Terminal Complement Complex (TCC) generation induced by the <i>Premolis semirufa</i>’<i>s</i> bristles extract.

<p>Samples of NHS (50 μL) were incubated with the bristles extract (175 μg/mL) or PBS, in the presence or absence of 10 mM 1,10-Phenanthroline (Phen), at 37°C for 30 min, and SC5b-9 complex present in human serum samples was measured using the MicroVue SC5b-9 Plus EIA Kit. Data are representative for two separate experiments, performed in duplicate, and the results are expressed as concentration of SC5b-9 complex per mL of human serum (ng/mL) ± SD. (***) <i>p</i> < 0.001: significant differences between the mean values obtained with the buffer and between the treatments.</p

Proteolytic action of the <i>Premolis semirufa’s</i> bristles extract on purified human C-components C3, C4 and C5.

<p>Samples of the bristles extract (2.0 μg for C3 and C4 or 3.0 μg for C5) were incubated, in the absence or presence of 10 mM PMSF or 1,10-Phenanthroline (Phen), with human C3 (3 μg) <b>[A]</b>, human C4 (3 μg) <b>[B]</b> and human C5 (3 μg) <b>[C]</b> at 37°C for 1 h. Proteolytic activity was examined on 10% polyacrylamide gel under reducing conditions and stained by silver. In the 1^st lanes of gels: electrophoretic separation of purified components incubated with PBS as a positive control; 2^nd lanes: incubation of purified components with the extract; 3^rd lanes: incubation of the mixture with PMSF; 4^th lanes: incubation of the mixture with Phenanthroline and 5^th lanes: electrophoretic separation of the extract. α (115 kDa) and β (75 kDa) for C3; α (93 kDa), β (75 kDa) and γ (33 kDa) for C4 and α (115 kDa) and β (75 kDa) for C5.</p

Chromatography of the <i>Premolis semirufa’s</i> bristles extract and isolation of the protease responsible for complement activation.

<p><b>[A]</b> Chromatography of 1 mg of the extract on a FPLC-GP-250 Plus system using a molecular exclusion column (Superdex 200 10/300 GL), in 0.05 M ammonium bicarbonate buffer, pH 7.4. <b>[B]</b> SDS-PAGE of fractions screened by their ability to cleave the component C3. <b>[C]</b> SDS-PAGE (12%) of 10 μg of the extract and 0.25 μg of the fraction 10, under reducing conditions, followed by silver staining.</p

Proteolytic action of Ps82 on purified human C components C3, C4 and C5.

<p>Samples of 0.1 μg of Ps82 were incubated, in the absence or presence of 10 mM PMSF or 1,10-Phenanthroline (Phen), with human C3 (3 μg) <b>[A]</b>, human C4 (3 μg) <b>[B]</b> and human C5 (3 μg) <b>[C]</b> at 37°C for 1 h. Proteolytic activity was examined on 10% polyacrylamide gel under reducing conditions and stained by silver. In the 1^st lanes of gels: electrophoretic separation of purified components incubated with PBS as a positive control; 2^nd lanes: incubation of purified components with Ps82; 3^rd lanes: incubation of the mixture with PMSF and 4^th lanes: incubation of the mixture with Phenanthroline. α (115 kDa) and β (75 kDa) for C3; α (93 kDa), β (75 kDa) and γ (33 kDa) for C4 and α (115 kDa) and β (75 kDa) for C5.</p

Action of Ps82 on the complement pathways.

<p>Samples (50 μL) of normal human serum (NHS) were pre-incubated with 0.25, 0.33 or 0.5 μg/mL of Ps82 or with 175 μg/mL of the <i>Premolis semirufa</i>’s bristles extract. The mixtures were pre-incubated for 30 min for the alternative pathway <b>[A]</b> or 1 h for the classical pathway <b>[C]</b> at 37°C, and then assayed for the residual complement activity in haemolytic tests. For lectin pathway <b>[B]</b>, samples of 50 μL of NHS were pre-incubated, in the presence or absence of 10 mM 1,10-Phenanthroline, with 0.5 μg/mL of Ps82 or 175 μg/mL of the <i>Premolis semirufa</i>’s bristles extract for 30 min at 37°C and then evaluated for the residual complement activity by ELISA. Alternatively, samples of NHS incubated with Ps82 or the <i>Premolis semirufa</i>’s bristles extract were evaluated in conditions for activation of the classical pathway, by the deposition of complement component C4b by ELISA <b>[D]</b>. Data are representative for three separate experiments, performed in duplicate, and the results are expressed as percentage of haemolysis (%) ± SD (for A and C) and percentage of C4b deposition (%) ± SD (for B and D) in relation to the control samples (NHS + buffer) and between the treatments. (*) <i>p</i> < 0.05 (**) <i>p</i> < 0.01 and (***) <i>p</i> < 0.001. The symbol (#) indicates significant differences between the extract and Ps82.</p

Human serum anaphylatoxins generation induced by Ps82.

<p>Samples of NHS (50 μL) were incubated with Ps82 (0.33 μg/mL) or the <i>Premolis semirufa’s</i> bristles extract (175 μg/mL), in the presence or absence of 10 mM 1,10-Phenanthroline (Phen), for 30 min at 37°C. The generation of the anaphylatoxins (C3a, C4a and C5a) was measured using the kits "Human C3a ELISA Kit, Human C4a ELISA Kit and Human C5a ELISA Kit". Data are representative for two separate experiments, performed in duplicate, and the results are expressed as concentration of each anaphylatoxin per mL of human serum (ng/mL) ± SD. (*) <i>p</i> < 0.05 (**) <i>p</i> < 0.01 and (***) <i>p</i> < 0.001: significant differences between the mean values obtained with the Buffer and the treatments.</p

Anaphylatoxins generation from purified human C5 induced by the <i>Premolis semirufa’s</i> bristles extract and Ps82.

<p>Samples of 3 μg of the extract or 0.1 μg of Ps82 were incubated, in the absence or presence of 10 mM PMSF, with human C5 (3 μg) for 30 min at 37°C. The generation of the anaphylatoxin C5a was measured using the "Human C5a ELISA Kit". Data are representative for two separate experiments, performed in duplicate, and the results are expressed as concentration of anaphylatoxin per mL (ng/mL) ± SD. (**) <i>p</i> < 0.01 and (***) <i>p</i> < 0.001: significant differences between the mean values obtained with the Buffer and the treatments.</p

Human serum anaphylatoxins generation induced by the <i>Premolis semirufa’s</i> bristles extract.

<p>Samples of NHS (50 μL) were incubated with the bristles extract (175 μg/mL) or PBS, in the presence or absence of 10 mM 1,10-Phenanthroline (Phen), at 37°C for 30 min. The generation of the anaphylatoxins (C3a, C4a and C5a) was measured using the <i>Human Anaphylatoxin Cytometric Bead Array</i> (CBA). Data are representative for two separate experiments, performed in duplicate, and the results are expressed as concentration of each anaphylatoxin per mL of human serum (ng/mL) ± SD. (*) <i>p</i> < 0.05 and (**) <i>p</i> < 0.01: significant differences between the mean values obtained with the buffer and between the treatments.</p

Activity of the <i>Premolis semirufa’s</i> bristles extract on the alternative and lectin pathways.

<p><b>[A]</b> Samples (50 μL) of normal human serum (NHS) were pre-incubated with increasing concentrations of the bristles extract or PBS, at 37°C for 30 min, and the residual haemolytic activity of the mixtures was measured. <b>[B]</b> The residual lectin pathway complement activity was assessed by incubation of NHS with the bristles extract (175 μg/mL), in the presence or absence of 1,10-Phenanthroline (10 mM), at 37°C for 30 min, and added to plates coated with mannan (100 μg/mL). Data are representative for three separate experiments, performed in duplicate, and the results are expressed as percentage of AP₅₀ reduction (%) ± SD (for A) and percentage of C4b deposition (%) ± SD (for B). (*) <i>p</i><0.05, (**) <i>p</i> < 0.01 and (***) <i>p</i> < 0.001: significant differences between the mean values obtained with PBS and the treatments.</p

Proteolytic activity of Ps82 analyzed by zymography.

<p>Samples of 0.05 μg of Ps82 or 0.5 μg of the extract were incubated in the absence (Lane 1) or presence of 10 mM 1,10-Phenanthroline (Lane 2) or PMSF (Lane 3), inhibitors of metallo- and serineproteases, respectively, submitted to electrophoresis and subsequently incubated, at 37°C for 12 h, in 50 mM Tris-HCl, pH 8.3. Following incubation, the gel was stained with 0.2% Coomassie Brilliant Blue R-250 and the gelatinolytic activity was detected as unstained bands on a dark background. The figure represents the negative of the original image.</p