Ultrastructure of <i>Giardia duodenalis</i> by TEM.

Untreated G. duodenalis trophozoites showing normal structure and morphology (a). Trophozoite coronal section (c). A coronal view of a trophozoite demonstrates the nuclei (N), endoplasmic reticulum (ER), flagella (F), vacuoles (V), ventral disk (VD), lateral crest (LC) and ventrolateral flange (VLF). The same sections (b, d) of treated parasites show swelling trophozoites, with an increased size, and evident alterations of their cellular membrane and with a vacuolar degenerative pattern (x6,700). Note in the coronal section the severely damaged nuclei, nuclear membrane rupture, loss of the chromatin, flanges and ventral disk rupture (x6,700).</p

Statistical evaluation of different parameters in <i>ex-vivo</i> duodenal tissue cultures, before and after treatment.

(a) Significant reduction in the number of viable Giardia cells counted in biopsies treated with Slab51 ultrafiltered supernatant at 12h and 18h. (b) Statistical comparison of TUNEL positive nuclei before and after the same treatment at 24h and 48h. (c) Statistical comparison of level of cellular viability and replication by Ki67 nuclear assessment at 12h and 18h. (d) Statistical confrontation of Caspase3 positive cells before and after the same treatment at 12h and 18h. Caspase3 expression in association with the level of the TUNEL expression, as showed in Fig 2B, indicate the entire fraction of apoptotic cell because these two apoptotic markers are expressed in subsequent time.</p

<i>In vitro</i> and <i>ex vivo</i> evaluation of the anti-<i>Giardia duodenalis</i> activity of the supernatant of Slab51 (SivoMixx) - Fig 1

(a) Growth inhibition of Giardia duodenalis trophozoites by fresh (Slab51 FS) and 56°C (Slab51S 56°C) and 90°C (Slab51S 96°C) heat-treated Slab51 supernatants. The number (n x104) of G. duodenalis trophozoites are expressed as average and standard deviation of trophozoites counted in three replicates after 24 and 48 h observation periods.; (b) Adhesion inhibition of G. duodenalis trophozoites by fresh (Slab51 FS) and 56°C (Slab51S 56°C) and 90°C (Slab51S 96°C) heat-treated Slab51 supernatants. Attached trophozoites have been expressed as the mean percentage of attached G. duodenalis trophozoites in relation to the total number of G. duodenalis trophozoites counted after 24 and 48 hours in each culture and in three replicates.</p

<i>Giardia duodenalis</i> in negative control (NC) group.

G. duodenalis trophozoites showed an intact morphology also after 18h (a), and the apoptosis rate of enterocytes (arrows), as demonstrated in b, increased progressively during the experiment, by the combined effect of the infection and the ex-vivo condition. (H&E, scale bar: a, 10 μm–b, 50 μm).</p

<i>Ex-vivo</i> intestinal tissue, from mice treated with Slab51 ultrafiltered fresh supernatant.

At 18h post-infection with Giardia duodenalis, biopsies showed a preserved morphology and viability as demonstrated by H&E stain (a) and Ki67 enterocytes expression (b). In these samples, a low number of TUNEL+ enterocytes is observed (c). A similar pattern of expression of Caspase-3 indicates a low apoptotic rate in these samples (d). Presence of inflammatory cells with a diffuse and non-polarized pattern of infiltration is also observed in these biopsies (H&E, and IHC with Mayer Haematoxilin nuclear counterstain, scale bar 400 μm).</p

<i>Ex-vivo</i> intestinal tissue, from negative control (NC) group.

Section stained with H&E revealed a diffuse epithelial loss with inflammatory cells infiltration polarized under the destroyed intestinal epithelium. Note the reinforcement of inflammatory cells around intestinal glands (a). Villi are partially damaged (arrows), while in part they are totally flat or in any case strongly tuned (arrowheads) due to the effect of the strong colonization-adhesion of Giardia duodenalis trophozoites on the surface of the epithelium, which has become detached in many areas of the mucosa. Note the reinforcement of inflammatory cells around intestinal glands (stars). Ki67 nuclear staining evidenced an apparently higher number of positive cells because many inflammatory cells showed a strongly nuclear positivity (b). Note that TUNEL (c) and Caspase-3 (d) are over-expressed in these explanted intestinal samples. (H&E, and IHC with Mayer Haematoxilin nuclear counterstain, scale bar 400 μm).</p

Adhesion inhibition of <i>Giardia duodenalis</i> trophozoites by fresh (Slab51 FS) and 56°C (Slab51S 56°C) and 90°C (Slab51S 96°C) heat-treated Slab51 supernatants.

Attached trophozoites have been expressed as the mean percentage of attached G. duodenalis trophozoites in relation to the total number of G. duodenalis trophozoites counted after 24 and 48 hours in each culture and in three replicates.</p

Growth inhibition of <i>Giardia duodenalis</i> trophozoites by fresh (Slab51 FS) and 56°C (Slab51S 56°C) and 90°C (Slab51S 96°C) heat-treated Slab51 supernatants.

The number (n x104) of Giardia duodenalis trophozoites are expressed as average and standard deviation of trophozoites counted in three replicates after 24 and 48 h observation periods.</p

Correlation of CFU/neutrophils in the BAL of susceptible A/J and resistant C3H/HeOuJ <i>P</i>. <i>aeruginosa</i>-infected mice.

<p>CFUs and neutrophils recovered in the BAL were plotted for the two murine strains during 18 hours of <i>P. aeruginosa</i> infection. Dots represent CFUs and neutrophils in individual mice. Blue dots represent A/J mice (n = 12, for 6 and 12 hours, n = 9 for 18 hours) and green dots C3H/HeOuJ mice (n = 12 for each time). The data are pooled from two independent experiment.</p

Cytokines and chemokines levels in lung homogenates of susceptible A/J and resistant C3H/HeOuJ mice infected with <i>P</i>. <i>aeruginosa</i> during a time course.

<p>Data are expressed as median of pg/500 ug lung.</p><p>Statistical analysis for comparison of A/J vs C3H/HeOuJ at each time point by the non-parametric Mann-Whitney U test (*p<0.05) is reported.</p><p>Nd: not detectable; ns: not significant.</p><p>Cytokines and chemokines levels in lung homogenates of susceptible A/J and resistant C3H/HeOuJ mice infected with <i>P</i>. <i>aeruginosa</i> during a time course.</p