P2 residues in peptides released from Hb following digestion by <i>Fhe</i>CL1.

<p>Analysis of frequency by which amino acids occur at the P2 position from the peptide bonds cleaved by <i>Fhe</i>CL1 in Hb α- and β-chains (corresponding to the 10 min reactions shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000369#pntd-0000369-g006" target="_blank">Figure 6</a>). The y axis represents the frequency of a particular residue at the P2 position of the haemoglobin substrates and the x axis shows the amino acids as represented by the one-letter code.</p

Characterisation of hydrolytic activity of <i>Fhe</i>CL1 on Hb.

<p>(A) Progress of digestion of Hb by recombinant <i>Fhe</i>CL1. Purified haemoglobin (Hb, lane 1) was digested by <i>Fhe</i>CL1 in 0.1 M sodium acetate buffer, pH 4.0, containing 1 mM GSH and 1 mM EDTA at 37°C. Reactions were stopped at time 0 and at various time-points (indicated on x axis) by the addition of the cysteine protease inhibitor E-64 and analysed on 4–12% Bis-Tris NuPage gels. The arrow indicates the position of <i>Fhe</i>CL1 (25 kDa) that was not degraded in the reaction. Molecular mass markers are shown on the left. (B) Map of Hb α- and β-chains indicating sites of <i>Fhe</i>CL1 cleavage the substrates. Cleavage sites within Hb present in 10 min reactions (arrows) compared to cleavages that occur with longer incubation times (120 min, arrowheads) as determined by nanoLc-MS/MS.</p

Influence of pH on zymogen pro<i>Fhe</i>CL1 autocatalytic activation and activity of mature <i>Fhe</i>CL1.

<p>(A) Analysis by SDS-PAGE of the activation of 0.2 mg/ml <i>Fhe</i>proCL1 to mature <i>Fhe</i>CL1 in 0.1 M sodium acetate buffer, pH 4.5. The zymogen, mature enzyme and degraded prosegment are indicated by arrowheads. (B) Kinetic study of the activation of 5 nM <i>Fhe</i>proCL1 between pH 4.0 and pH 7.0 in the presence of 2 µM Z-Phe-Arg-NHMec. (C) Relative k_cat/K_m values for the hydrolysis of 0.5 µM Z-Phe-ArgN-Mec by 0.14 nM mature <i>Fhe</i>CL1 at 37°C.</p

pH dependency of <i>Fhe</i>CL1 hydrolytic activity against protein substrates Hb and ovalbumin.

<p>(A) Hb incubated alone in solutions buffered in the ranges pH 3.5–pH 8.0; (B) Hb incubated with <i>Fhe</i>CL1 in the same buffers at pH 3.5–pH 8.0; (C) Ovalbumin incubated alone in solutions buffered in the ranges pH 3.5–pH 8.0, and (D) Ovalbumin incubated with <i>Fhe</i>CL1 in the same buffers at pH 3.5–pH 8.0. Digests were analysed by 15% SDS-PAGE. Molecular size markers are indicated on the left.</p

Stability of the zymogen pro<i>Fhe</i>CL1Gly²⁵ and mature <i>Fhe</i>CL1 at various pH values.

<p>(A) Far-UV CD spectra of 5.3 µM <i>Fhe</i>proCL1Gly²⁵ in 50 mM sodium acetate buffer, pH 4.0 and in 50 mM sodium phosphate buffer, pH 7.5. (B) Enzymatic stability of 6.0 µM mature <i>Fhe</i>CL1 at 37°C and in 0.1 M buffers over the pH range 2.5–9.0. Enzyme activity was monitored at various time-points by diluting aliquots of the reactions into 0.1 M sodium acetate buffer, pH 5.5, containing 3 mM DTT before addition of 5 µM Z-Phe-Arg-NMec.</p

Analysis of peptides released from Hb following digestion by <i>Fhe</i>CL1.

<p>Frequency (expressed as a percentage) of peptides of varying length released following proteolysis of Hb alpha and beta chains by <i>Fhe</i>CL1.</p

Treatment of NOD mice with FhES prevents the development of autoreactive immune responses.

<p>Female NOD mice (at four weeks of age; n = 5) were injected intraperitoneally with 10 µg of FhES (or PBS; vehicle control) on alternate days for a total of six injections. At (A) 24 h and (B) 7 weeks after the final injection spleen cells were isolated, stimulated with auto-antigen (insulin, 10 µg/ml) for 72 h and cell supernatants were assayed for IFN-γ. Data shown is representative of 3 independent experiments (C) Titres of insulin-specific IgG1 and IgG2a were measured in the sera of PBS- and FhES-treated mice (n = 15) at 15 weeks of age by ELISA. The data shown is representative of 3 independent experiments and displays the inverse of the end point serum dilution. (D) Ratios of IgG1 and IgG2a autoantibodies specific for insulin and (E) GAD in the sera of PBS- and FhES-treated mice (n = 15).</p

FhES treatment modulates the phenotype of immune cells in the pancreatic draining lymph nodes of NOD mice.

<p>Four-week old female NOD mice were treated with 10 µg of FhES or PBS intraperitoneally on alternate days for a total of six injections. The cellular composition within the PLNs was examined by flow cytometry 24 h after the final injection (n = 6; data representative of 5 independent experiments). (A) Absolute cell numbers and percentages of B220⁺ B cells and CD3⁺ T cells in the PLNs; (B) representative plots of the proportions of CD3⁺ T cells; (C) subsets of CD3⁺ T cells; (D) proportion and absolute numbers of CD4⁺CD25⁺Foxp3⁺ CD3⁺ T cells; (E) B220⁺ B cells; (F) representative dot plots of proportions (left panel), and absolute numbers (right panel) of IL-10 secreting CD19⁺ B cells within the CD19⁺ gate; (G) expression of Ym1, Retlna, Ear 11 and Arg-1 by quantitative realtime RT-PCR presented as fold change in expression, calculated compared to the average expression of the PBS cohort (each data point represents a single mouse; n = 6; data representative of at least 2 independent experiments).</p

Treatment of NOD mice co-incident with the initiation of autoimmunity prevents T1D.

<p>Four-week old female NOD mice were injected intraperitoneally with FhES (10 µg in 100 µl sterile PBS) or vehicle (PBS), on alternate days, for a total of six injections. (A) Blood glucose levels were monitored and animals were sacrificed when they became diabetic (as defined by two consecutive blood glucose concentrations ≥14 mmol/L). The graphs represent an analysis of the age at which each animal was sacrificed and was performed using survival analysis. Data shown is from one of three independent experiments, all of which produced the same outcome. (B–E) Pancreas isolated from mice at 10 (n = 13), 15 (n = 15), 23 (n = 16) and 30 (n = 16) weeks of age were graded for insulitis on a scale of 0–4; whereby 0 = healthy islet or mild peri-insular mononuclear cell infiltration, 1 = infiltration up to 25% of islet mass, 2 = infiltration up to 50% of islet mass, 3 = infiltration from 50% up to 75% of islet mass, and 4 = less than 25% of islet mass present. The proportion of islets with each grade of insulitis is shown.</p

Peritoneal FhES-induced regulatory M2 macrophages expand Foxp3⁺ regulatory T cells <i>ex vivo</i>.

<p>(A) Four-week old female NOD mice were treated with 10 µg of FhES, SEA or PBS intraperitoneally on alternate days for a total of six injections. Peritoneal macrophages were harvested 24 h after the final injection and co-cultured with splenocytes isolated from age matched naive NOD mice in the presence of anti-CD3 (2 µg/ml) for 72 h. (B) Splenocytes were co-incubated with FhES (20 µg/ml), SEA (50 µg/ml), or PBS in the presence of anti-CD3 antibody (2 µg/ml) for 72 h. Representative flow cytometry dot plots are shown with the numbers representing the percentage of cells expressing both CD25⁺ and Foxp3. Histograms show the means of triplicate samples ± SEM, and are representative of two independent experiments. (C) Pancreata were isolated from female NOD mice (n = 3) 24 h after the final (sixth) FhES or PBS treatment and the expression levels of Arg1, Ym1, Retlna and Ear 11 were determined by quantitative realtime RT-PCR. All fold changes in expression levels were calculated compared to the average expression levels of the PBS cohort. Data shown is representative of at least two repeat experiments.</p