Statistical analysis

The normality of the data was confirmed using an Anderson Darling test. To assess the effect of FIV on the proportion of cells undergoing apoptosis and necrosis in sections 3.2.1 - 3.2.3, a general linear model of ANOVA was used. The following statistics were assessed: geometric mean fluorescence intensity (MFI) of Annexin-V, MFI of 7AAD, percentage of viable (double

negative) cells, percentage of apoptotic (single positive) cells, and the percentage of necrotic (double positive) cells. Model factors included ‘time’ (day) and ‘treatment’.

Firstly, the 3 different control conditions were assessed for any differences in each parameter over time (fresh media, conditioned media, virus filtrate). Secondly, the overall effect of FIV infection (all isolates combined) compared to a pooled control was assessed for each parameter. This was assessed over time, as well as at the individual time points. Finally, differences among FIV isolates were examined over time, and at individual time points

(treatments – pooled results from all three control conditions, MUVTH002, MUVTH003,

CVK001, RVC009 and PetF14). Where the effect of treatment reached significance, a Bonferroni pairwise comparison was used to determine which means were different.

Differences in the cell concentration were assessed using one-way ANOVA at each time point to compare the mean cell flow rate (cells/sec) for each treatment. Tukey’s pairwise comparison was used to determine which means were significantly different.

The effect of virus on mitogen-induced cell proliferation was assessed using a general linear model of ANOVA, with the model factors of ‘treatment’ and the random variable ‘cat’. Within each treatment group, the mean absolute count per minute (cpm) for each cat was normalised by subtracting the mean cpm of unstimulated wells from the cpm of stimulated wells. The stimulation index (SI) was calculated for each treatment as the ratio of the cpm of the stimulated wells and the mean cpm of unstimulated wells for each cat. The normalised cpm and the SI were assessed separately as responses in the model.

All statistical analyses were performed using Minitab® _{Statistical Software (Version 17,}

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3.3.

Results

3.3.1.Development and optimisation of a flow-cytometric apoptosis assay 3.3.1.1.PBMC separation

Attempts to separate feline PBMC from whole blood using the CPT™ vacutainers were unsuccessful, with granulocyte contamination consistently occurring (as shown by a population of small, granular cells on the FSC/SSC profiles) (Figure 3-1). In contrast, separation of PBMC using Ficoll-Paque density gradient medium resulted in a clear population of PBMC with minimal to no granulocyte contamination. Consequently, Ficoll-Paque was used for PBMC separation in all subsequent experiments.

a) b)

Figure 3-1 Flow cytometry graph of PBMC separation.

FSC/SSC plots demonstrate PBMC separated from a single blood sample using two different methods a)CPT™ vacutainers and b) density gradient medium (Ficoll-Paque, GE Healthcare Life Sciences). The x-axis represents forward light scatter (FSC), and is proportional to cell size. Side light scatter (SSC) is represented on the y-axis and is a measure of internal complexity (i.e. granularity). Distinct populations of granulocytes and PBMC are circled separately and labelled. The numbers represent the percentage of all cells that belong to each population. Samples prepared using the CPT™ vacutainers were contaminated by granulocytes (62.8% of all cells), whereas samples prepared using the Ficoll-Paque density gradient medium showed minimal to no contamination with granulocytes. Using this latter method, 76.6% of cells isolated were PBMC, including populations of lymphocytes and monocytes.

3.3.1.2.Establishment of a positive control for the apoptosis assay using feline PBMC Treatment of PBMC with camptothecin resulted in apoptosis, but there was no difference in the percentage of cells undergoing apoptosis following treatment with the 2 different concentrations of camptothecin (p=0.681). There was also no significant difference over time

(within the 3 hour period measured) in the percentage of apoptotic cells (p=0.413) (Figure 3-2 and Figure 3-3).

a) b)

Figure 3-2 Flow cytometry graphs demonstrating camptothecin-induced apoptosis.

Graphs show Annexin-V fluorescence (FL4, x-axis) and 7AAD fluorescence (FL3, y-axis) of PBMC treated with 20 μM camptothecin for a) 1 hr and b) 3 hrs. Each dot represents a cell that is either viable (located in quadrant 4 – annexin-V negative / 7AAD negative), apoptotic (quadrant 3 – annexin-V positive / 7AAD negative) or necrotic (quadrant 2 – annexin-V positive / 7AAD positive). Numbers represent the percentage of total cells located within each quadrant. There were no significant differences between the two time points in either the percentage of apoptotic cells (p=0.413) or necrotic cells (p=0.545).

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Figure 3-3 The effect of time and concentration on camptothecin-induced apoptosis.

Feline PBMC were cultured in triplicate, in the presence of 2 different concentrations of camptothecin (6 and 20 μM) and harvested at 3 different time points (1, 2, 3 hrs). The percentage of cells undergoing apoptosis (annexin-V positive / 7AAD negative cells) was measured using a dual-colour flow cytometric assay. There was no difference in the percentage of apoptotic cells in cultures treated with 6 μM versus 20μM camptothecin (p=0.681).

3.3.1.3.Optimisation of cell culture conditions

3.3.1.3.i.The effects of media composition on PBMC viability

The effect of media composition on cell viability and apoptosis at different time points (days 1, 2 and 3) was assessed using the dual-colour flow cytometric apoptosis assay (Figure 3-4). All cultures except those treated with ConA showed >80% viability of PBMC on day 1. In all cultures, cell viability decreased by day 3, as shown by the reduced proportion of double negative cells in cultures on days 2 and 3. There was no effect of using an alternative media containing less serum, supplementing media with IL-2 or adding conditioned media to the cultures.

Figure 3-4 The effect of media composition on cell viability over time.

Feline PBMC were cultured in triplicate using 5 different media formulations (1-5) for a period of 3 days (with 1 culture harvested at each time point). Dual-colour flow cytometry was performed as described in section 3.2.1.2 to assess cell viability (defined as Annexin-V negative / 7AAD negative cells). The percentage of viable cells declined over time. Cultures containing media 5 had reduced cell viability compared to other media formulations. Media formulations consisted of: 1. RPMI with 10% FBS, 1% glutamine and 1% penicillin/streptomycin; 2. RPMI with 8.5% FBS, 0.5% glutamine, 0.5% penicillin/streptomycin, and 1% each of additional NEAA, HEPES and Na Pyruvate; 3. Formulation 1 + 10 units/mL of IL-2; 4. Formulation 1 + conditioned media (1mL added to 9mL of media 1); 5. Formulation 1 + 5 μg/mL of ConA (see Table 3-1 for details).

3.3.1.3.ii.The effects of ConA on cell viability

The effect of ConA on cell viability was inversely related to concentration, with cell viability decreased to <40% in media with >3 μg/mL of ConA (Figure 3-5). Cellular activation increased with the concentration of ConA, with 65.8% of cells expressing CD25 at a concentration of 5 μg/mL of ConA, versus 51.8% at a concentration of 1 μg/mL of ConA. Treatment of cells with ConA at a concentration of 1 μg/mL for 1 day was determined to be optimal for activation of lymphocytes with the least effect on cell viability.

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Figure 3-5 The effect of ConA on cell viability and cellular activation.

Feline PBMC were cultured for a period of 1 day in the presence of 3 different concentrations of ConA (1, 3 and 5 μg/mL). The negative control consisted of PBMC cultured in the absence of ConA (0 μg/mL). Dual-colour flow cytometry was performed as described in section 3.2.1.2 to assess cell viability (represented by the percentage of Annexin-V negative / 7AAD negative cells) and cellular activation (represented by the percentage of CD25 positive cells). Each square indicates the percentage of cells that became activated in the presence of each concentration of ConA. Each cross represents the percentage of viable cells at each concentration of ConA. The intercept of the two lines represents the optimal concentration of ConA required to activate cells yet maintain viability (1.0 μg/mL).

3.3.1.4.FIV-induced apoptosis in ConA stimulated feline PBMC

Stimulation of feline PBMC with ConA (1 μg/mL) resulted in increased apoptosis at day 7 when compared to unstimulated cells (26.8% versus 12.9%). Infection of ConA stimulated cells with FIV did not increase the percentage of apoptotic cells at day 7 (24.4% in FIV infected cultures versus 26.8% in uninfected cultures). At day 2, ConA stimulated cells infected with FIV showed the highest proportion of necrosis (27.3%), but this was only slightly higher than cultures of uninfected, ConA stimulated cells (24.8%). By day 7, all cells had undergone extensive necrosis (>40%), including those in the untreated cultures (Figure 3-6).

a)

b)

Figure 3-6 The effect of ConA and FIV infection on apoptosis and necrosis of feline PBMC.

Cells were cultured in triplicate, and stimulated with ConA (1.0 μg/mL) and infected with an FIV isolate (MUVTH002). Controls consisted of unstimulated / uninfected PBMC and ConA stimulated / uninfected PBMC. Dual-colour flow cytometry was performed as described in section 3.2.1.2 to assess a) apoptosis (represented by the percentage of Annexin-V positive / 7AAD negative cells and shown along the y-axis) at days 2 and 7, and b) necrosis (represented by the percentage of Annexin-V positive / 7AAD positive cells and shown along the y-axis) at days 2 and 7. There was no effect of FIV infection on the percentage of cells undergoing apoptosis or necrosis when compared to ConA stimulated / uninfected PBMC.

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3.3.1.5.FIV-induced apoptosis in unstimulated feline PBMC

Infection of unstimulated PBMC with FIV did not result in increased apoptosis or necrosis after 2 days when compared to uninfected, unstimulated cells (13.2% versus 12.6%).

3.3.1.6.FIV-induced apoptosis in MYA-1 cells

3.3.1.6.i.Establishment of a positive control for the apoptosis assay using MYA-1 cells Treatment of MYA-1 cells with 20 μM camptothecin resulted in increased apoptosis at all time points when compared to untreated cells. The percentage of apoptotic cells increased over time from 2 to 4 hrs, but there was no additional increase in apoptosis when cells were treated for 18 hrs (untreated 11.2%, 2 hrs camptothecin 18.3%, 4 hrs camptothecin 20.2%, 18 hrs camptothecin 20.5% apoptosis) (Figure 3-7 and Figure 3-8).

a) b)

Figure 3-7 Flow cytometry graphs demonstrating camptothecin-induced apoptosis in MYA-1 cells.

Representative graphs showing Annexin-V fluorescence (FL4, x-axis) and 7AAD fluorescence (FL3, y-axis) on a)

untreated MYA-1 cells and b) MYA-1 cells treated with 20 μM camptothecin for 2 hrs. Quadrant 4 represents viable cells, quadrant 3 represents apoptotic cells and quadrant 2 represents necrotic cells and figures indicate the percentage of cells in each quadrant. Treatment with camptothecin resulted in increased apoptosis and necrosis when compared to untreated cells.

Figure 3-8 The effect of time on camptothecin-induced apoptosis of MYA-1 cells.

Cells were incubated with 20 μM camptothecin for a period of 2 hrs, 4 hrs and overnight (18 hrs). Untreated cells were included as negative controls (0 hrs). Dual-colour flow cytometry was performed as described in section 3.2.1.2 to assess apoptosis in MYA-1 cells, and the percentage of Annexin-V positive / 7AAD negative cells (single positive, “sgl pos” are shown along the y-axis. Apoptosis was increased at 4 hrs versus 2 hrs of treatment, but there was no benefit of prolonged treatment beyond 4 hrs in this study.

3.3.1.6.ii.FIV-induced apoptosis in MYA-1 cells

In the initial pilot study using unquantified virus (supernatant from MYA-1 infected cells), FIV infection did not result in increased apoptosis and necrosis on day 1, when compared to uninfected controls (mean 13.2% versus 11.2%, p=0.245, Figure 3-9).

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a) b)

Figure 3-9 Flow cytometry graphs demonstrating the effect of FIV infection on apoptosis in MYA-1 cells. Representative graphs showing Annexin-V fluorescence (FL4, x-axis) and 7AAD fluorescence (FL3, y-axis) on a)

uninfected MYA-1 cells and b) FIV (RVC009) infected MYA-1 cells after 1 day. Quadrant 4 represents viable cells, quadrant 3 represents apoptotic cells and quadrant 2 represents necrotic cells and figures indicate the percentage of cells in each quadrant. Infection with FIV did not result in increased apoptosis and necrosis when compared to uninfected controls (p=0.245).

3.3.2.Comparison of apoptosis and necrosis of MYA-1 cells induced by different isolates of