Mycopathologia Vol. 63, 3 : 173-175
IMMUNOGLOBULIN CLASSES OF HUMAN SERUM ANTIBODIES IN VAGINAL CANDIDIASIS
David W. WARNOCK, J. Dorothy MILNE & Andrew M. F I E L D I N G Departments of Microbiology, Venereology and Chemical Pathology, Bristol Royal Infirmary, Bristol BS2 8HW, England
The titre and immunoglobulin class of antibodies against Candida albicans in serum from 60 non-pregnant women was determined. IgG titres up to 1 : 32, IgA titres up to 1 : 8, and IgM titres up to 1 : 4 were detected in 30 women with vaginal candidiasis. Similar titres were found in 20 women harbouring yeasts in the mouth or rectum, and in 10 women who were not harbouring yeasts in the vagina, mouth or rectum. Serum fractionation confirmed that antibodies to C. albicans are found in the three immunoglobulin classes and that these antibodies reside in highest titre in the IgG class. No secretory IgA antibodies against C. albicans were detected in the serum of these women.
Waldman et al. (6) reported the local production of IgA antibodies against C. albicans in the genital tract of normal women, albeit under artificial conditions. In a recent investigation (5), however, IgA antibodies to C. albicans were only detected in the genital tract secretions of 6.1 per cent of women with vaginal candidiasis. IgG antibodies were detected in the secretions of 21.2 per cent of these women and it was suggested that some of these antibodies might have been derived from the circulation. In view of these findings, we decided to investigate further the immunoglobulin classes of circulating antibodies against C. albicans, and to determine the incidence of SIgA antibodies in the serum of women with vaginal candidiasis and of other women.
Introduction
Pa.fients and methods
Abstract
There have been a number of reports concerning the immunoglobulin classes of circulating antibodies to Candida .albicans produced in patients with different forms of candidiasis. Lehner (3), using an indirect immunofluorescence (IF) method, found antibodies against surface 9antigens of C. albicans in all three major immunoglobulin classes in patients with different forms of oral candidiasis. Significant titres of IgG antibodies were detected in 78 per cent of the patients, of IgM in 51 per cent, and of IgA in 30 per cent. Mathur et al. (4) tested serum immunoglobulin fractions from three patients with vaginal candidiasis. Antibodies to C. albicans were found in fractions containing IgA, but not in fractions containing IgG or IgM without IgA. All fractions that contained significant titres of IgA antibodies also contained secretory component (SC). These findings led Mathur et al. to suggest that, in vaginal candidiasis, secretory IgA (SIgA) antibodies against C. albicans are produced in the genital tract and that a proportion of these antibodies then find their way into the circulation.
Subjects Altogether 60 selected non-pregnant women were investigated. These patients consisted of: 1. 30 '~vomen with vaginal candidiasis; these women had clinical signs of vaginitis Or vulvovaginitis (vaginitis was recorded if any part of the vaginal mucosa was reddened or granular; vulvitis was recorded if any part of the vulva was reddened, swollen, fissured or ulcerated) and a positive vaginal culture (29 C. albicans, 1 C. tropiealis); 28 of these women also had positive oral and/or rectal cultures (27 C. a[bicans, I C. tropicalis); 20 of these women had had vaginal candidiasis diagnosed on at least one previous occasion. 2. 20 women without clinical signs of vaginitis or vulvitis and a negative vaginal culture. These women all had positive oral and/or rectal cultures (20 C. albicans). 3. 10 women without clinical signs of vaginitis or vulvitis and negative vaginal, oral and rectal cultures. Five ml of blood were taken from each patient and the serum separated and stored a t - - 2 0 ~ until studied. 173
Preparation of antigen One strain of Candida albicans group A was used throughout this investigation. This strain was chosen because it contains all the antigens of C. albicans group B and C. tropicalis (2). A blastospore suspension was prepared using 0.4 per cent formal saline to harvest cells from 48-hour-old cultures on glucose peptone plates. After washing in saline, the blastospores wer~ diluted to 12,000 cells per ml and stored at 4 ~ until used. One drop of this suspension was placed on each spot of multispot PTFE-coated glass slides. The slides were dried at room temperature and fixed in methanol for 1 minute. Indirect IF test Serial dilutions of serum were made in phosphate buffered saline (PBS) pH 7.1. One drop of diluted serum was placed on each spot on each slide. After incubation for 30 minutes in a moist chamber at 37 ~ the slides were rinsed in two changes of PBS for 5 minutes each. One drop of the appropriate FITC-conjugated monospecific anti-human immunoglobulin was placed on each spot and left for 30 minutes at 37~ The slides were then rinsed in two changes of PBS for 5 minutes each and mounted in buffered glycerol. Fluorescein-conjugated sheep antisera to human IgG, IgA and IgM were obtained from Wellcome Reagents Ltd. A fluorescein-conjugated rabbit antiserum to human tgA secretory component (produced from anti-colostrum IgA by absorption with serum IgA) was obtained from Dakopatts AS, Copenhagen. The highest serum dilution giving a complete ring of bright green fluorescence around the blastospores was taken as the titre of the serum. Fractionation of serum Serum samples from four selected patients were fractionated. Each sample was first dialysed against 0.05 M trishydrochloride buffer pH 7.4 and then applied to a Bio-Gel A-1.5 m (Bio-Rad Laboratories) column equilibrated with the same buffer. The column was eluted with this buffer and fractions were collected and monitored at 280 nm (Fig. 1). Fractions were concentrated ten times using Minicon B-15 concentrators (Amicon Ltd) and then tested for IgG, IgA and IgM using radial immunodiffusion plates (Hoechst Pharmaceuticals). 174
I001
.,
,
,
,
L
I
ABSORBANCE
50I
0
I
I
i
I
I
I
fi
10
15
20
25
30
35
40
FRACTION NUMBER
Fig. 1. Fractionation of serum sample from patient 55. Fractions 10 to 15 contained IgM, fractions 13 to 21 contained IgA, e-~ fractions 17 to 27 contained IgC.
Results The titres of IgG, IgA and lgM antibodies against C. albicans are summarised in Table 1. IgG titres ranging from 1 : 4 to 1 : 32 and IgA titres from 1 : 4 to 1 : 16 were detected in the patients with vaginal candidiasis. Similar titres were found in the other women. Sera from 50 patients (including 24 patients with vaginal candidiasis) were tested for the presence of SIgA antibodies against C. albicans. No SIgA antibodies were detected. Sera from two patients with vaginal candidiasis and from two women harbouring C. albicans in the mouth and rectum were fractionated. The immunoglobulins present in the different fractions were determined by single radial immunodiffusion. The fractions were pooled according to the contents and tested for antibodies to C. albicans. The results (Table 2) demonstrate that the corresponding classes of antibodies were present in each of the fractions and confirm that these antibodies reside in highest titre in the IgG class. Table I. iF tTtres of antibodies to C, ~Zbleans in serum of womenwith vaginal candidlasls and of other women Diagnosis Number of IF titre of antibodies to C. ~Zbicqn8 patients IgG IgA IgM 1:4 1:8 "I:16 1:32 1:4 1:8 i:16 ~:4 1:8 Vaginal candidiasis 30 15 5 9 I 25 h 1 29 1 No signs of infectlon, yeastspresent ~n mouth and/or rectum
20
]2
3
5
0
13
4
3
20
0
No signs of infection, no yeasts present in vagina, mouth or rectum
IO
6
3
1
0
9
l
0
10
0
Table 2. The distribution of antibodies to C, a!bic~ns in separated fractions of serum from women with vaginal candidiasis and women harbouring C. aZbicans in the mouth and rectum
Serum
Fraction IgG
Patient 38 (vaginal candidiasis)
Patient 55 (vaginal candidiasis)
Patient 43
Patient
65
IF t i t r e IgA
lqM
IMMUNOGLOBULIN CLASSES OF HUMAN SERUM ANTIBODIES IN VAGINAL CANDIDIASIS
David W. WARNOCK, J. Dorothy MILNE & Andrew M. F I E L D I N G Departments of Microbiology, Venereology and Chemical Pathology, Bristol Royal Infirmary, Bristol BS2 8HW, England
The titre and immunoglobulin class of antibodies against Candida albicans in serum from 60 non-pregnant women was determined. IgG titres up to 1 : 32, IgA titres up to 1 : 8, and IgM titres up to 1 : 4 were detected in 30 women with vaginal candidiasis. Similar titres were found in 20 women harbouring yeasts in the mouth or rectum, and in 10 women who were not harbouring yeasts in the vagina, mouth or rectum. Serum fractionation confirmed that antibodies to C. albicans are found in the three immunoglobulin classes and that these antibodies reside in highest titre in the IgG class. No secretory IgA antibodies against C. albicans were detected in the serum of these women.
Waldman et al. (6) reported the local production of IgA antibodies against C. albicans in the genital tract of normal women, albeit under artificial conditions. In a recent investigation (5), however, IgA antibodies to C. albicans were only detected in the genital tract secretions of 6.1 per cent of women with vaginal candidiasis. IgG antibodies were detected in the secretions of 21.2 per cent of these women and it was suggested that some of these antibodies might have been derived from the circulation. In view of these findings, we decided to investigate further the immunoglobulin classes of circulating antibodies against C. albicans, and to determine the incidence of SIgA antibodies in the serum of women with vaginal candidiasis and of other women.
Introduction
Pa.fients and methods
Abstract
There have been a number of reports concerning the immunoglobulin classes of circulating antibodies to Candida .albicans produced in patients with different forms of candidiasis. Lehner (3), using an indirect immunofluorescence (IF) method, found antibodies against surface 9antigens of C. albicans in all three major immunoglobulin classes in patients with different forms of oral candidiasis. Significant titres of IgG antibodies were detected in 78 per cent of the patients, of IgM in 51 per cent, and of IgA in 30 per cent. Mathur et al. (4) tested serum immunoglobulin fractions from three patients with vaginal candidiasis. Antibodies to C. albicans were found in fractions containing IgA, but not in fractions containing IgG or IgM without IgA. All fractions that contained significant titres of IgA antibodies also contained secretory component (SC). These findings led Mathur et al. to suggest that, in vaginal candidiasis, secretory IgA (SIgA) antibodies against C. albicans are produced in the genital tract and that a proportion of these antibodies then find their way into the circulation.
Subjects Altogether 60 selected non-pregnant women were investigated. These patients consisted of: 1. 30 '~vomen with vaginal candidiasis; these women had clinical signs of vaginitis Or vulvovaginitis (vaginitis was recorded if any part of the vaginal mucosa was reddened or granular; vulvitis was recorded if any part of the vulva was reddened, swollen, fissured or ulcerated) and a positive vaginal culture (29 C. albicans, 1 C. tropiealis); 28 of these women also had positive oral and/or rectal cultures (27 C. a[bicans, I C. tropicalis); 20 of these women had had vaginal candidiasis diagnosed on at least one previous occasion. 2. 20 women without clinical signs of vaginitis or vulvitis and a negative vaginal culture. These women all had positive oral and/or rectal cultures (20 C. albicans). 3. 10 women without clinical signs of vaginitis or vulvitis and negative vaginal, oral and rectal cultures. Five ml of blood were taken from each patient and the serum separated and stored a t - - 2 0 ~ until studied. 173
Preparation of antigen One strain of Candida albicans group A was used throughout this investigation. This strain was chosen because it contains all the antigens of C. albicans group B and C. tropicalis (2). A blastospore suspension was prepared using 0.4 per cent formal saline to harvest cells from 48-hour-old cultures on glucose peptone plates. After washing in saline, the blastospores wer~ diluted to 12,000 cells per ml and stored at 4 ~ until used. One drop of this suspension was placed on each spot of multispot PTFE-coated glass slides. The slides were dried at room temperature and fixed in methanol for 1 minute. Indirect IF test Serial dilutions of serum were made in phosphate buffered saline (PBS) pH 7.1. One drop of diluted serum was placed on each spot on each slide. After incubation for 30 minutes in a moist chamber at 37 ~ the slides were rinsed in two changes of PBS for 5 minutes each. One drop of the appropriate FITC-conjugated monospecific anti-human immunoglobulin was placed on each spot and left for 30 minutes at 37~ The slides were then rinsed in two changes of PBS for 5 minutes each and mounted in buffered glycerol. Fluorescein-conjugated sheep antisera to human IgG, IgA and IgM were obtained from Wellcome Reagents Ltd. A fluorescein-conjugated rabbit antiserum to human tgA secretory component (produced from anti-colostrum IgA by absorption with serum IgA) was obtained from Dakopatts AS, Copenhagen. The highest serum dilution giving a complete ring of bright green fluorescence around the blastospores was taken as the titre of the serum. Fractionation of serum Serum samples from four selected patients were fractionated. Each sample was first dialysed against 0.05 M trishydrochloride buffer pH 7.4 and then applied to a Bio-Gel A-1.5 m (Bio-Rad Laboratories) column equilibrated with the same buffer. The column was eluted with this buffer and fractions were collected and monitored at 280 nm (Fig. 1). Fractions were concentrated ten times using Minicon B-15 concentrators (Amicon Ltd) and then tested for IgG, IgA and IgM using radial immunodiffusion plates (Hoechst Pharmaceuticals). 174
I001
.,
,
,
,
L
I
ABSORBANCE
50I
0
I
I
i
I
I
I
fi
10
15
20
25
30
35
40
FRACTION NUMBER
Fig. 1. Fractionation of serum sample from patient 55. Fractions 10 to 15 contained IgM, fractions 13 to 21 contained IgA, e-~ fractions 17 to 27 contained IgC.
Results The titres of IgG, IgA and lgM antibodies against C. albicans are summarised in Table 1. IgG titres ranging from 1 : 4 to 1 : 32 and IgA titres from 1 : 4 to 1 : 16 were detected in the patients with vaginal candidiasis. Similar titres were found in the other women. Sera from 50 patients (including 24 patients with vaginal candidiasis) were tested for the presence of SIgA antibodies against C. albicans. No SIgA antibodies were detected. Sera from two patients with vaginal candidiasis and from two women harbouring C. albicans in the mouth and rectum were fractionated. The immunoglobulins present in the different fractions were determined by single radial immunodiffusion. The fractions were pooled according to the contents and tested for antibodies to C. albicans. The results (Table 2) demonstrate that the corresponding classes of antibodies were present in each of the fractions and confirm that these antibodies reside in highest titre in the IgG class. Table I. iF tTtres of antibodies to C, ~Zbleans in serum of womenwith vaginal candidlasls and of other women Diagnosis Number of IF titre of antibodies to C. ~Zbicqn8 patients IgG IgA IgM 1:4 1:8 "I:16 1:32 1:4 1:8 i:16 ~:4 1:8 Vaginal candidiasis 30 15 5 9 I 25 h 1 29 1 No signs of infectlon, yeastspresent ~n mouth and/or rectum
20
]2
3
5
0
13
4
3
20
0
No signs of infection, no yeasts present in vagina, mouth or rectum
IO
6
3
1
0
9
l
0
10
0
Table 2. The distribution of antibodies to C, a!bic~ns in separated fractions of serum from women with vaginal candidiasis and women harbouring C. aZbicans in the mouth and rectum
Serum
Fraction IgG
Patient 38 (vaginal candidiasis)
Patient 55 (vaginal candidiasis)
Patient 43
Patient
65
IF t i t r e IgA
lqM
