German Primate Centre, Göttingen, 1 Institute of Clinical Chemistry and Pathobiochemistry of the University of Leipzig, Leipzig and 2 Dr Fooke Laboratories, Neuss, Germany
Correspondence and offprint requests to: Prof. Dr Thomas Mothes, Institute of Clinical Chemistry and Pathobiochemistry of the University of Leipzig, Paul-List-Str. 1315, D-04103 Leipzig, Germany.
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Abstract |
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Methods. The level of IgA-gliadin antibodies (IgA-AGA), of IgA-containing circulating immune complexes (IgA-CIC), and the degree of glomerular IgA deposits were compared between marmosets suffering from WMS and animals not affected by the disorder.
Results. Both IgA-AGA and IgA-CIC were demonstrable in all groups of monkeys investigated. IgA-AGA and IgA-CIC were significantly higher in monkeys with WMS than in non-affected animals. There was a significant correlation between the glomerular IgA-deposition and titre of IgA-AGA. The group of marmosets strongly positive for glomerular IgA deposits comprised significantly more animals suffering from WMS than the group without deposits. In the diet of the animals a considerable amount of gliadin-like cereal proteins was assayed.
Conclusions. There are several parallels between the human disorders (coeliac disease and IgA-nephropathy/Berger's disease) and the changes observed in WMS. It should be further investigated if WMS in marmosets is a suitable animal model for both human diseases.
Keywords: Wasting marmoset syndrome; gliadin antibodies; immune complexes; nephropathy
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Introduction |
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Chronic enteritis [4,5], diarrhoea [2], colon carcinoma [6], and glomerulopathy [7] with striking similarities to IgA-nephropathy in humans [8] have been described in various members of the callitrichidae. The diet of marmosets in captivity is considered as one possible cause for enteritis and WMS [4,9]. Recently, a beneficial effect of a gliadin-free diet on animals with WMS was reported [10].
In susceptible humans, gliadins from wheat and related proteins from other cereals represent the aetiological factor for coeliac disease [11,12] with damage of the small intestinal mucosa, diarrhoea, increased anti-gliadin (AGA) and connective tissue antibodies [13], and frequently enhanced circulating immune complexes (CIC) [14]. The antibodies involved include the IgA class. The incidence of glomerular IgA deposits in humans with coeliac disease is increased [15]. Furthermore, in man a relation between gliadins and primary IgA nephropathy is considered [16,17]. In this disorder, IgA is deposited in the glomerular mesangium, which was found positive for food antigens in a significant fraction of patients [18], and frequently IgA-CIC and antibodies against dietary antigens including AGA are present in the serum [1921]. Intestinal permeability is often enhanced [22]. In a group of patients with elevated IgA-CIC and food antibodies, gluten-free diet decreased in most cases CIC and antibody levels and reduced proteinuria and microscopic haematuria [17,23,24].
As yet there is no evidence for a causal involvement of gliadins in WMS and if IgA-AGA and IgA-CIC occur in marmosets with WMS. In this study, IgA-AGA and IgA-CIC class in marmoset with and without WMS were measured and IgA-AGA correlated to the degree of renal IgA deposition in marmosets.
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Materials and methods |
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The diagnosis of WMS was made according to history (recurrent diarrhoea), clinical findings (weight loss, dehydration, poor hair), and necropsy reports (chronic enteritis). Marmosets having developed typical signs of WMS were classified as `wasters'.
For comparison, three rhesus monkeys (Macaca mulatta), three cynomolgus monkeys (Macaca fascicularis), two baboons (Papio hamadryas), one mandrill (Mandrillus sphinx), and one langur (Presbytis entella) were included, all from GPC. These Old World monkeys ranged in age from 10 to 25 years. They were fed on Ssniff-Pellets, supplemented by fruits, vegetables, and milk products.
Venous blood was collected immediately before sacrificing the animals and sera were stored at -20°C until use.
Anti-IgA antibodies
Since no anti-marmoset and anti-Old World monkey IgA antibodies were available, rabbit anti-human IgA, -chain specific (Dako, Hamburg, Germany) was tested for reactivity with monkey IgA using agarose immunodiffusion techniques [25]. Specificity of rabbit antibodies for IgA indicated by the supplier was checked by comparison with reactivity of rabbit anti-human IgM and rabbit anti-human IgG (Dako) in immune electrophoresis [26]. Marmoset immunoglobulins precipitated by rabbit anti-human IgA were comparable in position with the respective human immunoglobulins and clearly different from those recognized by rabbit anti-human IgM and rabbit anti-human IgG. There was no cross reaction of rabbit antibodies against IgA with IgM and IgG.
Determination of IgA-AGA
A modification of a method previously described was applied [27]. In brief, microplates (Maxisorb, Nunc, Wiesbaden-Biebrich, Germany) were coated over night with a solution containing 50 µg gliadin/ml 70% ethanol, incubated 1 h at 37°C with 100 µl monkey serum (1:100) and 2 h at 37°C with 100 µl rabbit anti-human IgA conjugated with peroxidase (1:2000) (Dako, Hamburg, Germany). Colour was developed by use of Seramun Blue/TMB substrate solution (Seramun, Dolgenbrodt, Germany) and optical density (OD) was read at 450 nm. A calibration curve was constructed using the positive control provided by Labmaster Ltd (Turku, Finland). Gliadin was obtained from wheat variety `Kanzler' [28].
Determination of IgA-CIC
Maxisorb microplates (Nunc) were coated over night with a solution containing 10 µg bovine conglutinin (Dr Fooke, Neuss, Germany)/ml veronal-buffered saline (VBS, pH 7.2). After washing (0.1% Tween 20 in VBS) the plates were incubated 1 h at 37°C with monkey serum (1:10), washed again, and incubated 1 h at 37°C with anti-human IgA conjugated with peroxidase (1:2000) (Dako) and colour was developed as described above. Suitability of conglutinin based assays for detection of IgA-CIC was recently shown [17].
Estimation of glomerular immunoglobulin-deposits
For IgA-deposits, a modification of the immunofluorescence technique described previously [7] was used. Fresh renal tissues of marmosets from GPC obtained during necropsy were snap frozen in liquid nitrogen. Cryostat sections of these tissues were stained with FITC labelled polyclonal rabbit anti-human IgA (Dako) (1:20 in phosphate-buffered saline). IgM-deposits were estimated according to [7]. For negative control, reactivity of rabbit anti-human IgG, -chain-specific (Dako) was checked.
Determination of endomysium antibodies
These antibodies were assayed by indirect immunofluorescence methods using sections of monkey oesophagus (Mast Diagnostika, Reinfeld, Germany) [29]. For detection of bound monkey IgA, anti-human IgA conjugated with FITC (Dako) (1:20 in phosphate-buffered saline) was used.
Determination of gliadin
Pellets were milled and 100 mg dry powder were extracted with 5 ml 60% ethanol. Then 50 µl of the extract (diluted 1:1000) was incubated in the wells of microtitre plates (Flow, No. 76-381-04) over night. Gliadins and other ethanol soluble proteins coated to the wells were fixed with 100 µl formaldehyde (10%). Wells were then washed with buffer A (50 mM Tris, 150 mM NaCl, 5 mM NaN3/l, 0.05% (w/v) Tween 20, pH 10.2), blocked with buffer B (buffer A containing 0.5% Tween 20 and 10 g bovine serum albumin/l), rinsed again with buffer A, and incubated for 1 h with 100 µl antigliadin serum (1:400) obtained from rabbits after immunization with gliadin isolated from flour of wheat var. `Kanzler'.
After incubation, wells were washed with buffer A and incubated 2 h with 100 µl pig anti-rabbit immunoglobulins conjugated with peroxidase (Dako, 1:2000). Wells were rinsed again with buffer A, and colour was developed by adding a solution containing o-phenylene diamine. The reaction was stopped by adding sulphuric acid and OD was read at 492 nm. For calibration of the assay, gliadin extracted from wheat cultivar Kanzler was solubilized in 60% ethanol and diluted to concentrations between 10 and 800 ng/ml.
Expression of results and statistical evaluation
Results are presented in box plots extending from 25th to 75th percentiles and showing medians (solid lines). Tenth and 90th percentiles are indicated by capped bars. All data outside of these regions are presented.
Statistical differences between the different monkey groups (P<0.05) were evaluated by one-way analysis of variances to compare more than 2 groups amplified by Dunn's test for pairwise multiple comparison procedures. Fisher's Exact Test was used to evaluate differences in the proportion of observations in different categories which define the contingency table.
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Results |
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From 72 marmosets investigated, 12 suffered from WMS. IgA-AGA as well as IgA-CIC were demonstrable in all groups of monkeys investigated. IgA-AGA were higher in marmosets suffering from WMS than in normal marmosets (Figure 1). Antibody levels of marmosets without WMS were very close to that of the group comprising Old World monkeys not affected by the disorder. IgA-CIC were significantly higher in marmosets with WMS than in non-affected animals (Figure 2
). IgA-CIC levels of marmosets without WMS were also very close to that of the group comprising Old World monkeys not affected by the disorder.
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Discussion |
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In the wild, callitrichids basically feed on tree exudates, fruits, leaves, insects, and small vertebrates [31,32]. Of the protein sources ingested in the wild, gliadins do not appear to be present. In captivity, however, the diet is usually based on commercial pellets supplemented with fruits, vegetables, animal protein, and cereal products [33,34]. The marmoset diet at GPC contains a significant amount of gliadin-like proteins in pellets of different suppliers and in several of the diet supplements (biscuits).
For coeliac patients, a daily amount of as low as 100 mg of gliadin has been shown to induce toxic effects in the small intestine [35], and the gliadin content of so called `gluten-free' diet should not exceed 10 mg per 100 g food, provided that not more than 100 g are consumed daily [36]. At present, there is no information on the quantity of gliadin which may possibly induce adverse renal effects in humans. Considering the low body weight of marmosets a critical amount of gliadins may already be exceeded if only a small quantity of pellets are consumed daily, not taking into account gliadins in food supplements additionally offered to the animals.
The results of the present study are the first to show increased levels of IgA antibodies against gliadins and of IgA-CIC in marmosets with WMS. It is known from humans that AGA may be raised because of changes in the intestinal mucosa leading to increased permeability and enhanced access of the wheat peptides to the gastrointestinal immune system [37]. Thus, increase in AGA in WMS could indicate a damage of the monkey small intestinal mucosa a conclusion which agrees well with the finding that WMS is often accompanied by diarrhoea [2,38] and chronic enteritis [39]. Once damaged, the epithelial barrier should not only be permeable for gliadins but also for other food proteins. This may then induce the production of the respective antibodies, resulting in the formation of IgA-CIC, the glomerular deposition of which may be important in the pathogenesis of marmoset nephropathy.
Recently, glomerulopathy in marmosets of the GPC was reported [4]. The disorder was characterized by mesangial proliferation, glomerulosclerosis and interstitial fibrosis, electron dense deposits in the mesangium and in peripheral capillary walls, and glomerular IgM-deposits. Deposition of IgM in juvenile marmosets preceded histological signs of nephropathy in young adult and adult marmosets [4,7]. Because of IgM-deposition, the changes were described as IgM-nephropathy with similarity to IgA-nephropathy in humans [4]. Spreading of the typical mesangial immunoglobulin deposition to neighbouring capillary loops as observed in marmosets [4,7,8] was found in several cases of human IgA-nephropathy, too [40,41]. IgA-deposits were observed in marmosets as well [4]. However, due to the low degree of cross reactivity of anti-human IgA with marmoset IgA, these were faint and regarded as of minor importance.
Considering the weak reactivity of the antibodies with marmoset IgA, even a higher incidence of IgA than of IgM deposition was claimed recently [8]. Further, it was demonstrated that marmosets with glomerular IgA-deposits showed comparable histological and electron microscopy changes as described for IgM nephropathy, and that these animals had a higher prevalence of haematuria and proteinuria than not affected animals [8]. Taken together with the finding of increased frequency of WMS in animals with IgA depositions, data strongly argue for an association of these immunoglobulin deposits with disease.
The nature of antigen(s) in the deposits remains unknown. However, in rat experiments gliadins were shown to bind to mesangial cells and thus have been suspected to contribute to the pathogenesis of mesangial IgA nephropathy [42]. Renal deposition of IgA can be elicited experimentally in mice by introduction of gliadins into drinking water [43]. In man, introduction of a gluten-free diet decreased the titre of AGA and of CIC in IgA-nephropathy patients [17,23,24].
Data about immune complex deposition in other organs of marmosets like skin or intestine are not available until now but may be a promising field for future work.
As in human IgA-nephropathy [44,45], endomysium antibodies could not be found in the sera of marmosets suffering from WMS providing no conclusive evidence for a condition identical to coeliac disease in humans. However, lack of detection of endomysium antibodies might be due to low cross reactivity of the antibody against human IgA with marmoset IgA as well.
Thus, there are several parallels between coeliac disease and IgA-nephropathy in humans and the changes observed in WMS.
Further investigations should be promising to elucidate if these disorders comprise related pathogenetic events. Furthermore, future investigations should confirm whether a gliadin-free diet is beneficial for callitrichids suffering from WMS.
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Acknowledgments |
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References |
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