METODO DE INVESTIGACION
FOTO N° 10 Interior del Museo
3.11.3.3. Danzas y Tradiciones
Recipients of serum from OVA fed donor mice showed significant suppression of their systemic DTH responses when compared to recipients of serum from saline fed donors ( p < 0.0005; Figure 9.2), or when compared to recipients of unbound material from saline fed donor serum which had been passed through the mouse anti- OVA affinity column (p < 0.0005; Figure 9.2). In contrast, recipients of unbound material from the serum of OVA fed donors which has been passed through the mouse anti-OVA column, showed no significant suppression of their systemic DTH responses when compared to the recipients of unbound material from the serum of saline fed donor mice which had been treated identically i.e. group 2, (p=N S; Figure 9.2). Recipients of unbound material obtained from the serum of OVA fed donors which had been passed through the rabbit anti-BSA affinity column demonstrated significant suppression of their DTH responses when compared to recipients of serum from saline fed mice which had also been passed through the rabbit anti-BSA affinity column (0.005 < p < 0.0025; Figure 9.2).
F ig u re 9 .2 E ffe ct o f rem oving im m unoreactive OVA from serum and subsequent i.p. injection of this depleted serum into recipients. Groups of recipients (n = 8) were injected i.p. with (1) serum from saline fed mice, (2) serum from saline fed mice after passage through a MaOVA affinity column, (3) serum from saline fed mice after passage through a RaBSA affinity column, (4) serum from OVA fed mice, (5) unbound material from serum of OVA fed mice after passage through a MaOVA affinity colum n, and (6) unbound material from passage of serum from OVA fed mice through a RaBSA affinity column.
*: comparison with group 1, comparison with group 2, and ***: comparison with group 3
group 1
□
group 2 group 3 group 4■
group group 5 6 D o n o r s e r u m t r e a t m e n t ( g r o u p s ) Saline fedUnbound material after passage of serurn from saline fed mice through the MaOVA column Unbound material after passage of serum from saline fed mice through the RaBSA column OVA fed
Unbound material after passage of serurn from OVA fed mice through the MaOVA column
Unbound material after passage of serumi from OVA fed mice through the RaBSA column
m m 0 . 3 c <D s (D •- (/) a + CL c
8 ?
u <4— u <D Q_ CO0.2 4
0 . 1 -S yste m ic DTH re sp o n se s
0.0 * p < 0 . 0 0 0 5 * * p < 0 . 0 0 0 5 * * * p < 0 . 0 0 5A n ti- O V A IgG re sp on se s
1 7 5 0 r- 1 5 0 0 R 1 2 5 0 1000Comment The MaOVA affin ity colum n reduced by a fa c to r o f six the concentration of OVA in the serum of OVA fed mice. Such OVA depleted serum was unable to induce systemic DTH tolerance in BALB/c recipient mice suggesting that the dose of "tolerogen" is crucial for the successful transfer of oral tolerance. Surprisingly large concentrations of OVA were observed in the sera of OVA fed animals when compared to the levels measured in the time course study (see Chapter 5) (i.e. » 5 0 0 n g /m l at 60 minutes after feeding). This may have arisen from contamination by aerosols of the OVA solution used for feeding or, contamination when handling the OVA and bleeding mice during the tight time constraints in the experiment. However, the capacity of the affinity column appears to have been sufficient to cope with these increased levels of OVA.
The passage of serum from OVA fed donor mice through the RaBSA column, i.e. a column coupled to antibody raised against a unrelated protein antigen (BSA), had no effect on the tolerogenic properties of that serum on subsequent transfer into recipient BALB/c mice.
These experiments suggest that the factor in serum from OVA fed BALB/c mice, which confers antigen specific tolerance of DTH when injected into recipient BALB/c mice, is either native OVA or a form of OVA which has only undergone subtle alteration and which can still be recognised by polyclonal mouse anti-OVA antibodies.
9.3 Introduction of the Tolerogenic Fraction of Serum from
OVà Fed BALB/c Mice into Naive Recipient BALB/c Mice.
Aim To determine whether the purified immunoreactive OVA "tolerogen" from OVA fed BALB/c mice when introduced into BALB/c recipient mice can induce systemic DTH tolerance to OVA.
9.3. Adult BALB/c donor mice (n=24/group) were fed either 0.2ml of saline or 25mg of OVA in 0.2ml of saline. One hour after feeding, the donor animals were bled by cardiac puncture and the collected serum of each group pooled separately. Naive recipient mice were injected with 40/il/g body weight of the appropriate donor serum after passage through affinity columns, as follows
1. 7ml of serum from saline fed BALB/c mice after passage through the MaOVA affinity column, in PBS pH 7.3, for 1 hour at room temperature. The unbound material was collected by washing PBS through the column until the absorbance at 280nm reached zero. The material collected was then concentrated to its pre-column volume. The column was then eluted with IM glycine HCl pH 2.4 to remove any bound protein. The eluant was collected into tubes containing solid Trizma base in order to restore the samples to pH 7.0 and was then also concentrated to a volume of 7ml. When the absorbance at 280nm reached zero, the column was washed with PBS pH 7.3 before the application of further samples.
2. 7ml of serum from saline fed BALB/c mice were passed through the RaBSA affinity column equilibrated in PBS pH 7.3, for 1 hour at room temperature. The unbound material was collected and the bound fraction eluted with glycine HCl pH 2.4. The bound and unbound fractions were treated as previously described.
3. Serum from OVA fed donor mice (unmanipulated).
4. Unbound material obtained from the serum of OVA fed BALB/c mice after passage through the MaOVA column. The serum was circulated through the column for 1 hour, the unbound material was collected and the column eluted as before. The unbound material was then recirculated through the column for a fu rth er 1 hour at room tem p eratu re to rem ove any rem aining OVA. The unbound m aterial from the second run was then co n cen trated as b efo re. Residual bound material was eluted with IM glycine HCl pH 2.4.