SISTEMA DE SEGURIDAD ACTUAL DE PETROINDUSTRIAL

The fate of chromium labelled mature erythrocytes (^’CrRBCs) was followed in

uninfected and infected Balb/c, nude and SCID mice. This was to gain some insight into the premature removal of uninfected RBCs in infected mice. During a normal non-lethal

P. yoelii infection the parasite exclusively infects reticulocytes, therefore the elimination of the labelled cells is independent of direct destruction by the intra-cellular parasite. The pattern of removal of the injected ^^Cr RBCs, from the circulation of the three strains of mice, was studied to elucidate what would be the affect of the absence of B and/or T-cells. This was primarily to try and deduce the importance of antibody in this mechanism of premature removal of uninfected erythrocytes.

It was initially thought that SCID mice would not survive for very long once they were infected, consequently in the first experiments the ^'CrRBCs were injected 3 days post infection. The removal of the labelled cells under these conditions in infected SCIDs (CB-17s) did not significantly differ from the removal in uninfected SCID and Balb/c mice (Figure 4.1.a). The removal in infected Balb/cs, when compared to the uninfected controls , occurred at a significantly accelerated rate from day 7 post infection. Because the CPM in the SCID mice reached background levels before the infections became terminal (approximately 10 days before), the labelled erythrocytes in subsequent SCID experiments were injected 10 days post infection. This gave greater resolution at the latter time points, where CPM in the original experiments were low, and also revealed

— o— Balb/c uninfected — □— SCID uninfected —• — Balb/c infected — ■— SCID infected 9 0 - 7 0 - 6 0 - O 5 0 - 4 0 - 3 0 - T)- 2 8 0 4 6 t) 12 14 1B

Days post injection

Figure 4.1.a Clearance of autologous ^‘Cr labelled RBCs (injected 3 days (d=3) post infection) in uninfected and non-lethal P.yoelii infected Balb/c and SCID mice. Values are means ± SEM (n = 4-7).

When the radio actively labelled erythrocytes were injected 10 days post infection, a clear difference was revealed between the infected and uninfected SCIDs. The infected

Balb/cs, as before, removed the ^'CrRBCs rapidly (Figure 4.1 .b); the removal was significantly greater than uninfected Balb/cs from 14 days post infection (4 days post injection), reaching background levels at day 18. The infected SCIDs did not remove the labelled cells as rapidly as infected Balb/cs, however the removal did occur at an

enhanced rate when compared to uninfected SCID mice. The clearance of the ^^CrRBCs began at 15 days post infection (5 days post injection) in the infected SCIDs, showing a significant difference from uninfected SCIDs at day 15 and day 18 to 23 post infection. The removal of uninfected Balb/cs did not differ significantly from uninfected SCIDs in either of the two experiments (^^CrRBC injected d=3 or d=10).

— o— BaltVc uninfected — □— SOD uninfected — • — Balb/c infected — ■— SOD infected DO 9 0 - o 8 0 - 7 0 - 6 0 - O 5 0 - O 4 0 - 3 0 - 2 0- lO D - 8 8 0 2 4 D 12

Days post injection

Figure 4.1.b Clearance of autologous labelled RBCs (injected 10 days (d=l 0) post infection) in uninfected and non-lethal P.yoelii infected Balb/c and SCID mice. Values are means ± SEM (n = 8).

— o— Balb/c uninfected nude uninfected Balb/c infected nude infected DO —V- O 80 Û- 50 in 8 D 0 2 4 6

Days post injection

Figure 4.1.c Clearance of autologous ^^Cr labelled RBCs (injected 5 days (d=5) post infection) in uninfected and non-lethal P.yoelii infected Balb/c and nude mice. Values are means ± SEM (n = 5-8).

In the experiments using nude mice the radioactively labelled erythrocytes were injected 5 days post infection, which revealed a very early and rapid removal of these cells in infected nudes. Both infected Balb/c and nude mice showed an accelerated rate of removal of the ^^CrRBCs, this was significantly different from their respective uninfected controls from 7 days post infection (2 days post injection). There was no significant difference between the pattern of removal of the labelled cells in infected Balb/c and infected nude mice between day 5 and day 14 (day 0 and 11 post injection). The infected nude mice did show a slightly reduced rate of removal as their CPM approached background levels; there was a significant difference at day 15 post infection when compared to the infected Balb/cs. There was no significant difference between the uninfected nude and Balb/c mice throughout the period of ^’CrRBC removal.

Infected Balb/cs showed rapid removal of the radioactively labelled erythrocytes from very early time points (Figures 4.1.a and4.1.c), this continues through to the later stages of the infection (Figure 4. l b). When the rate of removal of the ^^CrRBC, compared to uninfected controls (CPM : % o f control), was related to the parasitaemia (Figure 4.1.d), Balb/c mice were seen to begin removing the uninfected labelled erythrocytes from the circulation at an enhanced rate with parasitaemias below 1% (approximately 0.3%). It was consistently observed with SCID mice that rate of removal did not increase until parasitaemias were approaching 10 % (approximately 8%). Preliminary experiments where the growth of the parasitaemias in SCID mice were forced to increase at a greater

rate, indicated that the removal of the labelled cells may begin sooner, in the time course of the infection, if the parasitaemia reaches 10% at an earlier stage (data not shown).

—• — Balb/c —■— SCID no-, no-

2

8

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Figure 4.1.d Relationship between enhanced rate of removal of ^^CrRBC(when compared to uninfected controls) and parasite levels in non-lethal

P.yoelii infected SCID and Balb/c mice. Values are means ± SEM (n=4-8).

The two mutant strains of mice used in this study were nudes (T-cell deficient) and SCIDs (T and B-cell deficient). In conjunction with normal immunocompetent Balb/c mice this provided all of the permutations of T and/or B-cell positivity, except for a mouse that was positive for T-cells but deficient in B-cells. Attempts were made to create a T-cell positive/B-cell negative mouse by reconstituting SCIDs with T-cells isolated from the spleens of normal Balb/c mice. Prior to attempting repopulating with purified T-cells, it was investigated whether or not the SCID mice could be successfully reconstituted with whole spleen cell preparations.

Four SCID mice were each injected (i.v.) with spleen cell preparations containing 1.6 x 10^ T-cells and 1x10^ B-cells. These mice, as well as a control group of four Balb/cs, were then injected (i.v.) with an inoculum of 10^ P. yoelii infected RBCs on the following day. The repopulated SCID mice were able to control and clear the parasite infection at approximately the same time as the Balb/cs. Having established that the SCID mice could be successfully repopulated with spleen cells, the next step was to purify a population of T-cells with which to reconstitute a group o f SCIDs.

Two techniques were tested to ascertain whether a suitable quantity of T-cells could be isolated to an acceptable degree of purity, both protocols relied on depleting spleen cell preparations of their B-cells. The use of nylon wool columns did not prove to be acceptable, the best cell recovery that could be achieved was poor at only 12%. When assayed by FACSscan the level of T-cells in the gated population had only risen from 39.2% (53.1% of the gated lymphocytes) to 53.2% (75.8% of the gated lymphocytes); the B-cells had been depleted from 36.6% ( 46.9% of the gated lymphocytes ) to 17% (24.2% of the gated lymphocytes). The use of Dynabeads proved to be much more successful (Figure 4.1.e), a cell recovery of 28% was achieved, the T-cells (assayed by FACSscan) increased from 33.8% (54.2% of the gated lymphocytes) to 75.7% (97.4% of the gated lymphocytes) and the B-cells were depleted from 28.6% (45.8% of the gated lymphocytes) to 2% (2.6% of the gated lymphocytes). An attempt was also made to purify B-cells by T-cell depletion, using anti-Thyl antibodies and guinea pig

complement. As with the nylon wool, this method o f cell depletion had a very low level of recovery (16%) and the B-cells (assessed by FACSscan) only increased from 36.3% to 61.6%, the T-cells were depleted from 42.4% to 25.2%.

BEFORE