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A histochemical study of the Nras/let-60 activity in filarial nematodes.

Geary JF, Lovato R, Wanji S, Guderian R, O'Neill M, Specht S, Madrill N, Geary TG, Mackenzie CD - Parasit Vectors (2015)

Bottom Line: An immunocytochemical approach using an antibody against human Nras was used to identify and detect changes in the nematode homolog let-60 that is associated with cell growth and maintenance.A statistically significant decrease in the general staining intensity of Nras/let-60 was observed in adult female O. volvulus treated with ivermectin when compared with parasites from untreated patients.Nras/let-60, as detected by immunocytochemical staining, is decreased in ivermectin-treated adult female O. volvulus relative to untreated control specimens, suggesting a suppressive effect of ivermectin on the overall biochemical activity of these parasites.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, MI, 48824, USA. gearyjam@msu.edu.

ABSTRACT

Background: Control and elimination of filarial pathogens is a central focus of major global health efforts directed at parasitic diseases of developing countries. Accomplishment of these goals would be markedly enhanced by the enhanced destruction of the adult stage of filariae. The identification of new, more quantitative biomarkers that correlate with mortality or chemotherapeutic damage to adult filariae, would greatly facilitate, for example, the development of new macrofilaricides.

Methods: An immunocytochemical approach using an antibody against human Nras was used to identify and detect changes in the nematode homolog let-60 that is associated with cell growth and maintenance. Single Onchocerca volvulus nodules were removed from each of 13 patients treated with ivermectin (as part of a community-wide mass drug administration programme), and from each of 13 untreated individuals; these 26 nodules were stained with the anti-Nras antibody. The localization and degree of positivity of Nras/let-60 staining were assessed subjectively and compared between the two groups; the positivity of staining was also quantified, using image analysis, in a subgroup of these nodules. In addition, the specific morphological association between Nras/let-60 and the Wolbachia endosymbiont present in these parasites was also observed in 4 additional filarial species using an anti-Wolbachia surface protein (WSP) antibody under light and confocal microscopy.

Results: Nras/let-60 is present in many structures within the adult female worms. A statistically significant decrease in the general staining intensity of Nras/let-60 was observed in adult female O. volvulus treated with ivermectin when compared with parasites from untreated patients. Nras/let-60 staining was frequently observed to be co-localized with WSP in O.volvulus, Brugia malayi, Litomosoides sigmodontis and Dirofilaria immitis. Nras/let60 is also present in Onchocerca ochengi.

Conclusion: Nras/let-60, as detected by immunocytochemical staining, is decreased in ivermectin-treated adult female O. volvulus relative to untreated control specimens, suggesting a suppressive effect of ivermectin on the overall biochemical activity of these parasites. Co-localization of Nras/let-60 and WSP suggests the possibility that the endosymbiont utilizes this nematode protein as part of a mutualistic relationship. Nras/let60 appears to be a useful biomarker for assessing the health of filariae.

No MeSH data available.


Related in: MedlinePlus

a LSM image of the hypodermis of an adult female O. volvulus demonstrating co-localization as evidenced by yellow color (Arrow). b DIC image of the site shown in 7C. c LSM image of the hypodermis of an adult female O. volvulus demonstrating co-localization as evidenced by yellow color (Arrow)
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Fig7: a LSM image of the hypodermis of an adult female O. volvulus demonstrating co-localization as evidenced by yellow color (Arrow). b DIC image of the site shown in 7C. c LSM image of the hypodermis of an adult female O. volvulus demonstrating co-localization as evidenced by yellow color (Arrow)

Mentions: Red fluorescence due to presence of red Alexa® Fluor 647 stain, indicating the presence of WSP, was confined to punctate areas in the hypodermis and germ line tissues (Figs. 6 and 7). In the hypodermal sections tested, this stain was co-localized with the green 488 stain for Nras/let-60 (Table 3), and thus was seen as yellow colour in the representative images (Fig. 7). Green (Nras/let-60) was the predominant colour in the hypodermal sections, indicating the presence of more abundant Nras/let-60 staining compared to WSP. Yellow coloured pixels, representing co-localization of Nras/let-60 and WSP, were observed in all hypodermal sections (Table 3); this supports the conclusion that Wolbachia is commonly co-localized with Nras/let-60 in O. volvulus. The hypodermal sections were analysed further using Manders’ Correlation Coefficient and Costes P-value [24, 25]. This conclusion is also supported by light microscopic analyses, in which structures with punctate Nras/let-60 staining appeared, from serial sections, to reside in the same anatomical location as those detected by anti-WSP (Fig. 5a and b).Fig. 6


A histochemical study of the Nras/let-60 activity in filarial nematodes.

Geary JF, Lovato R, Wanji S, Guderian R, O'Neill M, Specht S, Madrill N, Geary TG, Mackenzie CD - Parasit Vectors (2015)

a LSM image of the hypodermis of an adult female O. volvulus demonstrating co-localization as evidenced by yellow color (Arrow). b DIC image of the site shown in 7C. c LSM image of the hypodermis of an adult female O. volvulus demonstrating co-localization as evidenced by yellow color (Arrow)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4493820&req=5

Fig7: a LSM image of the hypodermis of an adult female O. volvulus demonstrating co-localization as evidenced by yellow color (Arrow). b DIC image of the site shown in 7C. c LSM image of the hypodermis of an adult female O. volvulus demonstrating co-localization as evidenced by yellow color (Arrow)
Mentions: Red fluorescence due to presence of red Alexa® Fluor 647 stain, indicating the presence of WSP, was confined to punctate areas in the hypodermis and germ line tissues (Figs. 6 and 7). In the hypodermal sections tested, this stain was co-localized with the green 488 stain for Nras/let-60 (Table 3), and thus was seen as yellow colour in the representative images (Fig. 7). Green (Nras/let-60) was the predominant colour in the hypodermal sections, indicating the presence of more abundant Nras/let-60 staining compared to WSP. Yellow coloured pixels, representing co-localization of Nras/let-60 and WSP, were observed in all hypodermal sections (Table 3); this supports the conclusion that Wolbachia is commonly co-localized with Nras/let-60 in O. volvulus. The hypodermal sections were analysed further using Manders’ Correlation Coefficient and Costes P-value [24, 25]. This conclusion is also supported by light microscopic analyses, in which structures with punctate Nras/let-60 staining appeared, from serial sections, to reside in the same anatomical location as those detected by anti-WSP (Fig. 5a and b).Fig. 6

Bottom Line: An immunocytochemical approach using an antibody against human Nras was used to identify and detect changes in the nematode homolog let-60 that is associated with cell growth and maintenance.A statistically significant decrease in the general staining intensity of Nras/let-60 was observed in adult female O. volvulus treated with ivermectin when compared with parasites from untreated patients.Nras/let-60, as detected by immunocytochemical staining, is decreased in ivermectin-treated adult female O. volvulus relative to untreated control specimens, suggesting a suppressive effect of ivermectin on the overall biochemical activity of these parasites.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, MI, 48824, USA. gearyjam@msu.edu.

ABSTRACT

Background: Control and elimination of filarial pathogens is a central focus of major global health efforts directed at parasitic diseases of developing countries. Accomplishment of these goals would be markedly enhanced by the enhanced destruction of the adult stage of filariae. The identification of new, more quantitative biomarkers that correlate with mortality or chemotherapeutic damage to adult filariae, would greatly facilitate, for example, the development of new macrofilaricides.

Methods: An immunocytochemical approach using an antibody against human Nras was used to identify and detect changes in the nematode homolog let-60 that is associated with cell growth and maintenance. Single Onchocerca volvulus nodules were removed from each of 13 patients treated with ivermectin (as part of a community-wide mass drug administration programme), and from each of 13 untreated individuals; these 26 nodules were stained with the anti-Nras antibody. The localization and degree of positivity of Nras/let-60 staining were assessed subjectively and compared between the two groups; the positivity of staining was also quantified, using image analysis, in a subgroup of these nodules. In addition, the specific morphological association between Nras/let-60 and the Wolbachia endosymbiont present in these parasites was also observed in 4 additional filarial species using an anti-Wolbachia surface protein (WSP) antibody under light and confocal microscopy.

Results: Nras/let-60 is present in many structures within the adult female worms. A statistically significant decrease in the general staining intensity of Nras/let-60 was observed in adult female O. volvulus treated with ivermectin when compared with parasites from untreated patients. Nras/let-60 staining was frequently observed to be co-localized with WSP in O.volvulus, Brugia malayi, Litomosoides sigmodontis and Dirofilaria immitis. Nras/let60 is also present in Onchocerca ochengi.

Conclusion: Nras/let-60, as detected by immunocytochemical staining, is decreased in ivermectin-treated adult female O. volvulus relative to untreated control specimens, suggesting a suppressive effect of ivermectin on the overall biochemical activity of these parasites. Co-localization of Nras/let-60 and WSP suggests the possibility that the endosymbiont utilizes this nematode protein as part of a mutualistic relationship. Nras/let60 appears to be a useful biomarker for assessing the health of filariae.

No MeSH data available.


Related in: MedlinePlus