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expression screen to be subtraction background (Fig. 2B). Their relative abundance may explain their incom- plete subtraction from the sporocyst library. The guide- lines of the technique provided by the manufacturer

Figure 2 expression screen to be subtraction background (Fig. 2B). Their relative abundance may explain their incom- plete subtraction from the sporocyst library. The guide- lines of the technique provided by the manufacturer

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Experimental groups of pigs Table 1
Experimental groups of pigs Table 1
Staining methods used for detection of various types of mucins in tissue sections
Staining methods used for detection of various types of mucins in tissue sections
Fig. 1. HID/AB staining in the duodenum of SPF pig (A) and conventional pig (B) infected with Trichinella spiralis, 14 days post-infection. Note that staining is confined to goblet cells of the villi and crypts and that sulphated mucin (black), carboxylated (blue) and mixed (sulphated and carboxyl- ated; black and blue)-containing goblet cells are visible. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 1. HID/AB staining in the duodenum of SPF pig (A) and conventional pig (B) infected with Trichinella spiralis, 14 days post-infection. Note that staining is confined to goblet cells of the villi and crypts and that sulphated mucin (black), carboxylated (blue) and mixed (sulphated and carboxyl- ated; black and blue)-containing goblet cells are visible. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. AB/PAS staining in the duodenum of SPF pig (A) and conventional pig (B) infected with Trichinella spiralis, 14 days post-infection. Note that staining is confined to goblet cells of the villi and crypts and that neutral mucin (magenta), acidic (blue) and mixed (neutral and acidic; purple)- contain- ing goblet cells are visible. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. AB/PAS staining in the duodenum of SPF pig (A) and conventional pig (B) infected with Trichinella spiralis, 14 days post-infection. Note that staining is confined to goblet cells of the villi and crypts and that neutral mucin (magenta), acidic (blue) and mixed (neutral and acidic; purple)- contain- ing goblet cells are visible. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Mean area of goblet cells containing sulphated mucins (labelled black with HID-AB stain) in the villi of the duodenum of experimental groups of pigs (TS-C: Group of conventional pigs infected with Trich- inella spiralis, TS-SPF: Group of SPF pigs infected with Trichinella spiralis, NT-C: Group of conventional pigs not infected with Trichi- nella spiralis, NT-SPF: Group of SPF pigs not infected with Trichi- nella spiralis).
Fig. 3. Mean area of goblet cells containing sulphated mucins (labelled black with HID-AB stain) in the villi of the duodenum of experimental groups of pigs (TS-C: Group of conventional pigs infected with Trich- inella spiralis, TS-SPF: Group of SPF pigs infected with Trichinella spiralis, NT-C: Group of conventional pigs not infected with Trichi- nella spiralis, NT-SPF: Group of SPF pigs not infected with Trichi- nella spiralis).
Histomorphometric parameters measured on duodenal and jejunal sections
Histomorphometric parameters measured on duodenal and jejunal sections
Colours defined according to grey values of red, green, and blue (pix- els) used by the histomorphometry system for detecting the colours of the stains ’ Colour mean = (red * 65.536) + (green * 256) + blue.
Colours defined according to grey values of red, green, and blue (pix- els) used by the histomorphometry system for detecting the colours of the stains ’ Colour mean = (red * 65.536) + (green * 256) + blue.
Fig. 5. Mean area of goblet cells containing sulphated mucins (labelled black with HID-AB stain) in the crypts of the jejunum of experimental groups of pigs (TS-C: Group of conventional pigs infected with Trich- inella spiralis, TS-SPF: Group of SPF pigs infected with Trichinellc spiralis, NT-C: Group of conventional pigs not infected with Trichi- nella spiralis, NT-SPF: Group of SPF pigs not infected with Trichi- nella spiralis). Glycosylation of mucins in the small intestine of the pig is affected by infection with T. spiralis with general increases in the staining of acidic mucins in goblet cells. The expression of neutral mucins (AB-PAS, magenta) was affected very little by the parasitic challenge, while acidic mucins (AB-PAS, blue) appeared to be involved more extensively in the intestinal response to parasitic infection. In contrast, acidic mucins in the small intestine decrease during the intestinal response to T. spiralis in the rat (Young, 1995). In addition, immediately after pri-
Fig. 5. Mean area of goblet cells containing sulphated mucins (labelled black with HID-AB stain) in the crypts of the jejunum of experimental groups of pigs (TS-C: Group of conventional pigs infected with Trich- inella spiralis, TS-SPF: Group of SPF pigs infected with Trichinellc spiralis, NT-C: Group of conventional pigs not infected with Trichi- nella spiralis, NT-SPF: Group of SPF pigs not infected with Trichi- nella spiralis). Glycosylation of mucins in the small intestine of the pig is affected by infection with T. spiralis with general increases in the staining of acidic mucins in goblet cells. The expression of neutral mucins (AB-PAS, magenta) was affected very little by the parasitic challenge, while acidic mucins (AB-PAS, blue) appeared to be involved more extensively in the intestinal response to parasitic infection. In contrast, acidic mucins in the small intestine decrease during the intestinal response to T. spiralis in the rat (Young, 1995). In addition, immediately after pri-
Fig. 4. Mean area of goblet cells containing sulphated mucins (labelled black with HID-AB stain) in the crypts of the duodenum of experi- mental groups of pigs (TS-C: Group of conventional pigs infected with Trichinella spiralis, TS-SPF: Group of SPF pigs infected with Trichi- nella spiralis, NT-C: Group of conventional pigs not infected with Trichinella spiralis, NT-SPF: Group of SPF pigs not infected with Trichinella spiralis).
Fig. 4. Mean area of goblet cells containing sulphated mucins (labelled black with HID-AB stain) in the crypts of the duodenum of experi- mental groups of pigs (TS-C: Group of conventional pigs infected with Trichinella spiralis, TS-SPF: Group of SPF pigs infected with Trichi- nella spiralis, NT-C: Group of conventional pigs not infected with Trichinella spiralis, NT-SPF: Group of SPF pigs not infected with Trichinella spiralis).
Fig. 6. Mean glycocalyx thickness of villus outline (labelled brown with Ulex stain) in the duodenum of experimental groups of pigs (TS- C: Group of conventional pigs infected with Trichinella spiralis, TS- SPF: Group of SPF pigs infected with Trichinella spiralis, NT-C: Group of conventional pigs not infected with Trichinella spiralis, NT- SPF: Group of SPF pigs not infected with Trichinella spiralis). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. Mean glycocalyx thickness of villus outline (labelled brown with Ulex stain) in the duodenum of experimental groups of pigs (TS- C: Group of conventional pigs infected with Trichinella spiralis, TS- SPF: Group of SPF pigs infected with Trichinella spiralis, NT-C: Group of conventional pigs not infected with Trichinella spiralis, NT- SPF: Group of SPF pigs not infected with Trichinella spiralis). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 7. Ulex immunolabelling in the duodenum of pig infected with Trichinella spiralis, 14 days post-infection. Note the increased thick- ness of the glycocalyx in SPF pigs (A) compared to conventional pigs (B). Some light-brown positive goblet cells are also visible.
Fig. 7. Ulex immunolabelling in the duodenum of pig infected with Trichinella spiralis, 14 days post-infection. Note the increased thick- ness of the glycocalyx in SPF pigs (A) compared to conventional pigs (B). Some light-brown positive goblet cells are also visible.
Fig. 8. Mean glycocalyx thickness of villus outline (labelled brown with Ulex stain) in the jejunum of experimental groups of pigs (TS-C: Group of conventional pigs infected with Trichinella spiralis, TS-SPF: Group of SPF pigs infected with Trichinella spiralis, NT-C: Group of conventional pigs not infected with Trichinella spiralis, NT-SPF: Group of SPF pigs not infected with Trichinella spiralis). (For inter- pretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 8. Mean glycocalyx thickness of villus outline (labelled brown with Ulex stain) in the jejunum of experimental groups of pigs (TS-C: Group of conventional pigs infected with Trichinella spiralis, TS-SPF: Group of SPF pigs infected with Trichinella spiralis, NT-C: Group of conventional pigs not infected with Trichinella spiralis, NT-SPF: Group of SPF pigs not infected with Trichinella spiralis). (For inter- pretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 1. Histamine release from HMC-1 stimulated with L. (V.) brazili- ensis promastigote lysates. HMC-1 at 1 x 10° cells/mL were incubated for 5 and 15 min at 37°C with L-Lb or S-Lb at concentration corre- sponding to 30 ug of total protein/mL. Control values were observed in medium-treated HMC-1 cells. The results are expressed as the per- centage of release of histamine (means + SEM of five experiments with two replicates in each experiment). *p<0.05, in comparison with medium-treated cultures. Lysate derived from L. braziliensis promastigotes induced release of histamine by HMC-1 cells (Fig. 1). Incubation with S-Lb at a 30 ug of protein/mL concen- tration induced a significant increase of histamine
Fig. 1. Histamine release from HMC-1 stimulated with L. (V.) brazili- ensis promastigote lysates. HMC-1 at 1 x 10° cells/mL were incubated for 5 and 15 min at 37°C with L-Lb or S-Lb at concentration corre- sponding to 30 ug of total protein/mL. Control values were observed in medium-treated HMC-1 cells. The results are expressed as the per- centage of release of histamine (means + SEM of five experiments with two replicates in each experiment). *p<0.05, in comparison with medium-treated cultures. Lysate derived from L. braziliensis promastigotes induced release of histamine by HMC-1 cells (Fig. 1). Incubation with S-Lb at a 30 ug of protein/mL concen- tration induced a significant increase of histamine
Fig. 2. Histamine release from HMC-1 cells stimulated with LPG. HMC-1 cultures were incubated at 1 x 10° cells/mL for 5 or 15 min at 37°C with LPG at concentration ranging from 10 to 200 ng/mL. The results are expressed as percentage of release of histamine (means + SEM of four experiments with two replicates in each experi- ment). *p < 0.05, in comparison with saline-treated cultures.
Fig. 2. Histamine release from HMC-1 cells stimulated with LPG. HMC-1 cultures were incubated at 1 x 10° cells/mL for 5 or 15 min at 37°C with LPG at concentration ranging from 10 to 200 ng/mL. The results are expressed as percentage of release of histamine (means + SEM of four experiments with two replicates in each experi- ment). *p < 0.05, in comparison with saline-treated cultures.
Fig. 3. The immunocytochemical demonstration of preformed media- tors in HMC-1 cells. In a representative experiment, the cells were incubated with anti-tryptase monoclonal antibody and the reaction was developed using AEC system. Intense and homogeneous cytoplas- matic staining for tryptase (A) is observed in medium-treated HMC-1 cells. After stimulation with L-Lb or S-Lb, HMC-1 cells exhibit mor- phological changes and an evident decrease of cytoplasmatic staining for tryptase (B). Magnification 400x.
Fig. 3. The immunocytochemical demonstration of preformed media- tors in HMC-1 cells. In a representative experiment, the cells were incubated with anti-tryptase monoclonal antibody and the reaction was developed using AEC system. Intense and homogeneous cytoplas- matic staining for tryptase (A) is observed in medium-treated HMC-1 cells. After stimulation with L-Lb or S-Lb, HMC-1 cells exhibit mor- phological changes and an evident decrease of cytoplasmatic staining for tryptase (B). Magnification 400x.
Fig. 4. Flow cytometry of HMC-1 cells fixed, permeabilized, and stained with mouse monoclonal antibodies to IL-4, IL-12 or an irrelevant control mouse isotype. The cells were stimulated by L-Lb or S-Lb at concentration of 3 or 30 wg of total protein/mL during 30 min (bold line). HMC-1 cells treated with medium alone were used as control (solid line). Dashed line shows the isotype match control. Data shown are one out of four experi- ments.
Fig. 4. Flow cytometry of HMC-1 cells fixed, permeabilized, and stained with mouse monoclonal antibodies to IL-4, IL-12 or an irrelevant control mouse isotype. The cells were stimulated by L-Lb or S-Lb at concentration of 3 or 30 wg of total protein/mL during 30 min (bold line). HMC-1 cells treated with medium alone were used as control (solid line). Dashed line shows the isotype match control. Data shown are one out of four experi- ments.
Fig. 5. Differential ability of logarithmic and stationary forms L. (V.) braziliensis promastigotes to induce IL-4 (A) and IL-12 (B) release in HMC-1 cultures. The cells were stimulated with L-Lb or S-Lb at con- centration of 3 or 30 1g of total protein/mL. Under similar conditions, logarithmic or stationary promastigotes were added to HMC-1 cul- tures at a 20:1 ratio. The supernatants were collected after 30 min, and cytokine concentration was determined by ELISA assay. Results are means + SE (n=6). *p<0.01, when compared with saline-treated cultures.
Fig. 5. Differential ability of logarithmic and stationary forms L. (V.) braziliensis promastigotes to induce IL-4 (A) and IL-12 (B) release in HMC-1 cultures. The cells were stimulated with L-Lb or S-Lb at con- centration of 3 or 30 1g of total protein/mL. Under similar conditions, logarithmic or stationary promastigotes were added to HMC-1 cul- tures at a 20:1 ratio. The supernatants were collected after 30 min, and cytokine concentration was determined by ELISA assay. Results are means + SE (n=6). *p<0.01, when compared with saline-treated cultures.
Sensitivity of long-term allopurinol-grown and control procyclics to purine analogues and selected trypanocides 3.2. Transport of [?H ]allopurinol by procyclic T. b. brucei
Sensitivity of long-term allopurinol-grown and control procyclics to purine analogues and selected trypanocides 3.2. Transport of [?H ]allopurinol by procyclic T. b. brucei
Fig. 1. (A) Effect of allopurinol on growth of T. b. brucei s427 procyclics. Trypanosomes were seeded at 10° cells/ml in PFTM with 200 uM of inosine as sole purine source and grown at 25 °C in the presence of 2mM (O), | mM (M), 15 uM (CL) or no allopurinol (@). (B) Alamar Blue assay with vari- ous concentrations of pentamidine (A), aminopurinol (¥), thiopurinol (@), and allopurinol (Q).
Fig. 1. (A) Effect of allopurinol on growth of T. b. brucei s427 procyclics. Trypanosomes were seeded at 10° cells/ml in PFTM with 200 uM of inosine as sole purine source and grown at 25 °C in the presence of 2mM (O), | mM (M), 15 uM (CL) or no allopurinol (@). (B) Alamar Blue assay with vari- ous concentrations of pentamidine (A), aminopurinol (¥), thiopurinol (@), and allopurinol (Q).
Fig. 2. (A) Transport of 1 1M allopurinol by T. b. brucei procyclics (Ml) was inhibited by 250 1M hypoxanthine (¥) or 1 mM allopurinol (©). (B) Allopurinol transport (1 1M; Mi) is inhibited 75% by guanosine (C1). The slope for guanosine inhibition was significantly non-zero (P = 0.025, F test; 7? =0.95). Inhibition was complete with 250 uM allopurinol (A) and 1 mM hypoxanthine (©). Units of transport are pmol (10’ cells)~! s~!. We have previously shown that T. 6. brucei procyclics express two hypoxanthine transporters, Hl and H4, with similar affinities for allopurinol: K; values are 5.0+0.9 and 2.5+0.4uM for HI and H4, respectively (Burch- more et al., 2003; De Koning and Jarvis, 1997a). Only the higher-affinity transporter, H4, is inhibited by gua- nosine or uracil (Burchmore et al., 2003). Consistent with [H]allopurinol uptake by both hypoxanthine transporters, IC.) values were determined using the Alamar Blue protocol as described in Section 2, using control procyclics cultured in SDM79 or procyclics that had been cultured in PFTM supplemented with inosine, in the presence of 3mM allopurinol for at least 12 months. Assay con- ditions were identical for both strains, using SDM79 as medium.
Fig. 2. (A) Transport of 1 1M allopurinol by T. b. brucei procyclics (Ml) was inhibited by 250 1M hypoxanthine (¥) or 1 mM allopurinol (©). (B) Allopurinol transport (1 1M; Mi) is inhibited 75% by guanosine (C1). The slope for guanosine inhibition was significantly non-zero (P = 0.025, F test; 7? =0.95). Inhibition was complete with 250 uM allopurinol (A) and 1 mM hypoxanthine (©). Units of transport are pmol (10’ cells)~! s~!. We have previously shown that T. 6. brucei procyclics express two hypoxanthine transporters, Hl and H4, with similar affinities for allopurinol: K; values are 5.0+0.9 and 2.5+0.4uM for HI and H4, respectively (Burch- more et al., 2003; De Koning and Jarvis, 1997a). Only the higher-affinity transporter, H4, is inhibited by gua- nosine or uracil (Burchmore et al., 2003). Consistent with [H]allopurinol uptake by both hypoxanthine transporters, IC.) values were determined using the Alamar Blue protocol as described in Section 2, using control procyclics cultured in SDM79 or procyclics that had been cultured in PFTM supplemented with inosine, in the presence of 3mM allopurinol for at least 12 months. Assay con- ditions were identical for both strains, using SDM79 as medium.
Fig. 3. PH]Allopurinol transport in S. cerevisiae strain MG887-1 expressing TbNBTI, over 2 min. Yeast cells were incubated for 0, 0.5, 1, or 2 min with 1 uM of PH]allopurinol in the presence (©) or absence (M) of 1mM unlabelled allopurinol. Rates were calculated by linear regression (7? > 0.99). Both slopes were significantly non-zero (F test), but transport of radiolabel was inhibited by 98% in the presence of excess allopurinol. this inhi allo 2.14 TbNBT1/H4-mediated PH]allopurinol transport in system, measured over 60s, was saturable, and bited by various concentrations of unlabelled purinol and hypoxanthine (Fig. 4), yielding a K,, of +0.4uM and V,,,, of 9.9+1.8pmol(10’ cells)! min! (n=3), and a K, value for hypoxanthine of
Fig. 3. PH]Allopurinol transport in S. cerevisiae strain MG887-1 expressing TbNBTI, over 2 min. Yeast cells were incubated for 0, 0.5, 1, or 2 min with 1 uM of PH]allopurinol in the presence (©) or absence (M) of 1mM unlabelled allopurinol. Rates were calculated by linear regression (7? > 0.99). Both slopes were significantly non-zero (F test), but transport of radiolabel was inhibited by 98% in the presence of excess allopurinol. this inhi allo 2.14 TbNBT1/H4-mediated PH]allopurinol transport in system, measured over 60s, was saturable, and bited by various concentrations of unlabelled purinol and hypoxanthine (Fig. 4), yielding a K,, of +0.4uM and V,,,, of 9.9+1.8pmol(10’ cells)! min! (n=3), and a K, value for hypoxanthine of
Fig. 4. Allopurinol transport by the TobNBT1/H4 nucleobase trans- porter expressed in S. cerevisiae strain MG887-1. Transport of 0.5 uM PH]allopurinol was assessed in the presence of various concentrations of unlabelled allopurinol (MM) or hypoxanthine (O). Inset: conversion of allopurinol inhibition data to a Michaelis-Menten plot.
Fig. 4. Allopurinol transport by the TobNBT1/H4 nucleobase trans- porter expressed in S. cerevisiae strain MG887-1. Transport of 0.5 uM PH]allopurinol was assessed in the presence of various concentrations of unlabelled allopurinol (MM) or hypoxanthine (O). Inset: conversion of allopurinol inhibition data to a Michaelis-Menten plot.
K,, values in pM, Vx in pmol (107 cells) s~!. Kinetic parameters of procyclic hypoxanthine transporters in control and long-term allopurinol exposed cells
K,, values in pM, Vx in pmol (107 cells) s~!. Kinetic parameters of procyclic hypoxanthine transporters in control and long-term allopurinol exposed cells
Fig. 5. Transport of 0.03 uM [H]hypoxanthine in long-term allopuri- nol-exposed procyclic trypanosomes. (A) Inhibition by up to 1mM unlabelled hypoxanthine. The data fitted a two-site competition equa- tion significantly better than an equation for one-site competition as defined by the Prism software package (P = 0.035, F test). Inset: con- version to an Eadie—Hofstee plot, showing two distinct transporters, with apparent K,, values of 1.1 and 26.0 uM in this experiment. v, the initial rate of hypoxanthine transport; s, the total hypoxanthine con- centration. (B) Inhibition by allopurinol. Data were fitted to a sigmoi- dal curve with variable slope (Hill slope = 0.67 + 0.1 (SE); 17 = 0.99).
Fig. 5. Transport of 0.03 uM [H]hypoxanthine in long-term allopuri- nol-exposed procyclic trypanosomes. (A) Inhibition by up to 1mM unlabelled hypoxanthine. The data fitted a two-site competition equa- tion significantly better than an equation for one-site competition as defined by the Prism software package (P = 0.035, F test). Inset: con- version to an Eadie—Hofstee plot, showing two distinct transporters, with apparent K,, values of 1.1 and 26.0 uM in this experiment. v, the initial rate of hypoxanthine transport; s, the total hypoxanthine con- centration. (B) Inhibition by allopurinol. Data were fitted to a sigmoi- dal curve with variable slope (Hill slope = 0.67 + 0.1 (SE); 17 = 0.99).
Fig. 6. Growth of long-term allopurinol-exposed procyclics on a single purine source. (A) Procyclics were grown in PFTM in the continued presence of 3mM allopurinol, using 100 uM of either inosine (Ml) or hypoxanthine (CJ) as purine source. (B) Procyclics were grown in PFTM without allopurinol, with 100M hypoxanthine as purine source, in the presence (I) or absence (C)) of 2mM uracil.
Fig. 6. Growth of long-term allopurinol-exposed procyclics on a single purine source. (A) Procyclics were grown in PFTM in the continued presence of 3mM allopurinol, using 100 uM of either inosine (Ml) or hypoxanthine (CJ) as purine source. (B) Procyclics were grown in PFTM without allopurinol, with 100M hypoxanthine as purine source, in the presence (I) or absence (C)) of 2mM uracil.
Effect of irradiation on B. malayi adult worm motility* Table 1
Effect of irradiation on B. malayi adult worm motility* Table 1
Fig. 1. Effects of irradiation on B. malayi male and female worm via- bility. Viability was measured by the MTT reduction test. Results are analysed as percent reduction in formazan production compared with nonirradiated control worms cultured in vitro and expressed as per- cent reduction in viability. * Motility was assessed visually by microscopy and scored as —1, immotile; 1, slightly active; 2, active and motile; 3, moderately active and motile; and 4, highly active and motile. The results are from two independent observations of duplicate cultures (containing 2 female and 2 male worms each) at day 10.
Fig. 1. Effects of irradiation on B. malayi male and female worm via- bility. Viability was measured by the MTT reduction test. Results are analysed as percent reduction in formazan production compared with nonirradiated control worms cultured in vitro and expressed as per- cent reduction in viability. * Motility was assessed visually by microscopy and scored as —1, immotile; 1, slightly active; 2, active and motile; 3, moderately active and motile; and 4, highly active and motile. The results are from two independent observations of duplicate cultures (containing 2 female and 2 male worms each) at day 10.
Fig. 2. Effects of irradiation on microfilariae (MF) release by B. malayi worms in vitro. Adult female worms (irradiated or nonirradiated) were incubated in vitro for 10 days, and the MF released in vitro on differ- ent days after culture were counted. Results shown are percent reduc- tion in MF release relative to control cultured worms. Irradiation inhibited MF release from female worms in a time- and dose-dependent manner (Fig. 2). After 6 days of culture (DPC), the minimum effective dose that reduced MF release by 50% was 25krad. Microfilaria release was significantly decreased by day10 in all irradi- ated worm groups (P<0.05), and MF release at 10 days was completely blocked in the worms irradiated at 45 and 7S krad.
Fig. 2. Effects of irradiation on microfilariae (MF) release by B. malayi worms in vitro. Adult female worms (irradiated or nonirradiated) were incubated in vitro for 10 days, and the MF released in vitro on differ- ent days after culture were counted. Results shown are percent reduc- tion in MF release relative to control cultured worms. Irradiation inhibited MF release from female worms in a time- and dose-dependent manner (Fig. 2). After 6 days of culture (DPC), the minimum effective dose that reduced MF release by 50% was 25krad. Microfilaria release was significantly decreased by day10 in all irradi- ated worm groups (P<0.05), and MF release at 10 days was completely blocked in the worms irradiated at 45 and 7S krad.
Fig. 3. Effects of irradiation on B. malayi female worm embryogenesis. Two worms were examined for each irradiation dose. The data shown are percentages of early developing embryos (DE), pretzels, and stretched microfilariae (MF) in worm homogenates. Differences between embryograms from control and irradiated worms were all sig- nificant by 7° (P< 0.05).
Fig. 3. Effects of irradiation on B. malayi female worm embryogenesis. Two worms were examined for each irradiation dose. The data shown are percentages of early developing embryos (DE), pretzels, and stretched microfilariae (MF) in worm homogenates. Differences between embryograms from control and irradiated worms were all sig- nificant by 7° (P< 0.05).
Effect of irradiation on Wolbachia DNA in B. malayi female worms
Effect of irradiation on Wolbachia DNA in B. malayi female worms
Fig. 4. Transmission electron micrographs (TEM) of B. malayi female worms. (A) TEM of control worm cultured in vitro for 10 days. Numerous Wolbachia were present in hypodermal tissues (arrows). (B) TEM of a female worm irradiated at 10 krad and cultured for 10 days in vitro. A few small bacteria-like bodies were present in the hypoder- mis (arrow). No significant damage to the cuticle or hypodermis was observed. (C and D) Sections of female worms irradiated at 25 and 45 krad. These show vacuoles in the hypodermis, where bacteria were seen in control worms (scale bar = | pm).
Fig. 4. Transmission electron micrographs (TEM) of B. malayi female worms. (A) TEM of control worm cultured in vitro for 10 days. Numerous Wolbachia were present in hypodermal tissues (arrows). (B) TEM of a female worm irradiated at 10 krad and cultured for 10 days in vitro. A few small bacteria-like bodies were present in the hypoder- mis (arrow). No significant damage to the cuticle or hypodermis was observed. (C and D) Sections of female worms irradiated at 25 and 45 krad. These show vacuoles in the hypodermis, where bacteria were seen in control worms (scale bar = | pm).
Fig. 5. Transmission electron micrographs of B. malayi microfilariae (MF) released from irradiated worms. Worms were cultured in vitro, and the MF released on day 8 were used for electron microscopy stud- ies. (A and B) Sections of MF released from control worms. Wolbachia were visible as small clusters or as individual bacteria (arrows). (C and D) Sections of MF released on day 8 from irradiated worms. Wolba- chia are degenerating within large vacuoles. Scale bars = | um (A, C, and D), 0.5 tm (B).
Fig. 5. Transmission electron micrographs of B. malayi microfilariae (MF) released from irradiated worms. Worms were cultured in vitro, and the MF released on day 8 were used for electron microscopy stud- ies. (A and B) Sections of MF released from control worms. Wolbachia were visible as small clusters or as individual bacteria (arrows). (C and D) Sections of MF released on day 8 from irradiated worms. Wolba- chia are degenerating within large vacuoles. Scale bars = | um (A, C, and D), 0.5 tm (B).
Primers used to measure gene expression by qPCR Table 1
Primers used to measure gene expression by qPCR Table 1
Table 2
Table 2
Fig. 1. Membrane array of E. paraensei sporocyst subtracted cDNA library probed with in vivo-derived sporocyst cDNA. The 72 cDNA dotblots represent 65 unique sequences of the sporocyst cDNA library, six redundant SSH-ESTs with minor variation from the respective cluster consensus (Table 2) and one transcript later excluded from study due to short sequence (c6). cDNA blotted onto the membrane was originally generated from in vitro cultures of spo- rocysts. Hybridization signals represented here confirm the transcrip- tion of these genes in vivo. Negative controls are transcripts corresponding to human testes specific protein (384 bp) and human semenogelin II (182 bp) (PCR-Select Differential Screening kit man- ual, Clontech), and are blotted at positions gl and g2, respectively. No hybridization was detected when an identical array was probed with cDNA derived from uninfected snail hearts.
Fig. 1. Membrane array of E. paraensei sporocyst subtracted cDNA library probed with in vivo-derived sporocyst cDNA. The 72 cDNA dotblots represent 65 unique sequences of the sporocyst cDNA library, six redundant SSH-ESTs with minor variation from the respective cluster consensus (Table 2) and one transcript later excluded from study due to short sequence (c6). cDNA blotted onto the membrane was originally generated from in vitro cultures of spo- rocysts. Hybridization signals represented here confirm the transcrip- tion of these genes in vivo. Negative controls are transcripts corresponding to human testes specific protein (384 bp) and human semenogelin II (182 bp) (PCR-Select Differential Screening kit man- ual, Clontech), and are blotted at positions gl and g2, respectively. No hybridization was detected when an identical array was probed with cDNA derived from uninfected snail hearts.
Fig. 2. Differential expression screen using duplicate membrane arrays from the E. paraensei sporocyst subtracted cDNA library. Sixty-five of the unique sequences from the subtracted library are represented (see Table 2). Probes used: (A) sporocyst cDNA subtracted with combined adult, rediz and cercaria cDNA using SSH; (B) reverse subtracted probe, combined adult redia and cercaria cDNA subtracted with sporocyst cDNA; (C) unsub tracted sporocyst cDNA; and (D) unsubtracted adult, redia and cercaria cDNA. Negative controls are transcripts corresponding to human teste: specific protein (348 bp) and human seminogelin II (182 bp) (PCR-Select Differential Screening kit manual, Clontech), and are blotted at positions g| and g2, respectively.
Fig. 2. Differential expression screen using duplicate membrane arrays from the E. paraensei sporocyst subtracted cDNA library. Sixty-five of the unique sequences from the subtracted library are represented (see Table 2). Probes used: (A) sporocyst cDNA subtracted with combined adult, rediz and cercaria cDNA using SSH; (B) reverse subtracted probe, combined adult redia and cercaria cDNA subtracted with sporocyst cDNA; (C) unsub tracted sporocyst cDNA; and (D) unsubtracted adult, redia and cercaria cDNA. Negative controls are transcripts corresponding to human teste: specific protein (348 bp) and human seminogelin II (182 bp) (PCR-Select Differential Screening kit manual, Clontech), and are blotted at positions g| and g2, respectively.
Fig. 1. Lymphocyte proliferation by C57BL/6 (A) and BALB/c (B) mice during L. infantum infection. Mice spleen leukocytes were cultured in vitro without exogenous stimulation (WS), stimulated with parasite crude antigen (Ag) and with the three groups of released proteins (High, Inter, and Low). Values represent mean of three independent experiments.
Fig. 1. Lymphocyte proliferation by C57BL/6 (A) and BALB/c (B) mice during L. infantum infection. Mice spleen leukocytes were cultured in vitro without exogenous stimulation (WS), stimulated with parasite crude antigen (Ag) and with the three groups of released proteins (High, Inter, and Low). Values represent mean of three independent experiments.
Fig. 2. Expression and production of IFN-y (A and B), IL-4 (C and D), IL-10 (E and F), and IL-12 (G and H) by BALB/c mice during L. infantum infection. Mice spleen leukocytes were cultured for 96 h in presence of parasite crude antigen (Ag), fractions High, Inter, and Low or without exoge- nous stimulation (WS). Cytokine expression and production were determined by real-time PCR (data are expressed in number of copies per 100 copies of HPRT) and ELISA (data are expressed in pg/ml), respectively. Values represent mean of three independent experiments.
Fig. 2. Expression and production of IFN-y (A and B), IL-4 (C and D), IL-10 (E and F), and IL-12 (G and H) by BALB/c mice during L. infantum infection. Mice spleen leukocytes were cultured for 96 h in presence of parasite crude antigen (Ag), fractions High, Inter, and Low or without exoge- nous stimulation (WS). Cytokine expression and production were determined by real-time PCR (data are expressed in number of copies per 100 copies of HPRT) and ELISA (data are expressed in pg/ml), respectively. Values represent mean of three independent experiments.
Fig. 3. Expression and production of IFN-y (A and B), IL-4 (C and D), IL-10 (E and F), and IL-12 (G and H) by C57BL/6 mice during L. infantum infection. Mice spleen leukocytes were cultured for 96h, without exogenous stimulation (WS) or stimulated with parasite crude antigen (Ag), High, Inter, and Low fractions. Cytokine expression and production were determined by real-time PCR (data are expressed in number of copies per 1000 copies of HPRT) and ELISA (data are expressed in pg/ml), respectively. Values represent mean of three independent experiments.
Fig. 3. Expression and production of IFN-y (A and B), IL-4 (C and D), IL-10 (E and F), and IL-12 (G and H) by C57BL/6 mice during L. infantum infection. Mice spleen leukocytes were cultured for 96h, without exogenous stimulation (WS) or stimulated with parasite crude antigen (Ag), High, Inter, and Low fractions. Cytokine expression and production were determined by real-time PCR (data are expressed in number of copies per 1000 copies of HPRT) and ELISA (data are expressed in pg/ml), respectively. Values represent mean of three independent experiments.
Fig. 1. Effect of growing temperature and nutrition on biomass of five isolates of D. flagrans cultivated in two liquid media (MM and LB). Isolates: (1) MUCL 28429, (2) CBS 143.83, (3) CBS 561.92, (4) CBS 565.50, and (5) CBS 583.91. Each data point represents the means + SE of 4-5 independent replications. The values for the biomass of CBS 143.83 isolate propagated in MM at 20°C versus 30°C are significant at P < 0.01. Values for the biomass of MUCL 28429 isolate propa- gated in LB at 20°C versus 30 °C are significant at P < 0.01 while val- ues for the isolates CBS 561.92 and CBS 565.50 cultivated in LB (20 °C versus 30°C) are significant at P < 0.05 and P < 0.01, respectively. Differences between biomass of all isolates cultivated in MM and LB are significant at P < 0.01. MM, poor in nutrients minimal medium; LB, rich in nutrients Luria broth. MUCL: Mycotheque de |’Universite catholique de Louvain, Belgium. CBS: Centraalbureau voor Schim- melcultures, Baarn, The Netherlands. From the data presented in Fig. 2 it appears that all tested fungal isolates poorly propagated on CMA plates compared to other solid culture media. After 3 weeks of cultivation at 20°C only 5—24% of CMA petri dish areas were covered by very fine white gossamery
Fig. 1. Effect of growing temperature and nutrition on biomass of five isolates of D. flagrans cultivated in two liquid media (MM and LB). Isolates: (1) MUCL 28429, (2) CBS 143.83, (3) CBS 561.92, (4) CBS 565.50, and (5) CBS 583.91. Each data point represents the means + SE of 4-5 independent replications. The values for the biomass of CBS 143.83 isolate propagated in MM at 20°C versus 30°C are significant at P < 0.01. Values for the biomass of MUCL 28429 isolate propa- gated in LB at 20°C versus 30 °C are significant at P < 0.01 while val- ues for the isolates CBS 561.92 and CBS 565.50 cultivated in LB (20 °C versus 30°C) are significant at P < 0.05 and P < 0.01, respectively. Differences between biomass of all isolates cultivated in MM and LB are significant at P < 0.01. MM, poor in nutrients minimal medium; LB, rich in nutrients Luria broth. MUCL: Mycotheque de |’Universite catholique de Louvain, Belgium. CBS: Centraalbureau voor Schim- melcultures, Baarn, The Netherlands. From the data presented in Fig. 2 it appears that all tested fungal isolates poorly propagated on CMA plates compared to other solid culture media. After 3 weeks of cultivation at 20°C only 5—24% of CMA petri dish areas were covered by very fine white gossamery
Fig. 2. Radial growth rates of five isolates of D. flagrans grown on four solid media (CMA, SAB, SAB-GM, and SAB-HP) and subjected to 20 and 30°C temperature regimes. Isolates: (1) MUCL 28429, (2) CBS 143.83, (3) CBS 561.92, (4) CBS 565.50, and (5) CBS 583.91. Each data point repre- sents the means + SE of 4-6 independent replications. Statistical analysis was performed within each isolate and the growth rates of fungal colonies propagated on various substrates in two temperature regimes were compared. Results of statistical analysis (consistent with an intuitive appraisal of data) are not shown in order to preserve clarity of the figure. CMA, corn meal agar; SAB, Sabouraud; SAB-GM, Sabouraud supplemented with homogenized G. mellonella larvae; and SAB-HP, Sabouraud supplemented with homogenized H. polygyrus larvae. Other details as in Fig. 1.
Fig. 2. Radial growth rates of five isolates of D. flagrans grown on four solid media (CMA, SAB, SAB-GM, and SAB-HP) and subjected to 20 and 30°C temperature regimes. Isolates: (1) MUCL 28429, (2) CBS 143.83, (3) CBS 561.92, (4) CBS 565.50, and (5) CBS 583.91. Each data point repre- sents the means + SE of 4-6 independent replications. Statistical analysis was performed within each isolate and the growth rates of fungal colonies propagated on various substrates in two temperature regimes were compared. Results of statistical analysis (consistent with an intuitive appraisal of data) are not shown in order to preserve clarity of the figure. CMA, corn meal agar; SAB, Sabouraud; SAB-GM, Sabouraud supplemented with homogenized G. mellonella larvae; and SAB-HP, Sabouraud supplemented with homogenized H. polygyrus larvae. Other details as in Fig. 1.
Fig. 4. Insecticidal properties of five D. flagrans isolates propagated at various growing conditions. Isolates: (1) MUCL 28429, (2) CBS 143.83, (3) CBS 561.92, (4) CBS 565.50, and (5) CBS 583.91. Each data point represents the means + SE of 4-6 independent replications. Sig- nificant differences (P < 0.05) between insecticidal activity of isolates cultured at two temperature regimes were found only in the case of CBS 143.83 and CBS 583.91. Other details as in Fig. 1. Fig. 3. Effect of two growing temperatures and four solid substrates on the production of chlamydospores by five D. flagrans isolates. Isolates: (1) MUCL 28429, (2) CBS 143.83, (3) CBS 561.92, (4) CBS 565.50, and (5) CBS 583.91. Each data point represents the means + SE of 4-€ independent replications. The values for the chlamydospores produc- tion by isolates cultured at 20°C on CMA, SAB and SAB-HP signifi- cantly differ from those noticed at 30°C (P < 0.05 or better). Other details as in Fig. 1.
Fig. 4. Insecticidal properties of five D. flagrans isolates propagated at various growing conditions. Isolates: (1) MUCL 28429, (2) CBS 143.83, (3) CBS 561.92, (4) CBS 565.50, and (5) CBS 583.91. Each data point represents the means + SE of 4-6 independent replications. Sig- nificant differences (P < 0.05) between insecticidal activity of isolates cultured at two temperature regimes were found only in the case of CBS 143.83 and CBS 583.91. Other details as in Fig. 1. Fig. 3. Effect of two growing temperatures and four solid substrates on the production of chlamydospores by five D. flagrans isolates. Isolates: (1) MUCL 28429, (2) CBS 143.83, (3) CBS 561.92, (4) CBS 565.50, and (5) CBS 583.91. Each data point represents the means + SE of 4-€ independent replications. The values for the chlamydospores produc- tion by isolates cultured at 20°C on CMA, SAB and SAB-HP signifi- cantly differ from those noticed at 30°C (P < 0.05 or better). Other details as in Fig. 1.
Fig. 5. Galleria mellonella larvae: (A) control and (B) infected with D. flagrans (isolate CBS 583.91 grown on SAB-GM medium at 20°C). Arrows indicate black spots on the cuticle at the fungal penetration site.
Fig. 5. Galleria mellonella larvae: (A) control and (B) infected with D. flagrans (isolate CBS 583.91 grown on SAB-GM medium at 20°C). Arrows indicate black spots on the cuticle at the fungal penetration site.
Fig. 6. Influence of preincubation with various BSA concentrations on the H. polygyrus motility. Each data point represents the means + SE of five independent assays. Values are significant at P < 0.01.
Fig. 6. Influence of preincubation with various BSA concentrations on the H. polygyrus motility. Each data point represents the means + SE of five independent assays. Values are significant at P < 0.01.
Fig. 7. Ability of D. flagrans metabolites to impair H. polygyrus motil- ity. Nematodes were preincubated for 18h with fungal homogenates of five isolates, briefly washed in PBS and assayed as described under Section 2. Control: nematodes were preincubated with BSA. Isolates: (1) MUCL 28429, (2) CBS 143.83, (3) CBS 561.92, (4) CBS 565.50, and (5) CBS 583.91. Each data point represents the means + SE of 3-5 independent assays. The mean rates of nematode motility inhibition between all tested homogenates and the control are significant at P < 0.01. Differences between the inhibition rates of isolates propa- gated in LB versus SAB-GM and SAB-HP are significant at P < 0.01. Differences between activity of isolates MUCL 28429, CBS 561.92, and CBS 565.50 propagated in LB are significant at P < 0.05 or better. Other details as in Fig. 1.
Fig. 7. Ability of D. flagrans metabolites to impair H. polygyrus motil- ity. Nematodes were preincubated for 18h with fungal homogenates of five isolates, briefly washed in PBS and assayed as described under Section 2. Control: nematodes were preincubated with BSA. Isolates: (1) MUCL 28429, (2) CBS 143.83, (3) CBS 561.92, (4) CBS 565.50, and (5) CBS 583.91. Each data point represents the means + SE of 3-5 independent assays. The mean rates of nematode motility inhibition between all tested homogenates and the control are significant at P < 0.01. Differences between the inhibition rates of isolates propa- gated in LB versus SAB-GM and SAB-HP are significant at P < 0.01. Differences between activity of isolates MUCL 28429, CBS 561.92, and CBS 565.50 propagated in LB are significant at P < 0.05 or better. Other details as in Fig. 1.
Fig. 1. Schematic representation of the MSP-1 of P. falciparum, show- ing the conserved, semiconserved, and variable domains defined by Tanabe et al. (1987) as white, gray, and black boxes, respectively. The four recombinant antigens, three derived from block 2 (3D7, MAD20. and RO33) and one derived from block 17 (Pf{MSP-1,9), are repre- sented. The location and orientation of primers used to type and sequence the gene fragment coding for block 2 are also shown. Block 17 codes for MSP-1j9, the 19-kDa C-terminal product of enzymatic processing of MSP-1 that remains anchored to the merozoite surface at the time of erythro- cyte invasion (Fig. 1) (Blackman et al., 1991). MSP-1,, has two cysteine-rich domains, analogous to epidermal growth factor (EGF) motifs, which are putatively required in interactions with erythrocytes (Blackman et al., 1991). MSP-1,, is a major target of naturally acquired human antibodies that are able to inhibit eryth- rocyte invasion by merozoites in vitro (O’Donnel et al.,
Fig. 1. Schematic representation of the MSP-1 of P. falciparum, show- ing the conserved, semiconserved, and variable domains defined by Tanabe et al. (1987) as white, gray, and black boxes, respectively. The four recombinant antigens, three derived from block 2 (3D7, MAD20. and RO33) and one derived from block 17 (Pf{MSP-1,9), are repre- sented. The location and orientation of primers used to type and sequence the gene fragment coding for block 2 are also shown. Block 17 codes for MSP-1j9, the 19-kDa C-terminal product of enzymatic processing of MSP-1 that remains anchored to the merozoite surface at the time of erythro- cyte invasion (Fig. 1) (Blackman et al., 1991). MSP-1,, has two cysteine-rich domains, analogous to epidermal growth factor (EGF) motifs, which are putatively required in interactions with erythrocytes (Blackman et al., 1991). MSP-1,, is a major target of naturally acquired human antibodies that are able to inhibit eryth- rocyte invasion by merozoites in vitro (O’Donnel et al.,
Fig. 2. Alignment of K1-type and MAD20-type tripeptide repeats in block 2 of MSP-1 among isolates from Brazil, and definition of local repea haplotypes. Each repetitive motif or allotype was assigned a number (1-3 for K1-type sequences and 5-8 for MAD20-type sequences). Underlinec numbers indicate alternative repeat allotypes showing synonymous nucleotide replacements (all found in the black-shaded central area of MAD20 type repeats). Here, we combine sequence data for 14 isolates from the state of Mato Grosso (numerals in parentheses indicate the number of isolate: with each repeat haplotype found among them) and 39 isolates from the state of Rondo6nia (data from Ferreira et al., 2003), both in northwestert Brazil.
Fig. 2. Alignment of K1-type and MAD20-type tripeptide repeats in block 2 of MSP-1 among isolates from Brazil, and definition of local repea haplotypes. Each repetitive motif or allotype was assigned a number (1-3 for K1-type sequences and 5-8 for MAD20-type sequences). Underlinec numbers indicate alternative repeat allotypes showing synonymous nucleotide replacements (all found in the black-shaded central area of MAD20 type repeats). Here, we combine sequence data for 14 isolates from the state of Mato Grosso (numerals in parentheses indicate the number of isolate: with each repeat haplotype found among them) and 39 isolates from the state of Rondo6nia (data from Ferreira et al., 2003), both in northwestert Brazil.
Fig. 3. Proportions of responders and levels of IgG subclass antibodies (measured by reactivity indices) to the block 2 antigens 3D7, MAD20, and RO33 and the block 17 antigen MSP-1,) among non-infected sub- jects (NI, n = 224), asymptomatic carriers of P. falciparum (A, n= 56) and symptomatic P. falciparum malaria patients (S, n=51). IgGl, IgG2, IgG3, and IgG4 subclasses antibodies are represented by open, hatched, closed, and dark-shaded boxes, respectively. Error bars indi- cate the upper limit of 95% confidence intervals of means. The propor- tion of responders represents the percentages of subjects, within each group, with detectable antibodies of at least one IgG subclass directed to a given antigen.
Fig. 3. Proportions of responders and levels of IgG subclass antibodies (measured by reactivity indices) to the block 2 antigens 3D7, MAD20, and RO33 and the block 17 antigen MSP-1,) among non-infected sub- jects (NI, n = 224), asymptomatic carriers of P. falciparum (A, n= 56) and symptomatic P. falciparum malaria patients (S, n=51). IgGl, IgG2, IgG3, and IgG4 subclasses antibodies are represented by open, hatched, closed, and dark-shaded boxes, respectively. Error bars indi- cate the upper limit of 95% confidence intervals of means. The propor- tion of responders represents the percentages of subjects, within each group, with detectable antibodies of at least one IgG subclass directed to a given antigen.
Fig. 4. Proportions of responders and levels of IgG subclass antibodies (measured by reactivity indices) to the block 2 antigens 3D7, MAD20, and RO33 and the block 17 antigen MSP-1,) among non-infected sub- jects stratified according to the levels of cumulative exposure to malaria measured by the length of residence in malaria-endemic areas: (a) <10 years, n = 70; (b) 10-20 years, n = 71; and (c) >20 years, n = 83. IgGl, IgG2, IgG3, and IgG4 subclasses antibodies are represented by open, hatched, closed, and dark-shaded boxes, respectively. Error bars indicate the upper limit of 95% confidence intervals of means. The pro- portion of responders represents the percentages of subjects, within each group, with detectable antibodies of at least one IgG subclass directed to a given antigen.
Fig. 4. Proportions of responders and levels of IgG subclass antibodies (measured by reactivity indices) to the block 2 antigens 3D7, MAD20, and RO33 and the block 17 antigen MSP-1,) among non-infected sub- jects stratified according to the levels of cumulative exposure to malaria measured by the length of residence in malaria-endemic areas: (a) <10 years, n = 70; (b) 10-20 years, n = 71; and (c) >20 years, n = 83. IgGl, IgG2, IgG3, and IgG4 subclasses antibodies are represented by open, hatched, closed, and dark-shaded boxes, respectively. Error bars indicate the upper limit of 95% confidence intervals of means. The pro- portion of responders represents the percentages of subjects, within each group, with detectable antibodies of at least one IgG subclass directed to a given antigen.
Fig. 5. IgG subclass antibody recognition of block 2 antigens (3D7, MAD20, and RO33) in 21 asymptomatic P. falciparum carriers and 16 symptom- atic malaria patients for whom block 2 typing data were available. Ages are given in years, and exposure to malaria is estimated as the time of residence in malaria-endemic areas. The repeat haplotypes (numbers in squared brackets refer to haplotypes described in Fig. 2) are indicated for K1- type and MAD20-type sequences, when available. Different levels of antibodies of each subclass are represented with different shading patterns.
Fig. 5. IgG subclass antibody recognition of block 2 antigens (3D7, MAD20, and RO33) in 21 asymptomatic P. falciparum carriers and 16 symptom- atic malaria patients for whom block 2 typing data were available. Ages are given in years, and exposure to malaria is estimated as the time of residence in malaria-endemic areas. The repeat haplotypes (numbers in squared brackets refer to haplotypes described in Fig. 2) are indicated for K1- type and MAD20-type sequences, when available. Different levels of antibodies of each subclass are represented with different shading patterns.
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