Assessment of IgA subclasses synthesis in children with selective and partial IgA deficiency

Introduction


The human IgA exists in two isotypic forms: IgA1 and IgA2, which differ both in their primary amino acid sequences and carbohydrate structures [1, 2]. IgA antibodies are found both in the blood and in the mucosal secretions. The IgA in human serum is primarily monomeric, with 90–95% being of the IgA1 subclass. Most of the IgA in secretions is polymeric and the concentrations of IgA2 is increased relative to serum. Serum IgA is derived primarily from the bone marrow, while IgA destined for external secretions is synthesized locally in mucosa-associated tissues and glands. The distribution of the two IgA subclasses in secretions is dependent on the mucosal site: IgA1-secreting cells predominate in the respiratory tract, in the upper gastrointestinal tract and in mammary glands (60–93%), whereas IgA2-secreting cells predominate in the lower gastrointestinal and in the female reproductive tracts [3, 4, 5]. Interestingly, in humans predominantly monomeric IgA from the systemic pool contributes little to external secretions, and vice-versa, locally produced polymeric IgA is selectively transported into external secretions, and only small amounts of this IgA enter the circulation [6].
An inability to produce antibodies of the IgA subclasses is the most frequently recognized form of primary immunodeficiency, having an incidence of approximately 1 in 600 individuals of European ancestry [7, 8]. IgA deficiency (IgAD) has been associated with an increased frequency of sinopulmonary infections, gastrointestinal disorders, autoimmune diseases and allergies [9, 10]. However, approximately two-thirds of IgAD individuals remain healthy - apparently even when living under poor hygienic conditions [11]. The reason for this difference in susceptibility to infections, as well as to other diseases, is not well understood.
The aim of our study was to disclose possible differences between serum and secretory levels of the two IgA subclasses in children with selective IgA deficiency (IgAD) and with partial IgA deficiency (p-IgAD) and to determine the failure degree of these subclasses in both groups of patients.


Materials and methods
Patients and controls


Twenty-one children (10 females and 11 males, aged 4–17 years) with selective IgA deficiency (IgAD) and eight children (6 females and 2 males, aged 4–18 years) with partial IgA deficiency (p-IgAD) were included in the study. Selective IgA deficiency was defined according to criteria of WHO [7] such as: serum IgA level was less or equal to 0.05 g/l, associated with normal serum levels of both IgM and IgG. And then, partial IgA deficiency was diagnosed as a serum concentration of IgA more than 0.05 g/l, but less than two standard deviations below normal serum level of IgA, with IgM and IgG levels within the normal range. The IgAD and p-IgAD groups involved children who were suffering from at least 8 respiratory infections during last year including pneumonia and/or bronchitis. The control group (C) comprised 32 sex- and age-matched healthy volunteers (15 females and 17 males, aged 6-14 years) with no history of primary and secondary humoral immunodeficiency. The characteristic of studied children is shown in Table 1.
All children were apparently healthy at time of collection of samples. None of them have been receiving any antibiotics for at least 3 weeks before the sample collection. All subjects were Caucasians. The study was approved by the Bioethical Committee of the Medical University of Wroclaw.


Serum samples

Venous blood samples were drawn into vacuum blood collection tubes without additives. The samples were allowed to clot and centrifuged at 450 x g for 10 min, and sera were immediately frozen and stored at -700C in multiple aliquots until analysis. Serum samples were frozen and thawed only once.


Saliva samples

Whole unstimulated saliva samples were collected by spitting directly into sterilized plastic tubes and placed in melting ice at once. The samples were stored at -700C until assayed following clarification at 9 000 x g for 10 min at 40C.


Fecal extracts

Approximately 1 g of fecal sample from one bowel movement was suspended in 7 ml PBS (BIOMED-Lublin). Subsequent mixing was done with a vortex mixer for 10 min, and the mixing process was repeated after 15 min, achieving a well-mixed suspension, even for some hard stool samples. The sample suspension was then centrifuged at 20 000 x g for 10 min at 40C. Supernatant was pipetted into a new tube and the mixing process was repeated. The sample suspension was then centrifuged again 12 000 x g for 10 min at 40C. The resulting clear supernatant was pipetted into Eppendorf reaction vessel. The vessel was stored at -700C until measurement.


Purification of S-IgA from colostrum

The purification of S-IgA from colostrum was established using a modified procedure described by Gregory et al. [12]. Briefly, human colostrum obtained from several healthy women 48 hours post partum was diluted (1:1) with 0.15 M NaCl and clarified by ultracentrifugation at 100 000x g for 60 min at 40C. Casein was precipitated at pH 4.5 by adding cold 2 M CH3COOH and then removed by ultracentrifugation at 100 000 x g for 60 min at 40C. The pH was adjusted to 7.0 with 0.5 M NaOH and the sample dialyzed overnight against 0.01 M sodium phosphate buffer (pH 7.4) containing 0.15 M NaCl and passed twice through a heparin-Sepharose CL-6B (Pharmacia, Uppsala, Sweden) column (1.5 x 5 cm) to remove lactoferrin and lysozyme. The column was washed with sodium phosphate buffer (pH 7.4) and the void volume collected and dialyzed against sodium phosphate buffer (pH 7.4) overnight. The heparin-Sepharose column was rejuvenated by washing with 1.65 M NaCl. The sample was then passed twice through an anti-human g chain (DAKO) conjugated to CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden) column (1.5 x 5 cm). The sodium phosphate buffer was used to wash the column and the void volume collected and dialyzed against sodium phosphate buffer (pH 7.4) overnight. Next, the sample was passed twice through an anti-human m chain (DAKO) conjugated to CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden) column (1.5 x 5 cm). After washing the column, the void volume was collected and dialyzed against sodium phosphate buffer (pH 7.4) overnight and concentrated by liofilization. The presence of protein in isolated fractions was examined using Bradford method [13].


Separation of S-IgA1 and S-IgA2 from purified S-IgA

The method of separation of S-IgA1 and S-IgA2 from colostral S-IgA has been described in detail [12]. Briefly, purified S-IgA from colostrum was passed through a jacalin-agarose (Vector) column (1.5 x 5 cm) in a 10 mM Hepes buffer (pH 7.4) containing 150 mM NaCl, 0.1 mM CaCl2, 20 mM galactose and 0.08% NaN3. The column was washed with the buffer and S-IgA2 was collected in the void volume. S-IgA1 was eluted with the same buffer containing 0.8 M galactose. Isolated fractions were analysed using the Laemmli system of SDS polyacrylamide gel electrophoresis [14] and assessed for purity and thus for suitability as S-IgA1 and S-IgA2 standards by means of Western blotting. The concentration of S-IgA was established using ELISA method.


ELISA for measurement of serum IgA1 and IgA2

The concentrations of IgA subclasses in the sera were measured by ELISA. Briefly, 96-well microtitre plates (MaxiSorp, NUNC) were coated with either jacalin (Sigma) for IgA1 or monoclonal anti-human IgA2 antibodies (CHEMICON or Southern Biotechnology) for IgA2 in appropriate dilution and kept for 2 h at 370C and overnight at 40C. After blocking the wells with Tris-HCl containing 0.05% Tween for IgA1 or PBS containing 3% BSA (Sigma) for IgA2, serially diluted test samples or standards were added to each well and cultured for 2 h at 370C. Purified human IgA1 and IgA2 standards (myeloma serum proteins) were purchased from CHEMICON. After washing, secondary antibodies were added and incubated for 2 h at 370C. For secondary antibody, horseradish peroxidase (HRP)-conjugated rabbit anti-human IgA (DAKO) was used. After washing, citrate buffer (pH 5.0) containing o-phenylenediamine substrate (Sigma) was added to each well and reacted for 15 min at room temperature. The optical density was determined with an automated ELISA plate reader (Dynatech 500). Standard curves of absorbance at 490 nm vs concentration of IgA1 or IgA2 were prepared and were used to determine the IgA1 or IgA2 concentration of the serum test samples, allowing for the fact that they had been diluted.


ELISA for measurement of S-IgA and S-IgA1

Immunoglobulins levels in saliva and fecal extracts were determined by ELISA methods, which were similar to the procedures measurement of serum IgA1 or IgA2 detailed above. Briefly, for measurement of S-IgA, 96-well microtitre plates were coated with rabbit anti-human SC antibodies (DAKO) and developed with (HRP)-conjugated rabbit anti-human IgA (DAKO). S-IgA1 were measured by ELISA with monoclonal anti-human IgA1 antibodies (ICN) - coated assay plates and developed with HRP-conjugated goat anti-rabbit Ig. Rabbit anti-human SC antibodies were utilized as the secondary antibody. The concentrations of S-IgA and S-IgA1 were calculated using the linear ranges of the dilution colostral S-IgA (Sigma) and prepared in-house S-IgA1 as standards, respectively.


Statistical analysis

The Mann-Whitney U test was used to compare IgA subclasses levels between the study groups. Coefficients of correlation (r) were calculated by the Spearman rank test. The p values less than 0.05 were considered as statistically significant.


Results

Standardization

Utilizing the commercially available IgA1 and IgA2 myeloma serum proteins, standard curves were generated for each subclass (Fig. 1). Graphs of protein concentration versus absorbance at 490 nm produced sigmoidal curves in the region between 0.0156 mg/ml and 2 mg/ml for IgA1, and 0.0156 mg/ml and 16 mg/ml for IgA2. These curves displayed linearity between 0.625 mg/ml and 0.5 mg/ml for IgA1, and between 0.625 mg/ml and 1 mg/ml for IgA2. Coefficients of variation values ranged from 0.52% to 20.65% for both standard curves.
Fig. 2. illustrates the typical standard curves for S-IgA and S-IgA1, using colostral S-IgA and prepared in-house S-IgA1 as the standards. Both standards curves plotted as protein concentration versus absorbance reading at 490 nm were sigmoidal in the region between 0.0156 mg/ml and 1 mg/ml. The curves was linear and steep in the range of 0.031 mg/ml to 0.25 mg/ml for S-IgA and in the range of 0.031 mg/ml to 0.5 mg/ml for S-IgA1. The S-IgA2 concentration was calculated as the difference between S-IgA and S-IgA1.

Serum IgA subclasses levels

The levels of IgA subclasses were determined by ELISA assays in the serum samples obtained from 21 IgAD children, 8 p-IgAD children and 32 healthy controls, with an age from 4 to 18 years. The results of serum levels of IgA1 and IgA2 in all studied groups of children are presented as scatter graphs in Fig. 3A and Fig. 3B, respectively. The mean concentration of serum IgA1 (0.0085±0.012 g/l for IgAD; 0.16±0.1 g/l for p-IgAD) was decreased above 167-fold in IgAD group and around 9-fold in p-IgAD group comparing to the healthy control (1.41±0.47 g/l), (Fig. 3A). On the other hand, the mean concentration of serum IgA2 was 17.6-fold lower in IgA-deficient patients (0.0026±0.0043 g/l) and 1.67-fold lower in p-IgAD children (0.027±0.026 g/l) in comparison with the control group (0.046±0.47 g/l), (Fig. 3B). Interestingly, the IgA2 level was within the normal range in 2 of the 21 IgA-deficient patients and in 6 of the 8 children with partial IgA deficiency (Fig. 3B). The normal range was defined as the mean plus two standard deviations and included about 95% observations on a normalized frequency distribution. As expected, when serum levels of IgA1 were determined, differences between healthy controls and both groups of patients were seen
(p <0.0000001 for IgAD; p <0.00005 for p-IgAD).
Although there was a great deal of variability in the levels of IgA subclasses in all individuals participating in the study, the concentration of IgA2 increased with increasing age (r=0.3; p <0.05). Furthermore, the mean concentration of serum IgA1 was slightly higher in healthy girls (1.63±0.52 g/l) than in healthy boys (1.22±0.33 g/l) and the difference was statistically significant (p <0.005). By comparison, no significant dependence was found between the serum levels of IgA subclasses and sex in both group of patients.
The percentage of IgA2 in the total IgA in serum was negative correlated with the percentage of IgA1 in the total IgA in the group with selective IgA deficiency (r=- 0.92; p <0.0000001). In his group, the percentage of serum IgA2 in the total IgA ranged from 0.94% to 62%. As shown in Fig. 4B, IgA2 constituted more than 50% of the IgA in 3 patients and less than 10% of the IgA in 5 patients. The percentage of serum IgA2 in the total IgA less than 10% stated also in 4 patients with p-IgAD.


Secretory S-IgA subclasses levels

The concentrations of secretory S-IgA subclasses were measured in saliva samples obtained from 15 IgAD children, 5 p-IgAD children and 23 healthy controls, whereas fecal S-IgA subclasses were detectable in 17 IgAD children, 5 p-IgAD children and 19 healthy controls. Both S-IgA1 and S-IgA2 were detectable in 35% of secretions (saliva and feces) from IgAD group and in 80% of secretions from p-IgAD.
The mean level of salivary S-IgA1 in children with selective IgA deficiency (0.87±2.09 g/l) was significantly decreased as compared with partial IgA-deficient children (54.27±46.51 g/l; p <0.05) as well as with healthy controls (137±78.4 g/l; p <0.0005). On the other hand, the mean concentration of salivary S-IgA2 (21.12±51.6 g/l) in such children was about 2.5-fold lower than that of both p-IgAD children (53.9±61.83 g/l; difference not significant) and control group (50.1±22.64 g/l; p <0.05). By comparison, the differences between the mean concentrations of fecal S-IgA subclasses in IgA-deficient patients and control group were statistically significant (24.15±28.14 mg/g vs 129.92±128.45 mg/g; p <0.05 for S-IgA1, 18.62±11.34 mg/g vs 147.07±267 mg/g; p <0.05 for S-IgA2). There were not observed any statistically considerable differences in both S-IgA1 and S-IgA2 levels between p-IgAD children and healthy individuals (154.82±174.04 mg/g vs 129.92±128.45 mg/g for S-IgA1, 112.21±121.1 mg/g vs 147.07±267 mg/g for S-IgA2).
Considering the mean percentage of the S-IgA1 in the total salivary S-IgA and the mean percentage of the S-IgA2 in the total salivary S-IgA in all studied groups, there were significant differences between IgAD children and healthy controls (p <0.005), (Fig. 5A and 5B). In contrast to above results, the differences in the percentage values of S-IgA1 or S-IgA2 in feces, were not statistically significant in both IgAD and p-IgAD children comparing to the group of healthy controls (Fig. 6A and 6B). There were, also, no statistically significant differences in this parameter in saliva obtained from p-IgAD children and healthy children. As shown in Fig. 5A and 5B in all children from IgAD group and in one out four children with p-IgAD, the percentage either S-IgA1 or S-IgA2 in the total salivary S-IgA did not reach the levels of the healthy controls.
Fig. 8. illustrates comparison of the distribution of the two IgA subclasses both in sera and secretions in all studied groups. It was noteworthy that the quantities of S-IgA2 exceeded the quantities of S-IgA1 both in saliva and feces as well as in sera of IgA-deficient children comparing to healthy controls.


Discussion

The main finding of our study was demonstration that systemic IgA1 synthesis was more impaired than systemic IgA2 production in the two tested groups of IgA-deficient children. Our study revealed that the level of serum IgA1 was significantly decreased in children with selective and partial IgA deficiency in comparison to the control group (Fig. 3A). On the contrary, the level of IgA2 was significantly decreased only in IgAD group. Moreover, we demonstrated that the concentration of IgA2 was on the level of control group in almost 10% of sera from IgAD group and in 75% of sera from p-IgAD (Fig. 3B). The reason for the differences in IgA subclass distribution found in this study is unclear. The IgA subclass shift found in this study may indicate that among these children there are cases with selectively impaired synthesis of only one subclass (defect of IgA1, normal IgA2). So far, such cases were present in closely related populations and as a general rule concerned the simultaneous deletion of heavy constant region genes different from Ca1 and Ca2 [15, 16, 17]. However, further studies are needed to confirm the presence of similar deletion in our patients. An increase in serum IgA2 levels to the normal range may also suggest that there are factors, which independently regulate the production of serum IgA1 and IgA2. Molecules regulating the expression of immunoglobulins would thus be expected to be involved in the pathogenesis of IgA deficiency. Interleukins are obvious examples of that molecules. However, it has recently been shown that the production of IgA1 by B cells from apparently healthy IgA-deficient patients may be efficiently up-regulated by IL-10 and IL-4 in vitro, whereas a corresponding up-regulation is not achieved in infection-prone IgAD subjects [18, 19].
Another important factor determining the different synthesis of various immunoglobulin class is fact that genes encoding a constant region of immunoglobulin are poliallelic. The reports by Litwin and Balaban [20] and Giessen et al [21] provided the suggestion that the total proportion of serum IgG that is IgG2 or IgG3 is related to the allotype of that IgG subclass. Except of g chain, differentiation of allotypic variants referred to e and a2 chain, as well. Among three IgA2 allotypes [A2m(1), Am2(2), A2(n)] over 90% of Caucasians have the allotype A2m(1) [2, 22]. Our studied groups included only Caucasians, therefore it is unlikely that the variation in the ratio of IgA1 to IgA2 seen in them can be attributed to allotype differences.
The differences in IgA subclass distribution found in this study could theoretically be the result of the slight differences in sex and age distribution in our study populations. Berth et al. [23] have recently shown that serum concentrations of both IgA1 and IgA2 were significantly higher in healthy men than in healthy women. In contrast to previous report, our present study revealed that the level of serum IgA1 was significantly higher in girls than in boys from the control group. The discrepancies between our results and those of others can be explained by the sex and have no effects on synthesis of IgA subclass, although, as well known, the gender of parents determined transmitting the defect: affected mothers are more likely to pass the defect on to their offspring than affected fathers [24]. Furthermore, this dependence may reflect the stronger B cell responses in females than in males [25].
The results of previous study, concerning the development of IgA during ontogenesis of human being, showed that the levels of IgA subclasses reach adult concentration in secretions - faster than in serum [26, 27]. Significantly increased the proportion of IgA2 in the total serum IgA both in IgAD and p-IgAD children comparing to the control group may suggest either more effective development of synthesis of intravascular IgA2 in such children or that the secretory surfaces do contribute significantly to serum IgA2 (Fig. 5B). According to Andre [28] if IgA production at mucosal surfaces with a relatively higher levels of IgA2 contributed significantly to serum pool of IgA, one might expect that in infant a higher proportion of the serum IgA2 would be derived from the secretory system. The results presented in this paper demonstrate a positive correlation between serum IgA2 and the age of all children. It probably excludes the involvement of secretory IgA2 in serum and confirms that serum and secretory pools of IgA derived from relatively independent sources. Similar association indicated both Conley et al. [29] who studied IgAD children and Berth et al. [23] who established reference values for serum concentration of the two IgA subclasses in Caucasians adults.

Observation of a negative correlation between the proportion of total serum IgA that was IgA1 and the proportion of total serum IgA that was IgA2 may suggest the compensative role of each other. This finding was accordant with previous study encompassing IgA subclass-deficient patients with deleted constant region genes [15]. The authors showed that the lack of IgA1 can be partially compensated by IgA2 antibodies, whereas the lack of IgA2 can be fully compensated by IgA1.
We also noted that the concentrations of both S-IgA1 and S-IgA2 in fecal extracts and S-IgA2 in saliva were significantly decreased in IgAD children as compared with the control group, whereas the salivary level of S-IgA1 in such children was considerably lower in comparison to
p-IgAD children as well as to control individuals. On the other hand, the normal values of both salivary and fecal
S-IgA subclasses together with the diminished systemic IgA1 synthesis observed in part children with partial IgA deficiency, may reflect the transient defect in such children followed by an increase and normalization of serum IgA level or a decrease and absence of the immunoglobulin. We can not also exclude that reduced IgA1 symthesis in such cases is an effect of redundant or insufficient antigen esposure.
Summing up, the normal IgA2 synthesis in mucosal system and “weaker failure” of its systemic synthesis in few children (in comparison to IgA1 production) on one hand, and a serious impairment of both local and systemic IgA synthesis in the other ones, confirm the heterogeneity of IgA deficiency. The obtained results of the research can constitute a contribution to the knowledge of pathogenesis of IgA deficiency, requiring certainly their further widening by elements of the immunoregulatory mechanisms.


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The work was done and should be attributed to Immunology Unit, Department of General Microbiology, Institute of Genetics and Microbiology, University of Wroclaw.

Correspondence: Grażyna Majkowska-Skrobek, PhD, Institute of Genetics and Microbiology, University of Wroclaw, Przybyszewskiego 63/77,
51-148 Wroclaw, Poland. Phone: +48 71 375 62 96, fax: +48 71 325 21 51, e-mail: majkosia@microb.uni.wroc.pl