless, 1988}. It is
an acidic complex sialoglycoprotein {Fleit, 1988} with a
31 molecular weight ranging from 50 - 70 kD p a rtly because of
heterogeneity of glycosylation {Kimberly, 1989}. It is a low
a ffin it y receptor which binds aggregates of IgG but not the monomeric form {Unkeless, 1988}. There are about 100,000 -
200,000 Fc R111 molecules per neutrophil {Huizinga, 1989}.
The receptor appears on the neutrophil surface at the
metamyelocyte stage {Unkeless, 1988} and is important in
regulating a number of functions including the differential
control of granule enzyme release, the modulation of super
oxide generation and the phagocytosis of opsonized particles
{Rosenberg, 1985} and soluble immune complexes {Unkeless,
1988}.
Polymorphic forms of FccRIII, as described by Ory and
colleagues {Ory, 1989}, have been found to co rre la te with
the two allotypes of the neutrophil antigen (NA) system.
People with the 29 and 33 kD form of the receptor expressed
both NA1 and NA2 antigens while others with only the 29kd or
33kd molecules were e ith e r NA2NA2 homozygotes or NA1NA1
homozygotes.
The neutrophil form of CD16 is attached to the plasma
membrane by a phosphatidylinositol-glycan linkage which is
affected in paroxysmal nocturnal haemoglobinuria (PNH), a
disease in which phosphatidylinositol-glycan biosynthesis is
defective {Lanier, 1989}. Neutrophils from these patients
express only about 7% of normal levels of FcRIII {Selvaraj,
1988}. The gene for FccRIII is closely associated with the
gene fo r FccRII {Peltz, 1989} and has been mapped onto
chromosome 1. It yields a 46 kD protein which folds to form
32 two immunoglobulin lik e domains which share considerable homology with other Fc receptors.
1.5.1.4 HUMAN FcaR
Receptors for the Fc portion of IgA (FcaR) have been id en tified on lymphocytes, neutrophils and monocytes, where they bind both monomeric and dimeric forms of IgA, possibly by the CH2 domain of the heavy chain {Fanger, 1981}. Recep tors have been found on 35% of blood neutrophils and on 62% of oral PMNs so it has been suggested that as the PMNs migrate to mucosal sites they express more receptors and can phagocytose IgA coated targets better than th e ir blood coun terparts {Fanger, 1983}.
1.5.2 COMPLEMENT RECEPTORS
Receptors for several complement components have been identified but only those which bind fragments of C3 namely, CR1, CR2, CR3 and CR4, are thought to be important in mediating opsonophagocytosis.
1.5.2.1 COMPLEMENT RECEPTOR TYPE 1
Complement receptor type 1 (CR1, CD35) is a membrane glycoprotein which is present on a variety of cells includ ing erythrocytes and neutrophils {Ross, 1986}. There are four known allotypes of CR1 with molecular weights of 190K
(type A), 220K (type B), 250K (type C) and 160K (type D)
{Ross, 1985} but in addition there are cell specific dif ferences in molecular size {Dykman, 1983}. Resting human
33 neutrophils express about 5000 CD35 molecules per cell
{Fearon, 1984}. These receptors are encoded by a gene lo cated on chromosome 1 which is part of a supergene family termed the REGULATOR OF COMPLEMENT ACTIVATION (RCA) {Wong,
1989}. CR1 binds to C3b p rin c ip a lly but also to fragments
iC3b and C4b {Fearon, 1984} either singly or in clusters
{Ross, 1985}. This is consistent with the presence of three distinct binding sites within CR1 near the N-terminal end of the molecule which can interact m ultivalently with C4b/C3b and C3b/C3b complexes { K lic k s te in , 1988}. Neutrophil CD35 mediates the ingestion of soluble immune complexes and par tic le s opsonized with C3b and C4b and enhances Fc dependent
phagocytosis {Fearon, 1984}.
An in tra c e llu la r pool of CR1 molecules has been found
in a membrane-enriched cytoplasmic fraction that is d is tin c t from primary or secondary granules. The upregulation of
plasma membrane expression of CR1 which follows PMN stimula
tion is due to the translocation of the CR1 receptors from
th is su b ce llu la r location to the plasma membrane {O'Shea,
1985}.
1.5.2.2 COMPLEMENT RECEPTOR TYPE 2
CR2 (CD21), the C3d receptor which is present on B
lymphocytes is also the B cell receptor for Epstein-Barr
virus and consists of a single glycoprotein chain of molecular weight 140-145 kD {Fearon, 1984}. This receptor
has separate binding sites for virus and ligand, and has
been shown to rosette with particles bearing C3dg and iC3b
34 ligands {Ross, 1985}. The gene for CR2 maps onto chromosome
1 and is separated from the CR1 gene by about 75kB.
1.5.2.3 COMPLEMENT RECEPTOR TYPE 3
The complement receptor type 3 (CR3, CDllb/CD18) con sists of two non-covalently linked glycoprotein chains, a
(165K) and b (95K) {Ross, 1986}, of which the b chain is also shared by other members of the CD11/CD18 family of adhesive glycoproteins. The receptor is found on monocyte/macrophages, NK and ADCC effector cells, some T cells and neutrophils and binds strongly to fixed iC3b and to a lesser extent with fixed C3d and C3dg {Ross, 1986}. In addition, the molecule also has a lectin binding site {Ross,
1986; Kuhlman, 1989} which can bind with substances such as baker's yeast (Saccharomyces cerevisiae).
The binding sites on this receptor have been iden tified on the a-chain, the first of which binds to iC3b,
C3dg and C3d and the second is sp e cific fo r b-glucan {Ross,
1986}. Both site s require calcium and magnesium ions fo r binding and the b-glucan site is inhibited by the sugar
N-acetyl-D-glucosamine (NADG) which suggests that its ligand binding s ite is an amino-sugar {Ross, 1985}. In addition, the iC3b segment which binds to CR3 contains the sequence
Arginine-Glycine-Aspartate (RGD) which is also recognized by other adhesion-promoting receptors such as the fibronectin receptor and the platelet glycoprotein Ilb/III {Wright,
1987}.
35 The resting neutrophil has about 4000 CR3 molecules per c e ll {Ross, 1985} but receptor upregulation occurs spon taneously at 37°C, can be induced by mechanical stress in curred during cell preparation {Hed, 1988}, or by exposure to chemotactic factors {Ross, 1985}. This upregulation is due to translocation of preformed CR3 molecules from an
intracellular pool on the specific granule membranes
{O'Shea, 1985}. This increased expression is calcium de pendent and reqires the m obilization of both in tra c e llu la r
and extracellular calcium stores {Berger, 1988}. However,
the 10-20 fold increase in surface expression of CR3 molecules seen after FMLP stimulation is not associated with
increased phagocytic activity of these receptors {Ross,
1986} and further activation to full phagocytic capacity can
be induced by the binding of fibronectin, serum amyloid
protein (SAP) and laminin to the neutrophils {Wright ,
198*}.
The study of patients with an inherited deficiency
of the CR3/LFA-1/CR4 fam ily of adhesion molecules, a condi
tion known as leucocyte adhesion deficiency (LAD), has
provided important insights into the in vivo functions of
CD11b/CD18. These patients suffer from recurrent, severe
bacterial infections primarily of the skin, mucous membranes
and intestinal tract though involvement of the upper and
lower respiratory tract also occurs commonly {Anderson,
1987}. Their phagocytes have defects of all adhesion-
dependent functions including impairment of in vitro and in
vivo movement tests and phagocytosis of iC3b coated par-
36 tid es. These defects were reproduced in vitro by treating normal c e lls with anti-CR3 antibodies {Anderson, 1987} con firming that the CD1lb/CD18 molecule is important in promoting phagocyte adhesion-dependent interactions such as cell-substrate, cell-cell, cell-endothelium or cell-organism interactions.
1.5.2.4 COMPLEMENT RECEPTOR 4
The complement receptor type 4 (CR4, CDllc/CD18) is the third member of the CR3/LFAl/pl50,95 family and is dis tributed on several cell types including neutrophils, monocytes and macrophages {Myones, 1988}. This receptor is expressed on freshly isolated neutrophils and monocytes in small amounts, binds to fixed iC3b, C3dg and C3d as well as soluble C3dg and has many functions in common with CR3 such as phagocytosis of iC3b and C3d coated particles and immune complexes {Ross, 1986; Myones, 1988}. The higher density of expression of CR4 on tissue macrophages is thought to be im portant in the removal of iC3b or C3d opsonised immune com plexes from the circu latio n {Myones, 1988}.
1.6 MECHANISM OF PHAGOCYTOSIS
Phagocytic ingestion occurs by circumferential ligand-receptor interaction which allows the progressive encroachment of cellular pseudopodia around the particle.
This "zipper" mechanism of phagocytosis (Griffin, 1975} was elegantly demonstrated using mouse B-lymphocytes, which were coated wholly or partially with anti-mouse IgG; the former
37 were completely ingested by the macrophage whereas the la t ter only adhered to the phagocyte. This process of inges tion consists of:- the recognition of the particle by the phagocyte, the transmission of a message from the plasma membrane to the e ffe cto r mechanisms of the c e ll, the as
sembly and movement of pseudopodia and f in a lly the fusion of the pseudopodia at the d istal surface of the p a rticle .
Close contact between the particle and phagocyte facilitates the process of particle recognition and involves
a number of factors, including the overall surface charge of
the particle and the cell, the hydrophobicity of the par
ticle and the opsonization of the particle, {Klebanoff,
1978}. A reduction in the net negative charge on the
neutrophil favours phagocytosis {Weiss, 1966}. However,
synovial fluid PMNs of patients with rheumatoid arthritis
have a low net negative charge but impaired phagocytosis
{Brown, 1988}. The hydrophobicity of p articles does however
correlate with susceptibility to phagocytosis and the more
hydrophobic a particle is relative to the neutrophil, the
more susceptible it is to phagocytosis {van Oss, 1972a}.
Furthermore, binding of specific anti-capsular antibodies to
the surface of bacterial capsules has the effect of render
ing the particle relatively more hydrophobic thereby
facilitating phagocytosis {van Oss, 1972b}.
The transmission of the phagocytic signal from the
surface of the PMN to the e ffe cto r mechanisms w ith in the
c e ll has not been f u lly elucidated but some clues have been
obtained from the study of membrane transduction following
38 chemotactic peptide-receptor binding (see 1.2.2). Similar mechanisms are thought to operate in Fc-receptor mediated phagocytosis {Gresham, 1987}. However, the transduction events following the phagocytosis of unopsonized and op sonized C.albicans are not associated with changes in the formation of in o sitol phosphates or a rise in in tra c e llu la r free calcium levels {Rossi, 1989}.
Signal transmission causes changes in the microfilamentous structures within the organelle-free hyaline ectoplasm which leads to the formation of pseudo podia {Stossel, 1976; Valeriu s, 1982}. To explain th is development Stossel and Hartwig {Stossel, 1976} in itia lly postulated a filament assembly and compression process in which perturbation of the plasma membrane at the point of particle contact resulted in activation of actin binding protein (ABP). This gave increased crosslinking of actin filaments to form a gel which was compressed by myosin and its cofactor to produce a pleat or pseudopod. This model was later reviewed {Valerius, 1982} to include the effect of lo calized increases of cytoplasmic calcium on the different actin binding proteins.
It was then postulated that in the resting phagocyte there are actin filaments of uniform lengths which are crosslinked by ABP to give a gel of uniform rigidity. Fol lowing stimulation of the plasma membrane by an opsonized particle, an influx of calcium into the subjacent cortical cytoplasm activates gel sol in, an actin severing protein, to shorten the actin filaments and cause a localized decrease
39 in the r ig id it y of the cytoplasm. The myosin molecules, in terspersed among the actin bundles, are also activated by the free calcium, thereby drawing the shortened actin f i l a ments and associated proteins from this "sol" like area to one of greater rigidity. The resultant cytoplasmic streaming produces a pseudopod which moves around the p a rtic le as a result of the sequential adherence of the cell membrane receptors to other ligands on the particle surface. This theory, though attractive, fails to explain how actin polymerization can take place in the absence of calcium
{Naccache, 1987; Lew, 1985}. Moreover, it is now known that signal transduction is also associated with other phenomena that may play a part in the initiation and assembly of actin filam ents namely, c y to so lic pH changes {Naccache, 1987}, changes in the composition of membrane lip id s and the phos phorylation of cytoskeletal components {Omann, 1987}.
The result of this process of ingestion is the forma tion of the PHAGOSOME with which the lysosomes coalesce to form a PHAGOLYSOSOME. Vacuoles are of two types, either fully sealed or unsealed; the latter account for 20-40% of the total phagolysosomes {Cech, 1984; Kemp, 1986}. Kemp and
Turner {Kemp, 1986} have suggested that the unsealed vacuoles are the consequence of the toxic effects of lysosomal granules and free radicals on the lining membrane.
Also, the reduced k illin g of the ingested p a rticle s within the unsealed as compared with the sealed vacuoles may be re lated to the failure of the unsealed vacuoles to acidify
{Cech, 1984}.
40 1.6.1 THE DIFFERENTIAL EFFECTS OF IGG AND COMPLEMENT DERIVED
OPSONINS ON PHAGOCYTOSIS
Experiments using particles specifically labeled with
IgG and C3b {Mantovani, 1975; Kaplan, 1977; Ehlenberger,
1977; Newman, 1979; Hed, 1982; Kemp, 1986} have shown that these opsonins effect phagocytosis differently. Thus, C3
derived opsonins caused p a rticle attachment without inges
tion in systems that used RBCs as the phagocytic targets,
but where yeast particles were utilized, there was evidence
of ingestion. Furthermore, binding of C3b-opsonized par
ticles to the phagocyte membrane did not appear to induce
superoxide generation {Hed, 1982} or stimulate the release
of b-glucuronidase or lysozyme {Newman, 1979}.
In contrast, IgG induces the ingestion of RBCs espe
cially at high IgG molecule/RBCs ratios {Ehlenberger, 1977}
and stimulates the respiratory burst and degranulation
{Newman, 1979; Hed, 1982}. These effects of IgG and C3b are
separate but complementary so that in the presence of the
C3b ligand, fewer molecules of IgG are required to induce
ingestion. Ehlenberger and Nussenzweig {1977} estimated
that fo r PMNs, one-tenth the amount of IgG normally needed
to induce IgG dependent phagocytosis of sheep RBCs was
enough when C3b was also present.
The importance of C3b mediated attachment in allowing
phagocytosis to occur is most marked when the cells and par
t ic le s are in suspension {Ehlenberger, 1977; Kemp, 1986}.
In this situation IgG coated particles are not ingested in
the absence of C3 derived opsonins, but the binding of C3
41 ligands to specific receptors overcomes the shearing forces between particles and cells and allows the internalization of IgG coated particles {Ehlenberger, 1977}.
Morphological evidence for differences in IgG and C3b mediated phagocytosis has been provided by Kaplan {1977} using scanning and transmission electron microscopy. He showed that IgG coated sRBCs were "ingested by means of thin membrane extensions risin g from the macrophage surface and enclosing the opsonized particles tightly in a cup-like structure protruding from the macrophage surface." But C3 opsonized sRBCs "were seen to sink d ire c tly into the macro phage cytoplasm without apparent involvement of membrane extensions.11 The author suggested that these results could be due to differences in the response of microfilaments to the two phagocytic stimuli.
1.6.2 SURFACE PHAGOCYTOSIS
Surface phagocytosis is the ingestion of particles by phagocytes in the absence of antibodies which occurs as a result of the particle being trapped against a rough surface such as glass fibre, fibrin strands or another leucocyte
(intercellular phagocytosis) {reviewed in Klebanoff, 1978}.
This mechanism is thought to be important in the e a rly phases of infection with encapsulated organisms before the development of type sp e cific antibodies.
42 1.7 TESTS OF PHAGOCYTOSIS AND OPSONIZATION
Tests of phagocytosis are broadly classified into two catergories: in the first, phagocytic cells and test par t ic le s are both suspended in known volumes of liq u id s , mixed, and if the shearing forces between the c e ll and par ticle are overcome, there can be effective phagocytosis. In the second type of assay, the phagocytes are allowed to adhere to a surface and the test p a rticle s are layered over the cells. An outline of the range of phagocytic assays that are available is given in Table 1.1.
In all phagocytic assays it is important to distin guish between true p a rtic le ingestion and accidental as sociation between cell and particle. In order to do this,
Stossel {1975} has suggested three criteria that should be fulfilled before a test can be said to measure true inges tion: Firstly, there should be little or no ingestion when the cells and particles are incubated at 0-4°C. Secondly, measurement of p a rtic le uptake should be n e g lig ib le in zero-time controls and thirdly, the system should be saturable with increasing particle to phagocyte ratios
{Stossel, 1975}. Additionally, several techniques have been used to distinguish between internalized particles and those that are merely adherent to the phagocytic surface for ex ample, electron microscopy, the removal of adherent staphylococci with lysostaphin or by exploiting the dif ferent fluorescent properties of living and dead bacteria after staining with fluorochromes.
43 T A B LE 1.1 METHODS FOR MEASURING OPSONIZATIONVPHAGOCYTOSIS
METHOD PARTICLE LABELNPROBE DETECTION REFERENCE
C.AIbicans Methylene blue Light microscope Lehrer.1969 MICROSCOPIC zym osan PASNmethyl green bacteria gram stain Phase contrast Mac,Farlane,1984
S.aureus Tritium Roberts,1982 E.coli 14-C arbon Hammer,1981 ISOTOPIC P.aeruginosa 45-Calcium, gamma counter Suzuki, 1971 C.botulinum 75-selenium Human albumin 131-Iodine Chang, 1969
EXTRACTION LPSXOil Red 0 spectrophotometry Stossel,1973
Yeast Yamamura,1977 PARTICLE 3H-labeled amino S.aureus gamma counter Foroozanfar,1976 REMOVAL or nucleic acids E.Coli Rajkovic,1985 S.epidermidls Harvey, 1986
Bacteria chemiluminescence luminometer Easmon,1981 INDIRECT zym osan iodination gamma counter Morris, 1981 METHODS Bacteria NBT reduction Elisa reader Rainard,1986 Yeast Heat production Therm ocell Hayatsu,1988
Yeast Acridine orange Wilson, 1985 FITCNtrypan blue Fluorescent Hed,1987 FLUORESCENT S.aureus Texas red activated cell Trinkle,1987 METHODS S.pneumonlae naphthalamide sorter (FACS) Sveum,1986 nanoparticles Ethidium bromide Rolland,1987
ELISA K.pneumoniae specific antibody Elisa reader Athamna,1988 METHODS
IN VIVO Bacteria 131-Iodine METHODS Blozzi,1961
PAS Periodic acid Schlff LPS Llpopolysaccharlde FITC Fluoreaceln laothlocyanate
44 1.8 DEFECTS OF OPSONIZATION AND PHAGOCYTOSIS
Defects of phagocytosis may be due to deficiencies within the phagocyte itself (CELLULAR DEFECTS) or to the in ability of the serum to opsonise the particle (HUMORAL
DEFECTS) either because of a lack of an opsonin or due to the presence of inhibitory substances within the serum. A l i s t of the c e llu la r and humoral causes of defective phagocytosis is given in Tables 1.2 and 1.3.
1.8.1 THE CLINICAL PRESENTATION OF PATIENTS WITH PHAGOCYTIC
DEFECTS
Patients with possible opsonic or phagocytic defects of clinical importance present with frequent bacterial and fungal infections usually starting in childhood {Axtell,
1988}. The infectio n s are often severe and p e rsiste n t despite a n tib io tic therapy and may be caused by unusual o r ganisms, for example Serratia sp. or Aspergillus. However, the usual pattern of infection is non-specific {Segal, 1985} and caused by ubiquitous pathogens lik e the pyogenic cocci and Gram negative organisms {Johnston, 1984}. In general the seriousness of the clinical disease correlates with the severity of the underlying defect.
45 TABLE 1.2 CELLULAR CAUSES OF DEFECTIVE PHAGOCYTOSIS
DISEASEDEFECTREFERENCE
Leucocyte adhesion CD11b\CD18 Anderson, 1987 deficiency (LAD) deficiency
Systemic lupus defective CR1 Mir,1988 erythematosus function
Factor I CR1 blockage Porteu,1986 deficiency
Autoimmune neutropenia Fc receptor Boxer, 1974 Felty's syndrome blockage Gupta, 1976
Bordeteila pertussis impaired signal reviewed by infection transduction Boxer, 1987
reviewed by Neutrophil actin defective actin Southwick, dysfunction assembly 1988 & 1989
Defective microtubule ? abnormal tubulin Gallin,1978 assembly
impaired ingestion Acatalasaemia Matsunaga, 1985 with H2Oz stress
46 T A B LE 1.3 DISEASES ASSOCTIATED WITH DEFECTIVE OPSONIZATION
DISEASE DEFECT REFERENCE
C2 Turner, 1978 C4 Clark, 1975 COMPLEMENT (C) C3 Ballow,1975 DEFICIENCIES FACTOR I Abramson, 1971 C5 dysfunction Jacobs, 1972 Factor B Stossel,1973
Agammaglobulinaemia Mickenberg, 1970 IMMUNOGLOBULIN Specific antibody Bra conier,19BH. DEFICIENCIES deficiencies Monteil,1987 Tuftsin Najjar, 1975
SLE Jasin,1974 RA McDuffie, 1972 C consumption and Severe Infections Alexander, 1976 immune complexes Thermal Injury Bjornson,1973
Newborns Low C values Stossel,1973
Sickle Cell Alternate pathway Johnston, 1973 Disease defect & Post-splenectomy ?Tuftsin deficiency Najjar, 1975
Mannose binding Super, 1990 protein deficiency
C complement SLE systemic lupus erythematous RA rheumatoid arthritis
47 Infections occur most commonly at sites with high microbial colonization {Segal, 1985} like the skin, the mucous membranes and gums, the sinuses and upper respiratory tract or at contiguous anatomical locations such as the middle ear and the lower respiratory tract. Lymphadenitis may accompany infections at any site. Septicaemia and in fe ctio n s of deeper structures such as the liv e r and bones may occur in more severe cases but meningitis is rare among this group of patients {Axtell, 1988}. In addition, delayed wound healing often complicated by secondary infections as well as failure of separation of the umbilical cord may be presenting complaints.
The findings at physical examination are also non specific but may give some indication of the severity of the underlying defect. For example, the number and size of scars give some measure of the frequency and extent of abscess formation. Other sequelae of infection which may be found include scarring of the tympanic membranes, loss of teeth, poor weight gain and short stature {Axtell, 1988}.
1.8.2 THE INVESTIGATION OF PATIENTS WITH PHAGOCYTIC DEFECTS
The investigation of patients with possible op- sonophagocytic disorders consists of two types of tests: firstly, tests are done to exclude other causes of im munodeficiency such as a) FULL AND DIFFERENTIAL BLOOD COUNTS and BLOOD FILM b) QUANTITATION OF IMMUNOGLOBULIN LEVELS and
ESTIMATION OF COMPLEMENT COMPONENT C3 and C4 +/- A TEST OF
COMPLEMENT FUNCTION eg (CH50). The second battery of tests
48 is aimed at defining the s p e c ific c e llu la r or humoral le sion. In our department the tests routinely used to assess opsonophagocytosis are the STAPHYLOCOCCAL INHIBITION ASSAY
{Foroozanfar, 1976} and the CANDIDA KILLING ASSAY which also measures the m icrobicidal capacity of the PMNs. Cross over experiments are used to test patient phagocytes and sera separately.
49 CHAPTER 2: THE COCCI
2.1 STAPHYLOCOCCUS AUREUS
2.1.1 BACTERIAL STRUCTURE
Staphylococcus aureus is one of the most successful
and versatile of pathogenic bacteria. Its infections are
found worldwide and date back to early man. It is a Gram
positive, catalase-positive, non-motile organism which forms
discrete creamish colonies on nutrient agar (Figure 2.1).
Staphylococcal cells are spheroid with a diameter of
approximately 1 ^m and are bounded by a plasma membrane, a
cell wall and a capsule in some strains. The constituents
of the cell wall and the presence of a capsule are
p a rtic u la rly relevant to host-bacterial interaction because
they contribute to the antigenicity and the protection of
the organism {Peterson, 1983}.
The c e ll wall is composed of three major components:
peptidoglycan (PG), teichoic acid (TA) and protein A (PA).
Peptidog 1 yean is a linear polymer of repeating
N-acetylglucosamine and N-acetylmuramic acid residues with
pentaglycine bridges between neighbouring polymers and
accounts for 50% of the weight of the cell wall. TA, a
negatively charged ribitol-phosphate polymer which is
covalently linked with peptidoglycan, comprises 40% of the
bacterial cell wall. PA accounts for only 5% of the cell wall weight but has the a b ilit y to bind onto the Fc portion
of IgG molecules making these unavailable to the Fc
receptors of phagocytes {Peterson, 1983}.
50 FIGURE 2.1 Overnight growth of Staphylococcus aureus on blood agar
i
2.1.2 VIRULENCE FACTORS
2.1.2.1 THE CAPSULE
Encapsulated staphylococci are more resistant to phagocytosis {Peterson, 1978} and killing {Adlam, 1984} than non-encapsulated forms. The capsule prevents efficient opsonization of the organism in non-immune sera by acting as a barrier between the opsonins deposited on the cell wall and the phagocytic receptors {Peterson, 1978;Edition, 1984}*
The ability to resist phagocytosis correlates with bacterial virulence so that unencapsulated strains are generally less virulent than encapsulated bacteria {Wilkinson, 1979}.
51 Freshly isolated organisms express capsular antigens suggesting that some staphylococci produce capsules in vivo
{Adlam, 1984}.
2.1.2.2 ENZYMES AND TOXINS
S.aureus produces several enzymes and toxins which may facilitate the development of staphylococcal infections
{Easmon, 1984}. The enzymes include staphylocoagulase, lipase and hyaluronidase while the toxins include four haemolysins, two types of epidermolytic toxins, enterotoxins
A-F, a leucocidin and a pyrogenic exotoxin. However, the role of some of these factors in staphylococcal infection is imprecisely understood {Adlam, 1984}.
Two of the better studied toxins are alpha haemolysin
(a-toxin) and the Panton-Valentine leucocidin. Alpha haemolysin has necrotizing, haemolytic, and lethal properties which may contribute to some of the features of staphylococcal septicaemia {Adlam, 1984}. Low levels of a n ti-to x in have been found in most normal sera and may protect the host from the lethal effects of a-toxin {Adlam,
1984}. The Panton Valentine leucocidin consists of two components, F and S, which are only biologically active when mixed together {Adlam, 1984}. It is c y to ly tic for human
PMNs and macrophages though its mode of action is s t i l l undetermined {Easmon, 1984}. The toxin is produced in vivo as evidenced by the detection of anti-leucocidin antibodies in patients with staphylococcal osteomyelitis {Adlam, 1984}.
52 2,1.3 STAPHYLOCOCCAL INFECTIONS
The basic lesion of staphylococcal infections is an abscess with a core of dead or dying bacteria and leucocytes surrounded by a relatively avascular wall. Such lesions can occur at almost any site in the body but are usually confined to superficial sites such as the skin and eyelids.
Indeed, deep seated staphylococcal infections are often a sign of an underlying defect in host defence {Easmon, 1984}
(Table 5.6). Other factors that predispose to serious
S. aureus in fectio n s include damage to normal skin, v ir a l in fe ctio n s which immunosuppress the host, fo r example influenza, and the presence of other diseases such as diabetes mellitus, cystic fibrosis, malignancy and alcoholism. The prophylactic use of antibiotics to which
S.aureus is not susceptible can alter the normal bacterial flo ra and enhance staphylococcal colo n izatio n {Shulman,
1970}.
S.aureus infections of the skin are the commonest of all bacterial infections and account for about 2-5% of the patients seen by general practitioners in Great Britain
{Shulman, 1970}. The infections usually begin around hair follicles and are more frequent in areas of high humidity for example, the axillae. The majority of patients presenting with skin lesions were found to be nasal carriers of the infecting organism. However, since it has been estimated that 25% of persons in the community are nasal c a rrie rs { P h illip s , 1987}, though only a portion of these have recurrent staphylococcal lesions, it follows that
53 there must be other predisposing factors, some as yet unidentified.
Deep-seated S.aureus infections can occur by contiguous spread from su p erficial sites, by the insertion of foreign materials like prostheses into the body or by the introduction of the organism directly into the bloodstream {Easmon, 1984}. Fortunately the spread of
S.aureus infection from superficial lesions to deeper tissues is uncommon and often due to the failure of the host phagocytic system to contain the in it ia l infection. However, the recently described staphylococcal toxic shock syndrome
(STSS) tends to occur in young previously healthy patients, particularly young women during a menstrual period. It is an acute illness characterized by high fever, hypotension, marked hyperaemia of the mucous membrane and conjunctivae, diffuse palmar erythema leading to desquamation of the skin of the hands and feet and multiorgan dysfuction or failure
{Davis, 1980}. It is associated with enterotoxin-producing strains of S.aureus, especially those producing Toxic Shock
Syndrome Toxin-1.
2.1.4 HOST RESISTANCE TO STAPHYLOCOCCAL INFECTIONS
The intact skin is a major deterrent to staphylococcal infection as evidenced by the rapidity with which the organism infects cuts and bruises. The sp e cific immunity of the skin is thought to be due to the secretion of lysozyme and immunoglobulins {Easmon, 1984} and the presence of other microbial flora which prevent staphylococcal colonization
54 {Verhoef, 1981}. The phagocytes, especially the PMNs, are
the principal subcutaneous defence once S.aureus has invaded
the integument so that deficiency of neutrophil number or
defects of opsonophagocytosis and killing are associated with staphylococcal septicaemia and deep-seated infections.
Both heat-stable and heat-labile opsonins contribute
to the efficient phagocytosis of S.aureus. In non-immune
sera, the C3 derived opsonins are the main opsonins and can
be generated by bacterial activation of the alternative
pathway {Peterson, 1983}. While in immune sera both sp e cific
antibodies and C3 derived opsonins are important, the
la tte r being formed as a re s u lt of c la s s ic a l pathway
activation by anti-staphylococcal antibody immune complexes.
Anti-staphylococcal antibodies are of two types, low potency
"natural" antibodies and IgG opsonins generated during
S.aureus infection {Koenig,1972}. The former are directed
against c e ll-w a ll components shared by many Gram-positive
organisms {Verhoef, 1981} but the immune antibodies have
heterogenous specificities including the bacterial capsular
antigens, the staphylococcal toxins and enzymes and the cell
wall components {Verhoef, 1981, Adlam, 1984}. Cell mediated
immunity (CMI) to S.aureus has also been demonstrated in
animals and man following repeated or prolonged exposure to
the organism {Adlam, 1984; Verhoef, 1981} and may give rise
to damaging hypersensitivity reactions.
55 2.2 GROUP A BETA-HAEMOLYTIC STREPTOCOCCI
2.2.1 STRUCTURE
Streptococcus pyogenes are Gram positive, bacitracin sensitve organisms that ape associated with a range of local, systemic and immunologically mediated diseases in man
{Jawetz, 1984} (Figure 2.2). Individual cocci are spherical and contain several antigenic structures that either contribute to microbial virulence or are important in the develpoment of host immunity. The hyaluronic acid capsule is not immunogenic but is a virulence factor while the surface M and T proteins are antigenic and in the case of the M protein, antiphagocytic {Krause, 1972}. There are more than seventy known M types among Group A organisms
{Wannamaker, 1982}.
FIGURE 2.2 Overnight growth of Streptococcus pyogenes on i blood agar
56 2.2.2 VIRULENCE FACTORS
2.2.2.1 SURFACE COMPONENTS
The streptococcal surface components which act as virulence factors are the fimbriae, the capsule and the M protein. The fimbriae project from the outer surface of the organisms and fa c ilit a t e bacterial adherence to e p ith e lia l cells by lipoteichoic acid constituents {Wannamaker, 1982;
Beachey, 1983}. Once the organism has penetrated the mucous membrane or skin, the hyaluronic acid capsule may influence
streptococcal survival. It is present in most epidemic
strains of streptococci {Wannamaker, 1982} and if removed causes a dramatic drop in the virulence of the test strain
{Krause, 1972}. Further resistance to the host defence is
provided by the M proteins which are a family of fibrillar
proteins {Bessen, 1989} with potent anti-phagocytic
properties {Wannamaker, 1982}. One mechanism by which the M
proteins exert their antiphagocytic effect is by binding to
Factor H which inhibits the formation of the alternate
pathway convertase and prevents the deposition of C3b molecules on the streptococcal surface -{Horstmann, 1988}.
Type specific antibodies for the M proteins opsonize the
streptococci and render them more susceptible to
phagocytosis {Wannamaker, 1982}.
2.2.2.2. ENZYMES AND TOXINS
The group A streptococci produce several e x tra ce llu la r
products but o f these o n ly the PYROGENIC EXOTOXINS
(erythrogenic toxins, SPE) and the HAEMOLYSINS S and 0 have
57 toxic properties {Wannamaker, 1983}. The three known SPEs designated A, B and C are low molecular weight proteins with several primary and secondary toxic effects such as pyrogenicity, skin reactivity, tissue damage and an enhanced s u s c e p tib ility to endotoxic shock {Kim, 1972}. D iffe re n t streptococcal strains can produce one, two or all of the
SPEs {Hallas, 1985}. Exotoxin A has been implicated in the fatal form of scarlet fever that was common before the antibiotic era {Hallas, 1985} and in the recently described
Streptococcal Toxic Shock-like syndrome (T515) {Stevens,
1989}. Haemolysins S and 0 are c y to ly tic toxins which damage a broad spectrum of mammalian cells. They bind to steroids in the c e ll membrane and induce swelling and death of the cells {Wannamaker, 1983}. Increased anti-streptolysin
0 antibodies in the host indicate recent streptococcal infection {Jawetz, 1984}.
2.2.3 STREPTOCOCCAL DISEASES
S.pyogenes causes a diversity of clinical diseases which can be c la s sifie d into:
1) Diseases caused locally or by invasion of the organism, for example, t o n s illit is , pyoderma, erysipelas and puerperal fever
2) Diseases attributed to streptococcal toxins, fo r example, the streptococcal toxic shock-like syndrome
3) Poststreptococcal diseases, notably rheumatic fever and acute glomerulonephritis
58 2.2.4 HOST RESISTANCE TO STREPTOCOCCAL INFECTION
Type-specific antibodies, directed against the streptococcal M-proteins and which enhance the phagocytosis of these organisms, provide the main host resistance to
S.pyogenes in fe ctio n . These antibodies protect against reinfection by the infecting strain but do not prevent in fe ctio n by other M-type streptococci {Jawetz, 1984}.
Moreover, only those antibodies directed against epitopes in the N-terminal part of the M-protein molecule have been shown to be opsonic {Jones, 1988}. Immunity to the pyrogenic toxins is provided by sp ecific antitoxins and indeed the presence of anti-erythrogenic toxin antibodies is inferred from a negative Dick test {Jawetz, 1984}.
59 2.3 AIMS OF THE PROJECT
Patients with clinically significant Staphylococcus aureus infections account for 40% of all patients attending the Department of Immunology, Westminster Hospital for investigation of neutrophil function. Since defects of antibody production or function could predispose to infection, the first aim of this project was to look for possible defects of opsonophagocytosis of S.aureus in these patients. Thus, opsonophagocytosis of a test strain,
S.aureus Oxford strain, by patient PMNs in autologous plasmas or sera was measured by the method of Foroozanfar
(1976) in a group of 31 patients with significant staphylococcal disease.
Secondly, ELISA tests for measuring specific a n t i- 5 .aureus IgG class and subclass antibodies were developed and applied in a comparative study of specific antibody levels in patients and controls. It was hoped that if defects in specific subclasses were contributing to patient in fe ctio n then these te sts might detect such a defect. Fc-dependent phagocytosis of the test organism was also measured to determine i f the levels of s p e c ific antibodies correlated with this form of opsonophagocytosis.
A third aim of the project was to modify the
Foroozanfar method to measure another common pathogen,
Streptococcus pyogenes. The modified assay was then used to study neutrophil phagocytosis of strains of invasive streptococci which are associated with a toxic shock-like syndrome.
60 CHAPTER 3: MATERIALS AND METHODS
3.1 SUBJECTS
The blood, plasma or serum samples used in these ex periments were obtained from patients, healthy laboratory workers and children from paediatric and surgical out patients' clinics. Most patients (aged 1-61 years) were seen when they were infection free and off antibiotic therapy though a small minority were investigated while convalescing in hospital. Acute and convalescent sera from patients with septic scarlet fever were taken by doctors at a district hospital, stored at -70°C for several weeks before being brought in liquid nitrogen to our department. Control blood samples were obtained from healthy adult laboratory workers (aged 20-45 years) and from 15 children (aged 2-15 years) with minor medical or surgical problems and from whom blood had been obtained for other investigations. Blood samples were usually collected between 9 to 10 am and were processed immediately.
3.2 BUFFERS AND MEDIA
3.2.1 MEDIUM 199
The neutrophil and bacterial suspensions used in the phagocytosis assays were prepared in Medium 199. Aliquots of medium 199 were freshly prepared from powdered concentrate
(Flow Laboratories) just prior to use and were buffered with
25 mMol/1 HEPES (BDH) and 300 mg/1 sodium bicarbonate (BDH) and the pH adjusted to 7.2.
61 3.2.2 PHOSPHATE BUFFERED SALINE (PBS)/ PBS-AZIDE-TWEEN
Phosphate buffered saline (PBS) was prepared by d is solving PBS tablets (Dulbecco "A", Oxoid) in sterile dis t ille d water (Ivex Pharmaceuticals). PBS-Azide-Tween (PBSAT) was prepared by adding 0.05% Tween 20 (BDH) and 0.01% sodium azide (BDH) to freshly prepared PBS. Fresh batches of PBS-
Azide-Tween were prepared weekly.
3.3 SEPARATION OF THE POLYMORPHONUCLEAR LEUCOCYTES
Neutrophils were separated from 10 ml a liq u o ts of lithium heparinised blood collected in Vacutainer tubes
(Becton-Dickinson). The tubes were spun at 400 x g for 10 minutes (min) and the plasma removed for further centrifugation at 800 x g for 30 min to precipitate out con taminating platelets. The cellular component was resuspended in medium 199, layered onto lymphocyte separation medium
(Lympho-paque, Nyegaard, Norway) and spun at 800 x g fo r 30 min. The mononuclear ce ll layer formed at the interface was discarded.
The cell layer of erythrocytes and granulocytes was resuspended in medium 199 containing 2% w/v dextran
(Dextraven 110, Fisons) and incubated at 37°C for 30 min to allow the erythrocytes to sediment. The supernatant, en riched with PMNs, was centrifuged at 400 x g and the cell pellet washed twice and adjusted to 107 cells/ml.
62 3.4 PLASMAS AND SERA
3.4.1 SEPARATION AND STORAGE
Plasmas were kept at room temperature during the preparation of the phagocytic assays and were then ali- quotted into small volumes for storage at - 20°C. Sera were obtained from blood samples which were allowed to clot at room temperature for one hour and then spun at 800 x g for
20 minutes. The sera were stored at - 70°C u n til used. Once thawed, the samples were not refrozen.
3.4.2 HEAT INACTIVATION OF PLASMA
Test plasmas and sera (300-500 /*1) were placed in d is posable borosilicate glass tubes (Corning Glassworks) and heated in a waterbath at 56°C for 35 minutes. The samples were cooled to room temperature before use. The e fficie n cy of complement inactivation in 6 samples was assessed by the
CH50 test (Brown,1981).
3.5 BACTERIA
3.5.1 STAPHYLOCOCCUS AUREUS
S.aureus Oxford strain was used as the test organism in all staphylococcal phagocytosis assays. This organism is weakly coagulase positive and sensitive to most antibiotics.
A formalin fixed preparation of S. aureus Wood 46 (Sigma), a protein A d eficien t strain, was used in the ELISA assays.
63 3.5.2 STREPTOCOCCUS PYOGENES
A total of 8 clinical isolates of S.pyogenes type
M1/T1/0F- were obtained from the Streptococcal Reference
Laboratory (Colindale). Three, designated S.pyogenes MB, JB and FS, were grown from the blood cultures of three patients with septic scarlet fever. The other 5 isolates were ob tained from d iffe re n t c lin ic a l situ a tio n s. They have been named according to the clinical situation with which they were associated:
1) S.pyogenes (tonsillitis)
2) S.pyogenes (asymptomatic)
3) S.pyogenes (abdominal pus)
4) S.pyogenes (pneumonia)
5) S.pyogenes (septicaemia)
3.6 BACTERIAL GROWTH MEDIA
3.6.1 TRYPTIC SOY AND TODD-HEWITT BROTHS
Tryptic soy broth was prepared by dissolving 26g tryp tic soy broth powder (Difco) in one litre of distilled water with continuous stirring. One hundred m illilitre volumes of the solution were poured into screw-topped glass bottles and autoclaved (Portaclave, Astell Scientific) at 120°C, 1.5 bars for 60 mins. The bottles were allowed to cool to room temperature and were stored at 4°C u n til required. Todd-
Hewitt broth was similarly prepared by dissolving 36.4g Todd
Hewitt broth powder (Oxoid) in 1 lit r e of d is t ille d water.
64 3.6.2 AGAR SLOPES AND AGAR PLATES
Cultures of S.aureus were maintained at 4°C on tryptic soy agar slopes. These slopes were prepared by dissolving 2% w/v agar (Difco) in tryptic soy broth and allowing 10 ml volumes to set in small glass bottles. The agar slopes were kept at 4°C t ill required. Subculturing was done at monthly intervals.
Cultures of S.pyogenes obtained from the Streptococcal
Reference Laboratory were immediately subcultured onto blood agar and then stored at -70°C in 300 aliquots of peptone broth with glycerol (BDH).
Nutrient and blood agar plates which were used to assess the purity of broth cultures and for colony counting, were ob tained from the Department of Microbiology.
3.7 METHODS OF ASSESSING BACTERIAL NUMBERS
3.7.1 COLONY COUNTING METHOD
Samples of overnight broth cultures were s e r ia lly diluted tenfold in freshly prepared PBS and 20 a 1 from each d ilu tio n was carefu lly pipetted onto blood agar plates. The plates were incubated overnight at 37°C. Duplicate or t r ip lic a t e determinations were made of each d ilu tio n . The plates were placed over a lig h t box and the colonies counted by eye. The average of duplicate or triplicate counts was used to calculate the approximate bacterial concentration in each broth (Table 3.1). An overnight growth of S.aureus usually yielded 1-2 x 109 colony forming units (cfu) /ml while only 2-4 x 108 cfu/ml of S.pyogenes were obtained.
65 TABLE 3.1 Duplicate colony counts of S.aureus from 3 con
secutive overnight broth cultures
BROTH NO.OF COLONIES DILUTION 1(A ) 19 10 6 (B) 22 2 (A ) 14 10"6 (B) 29 3 (A ) 25 10"6 (B) 17
3.7.2 SPECTROPHOTOMETRIC MEASUREMENTS
The absorbance of broth culture dilutions was measured
in itially at 620 nm and then later at 450nm on a Perkin-
Elmer Spectrophotometer. The o p tica l density measurements
were correlated with bacterial concentration.
3.8 PREPARATION OF BACTERIA FOR USE IN THE PHAGOCYTIC ASSAYS
Aliquots of staphylococcal or streptococcal broth
suspensions were washed in excess medium 199 p rior to use.
In the staphylococcal inhibition assay the bacterial con
centration was adjusted to give 107 cfu/ml but in the assays
in which heat inactivated sera/plasmas were tested, the bac
terial suspension was increased by approximately 33%. The
concentration of S.pyogenes was adjusted to 5 x 107 cfu/ml.
66 3.9 THE STAPHYLOCOCCAL INHIBITION ASSAY
3.9.1 THE PRINCIPLE
The staphylococcal inhibition test is a suspension
phagocytic assay developed by Foroozanfar (1976) which uses
liv e S.aureus (Oxford strain) as the test organism. In this
assay, the uptake of tritiated (3H) thymidine (specific ac
t iv it y , 26 Ci/mmol, Amersham International) by S.aureus in
the presence and absence of neutrophils is compared. The
thymidine is unavailable to ingested staphylococci since
only minimal quantities of it are taken up by the
neutrophils. Thus, a decrease in the radioactivity as
sociated with S.aureus in the presence of PMNs can be used
as a measure of bacterial uptake or % inhibition:
* com in the presence of PMNs 1 - cpm in the absence of PMNs x 100 = % in h ib itio n
* cpm = counts per minute
3.9.2 THE METHOD
The tests were done in triplicate in sterile, flat-
bottom 96 well microtitre plates (Cell-Cult). The reaction
mixtures consisted of 10 a ! te st plasma, 50 m 1 bacterial
supension with or without 50 Ml neutrophil suspension and
made up to 200 m ! with medium 199. This gave a ra tio of 1
cfu per neutrophil in a 10 % plasma concentration. The
m icrotitre plate was shaken for one hour at 37°C on a Vari-
shaker (Dynatech) at speed 7. Each reaction w ell was then
pulsed with 0.5 microCuries of 3H-thymidine and the plate
was incubated for another hour at 37°C without shaking. The
67 reaction mixtures were harvested (Minimash 2000, Dynatech) onto glass-fibre filte r papers (Whatman) and dried over night. Discs of glass-fibre paper were solubilised in liquid scintillant (Ecoscint A, National Diagnostics) and counted on a liquid scintillation counter (1212 Rackbeta, LKB
W allac).
In those assays in which heat inactivated control and patient plasmas were compared, the following modifications were made to the staphylococcal inhibition assay: the staphylococci: neutrophil ratio was increased to ap proximately 1.3 cfu:l and the initial incubation time was reduced to 15 min.
3.10 OPTIMISATION OF THE STAPHYLOCOCCAL INHIBITION ASSAY
Several optimisation experiments were done in order to maximise bacterial ingestion in the assay. Some of the results from these experiments are given in Figures 3.1,
3.2, 3.3, 3.4. A reaction mixture consisting of 1 cfu per
PMN in 10% plasma when incubated at 37° for 1 hour at speed
7 was found to give > 88% uptake in a ll the normal controls studied.
3.10.1 EFFECTS OF DIFFERENT PLASMA CONCENTRATIONS
The effect of plasma concentration on staphylococcal ingestion was measured by increasing the concentration of normal plasma in the reaction mixture from 1.25% to 40%.
Staphylococcal in h ib ition was maximal in 10% plasma (see Fig
3.1 on the following page).
68 FIGURE 3.1 The effect of increasing concentrations of normal plasma on the phagocytosis of S.aureus
% plasma in reaction mixture
3.10.2 EFFECTS OF DIFFEREHT STAPHYLOCOCCI:NEUTROPHIL RATIOS
Increasing the staphylococci: neutrophil ratios from 1 cfu :l to 8 cfu :l caused decreased uptake of the organisms as shown in Fig.3.2. A ratio of 1 cfu per PMN was optimal.
FIGURE 3.2 Effect of increasing S.aureus:PMN ra tio s on staphylococcal phagocytosis
69 3.10.3 EFFECTS OF SHAKER SPEEDS ON THE STAPHYLOCOCCAL IN
HIBITION ASSAY
Staphylococcal ingestion was also affected by the speed at which the m icro titre plate was shaken; higher speeds fa c ilita te d phagocytosis but caused sp illage. Speed 7 provided adequate PMN-bacteria contact with minimal sp illag e
(Fig 3.3).
FIGURE 3.3 The effects of shaker speed on S.aureus phagocytosis
3.10.4 TIME COURSE OF STAPHYLOCOCCAL PHAGOCYTOSIS
Incubation of the reaction mixtures for 5, 15, 30, 60 minutes showed that the rate of ingestion was greatest be
tween 5 and 30 minutes and then slowed to a maximum uptake
at 60 minutes. (Fig 3.4 shows uptake after 5, 15 and 30
minutes.)
70 ococcal inhibition iue icbto wt pams rm 0 ains n 10 and patients 10 from plasmas with incubation minutes IUE . % tpyoocl nhi ton atr . 5 n 30 and 15 5. after n itio ib h in % staphylococcal 3.4 FIGURE determ inations. The figu res are the medians fo r each time time each e r t a fo lic medians ip r the t are of point. average res figu the The is inations. point determ Each controls. healthy .05 SESET F H OSNC OTIUIN F COMPLEMENT OF CONTRIBUTION OPSONIC THE OF ASSESSMENT 3.10.5 INSTAPHYLOCOCCALINHIBITIONTHE ASSAY cla ssica l pathway, was required fo r maximal ingestion. (Fig (Fig ingestion. maximal r fo 3.5). required was pathway, l ssica cla showed that an in ta ct complement system, e s p e c ia lly the the lly ia c e p s e system, complement assay ct ta in is th an in that showed plasmas treated MgCl2.6H20,2.5mM) and lOmM h ue f et nciae o mgeimET (EGTA magnesium-EGTA or inactivated heat of use The 71
staphylococcal inhibition of normal and Mq-EGTA treated plasmas. Each point represents represents point Each plasmas. Mq-EGTA and treated normal of FIGURE 3.5 (a) Staphylococcal phagocytosis in the presence presence the in phagocytosis Staphylococcal (a) 3.5 FIGURE h aeae f eut otie fo 2 oml controls. normal 2 from obtained results of average the d iv id u a ls. Each point is the average of four separate separate four of average the is in point healthy results. Each 4 from ls. obtained a u id iv plasmas d heat-inactivated and b Sahlcca paoyoi i te rsne f normal of presence the in phagocytosis Staphylococcal (b) 72 3.11 MEASUREMENT OF BLOCKING ACTIVITY IN PATIENTS' PLASMAS
Cell directed blocking activity in the plasmas of
three patients with defective staphylococcal phagocytosis was measured as follows: 50 ^1 of normal PMNs were prein
cubated at 4°C for 60 minutes then at room temperature for
30 min, with mixtures consisting of 10 ^1 pure IgG solution
(125mg/l)(Sigma), 20 ^1 hypogammaglobulinaemic plasma
(complement source) and 20 ^1 patient or control plasmas.
To these reaction mixtures, 50 jil S.aureus suspension was
added and the inhibition assay performed as previously
described. Controls consisted of incubating the PMNs in
reaction mixtures in which each component was omitted in
turn. The results are given in Table 5.5.
3.12 STREPTOCOCCAL INHIBITION ASSAY
Streptococcal phagocytosis was measured as described
fo r S. aureus but 1 or 5 cfu S.pyogenes:PMN were used. The
absence of heat-labile opsonins reduced phagocytosis of in
vasive S.pyogenes by only 3-4% (Fig 3.6).
FIGURE 3.6 Phagocytosis of 5.pyogenes M1/T1/0F- in the
presence of normal and heat-inactivated plasmas.
100
control 1 control 2
73 3.13 ENZYME-LINKED IMMUNOSORBENT ASSAYS
Enzyme-linked immunosorbent assays (ELISA) were used to determine the levels of total IgG and IgG subclasses specific for S.aureus Wood 46 strain (Sigma) and to compare the binding of specific IgG to clinical isolates of
S.pyogenes M1/T1/0F-.
3.13.1 MEASUREMENT OF ANTI-S.pyogenes IqG LEVELS
Microassay plates (Nunc-Immuno Plate Maxisorp, In- terMed) were coated with 100 ^1 pasteurised S.pyogenes suspension (300mg/ml) in PBS-Azide and incubated overnight at 4°C. The plates were washed and incubated with 200 jil
PBSAT per well at 37°C for 20 min. Plasma samples diluted
2000-fold were added in 100 (il aliquots to triplicate wells and incubated at 37°C for 3 hr. After washing, 100 ^1 of alkaline phosphatase-conjugated goat anti-human IgG
(Atlantic) diluted 1000-fold was added and incubated at 37°C for 2 hr. Phosphatase substrate, para-nitrophenyl phosphate disodium (Sigma) (2mg/ml) was added in 200 jil volumes and the reaction allowed to develop in the dark at room tempera ture. It was stopped by the addition of 50 jil aliquots of 3M sodium hydroxide (BDH). Absorbance was read at 405nm on a
Titertek Multiscan MC (Flow Laboratories).
3.13.2 MEASUREMENT OF TOTAL IGG LEVELS SPECIFIC FOR S.aureus
Multi-well microassay plates were coated with formalin-fixed S. aureus suspension (Sigma) (500 ug/ml) in commercially prepared coating buffer (Whiteley Scientific
74 Limited), pH 9.6 and incubated at 4°C overnight. Thereafter, the assay procedure was essentially as described for measurement of the a n ti- S.pyogenes IgG levels except that the alkalin e phosphatase goat anti-human IgG was incubated at 37°C for 90 min. The intra- and interassay coefficients of variation (CV) were 2% and 13% respectively.
3.13.2.1 OPTIMISATION OF THE TOTAL IGG ASSAY
The concentration of S.aureus antigen and the dilution of plasma used in this assay were determined by checkerboard analysis. The results are shown in figure (3.7).
FIGURE 3.7 Chequerboard analysis of increasing d ilu tio n s of
S.aureus suspension and increasing d ilu tio n s of normal plasma. Each point is the result of triplicate determina- tions.
0.5 __
D
1/50 1/400 1/800 1/1600
dilution of S.aureus suspension (10% wet wt/vol)
75 absorbance at 405 3.13.2.2 DETERMINATION OF ASSAY SPECIFICITY ASSAY OF DETERMINATION 3.13.2.2 min and the supernatant added to a fresh bacterial p e lle t t lle e p bacterial fresh a to added 30 supernatant r fo g the and x 800 at min centrifuged was suspension The min. 20 for plasma absorbed out against against out absorbed plasma IUE . Anti n A FIGURE 3.8 repeated. procedure the and 3.8). B rie fly , 200 {il of plasma was added to a p e lle t of of t lle e p a to added was plasma of {il 200 109 2x , fly rie B 3.8). normal plasma and plasma absorbed against against absorbed plasma and plasma normal strain. sa specii t a dtrie b t tng normal g stin te by determined was ity ific c e p s Assay S. aureus -S.aureus raim ad otnosy oae a 37°C at rotated continuously and organisms IoG levels in doubling d ilu tio n s of of s n tio ilu d doubling in levels IoG S.aureus Wood 46 stra in (Fig (Fig in stra 46 Wood S.aureus Wood 46 76 3.13.3 MEASUREMENT OF IG6 SUBCLASS LEVELS OF ANTI-5.AUREUS
ANTIBODIES
The plates were coated with formalised S.aureus suspension (250 ^g/ml) and incubated at 4°C overnight. After washing and blocking with PBSAT, 100 *il plasma samples, diluted 16-fold with PBSAT, were added in triplicate and in cubated at 37°C for 90 minutes. Mouse anti-human IgGl (clone
HP6012; Oxoid), IgG2 (clone HP6014; Oxoid), IgG3 (clone
HP6050; Oxoid) and IgG4 (clones HP6011 and HP6013; Oxoid) monoclonal antibodies were diluted 1000-fold in PBSAT and added in duplicate or triplicate to the wells and incubated at 37°C fo r one hour. The plates were washed with PBSAT and affinity-purified peroxidase-conjugated sheep anti-mouse IgG
(Sigma) was added. A fter 60 minutes incubation and wash, substrate solution of para-nitrophenyl phosphate disodium
(2mg/ml) was added to each well and the reaction developed in the dark at room temperature. The absorbance was read at
405 nm. Concentrations of IgGl and IgG2 were estimated from a standard serum sample (SPS-01) by a m odification of the method described by Persson et al (1985). The interassay CV for IgGl and IgG2 was 34% and 25% respectively.
3.13.3.1 OPTIMISATION OF SPECIFIC SUBCLASS ASSAY
The S.aureus concentration and plasma dilution used in these assays were determined by checkerboard analysis. Some results from these experiments are given in the following graphs (Figs 3.9, 3.10).
77 absorbance at 405 nm absorbance at 405 nm mal plasma (1/16 d ilu tio n ). Each point is the average of of average the is point Each ). n tio ilu d (1/16 plasma mal 5 - i t n A 9 . 3 E R U G I F tr ip lic a te determinations. (Key: IgGl - inverse trian g les, les, g trian inverse - IgGl (Key: determinations. te a lic ip tr d ilu tio n s of of s n tio ilu d d ilu tio n s of normal plasma. Each point is the average of of average the is determinations. point Each te a lic plasma. rip t normal of s n tio ilu d s) ircle c - IgG4 triangles, - IgG3 diamonds, - IgG2 1.0 -/j\ -/j\ / 18 /6 /2 /4 /2 1/256 1/128 1/64 1/32 1/16 1/8 1/4 S.aureus .aureus upnin fe te diin f nor of addition the after suspension lsa dilution plasma ' ^ q subclass binding t doubling n i l b u o d to g n i d n i b s s a l c b u s IqG \ j / ~ .... • ------4^ 78 3.14 EQUAL POTENCY METHOD
Semi-quantitation of the specific anti-5 .aureus an tibodies of IgGl and IgG2 subclasses was done by a modifica tion of the method of Persson. Half a microtitre plate was coated with 100 ^1 S.aureus antigen (diluted 1/400) while the other half was coated with 100 jil polyclonal goat anti human IgG antibodies (diluted 1/500) and the plate was left overnight at 4°C. After rinsing, 100 jil of doubling dilu tions of a plasma sample, which was used as an internal con trol (IC) in all the ELISA experiments, were added to the staphylococcal antigen. In parallel, 100 ^1 of doubling dilutions of a standard serum SPS-01 [IgGl-6.31 g/1, IgG2-
3.95 g/1, IgG3-0.59 g/1, IgG4-0.37 g/1] (SAS Protein
Reference Units) were added to the wells coated with polyclonal antibodies and the plate incubated at 37°C for 90 min. After further washing the ELISA was continued as out lined in Section 3.13.3. The specific IgGl and IgG2 con centrations in the internal control sample were obtained from a standard curve of absorbance plotted against the
SPS-01 dilutions (Fig 3.11). The concentrations of specific antibodies in all plasmas tested were calculated as follows:
Absorbance (405) Test plasma x subclass conc.IC Absorbance (405) IC
79 absorbance at 405nm absorbance at 405 d ilu tio n s . The dotted lin e is the absorbance of a 1/32 1/32 a 1 0 of - S P S absorbance t s n i a the g a is e c n a b r e o lin s b A of dotted The s e v r u C . s d n r a d tio n a ilu t d S 1 1 3. E R U G I F d ilu tio n of the internal control control internal the of n tio ilu d l i f h itra control internal the of n tio ilu d 0.20 (A) (B) IqG2. The dotted lin e is the absorbance of a 1/2048 1/2048 a of absorbance the is e lin dotted The IqG2. (B) I q GI 80 The equal potency method offered, at best, a semi- quantitative method of measuring the re la tive concentrations of IgG subclasses in the internal control sample. The method is based on the assumption that binding of the standard serum, here SPS-01, to polyclonal anti-IgG antibody is equivalent to the binding of normal plasma to S.aureus an tigen. It does not take into account differences in the ac cessibility of antigenic determinants in these two parallel systems. Despite its obvious limitations, the method is easy to use and does allow a comparison of the re la tiv e levels of sp e cific subclasses in a single sample.
3.15 MEASUREMENT OF IMMUNOGLOBULIN AND COMPLEMENT CONCENTRA
TIONS
Concentrations of a ll immunoglobulin classes, IgG sub classes and C3 and C4 were measured by the Protein Reference
Unit. The Immunoglobulin classes and complement components were measured by rate nephelometry on a Beckman Array
Protein System. Serum IgG subclass concentrations were measured by an ELISA method.
3.16 MEASUREMENT OF THE EFFECT OF 4 DRUGS ON S. aureus
PHAGOCYTOSIS
Neutrophils (600^1) from a healthy volunteer were pre-incubated at 37°C for 1 hour with medium 199 (control) or increasing concentrations of ampicillin (2.5 and
25^g/ml)(Beecham Research Laboratories), clindamycin (5 and
50jig/ml) (Upjohn Limited), erythromycin (1.5 and 15 jig/ml)
81 (Abbott) and rifampicin (10 and 100 ^g/ml) (Merrell Phar maceuticals Lim ited). The drug concentrations used were equivalent to the average blood levels of these drugs
{Garrod, 1973} and 10-fold concentrations of those levels.
The PMNs were washed twice in excess medium 199 and the
staphylococcal inhibition assay performed as previously described. The results of this experiment are shown in
Figure 6.1.
3.17 STATISTICAL ANALYSIS
Coefficients of variation were calculated by dividing
the standard deviation, obtained from at least 6 different
lots of t r ip lic a te results on the same plasma sample, by the
sample mean and expressing the result as a percentage.
In this thesis all other data were analysed by the
non-parametric statistical test, the Mann-Whitney U Test
(S ie g e l,1956).
82 CHAPTER 4: DETAILS OF PATIENTS STUDIED
4.1 OVERVIEW
Three groups of patients were studied. The f i r s t group consisted of four patients, aged 3 months to 35 years, with deep-seated or chronic S. aureus infections and persistently
low staphylococcal inhibition assays. The second group is composed of 27 patients, aged 1-61 years, with clinically
significant S.aureus in fectio n s and normal staphylococcal
inhibition tests. Here, the possibility of a defect in the
production or function of specific IgG subclass antibodies was assessed by a combination of antibody measurement by
ELISA and measurement of Fc-mediated opsonophagocytosis of
S.aureus. F in a lly , the phagocytosis by normal PMNs of
clinical isolates of S.pyogenes M1/T1/0F-, obtained from
three cases of septic sca rle t fever, was compared using a
streptococcal inhibition assay.
4.2 PATIENTS WITH ABNORMAL STAPHYLOCOCCAL INHIBITION TESTS
4.2.1 PATIENT 1: L.C.
L.C. was first admitted to the Westminster Children's
Hospital (WCH) at 3 years of age with a septic arthritis of
the left knee. He had had recurrent boils during the
previous two years and was mildly eczematous. His father and
older brother were also atopic and prone to b o ils though
neither had had any deep-seated infections. On examination,
he was found to be underweight (at 10th cen tile) and small
for his age, with healing abscesses on the buttock and right
leg. Initial investigations revealed a normal white cell
83 count with normal immunoglobulin and complement levels
(Table 4.1). Nasal swabs and swabs of the skin lesions all grew S.aureus though the organism was not isolated from the pus aspirated from the septic knee. Investigation of his phagocyte functions (Table 4.1) revealed a defect of
staphylococcal opsonization which was corrected in vitro by the addition of IgG even at concentrations of 250mg/l (Table
5.2). The patient was started on IgG therapy (75mg/kg/ monthly) and after three months had gained 2 kgs (50th centile) and had not had further crops of boils. Tests of other fam ily members were e n tire ly normal.
TABLE 4.1 Summary of haematoloqical and immunological results of patient L.C.
TEST RESULT NORMAL RANGE Hgb 10.8 g/dl 12-14 g/dl WBC 10.7x 109 g/l 5-15x109 g/l PMN 6206 /ul 2000-6000 /ul Lymphs 4387 /ul 1500-4000 /ul IgG 11.9 g/l 3.5-14.0 g/l IgA 0.9 g/l 0.4-1.5 g/l igM 0.7 g/l 0.5-2.0 g/l igG2 1.34 g/l 0.3-2.9 g/l C 3 / C 4 normal NBT positive chemotaxis normal C.albicans killing* 30% > 25% S.aureus phagocytosis* 64% > 88%
* result of patient's PMN in autologous plasma
84 4.2.2 PATIENT 2: I.E.
Patient I.E. was first admitted to the WCH at 2 years of age with weeping lesions on the scalp and crops of b oils on the thighs, buttocks, groin, axillae and mouth which had failed to respond to 3 weeks of antibiotic treatment. He had a past history of frequent upper respiratory tract in fe ctio n s since the age of 3 months and numerous skin abscesses in the year prior to admission. He was an only child and there was no significant family history of undue susceptibility to infection. Swabs of the various skin lesions all grew S.aureus and he was s ta r te d on flucloxacillin. However, on discontinuing treatment, more pustules and indurated lesions appeared on his buttocks and he developed a purulent conjunctivitis. Swabs of these lesions also grew S.aureus. He had a normal white c e ll count and differential as well as normal levels of complement component 4 (C4), IgG and IgM but s lig h t ly low concentrations of complement component 3 (C3) and IgA (see
Table 4.2). Investigations of his phagocyte functions revealed defective staphylococcal opsonization which could be corrected in vitro with IgG. He was started on gammaglobulin therapy which caused a dramatic reduction in the frequency and severity of his lesions.
85 TABLE 4.2 Summary of haematoloqical and immunological results of patient I.E.
TEST RESULT NORMAL RANGE
Hgb 12.6 g/dl 12 - 14 g/dl
W BC 7.2 x 109/l 5 - 15 x 109/l
PMN 4392 /ul 2000 - 6000 /ul
Lym phs 1728 /ul 1500 - 4000 /ul
IgG 6.3 g/l 3.5 - 14.0 g/l
IgA 0.3 g/l 0.4 - 1.5 g/l
IgM 0.7 g/l 0.5 - 2.0 g/l lgG2 0.87 g/l 0.3- 2 3 g/l
0.5 g/l 0.7 - 1.7 g/l ^ CO O O normal
NBT positive chem otaxis normal
C.albicans killing* 30% > 25% S.aureus phagocytosis* 61% > 88%
* result of patient's PMN In autologous plasma
4.2.3 PATIENT 3: R.J-S.
R.J-S, a salesman and keen rugby player, was relatively well until the age of 33 years when he suffered two bouts of staphylococcal septicaemia. The first septicaemic episode followed bony and muscular injuries incurred during a rugby match while the second was associated with infection of the right sternoclavicular joint. In the past, he had also been treated for recurrent oral herpes infections and erythema multiforme. He had a
86 normal neutrophil count and normal complement and
immunoglobulin levels (Table 4.3). His PMNs were able to move and k i l l C. albicans adequately (Table 4.3) but his plasma failed to support the phagocytosis of S.aureus. There was no significant family history of an increased
susceptibility to viral or bacterial infections.
TABLE 4.3 Results of haematoloqical and immunological
investigations of patient R.J-S.
TEST RESULT NORMAL RANGE Hgb 13.8 g/dl 13-18 g/dl
WBC 5.7 x 109/l 4-10.5 x 109/l Neutrophils 3300 / ul 2000-7500 /ul
Lymphocytes 1760 /ul 1500-4000 /ul IgG 13 g/l 5.4-16.1 g/l
IgA 1.6 g/l 0.9-3.4 g/l IgM 1.7 g/l 0.5-2.0 g/l lgG2 6.0 g/l 1.2-6.6 g/l C3 normal
C4 normal PMN movement normal Candida killing* 47% > 25% S.aureus phagocytosis* 49% > 88%
* = patient's PMNs in autologous plasma
87 4.2.4 PATIENT 4: R.T.
R.T., a male patient of Kuwaiti descent, was referred
to the Department of Dermatology at the Westminster Hospital
with a history of recurrent, weeping staphylococcal lesions
of the scalp which appeared to fla re up whenever he was "run
down" or had cuts and grazes elsewhere. He had been
repeatedly treated by his GP with local and systemic
antibiotics but the lesions always recurred. Following a
diagnosis of chronic pyoderma by the Dermatologist, he was
treated with a combination of systemic and topical broad
spectrum antibiotics. His condition improved, but would
readily flare up once antibiotic cover was completely
removed. There was a fam ily h isto ry of atopy but not of
undue susceptibility to infections. He was found to have
impaired staphylococcal opsonization. Details of his
haematological and phagocytic function investigations are
given in Table 4.4.
TABLE 4.4 Results of investigations of patient R.T.
TEST RESULT NORMAL RANGE Hgb 12.1 g/dl 12-14 g/dl WBC 4.7 x 109 /I 5-15 x 109 /I PMN 2303 /ul 2000-6000 /ul Lymphs 1410 /ul 1500-4000 /ul lgG,A,M reported as normal** C3/C4 reported as normal C.albicans killing* 25% > 25% S.aureus phagocytosis* 79% > 88%
* = patient’s PMNs in autologous plasma ** = reported as normal by consultant immunologist
88 4.3 PATIENTS WITH RECURRENT STAPHYLOCOCCAL INFECTIONS AND
NORMAL STAPHYLOCOCCAL INHIBITION TESTS
The twenty-seven patients (16 males and 11 females, aged 1-61 years) in this study had either staphylococcal skin infections (22) or at least one episode of deep-seated infection caused by S.aureus (5). Their clinical details are outlined below and a summary of their immunological parameters are shown in Tables 4.5, 4.6, 4.7. The specific a n ti-5 .aureus IgG subclass results are given in Table 5.8.
4.3.1 CLINICAL SUMMARIES OF PATIENTS WITH NORMAL 5.AUREUS
INHIBITION BUT SIGNIFICANT STAPHYLOCOCCAL INFECTIONS
4.3.1.1 PATIENT Is G.B.
GB, a 46 year old female of East Indian descent, suffered from recurrent subcutaneous abscesses for two years. Crops of boils appeared intermittently over her chest and abdomen and were treated by incision and drainage with systemic antibiotic cover. Repeated cultures of purulent material all grew 5. aureus. She was not diabetic and had no other health problems. Lesions last appeared 4 months p rio r to her v is it to our department.
4.3.1.2 PATIENT 2: R.B.
RB, d.o.b 23-12-78, presented with an eight month
history of staphylococcal abscesses over the chest and legs
and about the nostrils. He had suffered at least six
episodes of abscesses which had been treated with systemic
89 a n tib io tics. His last lesions healed about 3 weeks before he visite d our department.
4.3.1.3 PATIENTS J.B., N.B., V.B.. Y.B.
These four patients are all members of a family of six. VB, aged 46 years and NB, aged 16 years suffered from recurrent boils that usually required incision and drainage before they could heal. JB and YB both aged 2 years are fraternal twins. JB was hospitalised with right sided cervical lymphadenopathy which settled with intravenous antibiotic treatment and YB had had three episodes of boils.
Despite the frequent use of topical and systemic antibiotics, S.aureus infections continued to occur among these family members. Interestingly, the mother and a brother aged 11 years, had not had any staphylococcal in fe ctio n s. Nasal swabs from VB and NB were p o sitiv e fo r
S.aureus.
4.3.1.4 PATIENT R.B,
RB, aged 33 years, suffered from severe eczema and recurrent skin infections since childhood. He had been hospitalised with a staphylococcal infection of his face which responded to flucloxaci11 in therapy. He was on topical steroids and emollient creams for his eczema.
90 4.3.1.5 PATIENT M.C.
MC aged 46 years, suffered from recurrent staphylococcal abscesses for many years. The lesions were frequently large and deep and usually required incision and drainage. They healed with scarring.
4.3.1.6 PATIENT D.C.
DC, d.o.b. 08-08-75, suffered from recurrent abscesses for about 7 years. During this time, he had over 12 crops of
boils which usually responded rapidly to antibiotic
treatment. He had no other health problems and his last
abscesses appeared a few months prior to his visit to our
department. A nasal swab was positive for S.aureus.
4.3.1.7 PATIENT D.H.
DH, d.o.b. 17-03-82 had at least three episodes of
S.aureus abscesses which had been associated with fever,
vomiting and malaise. He also experienced frequent upper
respiratory tract infections and had had his sinuses
irrigated at 2 years of age. His last episode of boils was
three months before he visite d our department. A nasal swab
was positive for S.aureus.
4.3.1.8 PATIENT L.J.
This 46 year old female had so many breast abscesses
that one breast was removed. She was not d ia b e tic though
there was a vague family history of diabetes mellitus. She
had previously suffered from gallstones but recovered after
91 cholecystectomy. Despite the mastectomy, she continued to
have frequent lesions over the other breast and was
intermittently treated with antibiotics and/or incision and drainage.
4.3.1.9 PATIENT D.M.
DM, d.o.b. 03-05-27, complained of recurrent abscesses fo r more than 30 years which seemed to have become more frequent. He was being intermittently treated with
antibiotics and the last lesion had healed about 2 months
prior to his visit to our department. He had no other
health problems.
4.3.1.10 PATIENTS A.N.,C.N.,R.N.
These three patients are all members of a family of
four. The patients all had at least two crops of boils over
a two year period despite treatment for nasal carriage of
S.aureus. Their lesions were usually as large as a 10 pence
piece and occasionally required incision and drainage.
There were no other health problems. Patient CN was found
to have a low C4 concentration with decreased neutrophil
chemotaxis but was healthy apart from the recurrent S.aureus
infections.
4.3.1.11 PATIENT S.P.
Patient SP, d.o.b. 05-06-81, had recurrent S.aureus
blepharitis with corneal ulceration. In addition, she once
92 had a large boil on the right cheek which took three months to heal.
4.3.1.12 PATIENT D.P.
This 22 year old patient had an 18 year history of eczema and asthma and a 2 year h isto ry of recurrent staphylococcal abscesses. Initially, the boils appeared over his back, shoulder, face and forehead. Nasal swabs grew S. aureus and he had been treated with fucidin but thereafter he developed further abscesses about his upper thighs. In total he had over 13 abscesses which usually had to be incised and drained and frequently healed with
scarring. He was on prophylactic minocycline when he
v isite d our department.
4.3.1.13 PATIENT S.R.
SR, a 24 year old female, had suffered from asthma
since childhood and was treated with inhaled steroids.
Following an appendicectomy, she suffered from recurrent
S.aureus infections of the wound site which necessitated
further explorative and repair operations and numerous
courses of antibiotic therapy.
4.3.1.14 PATIENT J.R.
JR was investigated by our department when he was 3
years of age. He had suffered with diarrhoea which resulted
in failure to thrive. Later he developed a large gluteal
abscess at an injection site and also suffered from
93 recurrent paronychia! infections. His neutrophils showed decreased chemotaxis in zymosan activated plasma.
4.3.1.15 PATIENT M.S.
MS, d.o.b. 24-11-71, complained of recurrent bouts
(2-4/year) of staphylococcal skin abscesses over an 8 year period with one episode of facial cellulitis. She also suffered from recurrent upper respiratory tra ct infections.
Her last abscess healed about three weeks before she was investigated at our department.
4.3.1.16 PATIENT G.S.
GS, a 41 year old male patient, suffered from recurrent skin abscesses which were treated with frequent courses of a n t ib io t ic s . He was re fe rre d to us fo r investigation due to the frequency of his lesions.
4.3.1.17 PATIENT J.S.
This young boy, aged 3 years, suffered from severe eczema which was c o n tro lle d w ith s te ro id creams and emollients. He had had several episodes of secondary
S. aureus infections which responded rapidly to systemic antibiotics. His neutrophils failed to move normally to zymosan activated plasma.
4.3.1.18 PATIENT F.C.
FC, a middle-aged man, had chronic myeloid leukaemia and was hospitalised with S.aureus septicaemia. Convalescent
94 plasma was obtained from th is patient while he was s t i l l in hospital but off all medication.
4.3.1.19 PATIENT R.C.
RC, d.o.b. 22-04-74, suffered from recurrent skin abscesses but developed S.aureus septicaemia and osteomyelitis. He also had frequent throat and ear
infections with poor enamel formation which necessitated removal of many teeth. The last abscess appeared about 4 months before he was seen at our department.
4.3.1.20 PATIENT P.D.
PD, d.o.b. 15-02-71, was referred to our department following an episode of indolent S.aureus arthritis of the
left hip which caused significant bony damage with shortening of the affected limb. Apart from mild childhood asthma he had no other health problems.
4.3.1.21 PATIENT E.F.
EF, a 1 year old female patient, was admitted to the
WCH with S.aureus septicaemia and pneumonia. There was no fam ily history of infection though the patient was prone to frequent "colds". She also was being investigated for
developmental delay.
4.3.1.22 PATIENT K.K.
KK, a middle-aged female, had a long history of red
cell aplasia which necessitated frequent blood transfusions.
95 In addition, she suffered from recurrent S.aureus infections of bones and joints which resulted in amputation of a lower limb. An IgG-kappa paraprotein had been id e n tifie d in her plasma.
TABLE 4.5 Immunoglobulin and complement concentrations in 27 patients with significant staphylococcal infections
IgG (g /l) IgA (g /l) IgM (g /l) PATIENT C3/C4 (g/l)
BG 11.9 3 .2 2 .0 1.5 1.4 RB 10.2 1.9 0 .8 1.3 0 .3 JB 10.7 0 .7 1.6 1.3 0 .4
NB 18.7 1.7 1.5 1.0 0 .2 VB 12.7 2 .0 1.6 0 .9 0 .3 YB 11.3 0 .9 0 .5 1.3 0 .3
RBN*NN NN MCN NN -- DC 12.7 0 .7 1.1 1.2 0 .4
DH 12.3 1.1 1.7 1.2 0 .4 LJ 10.9 1.3 2 .7 1.0 0 .4 DM 15.3 3 .5 0 .8 1.3 0 .5
CN 16.6 1.5 1.5 0 .7 0.1 RN 12.2 1.7 1.2 1.0 0 .2 AN 9 .0 0 .5 0 .6 1.0 0 .2
SP 7 .8 1.4 1.4 1.6 0 .2 DP N*NN - - SR 10.1 1.5 1.1 1.6 0 .6
JR 6 .6 0 .6 0 .3 * * - - MS 11.2 1.8 2 .4 1.9 0 .3 GS 17.8 3 .5 1.4 2 .0 0 .4
JS 8 .4 0 .6 1.2 1.2 0 .4 FC 6 .8 1.0 0 .7 2 .0 0 .4 RC 7 .4 2.1 0 .8 1.2 0 .3
PD 14.3 1.2 1.4 1.2 0 .4 EF 12.5 0 .7 1.7 1.5 0 .3 KK 2 3 .0 1.0 1.7 1.1 0 .2
* results reported as normal by senior physician, ** result
from another hospital
96 TABLE 4.6 Immunoglobulin 6 subclass levels in 27 patients with staphylococcal infections
PATIENT lgG1 g/l lgG2 g/l lgG4 g/l GB 6.8 3.7 0.24
RB - 2.5 - JB 7.6 1.2 0.24 NB 7.7 1.64 0.16 VB >6.8 0.68 0.28 YB 7.1 0.4 0.12 RB 6.6 1.8 0.78 MC - - - DC 8.5 0.5 0.28 DH 10.4 1.7 0.21 LJ 4.4 6.6 0.39 DM 10.1 3.9 1.73 AN 5.6 0.6 0.16
CN - - - RN 7.5 4.0 0.17 SP 6.34 1.67 0.07 DP 6.2 6.2 0.23 SR 5.8 0.6 0.65
JR - -- MS 6.6 3.0 0.23
GS - >6.0 - JS 6.2 1.0 0.53 FC 4.9 1.2 0.11 RC 6.1 2.4 0.72 PD 7.5 4.1 0.95 EF - 0.73 - KK - 0.5 0.12
97 TABLE 4.7 Results of phagocyte function tests in 27 patients
with significant S.aureus infections
CANDIDA S.AUREUS Nasal swab PATIENTCHEMOTAXIS* PMNS/ul KILLING PHAGOCYTOSIS for S.aureus
GB N 41 88 2,550 -v e RB nd nd 92 3,220 +ve JB low 45 99 -v e
NB low 44 99 2,700 +ve VB N 33 99 4,700 +ve YB low 56 98 3,700 -ve
RB N 38 97 9,640 MC nd 4 9 ** 99 DC N 32 94 4,312 +ve
DH low 56 89 2,016 LJN 53 94 4,560 +ve DM N 37 99 8,704
CN low 63 98 3,500 -v e RN N 48 96 5,700 -v e AN N 40 92 2,200 -v e
SP nd 86 DP N 74 92 3,400 SR N 35 97
JR low 36 96 2,331 MSN 65 97 3,750 +ve GS nd 71 ** 99
JS low 55 95 1,972 FC nd 92 RCN 34 95 2,679
PDN 58 98 4,356 EF low 28 88 5,828 KKN 33 99 2,400
N - normal, * - Adult only reference range ** patient plasma tested with control PMN nd - not done
98 4.4 THE PATIENTS WITH SEPTIC SCARLET FEVER
4.4.1 PATIENT 1: M.B.
MB, a healthy 39 year old engineer was admitted to a district hospital following a 24 hour history of a painful
left leg. Initial examination revealed a warm, tender calf associated with pyrexia and injected conjunctivae. He rapidly developed a diffuse erythematous rash with a vesicular eruption over the ankle, further swelling of the
leg, hypotension and multiorgan failure. Streptococcus pyogenes serotype M1/T1/0F- was isolated from the p atien t's blood, serous and vesicular fluids, but despite prompt treatment with large doses of benzylpenicillin, the patient died 19 days after admission. Post n.ortem examination
showed multiorgan involvement. The patient's immunoglobulin, complement and anti-streptococcal antibody levels are given
in Table 4.8 and IgG subclass responses in Table 4.9.
4.4.2 PATIENT 2: J.B.
This 57 year old woman was admitted with a five-day
history of right iliac fossa pain, increasing confusion and
swelling of the right thigh. On examination, she was found
to be jaundiced, febrile and hypotensive with a punctate erythematous rash over the leg and lower abdomen and
vesicles over the inner thigh. She was immediately started on antibiotic therapy but developed adult respiratory
distress syndrome and renal failure and died 12 hours after
admission. Blood cultures taken at admission and vesicle
fluid yielded S.pyogenes M1/T1/0F-. At post-mortem
99 examination, a ll tissues were found to be oedematous and haemorrhagic. The results of immunological investigations done on this patient are given in Tables 4.8 and 4.9.
4.4.3 PATIENT 3: F.S.
FS, a 34 year old fireman, was admitted to hospital with complaints of fever, a painful right leg following blunt trauma to the rig h t knee and a rash over the same
leg. On admission he was noted to have vesicles over the
lateral aspect of his rig h t knee. S.pyogenes M1/T1/0F- was grown from the patient's blood and later from wound fluid and subcutaneous tissu e. Despite prompt therapy with high dose benzylpenicillin, he deteriorated rapidly. He became markedly hypotensive with an erythematous rash which covered his entire body and later desquamated over the chest wall, palms and soles. He was also admitted to an intensive care u n it with adult re sp ira to ry d istre ss syndrome and renal failure. He eventually recovered over several weeks but required skin grafting to the leg. Details of immunological
investigations performed on samples of his acute and convalescent sera are given in Tables 4.8 and 4.9.
100 TABLE 4.8 Immunological investigations of patients with
septic scarlet fever
Patient FS Patient MB Patient JB P a ra m e te r s Acute Convalescent
IgG [N 5.4-16.1 g/L] 3.6 (5)* 4 .5 (5 )* 20.9 (9)* 50.8 (34)*
IgA [N 0.8-3.4 g/L] 1.1 0 .8 3 .7 3 .4
IgM [N 0.5-2.0 g/L] 0 .6 0 .4 5 .6 2 .6
C3 [N 0.7-1.7 g/L] 1.1 0 .6 1.8 3 .6
C4 [N 0.1-0.7 g/L] 0.5 0.2 0 .9 0 .9
CRP [N <10 mg/L] 2 0 0 36 124 53
Albumin [N 30-42 g/L] 28 41 16 27
A SO -a c u te 110 <50 >800 - -convalescent 2 0 0 (1 7 ) - - >800
ADB -acute <50 <50 4 0 0 -6 0 0 - -convalescent 1 2 0 0 -1 6 0 0 - - > 1600
AHT -acute 32 32 >4096 - -convalescent 128 - - 5 1 2
* Number in parentheses indicates the day after onset of illness when serum sample was collected N = normal range CRP = C-reactive protein ASO = Anti-streptolysin tltre [N <1:200] ADB = Anti-deoxyribonuclease B titre [N <1:250] AHT = Anti-hyaluronidase titre [N <1:128]
A X
101 TABLE 4,9 Immunoglobulin 6 subclass levels in acute and convalescent sera from patients with septic scarlet fever
Patient MB Patient FS igG Patient JB Normal Range Acute Convalescent Acute Convalescent
Total IgG (g /l) 3.6 3.6 4.5 20.9 50.8 5.0 - 16.0 lgG1 (g /l) 2.2 3.3 1.5 4.5 4.5 3.2 - 10.2 lgG2 (g /l) 0.8 1.4 0.8 1.4 1.6 1.2 - 6.6 lgG3 (g /l) 0.1 0.4 0.2 0.3 0.3 0.2 - 1.9 lgG4 (g /l) 0.1 >0.4 0.1 >1.6 >1.6 <0.1 - 1.3
102 CHAPTER 5: RESULTS AND DISCUSSION
5.1 PATIENTS WITH ABNORMAL STAPHYLOCOCCAL INHIBITION TESTS
Polymorphonuclear c e lls and autologous plasma samples obtained during infection-free intervals from four patients with significant S.aureus infections, gave subnormal staphylococcal inhibition results. The defect was in the plasma as incubation of th e ir c e lls with normal plasma raised the levels of bacterial phagocytosis from 49-79%
in h ib itio n to w ithin the normal range (>88% in h ib itio n )
(Table 5.1).
TABLE 5.1. Summary of staphylococcal inhibition results in 4 patients with defective S. aureus opsonization
SEX/AGE AT % STAPHYLOCOCCAL INHIBITION PATIENT ONSET PC,PP NC,PP PCjNP L.C. M / 3 64 68 100 I.E. M / 0.3 61 62 99 R.J-S. M / 33 49 51 100 R.T. M / 1 79 65 100
PC,PP = patient cells in autologous plasma NC,PP = control cells in patient plasma PC,NP = patient cells in control plasma
The concentrations of a ll immunoglobulin classes and
IgG2 subclass in these patients were w ithin the normal
ranges and th e ir plasmas could support the phagocytosis of
C. albicans via complement derived opsonins, suggesting a
103 defect in specific anti-staphylococcal antibodies. The clinical picture of predominantly S.aureus infections further supported an abnormality of specific S.aureus antibodies. The severity of the in vitro defect appeared to reflect the severity of the clinical presentation; patient
RT who had chronic pyoderma but no major infections, showed
79% staphylococcal inhibition whereas patient RJS, who suffered two bouts of staphylococcal septicaemia produced under 50% inhibition. The other patients, LC and IE, had
S.aureus infections of intermediate severity and produced inhibitions of 64% and 61% respectively (Table 5.1).
5.1.1 IN VITRO CORRECTION OF THE DEFECT
In vitro correction of the defect was readily achieved by the addition of purified normal IgG to the patient's plasma. As little as 250 mg/1 of IgG was sufficient to increase the level of phagocytosis to within the normal range (Table 5.2).
TABLE 5.2. In vitro correction of staphylococcal phagocytosis by the addition of pu rified immunoglobulin G
NEUTROPHIL PLASMA ^INHIBITION SOURCE SOURCE
NC NP 93
NCPP 53
NC PP + lgG(250mg/l) 90
NC PP + lgG(500mg/l) 95
NC PP + lgG(1000mg/l) 91
NC = control cells, NP = control plasma, PP = patient plasma
104 5.1.2 IN VIVO CORRECTION OF THE DEFECT
The frequency of infections in patients LC and IE with repeated hospitalisation and prolonged antibiotic therapy resulting in failure to thrive, justified a trial of IgG therapy. Both patients were started on monthly courses of purified IgG (75mg/kg/month) which decreased the frequency and severity of their infections and led to an improvement in their weights and heights. Cessation of treatment for p erio d s of over a month re s u lte d in a re la p se of staphylococcal infections and decreased staphylococcal inhibition (Table 5.3).
TABLE 5.3. Effect of IqG therapy on staphylococcal inhibition in patient LC
PATIENT S PLASMA % INHIBITION
Before treatment 64
On IgG 75mg/kg/month 93
Off IgG for 3 months 50
On IgG 75mg/kg/month 100
105 5.1.3 ANTI-5 .aureus IqG CLASS AND SUBCLASS ANTIBODIES IN
PATIENTS WITH DEFECTIVE STAPHYLOCOCCAL INHIBITION
Analysis by ELISA of pre-treatment plasmas from patients LC, IE and RJS, has shown that they a ll had anti-staphylococcal antibodies of IgG class and IgGl, IgG2 and IgG4 subclasses (Table 5.4). These patients have
a n ti-5 .aureus antibodies which fail to support efficient opsonophagocytosis of S.aureus. A humoral cause of defective opsonophagocytosis could arise from Fc-receptor blockade by
circulating immune complexes or by a deficiency or
dysfunction of opsonic antibodies. Circulating
IgG-rheumatoid factor complexes, which block Fc-receptors,
have been shown to decrease phagocytosis {Turner, 1973}, and
some antibody deficency states are associated with defective opsonization {reviewed by Klebanoff, 1978}.
TABLE 5.4. A n ti-S.aureus IqG class and subclass antibodies
in patients with defective staphylococcal inhibition
TOTAL SPECIFIC SPECIFIC SPECIFIC SPECIFIC PATIENT IgG* lgG1 (m g/l) lgG2 (m g/l) lgG4* L.C. 0.55 2.22 31.5 0.34 pre-treatment L.C. not done 1.94 46.99 0.39 post treatment I.E. not done 2.64 15.59 0.37 pre-treatment I.E. not done 1.87 12.75 0.35 post treatment
R.J-S. 1.17 1.42 66 1.18 REFERENCE 0.11 - 1.29 0.4 - 2.65 7.3 - 74 0.03 - 2.18** RANGES
* ELISA INDEX ** Adult reference range only
106 5.1.4 BLOCKING ACTIVITY IN THE PLASMAS OF PATIENTS WITH
DEFECTIVE STAPHYLOCOCCAL PHAGOCYTOSIS
Pre-incubation of normal neutrophils with plasma from patients RT, IE and RJS resulted in a drop in staphylococcal phagocytosis of up to 17% (Table 5.5). This blocking activity was maximal in patient IE but was also detectable in plasma from RJS. Decreased phagocytosis could be due to
Fc-receptor binding of immune complexes which are formed in all bacterial infections to facilitate the removal of bacterial antigens from the circu latio n {Mims, 1987}. Immune complex disease can follow acute bacterial infections and has been described in S.aureus subacute bacterial endocarditis {Adlam, 1984} and in shunt nephritis {Chapel,
1987}. Moreover, chronic immune complex formation can follow the administration of drugs which increase the bacterial antigen burden, for example, the 3-lactam antibiotics increase the release of cell wall components from S. aureus and cause increased levels of antibodies directed against these antigens {Verbrugh, 1983}.
However, these patients, despite recurrent infections and prolonged antibiotic therapy, did not manifest clinical features of an immune complex disorder such as a r t h r it is , skin rashes or glomerulonephritis. Additionally, a decrease
in phagocytosis of less than 20% caused by the patients' plasmas could not wholly account for a decreased staphylococcal inhibition to 61% and 49%. which were obtained with plasma samples from IE and RJS respectively, suggesting that another mechanism must be involved.
107 TABLE 5.5. Results of experiment to detect blocking activity
in patients' plasmas
PREINCUBATION MIXTURE % INHIBITION lgG(125mg/l) + Hp 8 8 + Nc
lgG(125mg/l) + Hp 9 2 .5 + N c + Np
lgG(125mg/l) + Hp 7 3 .2 + N c + lEp
lgG(125mg/l) + Hp 8 4 .4 + N c + R Tp
lgG(125mg/l) + Hp 7 9 .0 + N c + R JS p
Hp = Hypogammaglobulinaemlc plasma (Complement source) No = Control neutrophils Np = Control plasma lEp, RTp, RJSp = Patients’ plasmas
5.1.5 EVIDENCE FOR A SPECIFIC STAPHYLOCOCCAL OPSONIN
DEFICIENCY
Defective opsonophagocytosis of S.aureus may occur in
hypogammaglobulinaemia {Mickenberg, 1970} and can be
induced in vitro by absorbing out specific antibodies
{Wheat, 1974}. The patients of the present study had normal
levels of IgG and complement as well as sp e cific antibodies
yet failed to opsonize the test organism, suggesting a
specific defect of S.aureus opsonins. It has been shown from
work on the streptococcal M-protein that many antibodies are
formed against this antigen but only a few, directed against
epitopes in the N-terminal region, are opsonic {Jones,
1988}. T h e o re tica lly, there can be a normal response to
108 many streptococcal antigens except those which are important for opsonization. S.aureus has no M-protein but contains
Protein A and peptidoglycan which are thought to play a c r it ic a l ro le in the opsonization of the organism {Adlam,
1984}.
Peptidoglycan (PG) is present in most procaryotic micro-organisms but species specificity is determined by differences in the interpeptide bridges. S.aureus has penta- or hexaglycine interpeptide bridges {Verbrugh, 1983}. PG promotes phagocytosis by activating both complement pathways and by interacting with specific IgG {Peterson, 1983}.
PG antibodies, of IgG class, have been demonstrated in sera from virtually all healthy donors by sensitive ELISA and RIA though the levels vary considerably between in d ivid u a ls and with age. Three immunodominant s ite s have been identified using immune animal sera. These are the
N-acetyl-glucosamine residues of the glycan strand, the
C-terminal D-alanine-D-alanine of the peptide side chains and the pentaglycine interpeptide bridges {Verbrugh, 1983}.
The functional importance of antibodies directed against these immunogenic sites is still ill-defined. It is therefore possible that the patients lack antibodies against sp e cific epitopes on peptidoglycan which are important fo r phagocytosis. Their specific inability to ingest S.aureus suggests that the epitopes would be those unique to
S.aureus, such as the interpeptide glycine bridges.
Protein A is present in approximately 95% of pathogenic strains {Greenberg, 1990} and contributes to the
109 resistance of the organism to phagocytosis by binding to the
Fc portion of IgG and by blocking heat-labile opsonins for
S.aureus {Forsgren, 1974}. Species with high Protein A contents are phagocytosed at a slower rate than strains with low Protein A content {Dossett, 1969}. The role of Protein A specific antibodies in man has been difficult to define because of non-specific binding of IgG. However, high levels of anti-protein A antibodies have been found in patients with 5.aureus endocarditis and uncomplicated bacteraemia
{Greenberg, 1990}. It is conceivable that the 4 patients described in this study are unable to make specific Protein
A antibodies and so are more susceptible to strains with high protein A contents.
A specific lack of opsonins, predisposing to severe bacterial infection, could arise from clonal deletion or failure of maturation of specific B cell clones. In subclass deficiencies, there is failure of maturation of the
B cell clones capable of producing antibodies of a p a rtic u la r subclass(es) {Q uinti, 1989}. More s p e c ific antibody defects have been described in patients who fail to develop antibodies to Epstein-Barr Virus (EBV) in spite of documented infections {Ammann, 1987}. Braconier has described a patient with subclass deficiency and a reduced opsonic capacity fo r pneumococci even a fte r immunization
{Braconier, 1984}.
Recurrent staphylococcal infections are a feature of many disorders of phagocyte function (Table 5.6). S.aureus
is the commonest organism infecting patients with chronic
110 granulomatous disease (CGD) {Donowitz, 1983} and is the cause of significant morbidity in patients with leucocyte adhesion deficiency {Anderson, 1987}. Humoral deficiencies which predispose to significant staphylococcal infections include C3 dysfunction and d eficien cy syndromes { M ille r,
1969; Alper, 1972; Rosenfeld, 1976}, hypogammaglobulinaemia
{Mickenberg, 1970} and IgG subclass deficiencies {Schur,
1970}. An abnormal humoral response to S.aureus has been described in patients with hyperimmunoglobulin E syndrome
{reviewed Leung, 1988}. These patients have high levels of
IgE antibodies against staphylococcal antigens and suffer from recurrent staphylococcal abscesses because of poor neutrophil chemotaxis. A few patients with a specific defect in S.aureus killing or phagocytosis have been described
{Davis, 1968; Ito, 1980}. Davis' patient had many features of CGD but was also unable to mount a humoral response to
S.aureus despite repeated, severe infections caused by th is organism and he f in a lly succumbed at the age of 19 years.
Ito's patient, a young female infant with an intrinsic cellular defect was unable to phagocytose S.aureus though her polymorphs could ingest and kill E.Coli and
S. pneumoniae.
Ill TABLE 5.6 Differential diagnosis of recurrent staphylococcal
infection
DEFECT GENETIC ACQUIRED
Neutropenia C y c lic Iatrogenic
Hypogammaglobulinaemia
Secondary antibody Defective S.aureus Isolated IgG deficiency d e fic ie n c y opsonophagocytosis due to patient plasma lgG2 deficiency Immune complex diseases
Complement deficiencies
Chronic granulomatous disease Diabetes mellitus Chediak-Higashi syndrome Defective S.aureus Corticosteroid therapy phagocytosis & killing Homozygous G6PD deficiency due to patient cells Burns Leucocyte adhesion deficiency Malnutrition Pyruvate-kinase deficiency
Leiner's syndrome
Lazy phagocyte syndrome Other defects of plasma Viral impairment of neutrophil & neutrophil function fu n ction Complement deficiencies
Hyper-lgE syndrome
The four patients with defective staphylococcal
inhibiton tests differ from these other cases in that all
th e ir other phagocyte functions were normal as well as th e ir
levels of immunoglobulins and complement. The c lin ic a l
picture was one of only S.aureus infections and the defect
in phagocytosis was at the humoral le v e l. These four
patients have a unique defect in opsonization, namely a
specific inability to opsonize S.aureus. These patients are
112 very similar to four other cases investigated by Hobbs and
Citron {Monteil, 1987} who also failed to opsonize S.aureus, were repeatedly infected with th is organism and improved with IgG therapy.
5.2 PATIENTS WITH NORMAL STAPHYLOCOCCAL INHIBITION TESTS BUT
WITH RECURRENT OR SEVERE STAPHYLOCOCCAL INFECTIONS
Antibodies are important in protecting against
recurrent pyogenic infections {Mims, 1987} and patients with
quantitative or qualitative antibody abnormalities are prone
to bacterial infections including S.aureus. Since failure
to produce specific antibodies could result in host
s u s c e p tib ility to a particu lar pathogen, the concentrations
of a n ti- 5 .aureus IgG and IgG subclasses and Fc-dependent
phagocytosis of the organism were measured in 27 patients
with recurrent or deep-seated S.aureus infections and in 35
healthy adults and 15 children.
The median values of sp e cific IgG, IgG2 and IgG4 fo r
the two groups were not s t a t is t ic a lly different though IgGl
concentrations were significantly higher in the patient
group (p<0.00003) (Tables 5.7, 5.8 & Fig 5.1). Specific IgG3
was barely detectable in a ll the samples tested.
113 TABLE 5.7. Clinical details and specific anti-S .aureus IqGl and IqG2 concentrations of the 27 patients with
staphylococcal infections
anti-S.aureus anti-S.aureus PATIENT AGE/SEXDIAGNOSIS IgG1 (m g/l) lgG2 (m g/I)
GB 4 6 /F ABSCESSES 2 .4 5 6 7 .8 RB 1 0 /M “ 2 .2 7 5 8 .7 JB 2 /M " 1.71 3 6 .2
NB 1 6 /F “ 1.71 3 6 .0 VB 3 8 /M " 1.79 4 3.1 YB 2 /F M 1.4 3 8 .2
RB 3 2 /M » 1.81 4 9 .1 MC 4 6 /M - N O T DONE N O T D O N E DC 1 2 /M " 2 .8 4 4 5 .0
DH 6 /M « 1.00 3 7 .2 LJ 3 9 /F - 3 .4 6 6 9 .3 DM 6 1 /M ** 1.11 5 7 .9
AN 9 /M - 2 .6 3 2 4 .9 CN 1 1 /F ■ 3 .9 0 52.1 RN 3 5 /F 1.72 6 3 .3
SP 7 /F BLEPHARITIS 1.29 18.7 DP 2 2 /M ABSCESSES 1.98 7 1 .3 SR 24/F WOUND INFECTION 0 .9 8 3 4 .7
JR 3 /M ABSCESSES N O T DO NE N O T D O N E MS 1 6 /F - 1.68 6 4 .8 GS 4 1 /M " 1.23 6 6 .0
JS 3 /M 1.26 1 0.7 FC 60/M SEPTICAEMIA 2 .0 3 6 3 .3 RC 12/M OSTEOMYELITIS 1.05 6 1 .5
PD 17/M ARTHRITIS 1 .39 4 4 .8 EF 1/F SEPTICAEMIA 3 .7 5 3 7 .4 KK 5 6 /F ARTHRITIS 1.96 3 5 .6
114 TABLE 5.8. Median concentrations and ranges of anti-5 .aureus
IqG class and subclasses in patients and controls
Total IgG igGl lgG2 IgG4* GROUP (ELISA index) (mg/l) (mg/l) (ELISA index) 0.82 1.72 45.0 1.05 PATIENTS (0.22-1.16) (0.98-3.9) (10.7-71.3) (0.15-1.49) 0.81 1.00 46.9 0.90 CONTROLS (0.11-1.29) (0.4-2.65) (7.3-74) (0.03-2.18)
* lgG4 levels were measured in adult patients and controls only
FIGURE 5.1. IqGl anti-5 .aureus antibody concentrations in
patients and controls. Bars show median values.
5 T
CD E 4 - c o 4—' o 3 - c 0 o i c o 2- o 4 - o cn 1 - 1.00 mg/l I
0 normals patients
115 S.aureus phagocytosis 100T 5.2) suggesting an association between raised IgGl le ve ls ls ve le IgGl raised between association an suggesting 5.2) and increased Fc-dependent opsonophagocytosis by normal normal by opsonophagocytosis neutrophils. Fc-dependent (Fig (p<0.0002) increased group and patient the in higher tly n a ific n ig s ains n haty otos Br so mda values. show median Bars controls. healthy and patients FIGURE 5.2. Fc-dependent phagocytosis of of phagocytosis Fc-dependent 5.2. FIGURE Fc-mediated phagocytosis of the staphylococci was also also was staphylococci the of phagocytosis Fc-mediated omas patients als norm S.aureus in in 116 5.2.1 SPECIFIC ANTI-5.aureus IqG ANTIBODIES
Measurement of s p e c ific IgG directed against e ith e r whole S.aureus organism {Jarlov, 1985} or staphylococcal constituents such as lipoteichoic acid and peptidoglycan
{Granstrom, 1983; Wergeland, 1984} has been used in the diagnosis of suspected deep-seated S.aureus infections.
Generally, markedly raised levels have been found in patients with staphylococcal endocarditis or septicaemia but not in cases of osteomyelitis or superficial disease. Since most of the patients in th is study (82%) have had skin lesions, the level of total specific IgG would not be raised.
5.2.2 ANTI-5.aureus IqG2 SUBCLASS ANTIBODIES
A predominant IgG2 response to S.aureus was not surprising, as IgG2 is the main subclass response to polysaccharide antigens {Burton, 1986}. Hammarstrom {1984} has shown that anti-teichoic acid antibodies are mainly IgG2 subclass in normal adults and this polysaccharide comprises
40% of the staphylococcal cell wall {Peterson, 1983}. The
IgG2 levels were 25-47 times greater than IgGl in a ll samples tested, and even though the bacterial c e ll wall has many carbohydrate antigens, such a difference does not reflect the proportions of these subclasses in normal plasma; IgGl 60-71%, IgG2 19-31%, IgG3 5-8.4%, IgG4 0.7-4.2%
{Hamilton, 1987}. Perhaps the high levels of IgG2 induced in response to this organism confer some immunological advantage, since Givner {1987} has suggested that this
117 subclass may have a major role in modulating opsonophagocytosis of another Gram positive organism, the
Group B streptococcus (GBS). The IgG2 mediated opsonization of GBS activates the alternative complement pathway.
5.2.3 ANTI-5 .aureus IqG3 & IqG4 SUBCLASS ANTIBODIES
The absence of a measurable IgG3 response does not necessarily imply that antibodies of this subclass are not formed against S.aureus antigens since IgG3 molecules have a shorter h a lf - lif e (7 days) in the plasma than the other subclasses (21 days) and are more susceptible to the increased rate of catabolism which occurs when the serum IgG concentration is increased {Hamilton, 1987}. I gG 3 antibodies are formed in response to protein antigens such as the staphylococcal alpha toxin {Hammarstrom, 1984} and following vaccination with viral antigens {Hammarstrom,
1986; Ochs,1988}. IgG4 antibodies are increased when there is a prolonged antigenic challenge such as hyperimmunization
{Hammarstrom, 1986} and in a lle rg ic and p a ra sitic conditions with raised IgE concentrations {Heiner, 1984}. The absence of increased IgG4 levels in the patients with recurrent staphylococcal infections may be due to a selective hyposensitisation to S.aureus cell wall antigens noted in patients with chronic S.aureus colonization of the skin
{Hauser, 1985}.
118 5.2.4 ANTI-5.aureus IqGl SUBCLASS ANTIBODIES
The significantly raised IgGl levels found in our patient group may be attributable to their increased exposure to the pathogen and therefore the opportunity to develop a wider array of antibodies to other cell wall components, notably the peptide side chains and interpeptide bridges of peptidoglycan. The raised IgGl levels were associated with an increased ability to support opsonophagocytosis in the absence of complement. This is in agreement with the observation that the IgGl subclass binds avidly to Fc receptors {Burton, 1986}.
5.2.5 ANTI-5.aureus SUBCLASS ANTIBODIES IN CHILDREN AND
ADULTS
Adult levels of IgGl and IgG2 are attained in the paediatric population at 5-7 years and 8-10 years re sp e ctiv e ly {Hamilton, 1987}. S im ila rly , a n t i- S.aureus
IgG2 antibodies in non-infected and infected children were lower than the adult levels (Table 5.9) but the median concentration in infected children was higher but not statistically different (p>0.1) from that of the control paediatric group. The IgGl response in infected children was similar to that of infected adults and significantly higher than the adult (p=0.003) or childhood controls (p<0.001)
(Table 5.10). This pattern of a predominantly IgG2 response with markedly raised IgGl levels in the patients was also found in children aged 5 years or less (Table 5.11).
119 This subclass pattern is different from the pattern
seen in young children in response to pneumococcal
polysaccharide antigens {Freijd, 1984}. It was found that
normal ch ildren had higher amounts of IgGl, s ig n ific a n tly
exceeding those of the adults, and healthy children had
higher IgGl and IgG2 values than the o t it is prone children.
It was concluded that the generation of specific
anti-pneumococcal antibodies was not promoted by repeated
infection. In contrast, repeated S.aureus infections caused
a slight increase of IgG2 and a marked rise in IgGl values
in the infected children.
TABLE 5.9. Median values and ranges of a n ti- 5.aureus IqG2
antibodies in children and adults
Paediatric Paediatric Adult Adult controls (15)* patients (11) controls (35) patients (14) m g/l m g/l m g/l m g/l
Medians 28 37.4 51 57.9
Ranges 7.3 - 50.2 10.7 - 61.5 8.2 - 74 34.7 - 71.3
* number of patients or controls in each group
120 TABLE 5.10. Median values and ranges of anti-5.aureus IqGl
antibodies in children and adults
Paediatric Paediatric Adult Adult controls (15)* patients (11) controls (35) patients (14) m g/l m g/l m g/l m g/l
Medians 0.55 1.71 ** 1.12 1.79 Ranges 0.4 - 1.0 1.0 - 3.9 0.4 - 2.65 0.98 - 3.46
* Number of patients or controls In each group ** p<0.001 (compared with paediatric controls)
TABLE 5.11. Ant i-S.aureus IqG class and subclass
concentrations in healthy controls and patients aged 5 years
or under
Anti-S.aureus Anti-S.aureus Anti-S.aureus Anti-S.aureus Subject (Age) T o tal IgG* lgG1 (mg/L) lgG2 (mg/L) lgG4* CONTROLS
Samirahi(2) 0.13 0.40 9.4 0.26
Hodgson(4) 0.30 0.47 50.2 0.27 Clark (2) 0.48 0.91 7.3 -
Hicks (3) 0.98 1.01 40.2 - Jo rd a n (5) 0.13 0.75 27.7 0.29
Palmer (4) 0.11 0.44 12.7 0.24 MEDIANS 0.30 0.75 27.7 0.27 RANGES 0.11 - 0.98 0.40 - 1.01 7.3 - 50.2 0.24 - 0.29 PATIENTS JB (2) 0.74 1.71 36.2 0.004
Y B (2) 0.49 1.40 38.2 - JR (3) 0.35 - - -
JS (3) 0.30 1.26 10.7 0.26
EF (1) 0.34 3.75 37.4 0.34
LC (3) 0.55 2.22 31.5 0.34
IE (2) - 2.64 15.6 0.37 MEDIANS 0.49 2.22 36.2 0.34 RANGES 0.30 - 0.74 1.26 - 3.75 10.7 - 38.2 0.04 - 0.37
♦ELISA Index
121 5.2.6 ANTI-5 .aureus IqGl LEVELS AND OPSONOPHAGOCYTOSIS IN
PATIENTS WITH RECURRENT OR DEEP S.aureus INFECTIONS
The patients in this study have all had clinically s ig n ific a n t staphylococcal in fe ctio n s, but have normal levels of all immunoglobulin classes and IgG subclasses.
Measurement of their specific anti-staphylococcal subclass levels has not shown a deficiency of any subclass relative to the healthy control group, rather the patients have increased specific IgGl antibodies which were associated with an increased efficiency of Fc-mediated phagocytosis.
Therefore, it would appear that specific IgG subclass deficiency does not contribute to their increased susceptibility to staphylococcal infections.
5.2.7 OTHER POSSIBLE DEFECTS CAUSING RECURRENT OR DEEP
5.aureusIWFEClIOUS
These patients may be recurrently or severely infected with S.aureus, not because of any antibody defect but because of a defect in the ir a b ilit y to prevent the entry of the organism into the skin or deeper tissues. S.aureus attaches to nasal epithelial cells via teichoic acid and possibly fibronectin {Adlam, 1984-}. This organism is part of the normal flora of some people and is carried persistently by 25% of individuals {Phillips, 1987} which in those persons is thought to explain a greater susceptibility to staphylococcal in fe ctio n s {Chow, 1989; Luzar, 1990}.
Endothelial cells are also able to bind S.aureus {Tompkins,
1990} and perhaps individual differences in these sites may
122 predispose to bacterial invasion, bacteraemia and an increased likelihood of deep-seated infections.
5.3 PHAGOCYTOSIS OF CLINICAL ISOLATES OF S. PYOGENES TYPE
M1/T1/0F- OBTAINED FROM THREE PATIENTS WITH SEPTIC SCARLET
FEVER
5.3.1 THE RE-EMERGENCE OF STRAINS OF INVASIVE S.PYOGENES
WHICH CAUSE SEVERE INFECTION & A TOXIC SHOCK-LIKE SYNDROME
Invasive streptococcal infection associated with a to x ic shock syndrome caused by Group A 3-haem olytic streptococcus (GABHS) has recently been described in many patients in England {Shaunak, 1989; E d ito ria l, 1989;
MacLennan, 1990} and elsewhere, in particu lar the USA {Cone,
1987; Bartter, 1988; Stevens, 1989; Drabick, 1989; Thomas,
1989}, A u stra lia and Yugoslavia {Hansman, 1989; Begovac,
1990}. The causative agents have been of re s tric te d
M-protein type, mainly Ml and M3 strains {Stevens, 1989}, some of which produce the potent pyrogenic toxin A {Cone,
1987; Stevens, 1989}. This exotoxin was associated with the fatal cases of septic scarlet fever descibed in the first part of this century {Stollerman, 1988}.
5.3.2 DIFFERENCE IN THE PHAGOCYTOSIS OF 3 ISOLATES OF
INVASIVE S.PYOGENES
Clinical isolates, all type M1/T1/0F-, from three cases of septic scarlet fever were found to be resistant to phagocytosis. Figure 5.3 shows the phagocytosis of S(MB) and an untyped strain of S.pyogenes isolated from a patient with
123 a streptococcal skin lesion. Statistically significant differences in opsonophagocytosis of the three isolates were found when neutrophils and autologous plasmas from 13 laboratory workers were tested (Fig 5.4 & Table 5.12). The iso la te s from the two fa ta l cases S(JB) and S(MB) were phagocytosed less efficiently than the isolate from the only survivor S(FS) (p<0.001 & p<0.01 re sp e ctiv e ly ). In addition, S(JB) was the only organism which could be recovered from a laboratory rat after three animals were injected intraperitoneally with equal quantities of the three organisms S(JB), S(MB) & S(FS). The in vitro resistance to phagocyotsis of S(JB) reflected its in vivo virulence.
FIGURE 5.3. Phagocytosis of S(MB) M1/T1/0F- and an untyped clinical isolate of S.pyogenes
124 100 H S(JB)
S(FS)
2 S (M B)
FIGURE 5.4. Phagocytosis of three clinical isolates of
S.pyogenes M1/T1/0F- from cases of sceptic scarlet fever by
PMNs from healthy controls in autologous plasmas
C lin ical Median Range isolate S(JB) 19.9* 0.5 - 77.3 SCMB) 75.3** 66.1 - 92.4 S(FS) 92.0 80.0 - 98.6
* p <.001, * * p <.01 when compared with % phagocytosis of S(FS)
TABLE 5.12. Medians and ranges of phagocytosis of 3 isolates of S.pyogenes M1/T1/0F- by PMNs from 13 healthy controls
125 5.3.3 PHAGOCYTOSIS OF S.pvoaenes M1/T1/OF- FROM DIFFERENT
CLINICAL SITUATIONS
A comparison of phagocytosis of clinical isolates of
S.pyogenes M1/T1/0F-, obtained from other serious c lin ic a l situ a tio n s, namely streptococcal pneumonia, abdominal pus and a fatal case of septicaemia, did not show the statistically significant difference in bacterial uptake seen with strains causing septic scarlet fever (Table 5.13).
TABLE 5.13. Median values and ranges of the opsonophaqocytosis of S(septicaemia), S(abd.pus) and
$(pneumonia) by control PMNs in 4 different normal plasmas
MEDIAN ORGANISM RANGE (%) % inhibition
S.(SEPTICAEMIA) 52 5-68.4
S.(ABD.PUS) 60 4 9 -65
S.(PNEUMONIA) 67 46.2-78
126 5.3.4 ANTI S. pyogenes IqG ANTIBODIES
Resistance to group A streptococcal infection is dependent on the binding of type-specific opsonic antibodies to the M protein, which neutralizes its antiphagocytic e ffe c t { F is c h e tti, 1983}. The M protein is an a -h e lic a l coiled-coil fibrillar molecule on the surface of the organism and is the main anti-phagocytic factor {Wannamaker,
1982; Pancholi, 1989}. Since c lin ic a l iso la te s from the patients with septic scarlet fever were ingested to different extents, it followed that either the expression of
M-protein differed among the isolates or additional factors were adversely affecting phagocytosis. Since qualitative or quantitaive differences in the expression of the M-protein could have accounted for the observed differences in phagocytosis, an ELISA method was developed to measure the binding of anti-streptococcal antibodies to S.pyogenes
M1/T1/0F- (Table 5.14).
TABLE 5.14. Median values and ranges of anti-S.pyogenes IqG binding to 6 clinical isolates of S. pyogenes M1/T1/0F- measured by ELISA
POOL OF 100 CONVALESCENT ORGANISM SERA (A.R.)* SERUM (A.R.) S(asymptomatic) 0.94 >2.0 S(abd.pus) 0.89 >2.0 S(septicaemia) 0.66 >2.0 S(tonsillitis) 0.66 >2.0 S(pneumonia) 0.76 >2.0 S(JB) 0.94 1.68
A.R. = Absorbance readings at 405nm
127 There was no difference in binding of pooled sera from 100 donors and the convalescent plasma from patient FS, though the latter appeared to bind less well to isolate
S(JB) (Table 5.14). Failure to detect a difference could be due to the presence of 'natural antibodies' directed against the streptococcal cell wall carbohydrate and mucopeptide
{Krause, 1972} which could mask the binding of specific anti-M protein antibodies. Purified Ml protein would be required to compare the anti-phagocytic epitopes of these clinical isolates. Another M-protein, the M6 protein, has been isolated and cloned { F is c h e tti, 1976, 1977; Jones,
1988} and it has been shown that only those antibodies binding to specific epitopes in the N-terminal are protective {Jones, 1988}.
5.3.5 S.pvoaenes M1/T1/0F- VIRULENCE FACTORS
Other characteristics of invasive strains of
S. pyogenes include mucoid colony morphology and the production of pyrogenic toxin A {Stevens, 1989}. This is in keeping with previous observations that the presence of hyaluronic acid capsules and bacterial exotoxin production cause increased virulence and resistance to phagocytosis
{Wannamaker, 1982}. It was noted that cultures of
S(septicaemia) were very large and mucoid when compared with
S(asymptomatic) which grew small transparent colonies.
Hyaluronic acid capsules are not immunogenic presumably because they are chemically indistinguishable from the
128 hyaluronic acid of mammalian connective tissue. Removal of the capsule by hyaluronidase dramatically lowers the virulence of test strains in mice {Krause, 1972}. The presence of a capsule of low immunogenicity could facilitate the survival of the organism in the host.
The Streptococcal Reference Laboratory at Col indale did not detect the presence of erythrogenic toxin A from among 40 isolates of strain Ml which had been obtained from serious streptococcal disease between 1985 and 1988
{personal communication from Dr.G.Colman, Streptococcal
Reference Laboratory, Col indale}. However, 72% of the stra in s produced toxin B {Gaworzewska, 1989}. They have found an increased number of isolates of type M1/T1/0F- from cases of fa ta l in fection s since 1985 {Gaworzewska,
1988}. The streptococcal erythrogenic toxins A,B,C are small a c id ic p ro te in s (MWt 13-26 kD) {Cone, 1987} c lo s e ly associated with hyaluronic acid. They can induce fever, the scarlet fever rash, cytotoxic and tissue damage and lethal shock in test animals {Kim, 1972}.
The multi-system failure seen in cases of streptococcal toxic-shock lik e syndrome (TSLS) or septic scarlet fever may be toxin mediated since the clinical manifestations of this disorder are very similar to the staphylococcal toxic-shock syndrome {Cone, 1987}. Moreover, pyrogenic toxin A which has been implicated in some cases of
STSS share amino acid homology and biologic properties with toxic shock syndrome toxin-1 {Stollerman, 1988; Stevens,
1989}. The extent of tissue damage caused by these toxins
129 is partly due to the immunological state of the host as acquired hypersensitivity to the heat-stable portion of the toxin makes the tissues hyperreactive to extremely small q u a n titie s of these potent molecules {Kim, 1972}. The scarlet fever rash is a manifestation of skin reactivity to the toxins. The presence of astronomically high levels of
IgG4 in patient FS (see Table 4.10) are in keeping with an a lle rg ic reaction to the streptococcal components.
5.3.6 DEFECTIVE PHAGOCYTOSIS IN SEPTIC SCARLET FEVER
However, an iso la te of S.pyogenes M1/T1/0F- from a rapidly fatal case was found to be more resistant to phagocytosis by normal neutrophils than other isolates of the same type. This suggests that the presence of type-specific M-protein antibodies may not be sufficient to enhance the phagocytosis of very invasive strains. Failure to phagocytose the organism would facilitate its multiplication in affected tissues and the further production of lethal toxins. It may be that the toxin(s) in association with hyaluronic acid are also profoundly antiphagocytic since small amounts of erythrogenic toxin A can significantly depress reticuloendothelial system function fo r over 48 hours {Kim, 1972}. Convalescent serum from patient FS increased the opsonophagocytosis of S(JB) by normal PMNs (Table 5.15) suggesting that recovery from septic scarlet fever is associated with the production of protective antibodies directed against other anti-phagocytic fa c to r(s ).
130 TABLE 5.15. Phagocytosis of S(JB) by normal PMNs in the presence of normal plasmas and convalescent FS serum
% Inhibition Normal Convalescent PMN source plasma FS serum
Control 1 6 9 .6 7 1 .2
Control 2 7 7 .3 8 8 .6
Control 3 4 8 .0 7 1 .6
Control 4 7 7 .0 9 4 .0
5.3.7. VIRAL SUPPRESSION OF NEUTROPHIL FUNCTION AND INVASIVE
S. PYOGENES DISEASE
Failure of efficient phagocytosis leading to increased streptococcal survival in these patients may also arise from suppression of phagocytic function by antecedent or simultaneous v ira l illn e s s . Thomas and associates {1989} reported a simultaneous increase in Influenza virus Type B isolates and invasive streptococcal strains in Los Angeles
County in January-February 1989. Inluenza-1ike symptoms have also been reported in the patients of MacLennan and Hansman
{MacLennan, 1990, Hansman, 1989}. The influenza virus can suppress phagocyte chemotaxis, phagocytosis and killing
{reviewed by Abramson, 1988} and so increase the likelihoo d of bacterial invasion and proliferation.
131 CHAP 6: GENERAL DISCUSSION AND CONCLUSIONS
6.1 THE ABSCESS
Ford and associates {1989} measured the neutrophil and staphylococcal (free and phagocytosed) populations in experim entally induced abscesses over 5 days. They found that within the first day the host exerted control over the bacterial population at the abscess site, primarily through the influx and activity of phagocytic cells. However, the numbers of free S.aureus increased substantially on days 2 and 3 and then remained relatively constant. The second phase of containment was thought to be achieved by the limiting capsule in which the bacteria were trapped and nutritionally deprived. The consistent increase in bacteria at 2-3 days was not seen with S. epidermidis or E.Coli and the abscesses formed by these organisms were less intense.
Granulocytopenic mice were unable to contain the organisms and all died of systemic infection.
6.2 DEFECTIVE OPSONOPHAGOCYTOSIS AND FAILURE OF BACTERIAL
CONTAINMENT
This mouse model of the abscess is useful in that it attempts to define the role of host factors in the formation of this lesion. The host initially limits bacterial spread and replication by the action of phagocytic cells and follows this with structural containment of the bacteria.
Defects of opsonophagocytosis would compromise the in it ia l
132 containment of bacteria and increase the risk of systemic
spread by organisms, especially more virulent strains.
The failure of bacterial containment, because of
defective opsonization, could have caused the deep-seated
staphylococcal infections found in 3 of the patients in this
study. Other factors such as chronic illness or exposure to
large inocula of virulent staphylococci may explain why the
other patients with normal opsonization, in th is study,
s t i l l succumbed to serious S.aureus infection.
6.3 BACTERIAL HYPERSENSITIVITY AND ABSCESS FORMATION
An enhanced immune response to staphylococcal antigens
may account for the large abscesses, which heal with
scarring, found in some patients with recurrent S. aureus
skin lesions. Excessive formation of immune complexes at the
site of bacterial invasion could lead to significant tissue
damage by a ctiva tin g the complement pathway and enhancing
neutrophil secretion of potent enzymes. A lte rn a tiv e ly ,
immune complexes could impede phagocytosis and so increase
bacterial survival with consequent tissue damage.
6.4 OPSONIC DEFECTS AND RECURRENT INFECTIONS
Defects of opsonophagocytosis which predispose the
host to recurrent infections are not rare. Hosking et al
{1978} studied the immune function of children with
recurrent infections and found that approximately 20% of
tests for opsonins to S.aureus and C.albicans were outside
the control limits. Fifty five percent of 62 patients with
133 recurrent staphylococcal skin infections showed inadequate
neutrophil chemotaxis, defective post-phagocytic iodination
or opsonic d e ficie n cie s. More recently, Bondestam and
associates {1986} in an ongoing study of infection prone
children in Sweden found that defects in oxidative PMN
metabolism and defects in Fc-receptor-dependent phagocytosis were major determinants of susceptibility to infection and
that such defects were more common in patients with severe
multifocal infections.
Four (13%) of the 31 patients with staphylococcal
disease in this study had demonstrable defects of
opsonophagocytosis. This agrees well with Hosking {1978} who
found that 10% of the tests, in which S.aureus opsonins were
measured, were below his reference range. Eight (26%) of the
patients described in this thesis had low levels of
neutrophil chemotaxis (Fig 4.8). Six of these patients were
under 12 years of age and since young children are thought
to have lower levels of neutrophil migration compared with
adults {reviewed by Evans, 1990}, the low levels of movement
in these patients may be normal fo r th e ir ages. Hosking
{1978} also found that defective chemotaxis was common among
62 patients with recurrent S. aureus skin infections but he
too was comparing neutrophil movement in children with an
adult reference range.
6.5 PROPHYLAXIS AGAINST RECURRENT S.AUREUS INFECTIONS
Five (22%) of 22 patients with recurrent abscesses had
positive nasal swabs when they were investigated at our
134 department suggesting that persistent nasal carriage may
account in part for their predisposition to infection.
Drugs such as rifam p icin and clindamycin, which have been
shown to be effective against S. aureus nasal carriage
{reviewed in Editorial, 1988}, might be more useful in these
patients than the topical antibiotic preparations that were
commonly prescribed. Alternatively mupirocin, a new topical
antibiotic has been shown to be very effective in
eliminating nasal carriage among haemodialysis patients
{Chow, 1989}. Many of the patients in th is study were
treated only once for nasal carriage yet recolonization is
the norm in persistent nasal carriers. In these patients,
reculturing of the anterior nares after treatment and at
regular intervals might go a long way in decreasing the
frequency of their infections.
6.6 TREATMENT OF PATIENTS WITH RECURRENT S.AUREUS INFECTIONS
Most of these patients with S.aureus infections were
treated with broad spectrum antibiotics whenever the
abscesses appeared but a few were on prophylactic
anti-microbial therapy. Penicillinase-resistant penicillins were commonly prescribed. M e th ic illin , a p e n ic illin a s e
resistant drug was found to have some protective function
against abscess formation in the mouse model {Ford, 1989}
but penicillin derivatives usually do not achieve high
intraphagocytic levels {Forsgren, 198b}. This is in
contrast to rifampicin and clindamycin and ciprofloxacin which attain good intraphagocytic concentrations {Jacobs,
135 1983; Easmon, 1986}, and are e ffe c tiv e against systemic
S.aureus infection and in preventing abscess formation
{Ford, 1989}. A combination of a penicillinase-resistant preparation and another drug like rifampicin may be more effective in combating established infection. This two-pronged attack should give increased bactericidal activity but decrease the development of bacterial tolerance to rifampicin.
6.7 TREATMENT OF PATIENTS WITH SPECIFIC OPSONIC DEFECTS
Two of the 4 patients with defective S. aureus opsonization were treated with purified IgG (initially intramuscularly and then intravenously) which produced a dramatic reduction in the frequency and severity of their infections. The two other patients were lost to follow up.
Immunoglobulin therapy is used as replacement therapy in patients with acute or chronic infections who lack protective antibodies because of a congenital or acquired fa ilu r e in antibody synthesis {Barandun, 1985}. These patients had adequate levels of immunoglobulin classes, IgG subclasses and specific anti-5.awrews antibodies but can be assumed to have had a functional antibody deficiency as detected by the techniques used to investigated them.
Replacement IgG therapy provided these patients with the functional anti-5 .aureus antibodies of a large pool of donors which protected them from further infection. Most immunoglobulin preparations are known to have opsonic
136 a c tiv ity fo r most common pathogens including S. aureus { H ill,
1984}.
6.8 DEFECTIVE PHAGOCYTOSIS OF VIRULENT S.PYOGENES IN THE
PATHOGENESIS OF STREPTOCOCCAL TOXIC SHOCK-LIKE SYNDROME
Virulent strains of S.pyogenes, which cause a toxic shock-like syndrome associated with extensive soft-tissue
infection and a scarlet fever rash, have been the subject of
intense epidemiological and microbiological investigation within the last five years. Many of the patients have been young, fit adults without evidence of underlying
immunodeficiency, yet they were all rapidly overwhelmed by multi-organ failure and shock. The causative organisms have been described as mucoid toxin-producing strains of
5 .pyogenes, notably M-protein types Ml and M3. The potent toxins have been identified as pyrogeni c exotoxins especially type A. These toxins can induce the production of tumour necrosis factor-alpha (TNF-a) which may mediate
the shock symptoms of th is condition. A d d itio nally, TNF-a
can decrease neutrophil chemotactic activity {Fast, 1989}.
The three patients with septic scarlet fever were
notable fo r the absence of pus in th e ir so ft-tissues despite
severe infection with a pyogenic agent like S.pyogenes
{Shaunak, 1989}. This could be due to decreased neutrophil
influx into the tissues secondary to increased TNF
production. Alternatively, the neutrophils are unable to
phagocytose the bacteria so that there is little production
of toxic radicals and secretion of other phagocytic enzymes
137 into the tissues. Two of the three isolates from these patients were resistant to phagocytosis by normal neutrophils. Therefore the development of th is condition may be as follows: After the invasion of the soft-tissues by the streptococci, there is failure of the host to eradicate the
invaders because of the TNF induced decrease in PMN movement and the bacterial resistance to phagocytosis. The bacteria continue to m ultiply and produce toxins which are spread via the bloodstream to other organs. These toxins can induce myocardial damage which would worsen the toxic effects on
the circulation and various organs.
6.9 MANAGEMENT OF STREPTOCOCCAL TOXIC SHOCK-LIKE SYNDROME
The treatment of patients with streptococcal TSLS has
consisted of high doses of antibiotics mainly penicillin and
cephalosporin to eliminate the bacteria, surgical
debridement to remove severely infected soft-tissues and
supportive therapy for shock and multi-organ failure. Since
tissue damage may be mediated by TNF, anti-TNF antibodies
have been suggested as another therapeutic option
{ E d ito ria l, 1988}. In addition, possible fa ilu r e of
neutrophil function due to TNF inhibition of cell movement
or bacterial resistance to phagocytosis could cause
bacterial persistence and continued production of toxins.
The use of antibiotics like the penicillins which do not
achieve good intraphagocytic concentrations {Forsgren, 1985}
nor enhance phagocytosis (Figure 6.1) is questionable,
especially when these drugs have been reported to have
138 staphylococcal inhibition which are e ffe c tiv e against against e tiv c ffe e are which oe fetv i ti tp o srpoocl infection. streptococcal of type this in effective more erythromycin and am p icillin on the phagocytosis of of phagocytosis the on icillin p am and erythromycin erae efi n eee te oc cl nf i s n tio c fe in ccal co to strep severe in y c a ffic e decreased FIGURE 6.1 E ffects of preincubation of control PMNs with with PMNs control of preincubation of ffects E 6.1 FIGURE oducton n tio c u d ro p (Stevens,1988). Drugs lik e clindamycin and erythromycin erythromycin and clindamycin e lik Drugs (Stevens,1988). nrpaoyi lvl {aos 18; amn 18} ih be might 1986} Easmon, 1983; {Jacobs, levels intraphagocytic in creasin g concentrations of rifa m p ic in , clindam ycin, ycin, clindam , in ic p m rifa of concentrations g creasin in {Stevens, 1988} and and 1988} S.pyogenes, also attain high high attain also suppress toxin toxin suppress S.aureus 139 The pyrogenic exotoxins can suppress B-lymphocyte function and inhibit immunoglobulin production {Stevens,
1989; Fast, 1989}. This could account fo r the low IgG levels in the deceased patients, MB and JB. This is in contrast to survivor FS who had raised IgG, IgA and IgM concentrations even in the early stages of the infection
(Table 4.8). Low levels of IgG would lead to defective opsonophagocytosis of already resistant bacteria. Therefore, intravenous immunoglobulin therapy may be required in those patients who fail to maintain normal IgG levels.
Pyrogenic toxin A has only been shown to be produced by some clinical isolates but it shares considerable homology and biological properties with pyrogenic toxins B and C which have been associated with some other isolates of invasive S.pyogenes. Early use of specific anti-toxin, which could neutralize preformed toxin, may decrease the severity of organ damage. This type of treatment is w idely used in the management of diphtheria {Jawetz, 1980}. Specific anti-toxin could be induced in animals by repeated injection with small amounts of exotoxin though the p u rified exotoxins are not highly immunogenic {Kim, 1972}. Hyperimmune serum would be another good source of specific anti-toxin.
Certainly, convalescent serum from patient FS was able to enhance the phagocytosis of re sista n t S.pyogenes (Table
5.15) and contained high levels of antibodies to a range of streptococcal enzymes namely, anti-strepto1ysin, anti-deoxyribonuclease B and anti-hyaluronidase antibodies
(Table 4.9). The use of convalescent plasma would be better
140 than an animal derived preparation of anti-toxin as it would not cause serum sickness.
The present management of streptococcal TSLS is s till
associated with significant mortality. Shaunak {1989} had
two deaths out of three patients and Stevens {1989} had 6
deaths (30%) in a group of 20 patients. Bacterial
elim ination by drugs which enhance phagocytic ingestion and
killing plus the use of specific anti-toxin and
immunoglobulin therapy may be more useful therapeutic
adjuncts to surgical debridement than the present
approaches.
6.10 CONCLUSIONS
6.10.1 AIMS OF PROJECT
1) To identify possible opsonophagocytic defects in patients
with significant staphylococcal infections.
2) To measure sp ecific anti-Staphylococcus aureus IgG class
and subclass antibodies in patients and healthy volunteers
to see if specific subclass defects were predisposing these
patients to infection.
3) To modify the Foroozanfar phagocytic assay to measure
phagocytosis of another common pathogen, Streptococcus
pyogenes so as to compare the phagocytosis of virulent
isolates of this organism.
The project aims, as stated above, have a ll been
fulfilled in this thesis. Four patients with significant
S.aureus infections were found to have defective
141 opsonophagocytosis of th is pathogen and two of them were
successfully treated with purified IgG. Their defect, a
specific inablility to opsonise S.aureus has only ever been
identified in four other patients {Monteil, 1987}.
A comparative study of specific anti-5 .aureus IgG
class and subclass levels in patients with staphylococcal
infections and normal volunteers showed that the patients
had adequate levels of a ll antibodies tested. Thus, low or
absent levels of specific antibodies did not contribute to
their propensity to infection.
F in a lly , the phagocytic assay was successfully adapted
to measure in vitro phagocytosis of S.pyogenes and used to
compare the ingestion of clinical isolates of S. pyogenes
Ml/T1/OF-. Virulent organisms were found to be very
resistant to neutrophil ingestion. It was postulated that
such resistance could contribute to bacterial persistence in
patients with the streptococcal toxic shock-like syndrome.
6.10.2 THE FUTURE
The staphylococcal antigen(s) to which the four
patients with defective opsonization were unable to respond
are still to be identified. The glycine interpeptide bridges
that are specific to S.aureus are likely candidates.
Synthetic preparations of these are now available and could
be used to compare the binding of patient plasmas with that
of healthy controls by a sensitive ELISA or other technique.
The ELISA methods described in this thesis are limited
by the presence of antibodies directed against epitopes
142 common to all Gram positive organisms. These non-specific antibodies could obscure the binding of more specific antibodies. Therefore, these assays need to be repeated using sera from patients and controls that have been
absorbed out against another Gram positive organism such as
S.pyogenes. In addition, immunoblotting of patient and
control serum samples against S.aureus antigens that have
been separated may be a s t i l l more se n sitiv e method fo r
identifying any subtle specific antibody deficiencies.
Immunoblotting would also be a useful way of comparing
the antigenic determinants of different clinical isolates of
S.pyogenes M1/T1/0F- from asymptomatic cases and from
patients with serious invasive disease. This approach
should give some insight into the specific virulence factors
associated with invasive streptococci.
143 LIST OF REFERENCES
Adlam,C., Easmon,C.S.F. (1984) Immunity and hypersensitivity to staphylococcal infection. In: Staphylococci and Staphylococcal Infections vol.l pp 275-323. Easmon,C.S.F. (ed). Academic Press, London
Alexander,J.W., McClellan,M.A., Ogle,C.K., Ogle,J.D. (1976) Consumptive opsoninopathy: possible pathogenesis in lethal and opportunistic infections. Ann.Surgery 184:672-678
Allen,R.A., Traynor,A.E., Omann,G.M., Jesaitis,A.J. (1988) The chemotactic peptide receptor. A model for further understanding of chemotactic disorders. Haematology/Oncology C lin ic s of North America 2:33-59
Alpers,C.E., Colten,H.R., Rosen,F.S., Rabson,A.R., MacNab,G.M., Gear,J.S.S. (1972) Homozygous deficiency of C3 in a patient with repeated infections. Lancet 2:1179-1181
Ammann,A.J. (1987) Immunodeficiency diseases. In: Basic and Clinical Immunology, 6th Edition, pp 317-355. Stites,D.P., Stobo,J.D., Wells,J.V. (eds). Appleton & Lange, Norwalk, Connecticut
Anderson,D.C. (1987) Leukocyte adhesion deficiency: An inherited defect in the Mac-1,LFA-1,and p150,95 glycoproteins. Ann.Rev.Med. 38:175-194
Athamna,A., 0 fek,I. (1988) Enzyme-linked immunosorbent assay for quantitation of attachment and ingestion stages of bacterial phagocytosis. J.Cl in.Micro. 26:62-66
Athens,J.W ., Ra ab,S.0. , Ha ab , 0 .P. , Mauer,A.M., Ashenbrucker,H., Cartwright,G.E., Wintrobe,M.M. (1961) Leukokinetic Studies. 3. The distribution of granulocytes in the blood of normal subjects. J.Cl in.Invest. 40:159-164
A x te ll,R .A . (1988) Evaluation of the patient with a possible phagocytic disorder. Haematology/Oncology C lin ic s of North America 2:1-12
Ballow,M., Shira,J.E., Harden,L., Yang,S.Y., Day,N.K. (1975) Complete absence of the th ird component of complement in man. J.Cl in.Invest. 56:703-710
Bar-Shavit,Z., Goldman,R., Stabinsky,Y., Gottlieb,P., Fridkin,M., Teichberg,V. I., Blumberg,S. (1980) Enhancement of phagocytosis - a newly found a c tiv ity of substance P residing in its N-terminal tetrapeptide sequence. Biochem.Biophys.Res.Commun. 94:1445-1451
Barandun,S. (1985) Immunoglobulins: history, present trends and safety aspects. In: Intravenous Immunoglobulins in Immunodeficiency Syndromes and Idiopathic
144 Thrombocytopenic Purpura, pp 3-10. Waters,A.H.f Webster, A.D.B. (eds) Royal Soc. of Medicine, London
Bartter,T., Dascal,A., Carroll,K., Curley,F.J. (1988) Toxic Strep Syndrome. A manifestation of group A streptococcal infection. Arch Intern Med 148:1421-1424
Beachey,E.H, Simpson,W.A., 0fek,I., Hasty,D.L., Dale,J.B., Whitnack,E. (1983) Attachment of Streptococcus pyogenes to mammalian cells. Rev.Inf.Dis. 5, Suppl.4:s670-s677
Becker,E.L., Stossel,T.P. (1980) Chemotaxis Fed.Proc. 39:2949-2952
Begovac,J.( Gmajnicki,B., Kuzmanovic,N. (1990) Invasive streptococci. Lancet 335:109-109
B e lla n ti,J.A. (1985) Immunology III, 3rd Edition, pp 1-598. W.B.Saunders, Eastbourne
Berger,M ., B irx,D .L. (1988) Relationship between membrane depolarization and extracellular calcium influx during neutrophil activation J.Lab.Cl in.Med. 111:384-392
Bessen,D., Jones,K.F., Fischetti,V.A. (1989) Evidence for two distinct classes of streptococcal M protein and their relationship to Rheumatic Fever. J.Exp.Med. 169:269-283
Bio zzi,G ., S tiffe l,C ., Halpern,B.N., Le Minor,L., Mouton,D. (1961) Measurement of the opsonic effect of normal and immune sera on the phagocytosis of S.typhi by the RES. J.Immunol. 87:296-300
Bondestam,M., Hakansson,L., Foucard,T., Venge,P. (1986) Defects in polymorphonuclear neutrophil function and susceptibility to infection in children. Scand.J.Cl in.Lab.Invest. 46:685-694
Boxer,L.A., Harris,R.E., Baehner,R.L. (1979) Regulation of membrane peroxidation in health and disease. Pediatrics Suppl. 64:713-718
Boxer,L.A., Morganroth,M.L. (1987) Neutrophil function disorders. Disease-a-Month 33:681-755
Boxer,L.A., Smolen,J.E. (1988) Neutrophil granule constituents and their release in health and disease. Haematology/Oncology Clinics of North America 2:101-134
Boxer,L.A., Stossel,T.P. (1974) E ffects of anti-human neutrophil antibodies in vitro. Quantitative studies. J.Cl in.Invest. 53:1534-1545
Braconier,J.H., Nilsson,B., 0xelius,V.-A., Karup-Pedersen,F. (1984) Recurrent pneumococcal infections in a patient with lack of specific IgG and IgM pneumococcal
145 antibodies and deficiency of serum IgA, IgG2 and IgG4. Scand.J.Inf.Dis. 16:407-410
Brown,D., Hobart,M.J. (1981) Complement and complement fix a tio n . In: Techniques in C lin ic a l Immunology. 2nd E d itio n , pp 82-84. Thompson,R.A. (ed). Blackwell S c ie n tific , Oxford
Brown,E.J., Frank,M.M. (1981) Complement activation. Immunol.Today 2f No.7:129-134
Brown,K.A., Perry,J.D ., Black,C ., Dumonde,D.C. (1988) Identification by cell electrophoresis of a subpopulation of polymorphonuclear cells which is increased in patients with rheumatoid arthritis and certain other rheumatological disorders. Ann.Rheum.Dis. 47:353-358
Burton,D.R., Gregory,L., Jefferis,R. (1986) Aspects of the molecular structure of IgG subclasses. Monogr.Allergy 19:7-35
Cates,K.L. (1981) Evaluation of granulocyte adherence. In: Investigation of Phagocytes in Disease, pp 12-14. Douglas,S.D., Quie,P.G. (eds) Churchi11-Livingston, Edinburgh
Cech,P., Lehrer,R.I. (1984) Heterogeneity of human neutrophil phagolysosomes: functional consequences fo r candidacidal a c tiv ity . Blood 64:147-151
Chang,Y-H. (1969) Studies on phagocytosis 1. Uptake of ra d io -io d in a te d (1311) human serum albumin as a measure of the degree of phagocytosis in vitro. Cell Res. 54:42-48
Chapel,H.M. (1987) Abnormalities of the immune response. In: Oxford Textbook of Medicine, Vol 1, Section 4. pp 4.60-4.75. Weatheral1,D.J., Ledingham,J.G.G. , Warrell,D.A. (eds). Oxford Univ Press, Oxford
Chow,J.W., Yu,V.L. (1989) Staphylococcus aureus nasal carriage in hemodialysis patients. Arch Intern Med 149:1258-1262
Clark,R.A., Klebanoff,S. , 0chs,H.D., G i11i 1 and,B. , S ch a lle r,J., Wedgwood,R.J. (1975) C4 d eficien t human serum: opsonic and chemotactic activity. Clin. Res. 23:410A-0
Colditz,I.G. (1985) Margination and emigration of leucocytes. Surv.Synth.Path.Res. 4:44-68
Cone,L.A., Woodard,D.R., Schlievert,P.M ., Tomory,G.S. (1987) Clinical and bacteriologic observations of a toxic shock - like syndrome due to Streptococcus pyogenes. N.Engl.J.Med. 317:146-149
146 Davis,J.P., Chesney,P.J., Wand,P.J., La Venture,M. (1980) Toxic Shock Syndrome - epidemiologic features, recurrence, risk factors, and prevention. N.Engl.J.Med. 303:1429-1435
Davis,W.C., Douglas,S.D., Fudenberg,H.H. (1968) A selective neutrophil dysfunction syndrome: impaired killing of staphylococci. Ann.Int.Med. 69:1237-1243
Dobrina,A., Schwartz,B.R. , Carlos,T.M., 0chs,H.D., Beatty,P.G., Harlan,J.M. (1989) CDll/CD18-independent neutrophil adherence to inducible endothelial-leucocyte adhesion molecules (E-LAM) in v itro Immunol. 67:502-508
Donowitz,G.R., Mandell,G.L. (1983) Clinical presentation and unusual infections in chronic granulomatous disease. In: Advances in Host Defense Mechanisms, Vol.3. pp 55-75. Ga11in ,J . I ., Fauci,A.S. (eds). Raven Press, New York
D rabick,J . J . , Lennox,J.L. (1989) Group A streptococcal infections and a toxic shock-like syndrome. N.Engl.J.Med. 321:1545-1545
Dykman,T.R., Cole,J.L., Ii da,K., Atkinson,J.P. (1983) Structural heterogeneity of the C3b/C4b receptor (CR1) on human peripheral blood cells. J.Exp.Med. 157:2160-2165
Easmon,C.S.F., Cole,P.J. (1981) Lumino 1-dependent chemiluminescence. In: Techniques of Clinical Immunology, 2nd Edition, pp 294-299. Thompson,R.A. (ed). Blackwell Scientific Publications, Oxford
Easmon,C.S.F., Easman,C.A. (1984) Staphylococci and staphylococcal infections, V o ls.l & 2. Academic Press, London
Easmon,C.S.F., Crane,J.P., Blowers,A. (1986) Effect of Ciprofloxacin on intracellular organisms: in-vitro and in-vivo studies. J.Antimicrobial Chemotherapy. 18:43-48
Editorial. (1988) Recurrent staphylococcal skin infections. Lancet 2:1346-1347
E d ito ria l (1989) Invasive streptococci. Lancet 2:1255-1255
Ehlenberger,A.G., Nussenzweig,V. (1977) The role of membrane receptors for C3b and C3d in phagocytosis. J.Exp.Med. 145:357-371
Ekstedt,R.D. (1972) Immunity to the Staphylococci. In: The Staphylococci, Chapter 16. pp 385-418. Cohen,J.0. (ed). Wiley-Interscience, USA.
Evans,A.M. (1989) New approaches to the study of locomotion in polymorphonuclear leukocytes. PhD Thesis, Univ.London, Faculty of Medicine.
147 Fanger,M.W., Goldstein,S.N., S h e n , L . (1983) Cytofluorographic analysis of receptors for IgA on human polymorphonuclear cells and monocytes and the correlation of receptor expression with phagocytosis. Mol.Immunol. 20:1019-1027
Fanger,M.W., Pugh,J., Bernier,G.M. (1981) The specificity of receptors for IgA on human peripheral polymorphonuclear c e lls and monocytes. Cell.Immunol. 60:324-334
Fast,D.J.f Schlievert,P.M., Nelson,R.D. (1989) Toxic Shock Syndrome-associated staphylococcal and septococcal pyrogenic toxins are potent inducers of Tumor Necrosis Factor production. Infect.Immun. 57:291-294
Fearon,D.T. (1984) Cellular receptors for fragments of the th ird component of complement. Immunol.Today 5:105-110
Fischetti,V.A. (1977) Streptococcal M Protein extracted by nonionic detergent 2. Analysis of the antibody response to the multiple antigenic determinants of the M-Protein molecule. J.Exp.Med. 146:1108-1123
Fischetti,V.A. (1983) Requirements for the opsonic activity of human IgG directed to type Group A streptococci: net basic charge and intact Fc region. J.Immunol. 130:896-902
Fischetti,V.A., Gotschlich,E.C., Siviglia,G., Zabriskie,J.B. Streptococcal M Protein extracted by nonionic detergent 1. Properties of the atiphagocytic and type-specific molecules. J.Exp.Med. 144:32-53
F1e it,H.B., Kuhnle,M. (1988) Biochemical characterization of an Fey receptor purified from human neutrophils. J.Immunol. 140:3120-3125
Ford,C.W., Hamel,J.C., Stapert,D., Yancey,R.J. (1989) Establishment of an experimental model of a Staphylococcus aureus abscess in mice by use of dextran and gelatin microcarriers. J.Med.Microbiol. 28:259-266
Foroozanfar,N., Aghai ,Z., Ala,F., Hobbs,J.R. (1976) Inhibition of thymidine uptake by staphylococci, a new method for the investigation of phagocytosis J.Immunol.Meth. 11:345-353
Forsgren,A., Bellahsene,A. (1985) Antibiotic accumulation in human polymorphonuclear leucocytes and lymphocytes. Scand. J. Inf. Dis. Suppl. 44:16-23
Freijd,A., Hammarstrom,L., Persson,M.A.A., Smith,C.I.E. (1984) Plasma anti-pneumococcal antibody a c tiv ity of the IgG class and subclasses in otitis prone children. Cl in.Exp.Immunol. 56:233-238
F rid kin ,M ., G o ttlie b ,P . (1981) T u ftsin , Thr-Lys-Pro-Arg. Mol.Cell Biochem. 41:73-97
148 Gabay,J.E. (1988) M icrob icid al mechanisms of phagocytes. Current Opinion in Immunology 1:36-40
Gabay,J.E., Heiple,J.M., Cohn,Z.A., Nathan,C.F. (1986) Subcellular location and properties of bactericidal factors from human neutrophils J.Exp.Med. 164:1407-1421
Gallin,J.I., Malech,H.L., Wright,D.G., Whisnant,J.K., Kirkpatrick,C.H. (1978) Recurrent severe infections in a c h ild with abnormal leukocyte function: possible relationship to increased microtubule assembly. Blood 51:919-933
Ganz,T., Selsted,M.E., Szklarek,D., Harwig,S.SL., Daher,K., B ain to n ,D .F., Lehrer,R.I. (1985) Defensins. Natural peptide antibiotics of human neutrophils. J.C lin .In vest. 76:1427-1435
Garrod,L.P., Lambert,H.P., 0'Grady,F. (1973) Antibiotic and Chemotherapy, 4th Edition. Churchill Livingstone, Edinburgh
Gaworzewska,E., Colman,G. (1988) Changes in the pattern of infection caused by Streptococcus pyogenes . Epidem.Inf. 100:257-269
Gaworzewska,E.T . , Hallas,G. (1989) N.Eng 1.J .Med . 321:1546-1546
Givner,L.B., Baker,C.J., Edwards,M.S. (1987) Type III Group B streptococcus: Functional interaction with IgG subclass antibodies. J.In f.D is. 155:532-539
Goldman,R., Bar-Shavit,Z., Romeo,D. (1983) Neurotensin modulates human neutrophil locomotion and phagocytic cap ability. FEBS Lett. 159:63-67
Greenberg,D.P., Bayer,A.S., Turner,D., Ward,J.I. (1990) Antibody responses to Protein A in patients with Staphylococcus aureus bacteremia and endocarditis. J.Cl in.Micro 28:458-462
Gresham,H.D., Clement,L.T., Volanakis,J.E ., Brown,E.J. (1987) Cholera toxin and pertussis toxin regulate the Fc receptor-mediated phagocytic response of human neutrophils in a manner analogous to regulation by monoclonal antibody 1C2. J.Immunol. 139:4159-4166
Gresham,H.D., McGarr,J.A., Shackelford,P.G., Brown,E.J. (1988) Studies on the molecular mechanisms of human Fc receptor-mediated phagocytosis. Amplification of ingestion is dependent on the generation of reactive oxygen metabolites and is deficient in polymorphonuclear leukocytes from patients with chronic granulomatous disease. J.Cl in.Invest. 82:1192-1201
149 Griff in,F.M., Griffin,J. A., Leider,J.E., Silverstein,S.C. (1975) Studies on the mechanism of phagocytosis 1. Requirements for circumferential attachment of particle-bound ligands to specific receptors on the macrophage plasma membrane. J.Exp.Med. 142:1263-1282
GuptafR.C., Laforce,F.M ., M ills,D .M . (1976) Polymorphonuclear leukocyte inclusions and impaired bacterial killing in patients with Felty's syndrome. J.Lab.Clin.Med. 88:183-191
Hal las,G. (1985) The production of pyrogenic exotoxins by group A streptococci. J.Hyg.Camb. 95:47-57
Hakansson,L., H allgren,R ., Venge,P. (1980) Regulation of granulocyte function by hyaluronic acid. In vitro and in vivo effects on phagocytosis, locomotion, and metabolism. J.Cl in.Invest. 66:298-305
Hamilton,R.G. (1987) Human IgG sublcass measurements in the c lin ic a l laboratory. Clin.Chem. 33:1707-1725
Hammarstrom,L., Granstrom,M., Oxelius,M.A., Persson,A.A. (1984) IgG subclass distribution of antibodies against S.aureus teichoic acid and alpha-toxin in normal and immunodeficient donors. Cl in.Exp.Immunol. 55:593-601
Hammarstrom,L., Smith,C.I.E. (1986) IgG subclasses in bacterial infections. Monogr.Allergy 19:122-133
Hammer,M.C., Baltch,A.L., Sutphen,N.T., Smith,R.P., Conroy,J .V.(1981) Pseudomonas aeurginosa : quantitation of maximum phagocytic and bactericidal capabilities of normal human granulocytes. J.Lab.Clin.Med. 98:938-948
Hansman,D., Jarvinen,A. (1989) N.Engl.J.Med. 321:1546-1546
Harvey,D.M., Sheppard,K.J., Fletcher,J. (1986) A method for measuring rate of neutrophil phagocytosis of Staphylococcus epidermidis or Candida gui11 iermondii using uptake of tritiated uridine. J.Immunol.Meth. 93:259-264
Hauser,C., Wuethrich,B., Matter,L., Wilhelm,J.A., Schopfer,K. (1985) The immune response to S.aureus atopic dermatitis. Acta Dermatovenereol.(Stockholm.) 114:101-104
Hayatsu,H., Miyamae,T., Yamamura,M. (1988) Heat production as a quantitative parameter of phagocytosis. J.Immunol.Meth. 109:157-160
Hed,J., Berg,0., Forslid,J., Hallden,G., Larka-Rafner,G. (1988) The expression of CR1 and CR3 on non-modulated and modulated granulocytes of healthy blood donors as measured by flow cytofluorom etry. Scand.J.Immunol. 28:339-344
150 Hed,J., HalldenfG., Johansson,S.GO., Larsson,P. (1987) The use of fluorescence quenching in flow cytofluometry to measure the attachment and ingestion phases in phagocytosis in peripheral blood without prior cell separation. J.Immunol.Meth. 101:119-125
Hed,J., Stendahl,0. (1982) Differences in the ingestion mechanisms of IgG and C3b particles in phagocytosis by neutrophils. Immunol. 45:727-736
Heiner,D.C. (1984) Significance of Immunoglobulin G subclasses. Am.J.Med. 76 (3A):1-6
Henson,P.M. (1969) The aherence of leucocytes and p latelets induced by fixed IgG antibody or complement. Immunol. 16:107-121
Henson,P.M., Johnson,H.B., Spiegelberg,H.L. (1972) The release of granule enzymes from human neutrophils stimulated by aggregated immunoglobulins of d ifferen t classes and subclasses. J.Immunol. 109:1182-1192
H ill,H.R., Augustine,N.H., Shigeoka,A.O. (1984) Comparative opsonic activity of intravenous gamma globulin preparations for common bacterial pathogens. Am.J.Med. 76:61-66
H olzer,T.J., Edwards,K.M., Gewurz,H., Mold,C. (1984) Binding of C-reactive protein to the pneumococcal capsule or cell wall results in differential localization of C3 and stimulation of phagocytosis. J.Immunol. 133:1424-1430
Horowitz,J., Volanakis,J.E., Briles,D.E. (1987) Blood clearance of Streptococcus pneumoniae by C -reactive protein. J.Immunol. 138:2598-2603
Horstmann,R.D., Sievertsen,H.J., Knobloch,J., Fischetti,V.A. (1988) Antiphagocytic activity of streptococcal M protein: Selective binding of complement control protein factor H. Proc.Nat.Acad.Sci.USA. 85:1657-1661
Hosking,C.S., Fitzgerald,M.G., Shelton,M.J. (1978) The immunological investigation of children with recurrent infections. Australian Paed. J. 14 Supp1:1-107
Huizinga,T.W.J ., van Kemenade,F., Koenderman , L . , Dolman,K.M., von dem Borne,A.E.G.K., Tetteroo,P.A.T., Roos,D. (1989) The 40-kDa Fey receptor (FcRII) on human neutrophils is essential for the IgG-induced respiratory burst and IgG-induced phagocytosis. J.Immunol. 142:2365-2369
Humphreys,D.W., Wheat,L.J., White,A. (1974) Staphylococcal heat-stable opsonins. J.Lab.Cl in.Med. 84:122-128
Ito,S., Mikawa,H., Hirao,T., Yoshida,T., 0kuda,R. (1980) Defective phagocytosis confined to S. aureus in a female infant with recurrent infections. Eur.J.Pediatrics 133:11-16
151 Jacobs,J.C ., Mi11erfM.E. (1972) Fatal fa m ilia l L e in e r's disease: a deficiency of the opsonic activity of serum complement. Pediatrics 49:225-232
Jacobs,R.F., Wilson,C.B. (1983) Inracellular penetration and antimicrobial activity of antibiotics. J.Antimicrobial Chemotherapy 12:13-20
Jawetz,E., Melnick,J.L., Adelberg,E.A. (1984) Review of Medical Microbiology 16th Edition, pp 1-557. Lange, Los Altos
Johnston,R.B.Jr., Newman,S.L., Struth,A.G. (1973) An abnormality of the alternate pathway of complement activation in sickle-cell disease. N.Engl.J.Med. 288:803-808
Johnston,R.B.Jr. (1984) Recurrent b acte ria l in fe ctio n s in children. N.Engl.J.Med. 310:1237-1243
Jones,K.F., F isc h e tti,V .A . (1988) The importance of the location of antibody binding on the M6 protein for opsonization and phagocytosis of group A M6 streptococci. J.Exp.Med. 167:1114-1123
Kaplan,G. (1977) Differences in the mode of phagocytosis with Fc and C3 receptors in macrophages. Scand.J.Immunol. 6:797-807
Kemp,A.S., Turner,M.W. (1986) The ro le of opsonins in vacuolar sealing and the ingestion of zymosan by human neutrophils. Immunol. 59:69-74
Kim,Y.B., Watson,D.W. (1972) Streptococcal exotoxin. Biological and pathological properties. In: Streptococci and Streptococcal Diseases. Recognition, Understanding and Management. pp 33-50. Wannamaker,D.W., Matsen,J.M. (eds). Academic Press, New York
Kimberly,R.P., Tappe,N.J., Merriam,L.T., Redecha,P.B., Edberg,J.C., Schwartzman,S., Valinsky,J.E. (1989) Carbohydrates on human Fey receptors. Interdependence of the classical IgG ana nonclassical lectin-binding sites on human FcyRIII expressed on neutrophils. J.Immunol. 142:3923-3930
Klebanoff,S.J., Clark,R.A. (1978) The Neutrophil. Elsevier, Amsterdam
K1 ickstein,L.B., Bartow,T.J., Miletic,V., Rabson,L.D., Sm ith,J.A., Fearon,D.T. (1988) Id e n tific a tio n of distinct C3b and C4b recognition sites in the human C3b/C4b receptor (CR1, CD35) by deletion mutagenesis. J.Exp.Med. 168:1699-1717
Koenig,G.M. (1972) The phagocytosis of Staphylococci. In: The Staphylococci, Chapter 15. pp 365-383. Cohen,J.0. (ed). Wiley-Interscience, USA.
152 Krause,R.M. (1972) The Streptococcal cell: relationship of structure to function and pathogenesis. In: Streptococci and Streptococcal Diseases. Recognition, Understanding and Management. pp 3-18. Wannamaker,D.W., Matsen, J.M. (eds). Academic Press, New York
Kuhlman,M., Joiner,K., Ezekowitz,R.A.B. (1989) The human mannose-binding protein functions as an opsonin. J.Exp.Med. 169:1733-1745
La Fleur,M., Beaulieu,A.D., Kreis,C., Poubelle,P. (1987) Fibronectin gene expression in polymorphonuclear leukocytes. Accumulation of mRNA in inflammatory c e lls . J.Biol.Chem. 262:2111-2115
Lanier,L.L., Phil lips,J.H., Testi,R. (1989) Membrane anchoring and spontaneous release of CD16 (FCR III) by natural killer cells and granulocytes. Eur.J.Immunol. 19:775-778
Lanser,M.E., Saba,T.M. (1981) Fibronectin as a cofactor necessary for optimal granulocyte phagocytosis of Staphylococcus aureus. J.Reticuloendothel.Soc. 30:415-424
Law,S.K.A., Reid,K.B.M. (1988) Complement. IRL Press, Oxford
Laxdal,T., Messner,R.P., Williams,R.C.Jr., Quie,P.G. (1968) Opsonic, agglutinating, and complement-fixing antibodies in patients with subacute bacterial endocarditis. J.Lab.Cl in.Med. 71:638-653
Leff e ll,M.S., Spitznagel, J.K. (1975) Fate of human lactoferrin and myeloperoxidase in phagocytizing human neutrophils: effects of immunoglobulin G subclasses and immune complexes coated on latex beads. Infect.Immun. 12:813-820
L e h re r,R .I., C line,M .J. (1969) Interaction of C.albicans with human leukocytes and serum. J.B acte rio l. 98:996
Lehrer,R.I., Ganz,T., Selsted,M.E. (1988) Oxygen-independent b a cte ricid a l systems. Mechanisms and disorders. Haematology/Oncology Clinics of North America 2:159-169
Leung,D.Y.M., Geha,R.S. (1988) C lin ic a l and immunologic aspects of the hyperimmunoglobulin E syndrome. Haematology/Oncology C lin ic s of North America 2:81-100
Lew,D.P., Andersson,T., Hed,J., Di Virgi1io,F., Pozzan,T., Stendahl,0. (1985) Ca2+-dependent and Ca2+-independent phagocytosis in human neutrophils. Nature 315:509-511
Looney,R.J., Ryan,D.H., Takahashi,K., Fleit,H.B., Cohen,H.J. , Abraham,G.N., Anderson,C.L. (1986) Identification of a second class of IgG Fc receptors on human neutrophils. A 40 kilodalton molecule also found on eosinophils. J.Exp.Med. 163:826-836
153 Luzar,M.A., Coles,6.A., Faller,B., Slingeneyer,A., Dah Dah,G., Briat,C., Wone,C., Knefati,Y., Kessler,M., Peluso,F. (1990) Staphylococcus aureus nasal carriage and infection in patients on continuous ambulatory peritoneal d ia ly sis. N.Engl.J.Med. 322:505-509
MacFarlane,G.D., Herzberg,M.C. (1984) Concurrent estimation of the kinetics of adhesion and ingestion of S.aureus by human PMNs. J.Immunol.Meth. 66:35-49
MacLennan, A.C. (1990) Invasive Streptococci. Lancet 335:109-109
Mantovani,B. (1975) D ifferen t ro les of IgG and complement receptors in phagocytosis by polymorphonuclear leukocytes. J.Immunol. 115:15-17
Matsunaga,T., Seger,R., Hoger,P.f Tiefenauer,L., Hitzig,W.H. (1985) Congenital Acatalasemia: a study of neutrophil functions after provocation with hydrogen peroxide. Pediatric Res. 19:1187-1190
Metcalf,D. (1989) Haemopoietic Growth Factors. Lancet April 15:825-827
M etchnikoff,E. (1905) Summary. In: Immunity in Infectious Diseases, Chapter 27. pp 544-569. Cambridge University Press, Cambridge
Mickenberg, I .D ., Root,R.K., Wolff,S.M. (1970) Leukocytic function in hypogammaglobulinemia. J.Cl in.Invest. 49:1528-1538
Miller,R.G., Phil lips,R.A. (1969) Separation of cells by velo city sedimentation. J.C e ll Physiol. 73:191-202
Mims,C.A. (1987) The Pathogenesis of Infectious Disease, 3rd Edition. Academic Press, London
Mir,A., Porteu , F . , Levy,M., Lesavre , P . , Halbwachs-Mecarel1i,L . (1988) C3b receptor(CRl) on phagocytic cells from SLE patients: analysis of the defect and familial study. Cl in.Exp.Immunol. 73:461-466
Monteil,M., Hobbs,J., Citron,K. (1987) Selective immunodeficiency affecting staphylococccal response. Lancet 2,1987:880-883
M o rris,L ., Faulkner,J. (1981) The protein iodination and opsonisation tests. In: Techniques of Clinical Immunology, 2nd Edition, pp 299-305. Thompson,R.A. (ed). Blackwell Scientific Publications, Oxford
Mosesson,M.W., Amrani,D.L. (1980) The structure and biologic a c tiv itie s of plasma fibronectin. Blood 56:145-158
Movat,H.Z., Cybulsky,M.I. (1987) Neutrophil emigration and microvascu1ar injury. Patho1.Immunopatho1.Res. 6:153-176
154 MuddfS. (1970) A successful parasite: parasite-host interaction by S.aureus. In: Infectious Agents and Host Reactions, pp 197-227. Mudd,S. (ed). Saunders, Philadelphia
Myones,B.L.f Dalzel1,J.G., Hogg,N.f Ross,G.D.(1988) Neutrophil and monocyte c e ll surface p i50f95 has iC3b-receptor (CR4) activity resembling CR3. J.Cl in.Invest. 82:640-651
Naccache,P.H. (1987) Signals for actin polymerizaton in neutrophils. Biomedicine Pharmacotherapy. 41:297-304
Nahmias,A.J., Shulman,J.A. (1972) Epidemiologic aspects and control methods. In: The Staphylococci, Chapter 21. Cohen,J.O. (ed) pp 483-502. W iley-Interscience, USA
Najjar,V.A. (1975) Defective phagocytosis due to deficiencies involving the tetrapeptide tuftsin. J.Ped iatrics 87:1121-1124
Nakayama,S., Mold,C., Gewurz,H., Du Clos,T.W. (1982) Opsonic properties of C-reactive protein in vivo. J.Immunol. 128:2435-2435
Newman,S.L., Johnston,R.B.Jr. (1979) Role of binding through C3b and IgG in polymorphonuclear neutrophil function: studies with trypsin-generated C3b. J.Immunol. 123:1839-1846
Nickerson,D.S., Kazmierowski,J.A., Dossett,J.H., Williams Jr,R .C ., Quie,P.G. (1969) Studies of immune and normal opsonins during experimental staphylococcal infection in rabbits. J.Immunol. 102:1235-1241
O'Grady,J.H., Looney,R.J., Anderson,C.L. (1986) The valence for ligand of the human mononuclear phagocyte 72kD h ig h -a ffin ity IgG Fc receptor is one. J.Immunol. 137:2307-2310
0'Shea,J.J., Brown,E.J., Seligmann,B.E., Metcalf, J.A., Frank,M.M., Ga11in,J.I. (1985) Evidence for distinct intracellular pools of receptors for C3b and C3bi in human neutrophils. J.Immunol. 134:2580-2587
0chs,H.D., Fischer,S.H., Pyun,K.H., Wedgwood,R.J., Morel!,A. (1989) Antigen-specific IgG subclass production and metabolism In: IgG Subclass Deficiencies. pp27-35. Levinsky,R.J. (ed). Royal Soc. Med., London
0eding,P., Wergeland,H., Endresen,C., Natas,0.B., Aasjord,P. (1983) Antibodies to Staphylococcus aureus peptidoglycan and lipoteichoic acid in staphylococcal infections. Scand.J.Inf.Dis.Suppl. 41:126-129
0mann,G.M., Allen,R.A., Bokoch,G.M., Painter,R.G . , Traynor,A.E., Sklar,L.A. (1987) Signal transduction and cytoskeletal activation in the neutrophil. Physiological Rev. 67:285-322
155 Ory,P.A., Goldstein,I.M., Kwoh,E.E., Clarkson,S.B. (1989) Characterization of polymorphic forms of Fc receptor III on human neutrophils. J.Cl in.Invest. 83:1676-1681
Pancholi,V., Fischetti,V.A. (1989) Identification of an endogenous membrane anchor-cleaving enzyme fo r Group A streptococcal M protein J.Exp.Med. 170:2119-2133
Payan,D.G.f Goldman,D.W., G oetzl,E.J. (1984) Biochemical and cellular characteristics of the regulation of human leukocyte function by lipoxygenase products of arachidonic acid. In: The Leukotrienes - Chemistry and Biology, pp 231-245. Chakrin,L.W., Bailey,D.M. (eds). Academic Press
Peltz,G.A., Grundy,H.O., Lebo,R.V., Yssel,H., Barsh,G.S., Moore,K.W. (1989) Human FcyRIII: cloning, expression, and id e n tifica tio n of the chromosomal locus of two Fc receptors for IgG. Proc.Nat.Acad.Sci.USA. 86:1013-1017
Perry,G.S., Spector,B.D., Schuman,L.M., Mandel,J.S., Anderson,E., McHugh,R.B. (1980) The W iskott-A ldrich syndrome in the United States and Canada (1892-1979). J.Ped iatrics 97:72-78
Persson,M.A.A., Hammarstrom,L. , Smith,C.I.E. (1985) Enzyme-1 inked immunosorbent assay for subclass distribution of human IgG and IgA antigen-specific antibodies. J.Immunol.Meth. 78:109-121
Peterson,P.K . , Quie,P.G., Kim,Y., Wi1kinson,B.J. , Verbrugh,H.A., Verhoef,J. (1983) Recognition of Staphylococcus aureus by human p h a g o c y te s . Scand.J.Inf.Dis.Suppl. 41:67-76
Peterson,P.K., Wilkinson,B.J. , Kim,Y., Schmeling,D. , Quie,P.G. (1978) Influence of encapsulation on staphylococcal opsonisation and phagocytosis by human polymorphonuclear leukocytes. Infect.Immun. 19:943-949
P h i11ip s , I ., Eykyn,S.J. (1987) Staphylococci. In: Oxford Textbook of Medicine, Vol 1, Section 5. pp 5.191-5.199. Weatheral1,D. J ., Ledingham,J.G.G. , Warrell,D.A. (eds). Oxford Univ Press, Oxford
Pommier,C.G., 0'Shea,J., Chused,T., Yancey,K., Frank,M.M., Takahashi,T., Brown,E.J. (1984) Studies on the fibronectin receptors of human peripheral blood leukocytes: morphologic and functional characterization. J.Exp.Med. 159:137-151
Porteu,F. , Fischer,A. , Descamps-Latscha,B. , Haibwachs-Mecarelli,L. (1986) Defective complement receptors (CR1 and CR3) on erythrocytes and leukocytes of factor I (C3b- inactivator) deficient patients. Cl in.Exp.Immunol. 66:463-471
Quinti,I., Velardi,A., Guerra,E., Pesce,A.M., Di Sabatino,A., Aiuti,F. (1989) IgG subclass deficiency:
156 p a th o g e n e sis and tre a tm e n t. In: IgG S u b cla ss D eficiencies, Chapter 7. pp55-61. Levinsky,R.J. (ed). Royal Soc.Med., London
Rainard,P. (1986) A colorimetric microassay for opsonins by reduction of NBT in phagocytosing bovine polymorphs. J.Immunol.Meth. 90:197-201
R a jk o v ic ,I .A ., W illiam s,R. (1985) Rapid microassays of phagocytosis, bacterial killing, superoxide and hydrogen peroxide production by human neutrophils in vitro. J.Immunol.Meth. 78:35-47
R o b e rts ,P .J., Ford,J.M. (1982) A new combined assay of phagocytosis and intracellular killing of Escherichia coli by polymorphonuclear leukocytes. J.Immunol.Meth. 49:193-207
Rolland,A., Merdrignac,G., Gouranton,J., Bourel,D., Le Verge,R., Genetet,B. (1987) Flow cytometric quantitative evaluation of phagocytosis by human mononuclear and polymorphonuclear cells using flurescent nanoparticles. J.Immunol.Meth. 96:185-193
Rosenberg,J.S., Melnicoff,M.J., Wilding,P. (1985) Fc receptors for IgG on human neutrophils: analysis of structure and function by using monoclonal antibody probes. Clin.Chem. 31:1444-1448
Rosenfeld,S.I., Baum,J., Steigbigel,R.T., Leddy,J.P. (1976) Hereditary deficiency of the fifth component of complement in man. J.Cl in.Invest. 57:1635-1643
Ross,G.D. (1986) Opsonization and membrane complement receptors. In: Immunobiology of the Complement System. An Introduction for Research and Clinical Medicine, pp 87-114. Ross,G.D. (ed). Academic Press, Orlando
Ross,G.D., Medof,M.E. (1985) Membrane complement receptors specific for bound fragments of C3. Adv.Immunol. 37:217-267
Rossi,F., Della Bianca,V., Grzeskowiak,M., Bazzoni,F. (1989) Studies on molecular regulation of phagocytosis in neutrophils. Con A-mediated ingestion and associated respiratory burst independent of phosphoinositide turnover, rise in [Ca2+]i, and arachidonic acid release. J.Immunol. 142:1652-1660
Samanta,A.K. , Oppenheim,J.J . , Matsushima,K. (1989) Identification and characterization of specific receptors for monocyte-derived neutrophil chemotactic factor (MDNCF) on human neutrophils. J.Exp.Med. 169:1185-1189
Savill,J.S., Wyllie,A.H., Henson,J.E., Walport,M.J. , Henson,P.M., Haslett,C. (1989) Macrophage phagocytosis of aging neutrophils in inflammation programmed cell death in the neutrophil leads to its recognition by macrophages. J.Cl in.Invest. 83:865-875
157 Schur,P.H., Borel,H., Gelfand,E.W., Alper,C.A., Rosen,F.S. (1970) S elective gamma-G g lo bulin d e ficie n cie s in patients with recurrent pyogenic infections. N.Engl.J.Med. 283:631-634
Segal,A.W. (1980) Neutrophil function te sts. Hospital Update. 1043:1054
Segal,A.W. (1981) Phagocytic and b a cte ricid a l defects in neutrophils. C lin ic s Immunol.A1lergy 1:581-601
Segal,A.W. (1985) Testing neutrophil function. C lin ic s Immunol.A1lergy 5:491-512
Selvaraj,P., Rosse,W.F., Silber,R., Springer,T.A. (1988) The major Fc receptor in blood has a phosphatidyl inositol anchor and is deficient in paroxysmal nocturnal haemoglobinuria. Nature 333:565-567
Shalaby,M.R. , Aggarwal,B.B. , Rinderknecht,E., Svedersky,L.P., Finkle,B.S., Palladino Jr,M.A. (1985) Activation of human polymorphonuclear neutrophil functions by interferon-y and tumor necrosis factors. J.Immunol. 135:2069-2073
Shaunak,S., Wendon,J., Monteil,M., Gordon,A.M. (1988) Septic scarlet fever due to Streptococcus pyogenes c e llu lit is . Quar.J.Med. 69:921-925
Shephard,E.G., Anderson,R., Strachan,A.F., Kuhn,S.H., De Beer,F.C. (1986) CRP and neutrophils: functional effects and complex uptake. Cl in.Exp.Immunol. 63:718-727
Shulman,J.A., Nahmias,A.J. (1972) Staphylococcal infections: clinical aspects. In: The Staphylococci. Cohen,J.0. (ed). Wiley, New York
Siegel,S. (1956) Nonparametric statistics for the behavioral sciences, pp 1-312. McGraw-Hill, Tokyo
Smith,G.S., Lumsden,J.H. (1983) Review of neutrophil adherence, chemotaxis, phagocytosis and killing. Vet.Immunol. Immunopathol. 4:177-236
Smyth,E.G., Weinbren,M.J. (1988) Severe group A streptococcal infection. Lancet 2, 1988:452-452
Snyderman,R., Pike,M.C. (1984) Chemoattractant receptors on phagocytic c e lls . Annu.Rev.Immunol. 2:257-281
Southwick,F.S., Dabiri,G.A., Stossel,T.P. (1988) Neutrophil actin dysfunction is a genetic disorder associated with p artial impairment of neutrophil actin assembly in three fam ily members. J.Cl in.Invest. 82:1525-1531
Southwick,F.S., Howard,T.H., Holbrook,T., Anderson,D.C., S to s s e l,T .P ., Arnaout,M.A. (1989) The re la tio n s h ip between CR3 deficiency and neutrophil actin assembly. Blood 73:1973-1979
158 between CR3 deficiency and neutrophil actin assembly. Blood 73:1973-1979
Stevens,D.L., Gibbons,A .E ., Bergstrom,R., Winn,V. (1988) The Eagle effect revisited: efficacy of clindamycin, erythromycin and penicillin in the treatment of streptococcal myositis. J.Inf.Dis. 158:23-28
Stevens,D.L., Tanner,M.H., Winship,J., Swarts,R., Ries,K.M., S c h lie v e rt,P .M ., Kaplan,E. (1989) Severe group A streptococcal infections associated with a toxic sh o c k -lik e syndrome and s c a rle t fever to x in A. N.Engl.J.Med. 321:1-7
Stollerman,G.H. (1988) Changing group A Streptococci - The reappearance of streptococcal "Toxic Shock". Arch.Intern.Med. 148:1268-1270
S to ssel,T .P. (1973) Q uantitative studies of phagocytosis. Kinetic effects of cations and heat-labile opsonin. J.C e ll B io l. 58:346-356
Stossel,T.P. (1975) Phagocytosis: recognition and ingestion. Seminars Hematol. 12:83-116
Stossel,T.P. (1976) The mechanism of phagocytosis. J.Reticuloendothel. Soc. 19:237-245
Super,M., L e v in sk y ,R .J., Turner,M.W. (1990) The level of mannan-binding protein regulates the binding of complement-derived opsonins to mannan and zymosan at low serum concentrations. Cl in.Exp.Immunol. 79:144-150
S u zu ki,J.B ., Booth,R.R., Grecz,N. (1971) Evaluation of phagocytic activity by ingestion of labeled bacteria. J.In f.D is. 123:93-96
Sveum,R.J., Chused,T.M., Frank,M.M., Brown,E.J. (1986) A q u an titative fluorescent method fo r measurement of bacterial adherence and phagocytosis. J.Immunol.Meth. 90:257-264
Thelen,M., Peveri,P., Kernen,P., von Tscharner,V., Walz,A., Baggiolini,M. (1988) Mechanism of neutrophil activation by NAF, a novel monocyte-derived peptide agonist. FASEB J. 2:2702-2706
Thomas,J.C., Carr,S.J., Fujioka,K., Waterman,S.H. (1989) Community-acquired group A streptococcal deaths in Los Angeles County. J.In f.D is. 160:1086-1087
Todd III,R.F., Freyer,D.R. (1989) The CD11/CD18 leukocyte deficiency. Haematology/Oncology Clinics of North America 2:13-31
Tompkins,D.C. , Hatcher,V.B . , Patel,D., 0rr,G.A., Higgins,L.L., Lowy,F.D. (1990) A human endothelial cell membrane protein that binds Staphylococcus aureus in vitro. J.Cl in.Invest. 85:1248-1254
159 Tonnesen,M.G., Anderson,D.C., SpringerfT.A., Knedler,A., Avdi,N., Henson,P.M. (1989) Adherence of neutrophils to cultured human microvascular endothelial cells. S tim u la tio n by chem otactic peptides and lipid mediators and dependence upon the Mac-1, LFA-1, pl50,95 glycoprotein family. J.Cl in.Invest. 83:637-646
Trinkle,L.S., Wellhausen,S.R., McLeish,K.R. (1987) A simultaneous flow cytometric measurement of neutrophil phagocytosis and oxidative burst in whole blood. Diag.Clin.Immunol. 5:62-68
Turner,M.W., Mowbray,J.F., Harvey,B.AM., Brostoff,J., W ells,R .S., S o o th il1,J.F. (1978) Defective yeast opsonization and C2 deficiency in atopic patients. Clin.Exp.Immunol. 34:253-259
Turner,R.A., Schumacher,R., Myers,A.R. (1973) Phagocytic function of polymorphonuclear leukocytes in rheumatic diseases. J.C lin.Invest. 52:1632-1635
U nkeless,J.C., S cig lia n o ,E ., Freedman,V.H. (1988) Structure and function of human and murine receptors for IgG. Annu.Rev.Immunol. 6:251-281
Valerius,N.H., Stendahl,0.1., Hartwig,J.H., Stossel,T.P. (1982) D istrib u tio n of actin-binding protein and myosin in neutrophils during chemotaxis and phagocytosis. Advances Exp.Med.Biol. 141:19-28
Van de Water,L., Destree,A.T., Hynes,R.O. (1983) Fibronectin binds to some bacteria but does not promote th e ir uptake by phagocytic c e lls . Science 220:201-204
van 0ss,C.J., Gillman,C.F. (1972a) Phagocytosis as a surface phenomenon 2. Contact angles and phagocytosis of encapsulated bacteria before and after opsonization by specific antiserum and complement. J.Reticuloendothel.Soc. 12:497-502
van 0ss,C.J., Gillman,C.F. (1972b) Phagocytosis as a surface phenomenon 1. Contact angles and phagocytosis of non-opsonized bacteria. J.Reticuloendothel .Soc. 12:283-292
Verbrugh,H.A., Peterson,P.K., Nguyen,B.-YT., Sisson,S.P., Kim,Y. (1982) Opsonization of encapsulated Staphylococcus aureus: the role of specific antibody and complement. J.Immunol. 129:1681-1687
Verbrugh,H.A., Peterson,P.K., Smith,D.E., Nguyen,B.-YT., Hoidal,J.R., Wilkinson,B.J., Verhoef,J., Furcht,L.T. (1981) Human fib ro n e ctin binding to staphylococcal surface protein and its relative inefficiency in promoting phagocytosis by human polymorphonuclear leukocytes, monocytes and alveolar macrophages. Infect.Immun. 33:811-819
160 Verbrugh,H.A., Verhoef,J., Wilkinson,B.J., Peterson,P.K. (1983) Biology and clinical significance o f peptidoglycan antibody response in staphylococcal infections. Scand.J.Inf.Dis.Suppl. 41:117-125
Verhoef,J., Peterson,P.K., Kim,Y., Sabath,L.D., Quie,P.G. (1977) Opsonic requirements for staphylococcal phagocytosis. Heterogeneity among strains. Immunol. 33:191-197
Verhoef,J., Peter son,P.K . , Verbrugh,H.A . ( 1979) Host-parasite relationship in staphylococcal infections: the role of the staphylococcal cell wall during the process of phagocytosis. Antonie van Leeuwenhoek 45:49-53
Verhoef,J., Verbrugh,H.A. (1981) Host determinants in staphylococcal disease. Annu.Rev.Med. 32:107-122
Vik,D.P., Fearon,D.T. (1985) Neutrophils express a receptor for iC3b, C3dg, and C3d that is distinct from CR1, CR2, and CR3. J.Immunol. 134:2571-2579
Walsh,G.M., Kay,A.B. (1986) Binding of immunoglobulin classes and subclasses to human neutrophils and eosinophils. Cl in.Exp.Immunol. 63:466-472
Wannamaker,L.W. (1982) Virulence factors in Streptococci. Scand. J. Inf. Dis. Suppl. 31:22-27
Wannamaker,L.W. (1983) Streptococcal toxins. R ev.Inf.D is. 5,Suppl. 4:s723-s732
Wardle,E.N. (1986) Assessment of neutrophil function - I Postgraduate Med.J. 62:997-1000
Weiss,L., Mayhew,E., Ulrich,K. (1966) The effect of neuraminidase on the phagocytic process in human monocytes. Lab.Invest. 15:1304-1309
Wergeland,H., Endresen,C., Natas,0.B., Aasjord,P., 0eding,P. (1984) Antibodies to Staphylococcus aureus peptidoglycan and lipoteichoic acid in sera from blood donors and patients with staphylococcal infections. Acta Path.M icrobiol. Immunol.Scand. 92:265-269
Wheat,L.J., Humphreys,D.W., White,A. (1974) Opsonization of Staphylococci by normal human sera: the ro le of antibody and heat-labile factors. J.Lab.Cl in.Med. 83:73-78
Wilkinson,B.J., Sisson,S.P., Kim,Y., Peterson,P.K. (1979) Localization of the third component of complement on the cell wall of encapsulated Staphylococcus aureus M: implications for the mechanism of resistance to phagocytosis. Infect.Immun. 26:1159-1163
Williams,A.F., Barclay,A.N. (1986) The immunoglobulin superfam ily - domains fo r c e ll surface recognition. Annu.Rev.Immunol. 6:381-405
161 Wi11iams,R.C., Dossett,J.H ., Quie,P.G. (1969) Comparative studies of immunoglobulin opsonins in osteomyelitis and other established infections Immunol. 17:249-265
Williams,W.J., Beutler,E., Erslev,A.J., Lichtman,M.A. (1983) Haematology, 3rd Edition. McGraw-Hill, New York
Wilson,R.M., Galvin,A.M., Robins,R.A., Reeves,W.G. (1985) A flow cytometric method for the measurement of phagocytosis by polymorphonuclear leucocytes. J.Immunol.Meth. 76:247-253
Wong,L., Wilson,J.D. (1975) The id e n tifica tio n of Fc and C3 receptors on human neutrophils. J.Immunol.Meth. 7:69-76
Wong,W.W., Cahill,J.M., Rosen,M.D., Kennedy,C.A., Bonaccio,E.T., Morris,M.J., Wilson,J.G., K1ickstein,L.B., Fearon,D.T. (1989) Structure of the human CR1 gene. Molecular basis of the structural and quantitative polymorphisms and id e n tifica tio n of a new C R l-like a lle le . J.Exp.Med. 169:847-863
Wright,A.E., Douglas,S.R. (1903) An experimental investigation of the role of the blood fluids in connection with phagocytosis. Proc.Royal Soc. London 72:357-370 (Reprinted in Rev.Inf.Dis. 11:827-834, 1989)
Wright,S.D., Griff in,F.M.Jr. (1985) Activation of phagocytic cells' C3 receptors for phagocytosis. J.Leukocyte B io l.38:327-339
Wright,S.D., Reddy,P.A., Jong,M.TC., Erikson,B.W. (1987) C3bi receptor (complement receptor type 3) recognizes a region of complement protein C3 containing the sequence Arg-Gly-Asp. Proc.Nat.Acad.S ci.USA. 84:1965-1968
Yamamura,M., Boler,J., Valdimarsson,H. (1977) Phagocytosis measured as in h ib itio n of uridine uptake by Candida albicans. J.Immunol.Meth. 14:19-24
Yancey,K.B. (1988) B io lo g ica l properties of human C5a: selected in vitro and in vivo studies. Clin.Exp.Immunol. 71:207-210
Yeaman,G.R., Kerr,M.A. (1987) Opsonization of yeast by human serum IgA anti-mannan antibodies and phagocytosis by human polymorphonuclear leucocytes. Cl in.Exp.Immunol. 68:200-208
162 Normal Ranges 1 IgG, IgA, IgM
AGE (years) IgG g/l IgA g/l IgM g/l 1-2 3 .1 -1 3 .8 0 .3 -1 .2 0 .5 -2 .2 2 -3 3 .7 -1 5 .8 0 .3 -1 .3 0 .5 -2 .2 3 -6 4.9-16.1 0 .4 -2 .0 0 .5 -2 .0 6 -9 5.4-16.1 0 .5 -2 .4 0 .5 -1 .8 9 -1 2 5.4-16.1 0.7-2.5 0.5-1.8 12-15 5.4-16.1 0.8-2.8 0.5-1.9 15-45 5.4-16.1 0.8-2.8 0.5-1.9 45 5 .3 -1 6 .5 0.8-4.0 0.5-2.0
Normal ranges 2: IgG subclasses
AGE (years) lgG1 g /l lgG2 g /l lgG3 g /l lgG4 g /l
6 months 1.5-3.0 0.3-0.5 0.1-0.6 < 0.1
2 2.3-5.8 0.3—2.9 0.1-0.8 < 0.5 00 " V o 0 V 1 ■ 5 2.3-6.4 0.7-4.5 0.1-1.1 ■
10 3.6-7.3 1.4-4.5 0.3-1.1 < 0.1-1.0
15 3.8-7.7 1.3-4.6 0.2-1.2 < 0.1-1.1 O CM T“ 0) a Adult 3.2-10.2 1.2-6.6 ■ <0.1-1.3
163 Normal Ranges 3: White Blood Cells, Neutrophils and Lymphocytes
AGE (years) W B C (x 109/l) Neutrophils/ ul Lymphocytes/ ul
1 12+ 6 1500-7000 2000-5000 0
4-7 + 5 2000-6000 2 50 0-8500 1
Adults 7.5+ 3.5 2000-7500 1500-4000
164 880 THE LANCET, OCTOBER 17, 198?
peripheral blood mononuclear cells* may help to elucidate SELECTIVE IMMUNODEFICIENCY the degree of in-vivo inactivation of the interferon syttem in AFFECTING STAPHYLOCOCCAL RESPONSE children who are HBsAg carriers. However, if r-IFN treatment is limited only to subjects with raised A S T and M ic h e le Mo n t b il Jo h n Hobbs A L T levels, the majority of the Chinese HB&Ag carriers, Department of Chemical Immunology, Charing Crou and whether children or adults, would be excluded from Westminster M edical School, London treatment; in our experience with over 3000 Chinese HBsAg
carriers, 80-85% had normal A S T and A L T levels. K e n n e t h C i t r o n The early age at which our study population acquired Brampton Hospital, London their HBV infection probably reflects the immaturity of their immune system. This immaturity may allow the develop S u m m a r y Eight patients with recurrent ttaphylo- ment of immunological tolerance such that the host docs not • . coccal infections, necessitating up to 213 recognise the HBV as foreign which may explain why r-1FN hospital admissions in one patient, gave normal results had little or no effect in elimination of the virus. with the usual immunological investigations, including Genetic differences between Chinese and Caucasians measurement of serum IgG and IgG,. In the staphylococcal may also acoount for the non-responsivencss in the Chinese inhibition test all showed persistently subnormal results, HBsAg carriers. However, so far no specific HLA type has corrected by the addition of compatible normal plasma or been shown to be predominant in causcasian HBV carriers, normal IgG therapy for 6 months to 21 years, and one died (refs 21,23, and personal observations). from staphylococcal septicaemia 6 months after withdrawal Sidc-cffccts of r-IFN occurred much less frequently of treatment. The impairment in anti-staphylococcal among the children in this trial than among adults.7 " Very response, with failure to produce adequate antibodies, was mild fatigue in 1 patient and the “flu" syndrome, which probably acquired in utcro in four patients and inherited in showed tachyphylaxis and did not cause any febrile two. In these six patients symptoms startedsoon after 4 convulsions, were the only side-effects reported. Side- months. In the remaining two the syndrome was acquired cfTects have also been uncommon among children given later in life. r-IFN for other diseases. Perhaps the high regenerative power in children may prevent alopecia and bone-marrow INTRODUCTION suppression. Although a 12-week course of 10 * 10* IU/rrF r-IFN given thrice weekly did not have any long-term R e c u r r e n t staphylococcal disease has many beneficial effect in Chinese HBsAg carrier children, the determinants' but is often a feature of prim ary or secondary transient suppression of HBV DNAp levels and HBV DNA phagocytic dysfunction.^' scores that occurred during the course of treatment and the The polymorphonuclear neutrophil leucocytes (PMN) scarcity of sidc-cffects may make it worthwhile investigating play a key part in the host defence against staphyhxroccF4 yet the effectiveness of combination therapy— some recent without opsonins (fig 1) they inhibit Staphylococcus aureus work14 suggests that steroid withdrawal followed by r-IFN to a limited extent. 10% plasma must be present (the may be valuable. concentration expected in extracellular fluid), as a source of opsonins. Although complement clearly augments \X'c thank Hoffmann 1j Roche, Basic, Sw itzerland, both lor supplying the recombinant ■Sj tnicrfcnm ('Rolcmn') and lor financing the study. Dr VX' K inhibition,' alone it is not impressive: normal polyclonal Oxang for help with *ome of the serological tests. Miss Elsie l-cung for IgG is essential even at a concentration of 25 mg/dl (fig 1 and nursing help.
T his paper has been presented at the annual meeting of the American Association for Study o f Liver Disease in Chicago, November, 1986, and at < :-L Ij M AND OTHERS Rl-.HERKNCES— c o n tin u e d
the International Symposium on Viral Hepatitis and Liver Disease in 12 Ofcabe M .Q n h a Y .O ittw S .et al Socvrsaful treatment of a child w ith H H v A f positive tjondon, 1987. chnmK »tit\e hepatitis h\ human leuktvyic mtcrlcnn 7 <-7 i»i /YJiurr 1 9 7 9 .2 7 :1 2 1 27 Qirrcspondence should be addressed to I. 1. 1 3 Ki\hkLi I . K
TABLE I—CORRECTION OF STAPHYLOCOCCAL KILLING BY A. Without phagocytes B.With patient's phagocytes ADDITION OF IMMUNOGLOBULINS
Phagocyte source Plasma source % Inhibition
N C NP 93 N C PP 53 NC PP + IgG (25 mg/dl) 90 NC PP + IgG (50 mg/dl) 95 NC PP + Ig G (100 mg/dl) 91
table I). Deficiencies in specific antibodies, even with normal serum immunoglobulin levels, have been reported.6'9 This paper describes eight patients with recurrent staphylococcal diseases due to selective immunodeficiency of staphylococcal response.
Patients and Methods
Patients Fig I—Effects of blood components and IgG on staphylococcal uptake of thymidine. T he clinical data of the eight subjects (six males, two females) are summarised in table il. Major infections required hospital C M ■ = culture medium (Tc 199 without antibiotics), PP = patient’s plasma, N P = normal plasma, P P + IgG = PP + plus IgG added toa final admissions and parenteral therapy, whereas minor infections were concentration o f 25 mg I, Treated PP = PP after 24 days of intravenous IgG treated only with oral antibiodcs.10 Recurrent skin sepsis was therapy. common; systemic disease occurred to a variable degree. Patients’ samples were taken during infecuon-free intervals, when they were off treatment. Other Tests Staphylococcal Test T he in-vitro cidal activity of neutrophils against Candida albicans This was done as described by Foroozanfar et al.11 5 aureus was measured according to the method of Lehrer and Cline.13 (Oxford) were incubated in medium T C 199 alone, with normal Bactericidal capacity was measured with the spontaneous nitroblue plasma (NP), or with padent’s plasma (PP), first in the absence of tetrazolium (N BT) test.14 Other variables measured were total and phagocytes, to check for any andbiodc or other inhibitor. Later differenual leucocyte counts, serum immunoglobulin, IgG 2 padent’s PM N (PC) were compared with those of a normal subclass, and complement levels (including CHW). blood-group-compatible volunteer (NC). Results were expressed as Mouse protection tests3 were done in six pauents immunised % inhibiuon and the 2 SD lower limit of the normal range was with tetanus toxoid (Dr D. McSwiggan, Colindale Reference found to be 88% for eight-one subjects. Laboratory). Schick tests were done in five padents and IgG response to cholera vaccine was tested in one patient. Patient 1 was Chemotaxis and Random Mobility tested for antibodies against brucella. Chemotaxis was assessed at 3 h by the use of a modified Boyden Results and Discussion chamber assay12 with 3 pm filters. Plasma (heparin with no preservative) from padent or control was subjected to zymosan Convalescent samples from the eight patients showed acuvadon of the alternate pathway (ZAP). Random mobility (RM ) subnormal staphylococcal inhibition. Normal subjects was assessed through 8 pm filters with no chemotacdc gradient. usually show 95-100% inhibition during convalescence; in
TABLE II—CLINICAL FEATURES*
Past number % Staphylococcal inhibition Patient/sex/ Age (yr) Minor and major of major Neutrophils Pretreatment Years on year of birthat onset infections infections /Hi PC, PP NC, PP PC, NP serum Ig (g/1) IgG l/M /1931 08 Boils URTI Pneumonia Abscesses 213 4300 < 2 0 + <20+ 100 6-4 21 2/F/1934 08 Abscesses Carbuncles Pneumonia Septicaemia 35 6400 30 32 98 7 0 20 3/F/196I 0 3 Carbuncles, Pneumonia Septicaemia 5 5800 < 2 0 + < 2 0 t 100 7-4 0 5 4/M/1971 0-4 Boils abscesses Osteomyelitis 12 4900 30 35 100 6 8 16 5/M /1978 0 3 Boils Purulent conjunctivitis 2 5100 61 62 99 6 3 5 6/M/1976 3 Boils Septic arthritis 3 4800 68 64 100 9 6 4 7/M /1970 1 Chronic pyoderma 0 3100 79 65 100 7-8 — 8/M /1949 20 Erythema multiforme Septicaemia 2 3300 49 51 100 13 0 —
PC = patient’s cells. ‘ Findings as at diagnosis except for years on IgG. tBy a colony counting method. U R TI = upper respiratory tract infection. 882 THE LANCET, OCTOBER 17, 1987
M I H U M i l I Ml M TAIILU III-C F H iC r OF IgOTHUtATYON STAPHYMXXKXIAL IgG 1-Sg/Vk INFBCnON INFAT1KNT6 *---- — r*0 Patient's plasma % Inhibition
Before tratm ent 64 On IgG 75 mg/kg/month 93 O ff IgG for 5 months 50 On IgG 75 mg/Vg/month 100 Plasma aampki from patient 6 had been collected in the previous 2 yean a? during period* when he wat ofT antibodie*. The stored tamplei were tested M simultaneously with the aamc washed, compatible edit.
Six of the patients had illnesses severe enough to justify a trial a f Ig G therapy, whichenabled them to lead healthy norm al lives within a month of start of therapy: patient 2, A till who had twice been referreda topsychiatrist and was about to be dismissed from her post as an operating theatre sister, was n o t o n ly reinstated but became a chief nursing officer. Because continuing somatic mutations within the immune S * rruHx infection with 5 attrtus. system might eventually confer adequate responsiveness to this paper8 0 -1 0 0 % is taken as the normal range to include staphylococci, sera from our patients have been retested just normal subjects who have not had staphylococcal disease forbefore the next dose of IgG or after a period of treatment (fig years. Paired sera from patient 2 (who was the only one 2, tableIII). For patients 1, 2, and 6, no spontaneous examined for all these antibodies) showed no antibodies to correction has been detected; patients 2 and 6 had relapses of Icucocidin, staphylolysin, or wall or capsule antigens (tested staphylococcal infection while off IgG therapy. The others by the late Prof Mary Barber, Royal Postgraduate Medical could not be examined for spontaneous correction. Patient 3 School, I^ondon). H er sera also did not show the capsule had her IgG treatment withdrawn by another consultant on formation found in hyperimmune subjects,*' despite 35 the grounds that her pretreatment IgG level was normal; 6 proven episodes of 5aureus infection. The in-vitro months later she died suddenly from her third attack of assessment of immune function seemed to be related tostaphylococcal septicaemia. With gammaglobulin treatment clinical severity— patient 7, who had chronic pyoderma butpatients 1 and 2 have led normal lives for 21 and 20 years, no major infections, showed 79% staphylococcal inhibition, respectively, quite unlike patients treated for acquired adult whereas patients 2, 3, 4, and 8, who had had septicaemia, hypogamrruglobulinacmia, who usually do not survive 10 produced under 50% inhibition; patients 5 and 6, whose years. Patients 4 and 6 have grown normally. staphylococcal infections were of intermediate severity, produced inhibitions of61 % and 68% . Differential Diagnosis In all eight patients (table n , fig 1) the defect was confined Among over a hundred other patients investigated for to their plasma and was corrected by the addition of normal recurrent staphylococcal infections, about twenty have compatible plasma to their cells; the addition of shown subnormal staphylococcal inhibition, but it has not incompatible plasma may not always correct the defect. For been due to a selective staphylococcal opsonin deficiency all eight patients, addition of their plasma to normal compatible cells (NC) produced defective staphylococcal t a b u ; iv —differential d ia g n o sis o f r e c u r r e n t STAPHYLOCOCCAL INFECTION inhibition (table II). Correction was also achieved by addition of purified normal human IgG to the patient’s Genetic Acquired plasma (tableII, fig 1) to a final concentration of25 mg/dl Neutropenias Cyclic (the concentration expected in extracellular fluid). In all Persistent Iatrogenic eight patients almost all the other tests of immune function I>efectivc Panh)-pogammagiobulinaemiaSevere secondary yielded results within normal ranges for their sex and age.staphylococcal Isolated IgG deficiency' deficiencies-11 inhibition due to IgG + IgA deficiency' AIDS Total levels of IgG were acceptable (table I), as were theirpatient's plasma* IgG, deficiency (IgA Immune complex antibody responses against tetanus toxoid, diphtheria toxin, deficiency) diseases''' and cholera. Patient 1 had no IgG antibodies against Complement deficiencies'* brucella but IgM agglutinins were present. During Defective Chronic granulomatous disease Prematurity staphylococcal Chcdiak-Higashi syndrome Diabetes mcllitus infections most patients had raised levels of C3, IgA or IgM , inhibition due to Homozygous G6PD deficiency Excess or CRP (C-rcactivc protein), together with a raised ESR. patient's cells Adhesive protein deficiency'* conicusieniids The expected rise in serum IgG during convalescence was Severe pyruvate-kinase Malnutrition' often not observed. T-cell subsets and lymphocyte function deficiency Deficiencies of iron, Others' zinc, sitamins A tests were normal (patient 3 could not be investigated). The o rC ’ findings thus indicate a selective IgG defect against Iron overload staphylococci, in keeping with the clinical histories of Bums predominantly recurrent staphylococcal infection. Chronic myeloid leukaemia Thallium poisoning, Response to IgG Therapy &c With normal in- Leuser's syndrome* Viral impairment of Over the years, our own IgG standard (purified by D EAE vitro Lazy phagocyte syndromes'* chemotaxis1 chromatography) and gammaglobulin preparations from staphylococcal Complement deficiencies'* the National Blood Transfusion Service (Elstrcc and inhibition ‘ Edinburgh), KABI, Sandoz, and Immuno have been shown ‘ Genetic and acquired disorders listed may be present singly or as pan of a to restore staphylococcal inhibition to normal. multiple deficiency. Till-: LANCET, OCTOBER 17, 1987 883
since other abnormalities of immune function are present ATRIAL NATRIURETIC PEPTIDE AND BLOOD (table IV). PRESSURE IN A GEOGRAPHICALLY DEFINED Some complement deficiencies16 were correctable by the POPULATION off-treatment plasma of patient 2, who had normal complement. Other patients had immune complexes P e t e r N ilsso n 1 L ars L in dh o lm 1 (rheumatoid arthritis, even IgA myelomatosis) which BENGT SCHERSTfeN1 RODIGER HORN2 presumably occupied the receptors of the PC or N C , as has Ar n e M e la n d er 1 R o l f D iet er H esch 2 been shown for Fclty’s syndrome.17 Two patients with eventually fatal staphylococcal Health Sciences Centre, Lund University, Dalby, Sweden1; and infection due to PC were shown to have had thallium Department of Endocrinology, Medtzinische Hochschule, Hannover, poisoning (when forced by Secret Police in the Middle East West Germany to drink tasteless fluids). On three occasions the impairment of staphylococcal S u m m a r y Plasma atrial natriuretic peptide (ANP) inhibition has been due to a combined defect, corrected only ' was measured in 717 subjects in a cross- partly by adding N P to PC, or N C to PP. One of the patients sectional study of patients with hypertension (treated and was a premature baby who by 7 months had shown untreated) and normotensive controls, representatives of a spontaneous correction of both defects. Cases with geographically defined population. ANP concentrations did combined defects illustrate the need for repeated tests not differ between the groups and there was no correlation during infection-free intervals to fully establish the correct with blood pressure. These results do not support the view diagnosis, which should not be based solely on the that hypertensive and normotensive subjects have different staphylococcal inhibition test. ANP concentrations; they also call into question the role of Some patients with recurrent staphylococcal infections ANP in the development of high blood pressure. can have normal staphylococcal inhibition tests. In these cases defects in chemotaxis (due to failure to generate C5a, as Introduction in Leiner’s disease20) or the lazy phagocyte syndromes19 THE role of atrial natriuretic peptide (ANP) in mean that the polymorphonuclear cells do not home in hypertensive patients is widely debated. Some workers1 1 adequately and eradicate staphylococci. have reported increased plasma ANP concentrations, whereas others have found no differences between Aetiology of Specific Staphylococcal Opsonin Deficiencyhypertensive and normotensive subjects.4-s Laragh Patients 2-5 had recurrent staphylococcal infections suggested that ANP might act as a safety mechanism to starting soon after birth, at about the time that they could be protea the body from excessive activity of the renin- expected to have run out of maternal IgG. Their mothers aldostcronc system, with an important funaion in certain had had severe staphylococcal infections at 6-16 weeks of subgroups of hypertensives.6 We have now compared ANP pregnancy— two had had carbuncles; another, multiple boils; and the fourth a dental abscess associated with staphylococcal endocarditis. They had not previously had M. MONTHIL AND OTHERS: REFERENCES— continued recurrent staphylococcal infection. Presumably, excess 3. Hobbs JR , Foroozanfar N. Neutrophil funaion and dysfunction. In: Truclove S(', staphylococcal antigens conferred high-dose tolerance on Willoughby C l*, cds. Topics in gastroenterology: 7. Oxford: Blackwell, 1979; 193-203. the fetal immune systems. For such patients we believe the 4. Cx>hen M L . Staphylococcus aureus: biology, mechanisms of virulence, epidemiology. syndrome was acquired in utero. For patients 1 and 7, whose J Pediatr 1986; 108: 796 - 99. mothers did not have infection during pregnancy we suggest 5. Verburgh HA, Van Dijk WC, Kmc Mli, Peters R, Peterson PK, Vcrhoef J. Quantitation o f the third component of human complement attached to the surface a genetic defect, such as the absence of idiotypes needed to o f opsonised bacteria: opsonin-dcfician sera and phagocytosis-resinstant strains. develop an adequate antistaphylococcal response. Without Infect Immun 1979; 26: 808-12. 6. Hobbs JR . Disturbances of the immunoglobulins. In:'The scientific basis of medicine antileucocidin,9 antigen-processing cells might never Annua] Review. I>ondon: Athlone Press, 1966; 115. survive their encounter with staphylococci; with 7. Hobbs JR , Russell A,Worlledge SM. Dysgammaglobulinaemia type IVc. C lm T.xp Immunol 19t>7; 2: 589-99. gammaglobulin therapy, protected leucocytes might 8. Hobbs JR . Primary immune paresis. In: Adinolfi M ,ed . Developmental immunology. produce responses to other staphylococcal antigens. A London: Heincmann, 1969; 142. 9. Davis WC, Douglas SD, Fudcnberg H1L A selective neutrophil dysfunction similar process might explain the occasional amazing syndrome: impaired killing o f staphylococci. Ann Intern Med 1968; 69: 1237 - 43. response to autogenous vaccine observed in the early half of 10. Donabedian H, Gallin JI. 'Die hyperimmunoglobulin It recurreni-infeaion (Job's) this century. syndrome. M edicine 1983; 62: 195 - 208. 11. Foroozanfar N , Aghai Z, Hobbs JR . Inhibition of thymidine uptake by staphylococci, The late onsets of recurrent staphylococcal infections, as a ne^T method for the investigation of phagocytosis. J Immunol Mcduxb 1976; 11: occurred in patients 6 and 8, cannot be explained by the 3 4 5 -5 3 . 12. Boyden S. 'I*he chcmotactic effect o f mixtures o f antibody and antigen on above mechanisms. Jem e’s network theory suggests that an polymorphonuclear leucocytes. J iixptl Med 1962; 115: 453 - 66. autoimmune anti-idiotype reaction (mirror-image antibody; 13. Lchrer R , Cline M J. Interaction of Candida albicans with leucocytes and serum. J suppressor T-cell) might eliminate an adequate Bacterial 1969; 98:996 - 1004. 14. Park BH , Fikrig SM , Smithwick EM. Infection and nitroblue tetrazolium reduction staphylococcal antibody response, but this has yet to be by neutrophils, la n ce t 1968; ii: 532 - 34. demonstrated. 15. Stamp Lord, Hobbs JR . A natural “antibody” in rabbit serum producing a capsular reaction with staphylococcus pyogenews and related to immunity. B r J lixptl Path This work was supported by the Dobson and Immunology Research 1967; 48: 1 - 10. Funds of the Westminster Medical School Research Trust. 16. Lachmann RJ. Inherited complement deficiencies. Phil Trans R Sac Ijcmd B 1984; 306:419-30. Correspondence should be addressed to J. H., Department of Chemical 17. Gupta RC, LaForce FM , Mills DM. Polymorphonuclear leucocyte inclusions and Immunology, Westminster Hospital, Page Street Wing, London SW1P impaired bacterial killing in patients with Felty’s syndrome. J I Mb Clin M ed 1976; ZAP 88: 183 -91. REFERENCES 18. Fischer A , I’ham Huu Trung, Durandy A, et al. Bone marrow transplantation in two patictns with granulocytopathies. Hxptr Hanatal 1983; 11 (supple 13): 93 - 94. 1. Verhoef J, Verbrugh HA. Host determinants in staphylococcal disease. A m Rev M ed 19. Foroozanfar N ,G rohm ann PH, Hobbs JR . Abnormal fluorescent actin pattern o f lazy 1981; 32: 107-22. phagocyte syndromes. I hag Immunol 1984; 2: 25 - 29. 2. Quie E G , Hill H R , Todd-D avis A. Defective phagocytosis o f staphylococci. A m \'Y 20. Miller M E, Koblenzer PJ. Leiner’s disease and deficiency of C5. J Pediatr 1972; 80: A c a d S ci 1974;236:233-43. 8 7 9 -8 0 . References continued at foot of next column 21. Hobbs JR . Secondary antibody deficiency. I^roc Roy1 S oc A ied 1968; 61:883 - 87. Quarterly Journal of Medicine, New Series 69, No. 258, pp. 921-925, November 1988
Septic Scarlet Fever due to Streptococcus pyogenes Cellulitis
SUNIL SHAUNAK, JULIA WENDON, MICHELE MONTEIL* and A. M. GORDONt
From the Medway Hospital, Gillingham, Kent, * Charing Cross and Westminster Medical School, London, and tA ll Saints Hospital, Chatham, Kent
Accepted 26 June 1988
SUMMARY We report three cases of septic scarlet fever due to Streptococcus pyogenes Group A (serotype M 1/T1/O F—) cellulitis in healthy young adults. Despite prompt treatment two of the patients died. Such cases of cellulitis associated with scarlet fever, severe toxaemia and septicaemia have not been reported in the post-antibiotic era.
INTRODUCTION Since the introduction of penicillin, mortality from cellulitis due to Streptococcus pyogenes has fallen considerably. Furthermore, in our own district Streptococcus pyogenes Group A accounted for only 1.4 per cent of clinically significant episodes of bacteraemia encountered between 1983 and 1987. Of four episodes in adults, three were associated with cellulitis and all recovered. More recently, we have seen three previously fit adults who presented with cellulitis due to Streptococcus pyogenes serotype M 1/T 1/O F - and a rash typical of scarlet fever, who developed bacteraemia, acute renal failure, adult respiratory distress syndrome and dissemi nated intravascular coagulation despite prompt treatment with large doses of benzylpenicillin. Two of these patients died.
CASE HISTORIES CASE 1
A 39-year-old white engineer presented with a 24-h history of a painful left leg. He was pyrexial (38.5°C), and had a warm tender calf. There were no signs of injury or infection of the skin. Both the bulbar and palpebral conjunctivae were intensely injected. Full blood count, electro lytes and venographical examination were normal. Twenty-four hours later, a diffuse erythematous rash developed over the calf and spread rapidly up the thigh. High-dose intra venous benzylpenicillin (12 million units (MU)/day) was started. Three sets of blood cultures taken within 48 h of admission showed no growth, but Streptococcus pyogenes Group A was
Address correspondence to Dr Sunil Shaunak, Infectious Diseases Fellow, Box 3537. Duke University Medical Center, Durham. NC USA 27710. © Oxford University Press 1988 922 Sunil Shaunak and others
isolated from one of six blood culture sets taken over the subsequent 48 h. The leg continued to swell and vesicles began to appear over the lateral malleolus. A punctate erythema now covered the entire body and was most intense in the folds of the axillae, elbows and knees. Petechial haemorrhages were present in both axillae. The temperature rose to 40°C and he became confused, hypotensive and anuric. White cell count was 22 x 10.9/1 with a toxic neutrophilia, there were non-specific increases in serum liver-related enzymes, corrected calcium level was 2.07 mmol/1, serum phosphate was 0.5 mmol/1, and albumin 28 g/1. There was evidence of disseminated intravascular coagulation. Cefotaxime and metronidazole were added to benzylpenicillin. Fasciotomies of the medial and lateral compartments of the thigh and leg were performed. Yellow serous fluid was noted in the subcutaneous tissues overlying the soleus, with no necrotizing fasciitis or myositis. Streptococcus pyogenes serotype M 1/T 1/O F - was isolated from the serous fluid, vesicle fluid, and from biopsies of macroscopically normal muscle taken from both the medial and lateral compartments of the calf. Haemofiltration was commenced, and the patient required ventilation because of the development of adult respiratory distress syndrome. No streptococci were isolated from any other site. Extensive desquamation of the skin of the face and trunk occurred over the next two days. During the ensuing three weeks, his clinical course was complicated by a Staphylococcus epidermidis bacteraemia acquired from a central line site. His condition deteriorated rapidly, and he died 19 days after admission. At post-mortem examination, there was a slightly turbid pericardial effusion (70 ml), visceral pericardial and sub-endocardial petechial haemorrhages and myocardial pallor with small haemorrhagic foci. Histological examination showed a patchy myocarditis with an infiltrate of lymphocytes and plasma cells. The changes in the lungs were consistent with the adult respira tory distress syndrome. The liver was enlarged (3100 g), with focal centrilobular hepatocellular necrosis and a patchy, mild lymphocytic portal tract infiltrate. The spleen was enlarged (890 g). and showed extensive infarction. The pancreas had frequent foci of yellow necrosis, and there were numerous scattered petechiae throughout most other organs.
CASE 2 A 57-year-old white woman was admitted with a five-day history of pain in the right iliac fossa, increasing confusion, and a swollen right thigh. She was jaundiced, pyrexial (39.5°C) and hypotensive (systolic 60 mmHg). Vesicles were present over the inner aspect of the thigh, and on a hyperaemic buccal mucosa. The skin in the right iliac fossa was tense and indurated. A punctate erythema was present over the leg and lower abdomen, which rapidly spread over the entire body. No fluid or pus was obtained from these sites on fine needle aspiration, and radiological as well as ultrasound examination did not demonstrate a gas or fluid collection. White cell count was 6.1 x 109/1 with 87 per cent neutrophils, and evidence of severe dissemi nated intravascular coagulation. She required large volumes of colloid solutions (which increased the plasma albumin concentration to 41 g/1), and inotropic support. Ampicillin, flucloxacillin, cefotaxime and metronidazole were started. Over the next six hours, she became anuric, developed adult respiratory distress syndrome and a rapidly worsening metabolic acidosis. Despite ventilation and haemofiltration she died 12 h after admission. Blood cultures taken on admission and the vesicle fluid yielded Streptococcus pyogenes serotype M 1/T1/O F— sensitive to penicillin. All other cultures were negative. At post-mortem examination all tissues were oedematous and haemorrhagic. There was one litre of blood-stained fluid in each pleural cavity, and 200 ml of blood-stained fluid in the peritoneal cavity. Septic Scarlet Fever due to Streptococcus pyogenes C ellulitis
CASE 3 A 34-year-old white fireman was admitted with a seven-day history of a painful right leg. Trrrc had been initial blunt trauma to the right knee but no external signs of injury. Four days :asr he became feverish and developed an erythematous rash over the leg which became tense am tender. On admission, vesicles were present on the lateral aspect of the knee. White cell count was 11.2xl09/l, with toxic neutrophilia, plasma urea concentration wnv-L mmol/1, plasma creatinine was 165 amol/1, corrected calcium 1.90 mmol/1, serumphosrran: level was 0.89 mmol/1, serum albumin level was 16 g/1, and urinary nitrogen excrenon wa-JLl g/day. Excision of the skin and subcutaneous tissues, as well as fasciotomy of the lateral ccmnzr - ments of the thigh and leg were immediately performed. There was yellow serous fluic ~ subcutaneous tissues, with no necrotizing fasciitis or myositis. High-dose benzylpeniaihr. .1- million units/day), flucloxacillin and metronidazole were started and continued for 1 m- Streptococcus pyogenes serotype M 1/T 1/O F - was isolated from cultures of blood ta^er r admission, wound fluid and subcutaneous tissues. No other organism was isolated fron ~ site. Over the next four days, he became confused and hypotensive, and required ven::—rr~ because of the development of adult respiratory distress syndrome. A punctate erythema re covered the entire body, and he remained pyrexial (39°C). On the ninth day after the ome: illness, there was desquamation of the skin over the chest wall. Both the bulbar and rarx-~_ conjunctivae became intensely injected, but swabs from these were sterile. By day IA there -a. desquamation of the skin over the palms of his hands and subsequently over the soles of rm tax His tongue underwent desquamation and became red and painful. He made a gradual rear rr over several weeks, eventually requiring skin grafting to the leg. His hospital -ta> wa- months.
DISCUSSION
In Britain today, scarlet fever is a mild disease. Its main features are a punctate enrherra • ate is often associated with a streptococcal sore throat, a strawberry tongue and re ~ '_ lymphadenopathy [1]. The incubation period is usually one to two days, with periods o :.. •<-=. or more being rare. Both Christie and Bisno note that cases of extra-faucial scarlet :ev-r: those associated with septicaemia (septic scarlet fever) used to carry a grave prognosis, ru: =_ such cases have rarely if ever been seen in the post-antibiotic era [2. 3J. Case reports of severe and fatal Streptococcus pyogenes serotype M l/T l bacteraerr.u. r r : appeared recently, but features of toxic scarlet fever were not present [4], In other report toxic scarlet fever, there was no associated septicaemia and cellulitis [5, 13] and in the fe«. uu.. of scarlet fever after surgery, the organism was isolated from the surgical wound orJ\ t' Tue* are only two reports of septic scarlet fever in the English literature, neither of wmcr. associated with an initial cellulitis [7, 8]. Both patients survived, although one Je'r:.~T^ hypotension and acute renal failure. Our three cases with community-acquired infect or. r notable for several reasons: the age of the patients, the lack of an underlying chrome iilne*>.n. the median delay of five days before admission. The severity of toxaemia and organ :_ r r which developed despite prompt treatment was remarkable. No patient gave a history o: _ >rr throat, and no streptococci were isolated from throat cultures. The punctate erytherr- followed by desquamation of the skin over the face and trunk, as well as that of the paim> " " hands, soles of the feet, and tongue. The findings at autopsy in Case 1 of an enlarged i:\-er spleen, as well as subendocardial petechial haemorrhages were typical of those desrr.rx X**
924 Sunil Shaunak and others
earlier this century in patients who had died of scarlet fever. Tissue infiltration by lymphocvic* I and plasma cells was a characteristic finding [9]. % [n a recent review of Streptococcus pyogenes bacteraemia in Nottingham, it was noted th«f shock was present in 40 per cent of 40 cases, and carried a mortality of 60 per cent (inj. \ generalized rash consistent with scarlet fever was noted in two cases, but no further detail* m given and the streptococci in these cases were not serotyped. Of nine cases due to Ml Mram*. only one died. In two previous studies shock was noted in 21 percent (unselected) [l 11. md l* per cent (cancer) [12] of patients; other studies did not comment on the incidence ot >h«xk. No mention is made in any of the studies of the number of patients, if any. who developed lo t* scarlet fever. There has been a recent increase in the number of M 1/T 1/O F- strains of StrepuKtHiuM p y o g en es referred to the Streptococcus Reference Unit of the Central Public Health Labora tory Colindale from 2.4_per cent of all strains submitted in 1983 to more than 30 per cent in the first six months of 19fl7 (G. Colman, personal communication). The proportion M 1/T1/O F— strains received from cases of serious invasive disease has doubled, and no* represents about half of all Group A strains handled by the Streptococcal Reference Labora tory. Notably, the number of strains from fatal episodes of septicaemia has risen from an average of two per year to 40 by mid-1987, and of these, representatives of M l/T l h.oc been the most numerous. In a recent American report of two patients with Group A streptococcus-mediated 'owe shock-like syndrome, without bacteraemia, the organism produced streptococcal exotoxins Type A in the patient who died, and Type B and Type C in the one who >um\ cd : '! The serological sub-typing of the organism was not reported. An M l/T l/O F - strain ^auMn* scarlet fever in the United Kingdom from before 1940 has been reported to produce MrcrtiM*- cal pyrogenic exotoxin Type A [14]. The Streptococcus Reference Unit of the Public Health Laboratory Colindale is currently unable to evaluate streptococci for production ot '.trcpti***- cal pyrogenic exotoxins (G. Colman. personal communication). It is possible that there was a pre-existing IgG deficiency in Patient l . in whom a low f-b • '1'5c (3.6 g/1) was associated with low levels of all IgG sub-classes (Table 1). IgA and IgM titte>* cjc
TABLE 1. Serum immunoglobulin, complement and antistreptococcal antibody levels __
Normal C ase l Case 2 Case 3 range Acute s i ;a» IsG 5 .4 -1 6 .1 g/l 3 .6 (5 ) 4.5 (5) 20.9 (9) t.4 liA 0 .8 -3 .4 g/l 1.1 0.8 3.7 J o IiM 0 .5 -2 .0 g/l 0.6 0.4 5.6 s r» C? 0 .7 -1 .7~gj\ 1.1 0.6 1.8 C-i 0.1-0.7 g/l 0.5 0.2 0.9 0 •» > > CRP <10 mg/1 200 36 124 A bum in 30-42 g/l 28 41 16 - Antistreptolysin titre < 1 :2 0 0 A cute 110 < 5 0 > 8 0 0 - Ml) Convalescent 200 (17) - - Antideoxyribonuclease B titre < 1 :2 5 0 Acute < 5 0 < 5 0 400-61X) - > | r4 AJ Convalescent 1200-1600- - Anuhyaluronidase titre < 1 :1 2 8 Acute 32 32 > 4 0 9 6
wui cuiicvlc tL Number in parentheses indicates the day after onset of illness serumwhen sample Septic Scarlet Fever due to Streptococcus pyogenes C ellulitis 925
normal. Patient 2 had low levels of immunoglobulins and plasma proteins reflecting a hyper- catabolic state. Patient 3 had raised levels of immunoglobulins by the ninth day of his illness, and they continued to rise. In each of our patients, organ failure occurred despite the prompt institution of appropriate antibiotic chemotherapy, and when applicable surgical debridement. Toxin production undoubtedly contributed to the epithelial changes, conjunctivitis and multi-organ failure making it difficult to evaluate the success of treatment with antibiotics and surgery. Cases of Streptococcus pyogenes serotype M 1/T 1/O F - cellulitis associated with scarlet fever, severe toxaemia and septicaemia have not been reported in the post-antibiotic era; our cases serve to emphasize how severe the illness can be. With the re-emergence of highly virulent streptococci, a high index of suspicion and early referral to hospital may help to reduce mortality.
ACKNOWLEDGEMENTS We gratefully acknowledge permission given by the following consultant physicians and surgeons to report patients under their care: Dr G. Smith-Laing, Dr D. S. Thompson and Mr P. Webb. Dr T. Telfer, and Dr A. Azzopardi kindly supplied relevant post-mortem summaries. We are also grateful to Dr G. Colman. Streptococcal Reference Laboratory, Public Health Laboratory, Colindale, Professor J. Hobbs. Department of Chemical Immunology, Charing Cross and Westminster Hospital Medical School. London and Dr Harry Gallis, Division of Infectious Diseases, Duke University Medical Center, Durham. North Carolina for their invaluable help. Mrs Janet Routten kindly typed the manuscript.
REFERENCES 1. Dick GF. Scarlet fever. Chicago, Illinois: The Year Book Publishers. Inc.. 1938: 34—52. 2. Christie AB. Infectious diseases. London: Churchill Livingstone. 1987: 1281-1288. 3. Bisno AL. Streptococcus pyogenes. In: Mandell GL, Douglas RG. Bennett JE, eds. Principles and practice of infectious diseases. New York: John Wiley and Sons, 1985: 1124-1133. 4. Cruickshank JG, Hart RJC, George M. Feest TG. Fatal streptococcal septicaemia. Br Med J 1981: 282: 1944-1945. 5. Fishbein WN. Jaundice as an early manifestation of scarlet fever. Ann Intern Med 1962; 57: 60-72. 6. Richman DD, Breton SJ, Goldmann DA. Scarlet fever and group A streptococcal surgical wound infection traced to an anal carrier. J Paediatrics 1977; 90 (3): 387-390. 7. Robbens E, DeMan M, Schurgers M, Boelaert J, Lameire N. Systemic complications of streptococcal scarlet fever: two case reports and a review of the literature. Acta Clin Belg 1986; 41 (5): 311-318. 8. Stein DS, Nelson KE. Pyle KR. Kelly JJ. Septic scarlet fever in the 11th decade of life. South Med J 1987; 80 (6): 798-799. 9. Mallory AK, Keefer CS. Tissue reactions in fatal cases of streptococcus haemolyticus infection. Arch Pathol 1941; 32: 334-355. 10. Ispahani P, Donald FE, Aveline AJD. Streptococcus pyogenes bacteraemia: an old enemy subdued, but not defeated. J Infect 1988; 16: 37—46. 11. Duma RM, Weinberg AN, MedrokTF. Kunz LJ. Streptococcal infection: a bacteriological and clinical study of streptococcal bacteraemia. Medicine (Baltimore) 1969; 48: 87-127. 12. Henkel JS, Armstrong D, Blevins A, Moody MD. Group A beta-haemolytic streptococcus bacteraemia in a cancer hospital. JAMA 1970; 211: 983-986. 13. Cone LA, Woodard DR, Schlievert PM. Tomory GS. Clinical and bacteriologic observations of a toxic shock-like syndrome due to Streptococcus pvogenes. N Engl J Med 1987; 317 (3): 146— 149. 14. Hallas G. The production of pvrogenic exotoxins by group A streptococci. J Hyg (Camb) 1985; 95: 47-57. 97
HHEBoe or ncRAnxncr i q l l o n d o b o k b -m a r o * TOMBPIAWMiai
P. G. Riches, M. Hancock, B. Jurgee, I. Ruxobanm, C. Gray*, M. A. Montail and J. R. Hobbe
Department of XtaunoLogy, Charing Czoas and Neetmlnster Nadioal School, 17 Page Street, London SNIP SAP, and *Cytogenetics Unit, St Mary's Hospital Medical School, London W2 1NY, UK
INTRODUCTION
Ourlng bone-marrow transplantation (BMT) there are coeplex reactions between host tissues, the donor Immune system and the residual host immune system. An analysis of the respective contributions of donor and recipient cells to the lympho-haematopoietic system is critical to the events following transplantation. A number of markers can be used to determine engraftment of donor cells or persistence of recipient cells. These include red cell antigens, chromosome markers (sex chromosome or metaphase banding patterns), enzyme activity in deficient patients, immunoglobulin allotype markers, DNA sequence polymorphism (RFLP) and, in non-HLA-identical transplants, the HLA markers. In the present study various markers have been used to assess engraftment patterns in patients undergoing displacement BMT for the correction of inborn errors of metabolism.
PATIENT POPULATION
50 patients were studied, all of whom survived for more than 100 days post-graft. The patients were transplanted between 1980 and 1989 for correction of beta thalassaemia major (n-15), Gaucher's disease (n»8), Hurler's disease (n-14), Hunter's disease (n-4), Morquio's disease (n-3), San Fillipo A (n-2), San Flllipo B (n-1), GM1 gangliosldase deficiency (n-1), Niemann-Pick disease type B (n-1) and Maroteaux-Lamy disease (n-1). A number of patients showed early graft failure and required a second transplant. In 5 patients (1 Gaucher's and 4 Hurler's) the first graft was only slowly rejected and persisted for more than 100 days; in these patients the first and second grafts have been included In the study giving a total of 55 grafts analysed.
METHODS
Red cell antigens were Identified during routine haematologleal Investigations of the recipient and donor In the pre-graft period and in the recipient at frequent Intervals during the grafting procedure and thereafter at routine out-patient visits. Chromosomes were analysed In peripheral blood or bone-marrow using sex differences or characteristic banding patterns 1n the absence of gonosomal
I m Hobbe JR ed. Correction of certain genetic dlee by transplantation. 1969. Londoni COGENT. 1909i 97-102. 98
Markers. T cell chromosome patterns were Investigated following phytohaemagglutlnation (PHA) stlaiulatlon and B cell following poke-weed Mitogen (PWM) stimulation of peripheral blood lyMphocytes. Enzyme activity was assessed using peripheral blood leukocytes using artificial substrates appropriate to the eniyme Involved. Activities were standardised to the protein concentration of the leukocyte preparation. Immunoglobulin allotypes were determined by haemagglutlnation Inhibition. Agglutination of D-pos1t1ve red cells coated with antl-D of known allotype, by reference anti-allotype serum, Is Inhibited when a patient's serum with the same allotype Is added to the reaction mixture.
RESULTS
Using markers of engraftment, In 25/55 grafts It appeared that there was full donor engraftment. In a further 8 Instances engraftment could be assumed by clinical Improvement but markers were not available to characterise the donor and recipient contribution. An example of this type of case would be a beta thalassaemlc, who maintained haemoglobin levels post-graft, but had an Identical blood group to the donor, with no distinguishing chromosome banding and no difference In Immunoglobulin allotypes. In 6 grafts all parameters Indicated a complete rejection. A high proportion of these grafts had been subjected to T cell stripping. The remaining 16/55 grafts showed evidence of chlmerlsm In one or more marker parameters. More detailed analyses were made of markers In this group. In 21 patients there was a blood group antigen difference between donor and recipient (20 ABO and 1 Jk). In all patients, whether engraftment was partial or complete the blood group changed to that of the donor. In most patients conversion occurred between 3 weeks and 2 months post transplant. In 2 patients conversion was not seen until after 3 months post-BMT. In none of the patients, who were apparently chimeric by other markers, did the blood grouping indicate chimerism. Chromosome markers were available for 34/55 of the grafts. Donor and recipient types could be detected post-graft in 9 cases. The contribution of recipient cells varied from 2% - 78%. In all 3 patients with recipient cells greater than 30%, the graft was slowly rejected. A single patient has 30% recipient cells 4yrs post-BMT. In all other cases the recipient cells accounted for 10% or less of the total. In 3 patients the chromosome marker was the only Indicator of chlmerlsm, although in 2 of these patients the blood group had converted to donor type. In 4/9 of the patients, assessed to be chimeric by chromosome analysis, there was a subnormal enzyme level (with reference to normal or donor range). Immunoglobulin allotyplng was started later In the study and was only tested on 20 donor/recipient pairs of which 10 showed one or more differences. In general, where a marker was available to distinguish donor from recipient, donor allotype could be detected early In the post-BMT period (2-3 weeks). Where synthesis of recipient type ceased this occurred anywhere from 4-36 weeks post-BMT. In 6 patients the allotyplng Indicated chlmerlsm based on presence of recipient and donor type or alternatively, persistence of recipient Immunoglobulin allotype with another marker Indicating donor engraftment e.g. 99
chromosomes of donor origin, again 2 patients 1n this group showed subnormal enzyme level. Finally a single patient acquired only subnormal enzyme levels Indicating a probable chimera. No other markers were available to assess this patient. Chimeras may be stable, with Inadequate enzyme levels as Illustrated In Figure 1 or adequate enzyme levels as shown In Figure 2. Most other chimerism Is unstable and an early Indicator of graft failure (see Figure 3); note how different markers Indicated graft loss at different times post-graft; the enzyme (phagocytes) fell early while T cells (PHA chromosomes) rose slowly (T-depleted marrow) to fall later.
Fig 1: Leukocyte N-acetylgalactosamine Fig 2: Leukocyte beta-glucocerebro- 6-sulphate sulphatase activity in sidase activity in patient SE patient ZB (Morquio) when both donor (Gaucher) when both donor and HLA and recipient 6m immunoglobulin recipient 6m type and donor type was detectable chromosome type was detectable
60 v) v £ o
2 Fig 3: Leukocyte beta- u glucocerebrosidase activity in 20v patient LS (Gaucher) with c Incomplete engraftment followed <5 by gradual loss of graft judged by % donor chromosomes and loss * of enzyme activity (enzyme reference range as In Fig 2) 100
Finally the number of chimeras was compared with the total number of grafts for each disease type and the results are shown In the table.
Table: Chlmerlsm compared with total grafts In various disease categories
Beta thalassaemla major 3/15 Gaucher's disease 4/9 Hurler's disease 5/18 Hunter's disease 1/4 Morquio's disease 1/3 Others (see methods) 2/6
The numbers of patients In each group 1n the table Is too small to allow assessment of statistical significance but 1t would appear that a greater proportion of transplants In patients In the enzyme-deficient-storage disease group (13/40 or 32.5%) result in chimerism than do those in thalassaemic patients (3/15 or 20%).
DISCUSSION
The aim of displacement bone marrow transplantation (DBMT) 1s to achieve 100% donor-type marrow (1). Despite optimisation of the induction chemotherapy, in 16/55 (29%) of grafts some degree of haematopoietic chimerism was apparent. The red cell antigens were Insensitive markers of chimerism. No attempt was made to use sensitive techniques to look for low levels of recipient antigen as used by other workers who have found red cel! chimerism (2). Chromosome analysis and 6m typing were the main Indicators of chimerism and where stable chimerism was achieved the contribution of recipient cells, judged by these markers, was usually less than 10% of total lymphocytes. Although this might seem an acceptable level there are several points to consider. Firstly, persistent recipient lymphocytes may have the potential to produce neutralising antibody to the newly introduced enzyme as such cells would not have been previously tolerised due to absence of that enzyme. Usually the Westminster Bone Harrow Team practise Immunoprophylaxis (see pi), but 1n the patient shown In Figure 1 who acquired very low enzyme activity, the HIA type was all that of the donor suggesting full engraftment but 6m typing Indicated persistence of recipient B cells. An IgG fraction prepared from the post-graft serum of this patient was able to Inhibit normal enzyme activity whereas the pre-graft fraction did not. It was then discovered that the donor's buffy coat had not*been given to the recipient on day -6, so that no abrogation of a recipient primary response had occurred. This patient thus had full engraftment but the recipient antibody formation that followed and persisted prevented any delivery of active enzyme to the deficient tissues. The patient showed no clinical Improvement and died from complications of the 101 primary disease. In contrast, patient SE (see figure 2) did receive donor buffy coat and although she retained B cell chlmerlsm no antibodies have been found that either bind to or Inactivate beta-glucocerebrosldase. These findings emphasise the need for Immunoprophylaxis when the antigenicity of donor enzyme to the recipient Is unknown. Secondly there Is the risk of loss of the graft as the recipient narrow, 1n It's own environment, usually has the advantage over the donor narrow, as illustrated by the patient shown 1n Figure 3.; this patient went on to completely lose her graft and required a second transplant. Finally poor functional antibody responses in the post-graft period have been described (3) and chlnerlsn could aggravate this problem If donor T cells were to cooperate poorly with recipient B cell due to minor histocompatibility differences. No single marker Is able to distinguish complete or partial engraftment as patients with non-malignant disorders who have been conditioned with regimens not Including radiotherapy have experienced partial engraftment of one or more cell lineages (4). The patient shown In Figure 3 had a low enzyme level (mainly neutrophil) at a time when chromosomes showed the lymphocytes were greater than 50% donor type. Such discordance of markers of engraftment has been reported by other workers (5). RFLP analysis was not used in this study but reports from other workers would indicate that this will be a sensitive technique for distinguishing the donor or recipient origin of the various cell lineages (6).
ACKNOWLEDGMENT
We wish to thank Ms Yvonne Stoddard for providing chromosome analysis on some or the earlier patients in this series.
REFERENCES
1. Hobbs JR. Correction of 34 genetic diseases by displacement bone marrow transplantation..Plasma Ther Transfus Technol 1985;6:221-46. 2. Janssen J, Drenthe-Schonk A, van Oijk B, Bloo J, Kunst V, de Witte T. Erythrocyte repopulation after allogeneic bone marrow transplantation using lymphocyte depleted marrow from histocompatible siblings. Bone Marrow Transpl 1986;1:239-40. 3. Lum 16. The kinetics of immune reconstitution after human marrow transplantation. Blood 1987;69:369-80. 4. Blazer BR, Ramsey NKC, Kersey JH, Krivlt W, Flllpovich AH. Pretransplant conditioning with busulphan (myleran) and cyclophosphamide for non-malignant disease: assessment of engraftment fdllowing histocompatible allogeneic bone marrow transplantation. Transplantation 1985;39:597-603. 5. Blazer BR, et al. Comparison of enzymatic activity with evidence of engraftment in patients with Inborn errors of metabolism receiving allogeneic marrow transplantation. Birth Defects: Original Article Series 1986;22:135-52. 6. Know 1 tonR6, et al. Use of highly polymorphic DNA probesfor genetyplc analysis following bone marrow transplantation. Blood 1986;68:378-85. 102
SUMMARY OF PISCUSSIOM AFTER THE PAPER
Others confirmed the chlmerlsm that can occur after DBMT for red cell errors and of particular Interest was the observation In one patient by Dr. Vermylen that the recipient sickle red cells had persisted for 60 days before any donor red cells could be detected, and yet the graf$ had then completely taken to eventually replace the recipient's red blood cells. This had Important Implications 1n judging Intervention postgraft, where allowance would have to be made for the occasional late engraftment, but sometimes platelets and white blood cell values would dictate rescue with autologous marrow. Dr. Poynton had been using molecular biology techniques to study tne recovery from BMT for aplasia and had also observed mixed and unstable chlmerlsm 1n some patients. Similarly the Italian group had observed In some of their patients given BHT for thalassaemla major that some patients had been transfusion-dependent for several months, to then finally recover; thus mixed chlmerlsm did not always end up with rejection, but 1n the occasional patient the donor marrow gained a true advantage. Clearly, if platelets and WBC were not a problem, 1t would be wise to wait and see, and in any case one would not normally attempt a second DBMT for at least 3 months. Another example of the dissociation of markers had been the finding in one Infant transplanted from a matched sibling at three months of age,who had shown 22/22 donor type chromosomes after PHA, but whose enzyme slowly disappeared, and whose own chromosomes then completely dominated the PHA response 1 year later. MLCs done on 2 occasions showed no reaction of the recipient against her brother, and this was proven by the repeat graft being done from the same donor; again 100% donor chromosomes and the full enzyme level were achieved, but on this occasion have remained stable. This indicates that the resilience of the recipient's stem cells at 3 months was much greater (perhaps because Busulphan may then have had a shorter half-life) than at the age of 15 months (admittedly after a second course of Busulphan). This patient was the only Westminster patient who achieved apparently 100% donor engraftment who had then subsequently lost the graft. Professor Ringden suggested that ADCC studies were worth undertaking, but Professor Krivit stated that there was a limit as to how much you could do where the patient had to pay for the investigations, and that his priority would be the enzyme level.
Professor Yamamura suggested that foetal transplants, particularly those in utero, would need different evaluations (AFP would be measured for some) and raised the issue of whether tolerance might be more inducible. In numerous early foetal transplants, the majority had been unstable with little evidence of acquiring a tolerant state. However, the use of Isologous foetal liver and foetal thyumus by Touralne's group had achieved both stable chlmerism and tolerance. The very unusual feature of donor lymphocytes bearing a total mismatch on the HLA and yet apparently being able to maintain normal Immune defences in the patient, at least 5 and 6 years later. It was believed that the isologous foetal thymic tissue had probably introduced the host's cell surfaces to the donor Immune cells to Impose tolerance; and this was supported by the finding that Busulphan/Cyclophospnamlde Inductions which did not appear to destroy the thymus had produced longterm stable chlmerism after transplants where only 1-genetIc-Haplotye was shared between the recipient and donor, whereas total body irradiation which did destroy the thymus could rarely achieve this'.
REfEREttCS 1. Hobbs JR, Williamson S, Chambers JD, et si (1985) Use of donors sharing one genetic haplotype for bone marrow transplantation. Tokal J Exp Clin Med Ifi: 207-214.