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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 Anti-Nras staining of host cellular reaction associated with adult O. volvulus demonstrating the intensity of Nras staining in the plasma cells and the general absence of staining in other host cells. b 8-bit grayscale converted image of host plasma cell zone, showing the selection of positive cells (arrow) as control values for quantification purposes
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Fig1: a Anti-Nras staining of host cellular reaction associated with adult O. volvulus demonstrating the intensity of Nras staining in the plasma cells and the general absence of staining in other host cells. b 8-bit grayscale converted image of host plasma cell zone, showing the selection of positive cells (arrow) as control values for quantification purposes

Mentions: The computer programme ImagePro® (Media Cybernetics, Rockville, MD) was used to perform the image analysis. An image of each nodule was taken using an Olympus DP71 microscope mounted camera (Olympus, Center Valley, PA) captured at the highest resolution available and maintained in .tiff format to avoid degradation through file compression. Host plasma cells were selected by visual comparison to establish the most densely stained entities to act as the internal controls. No statistically significant difference was observed between the plasma cell controls of ivermectin-treated and the untreated nodules; this concurred with the subjective visual assessment; 5 cells was determined to be statistically a sufficient number to count (p > 0.05). Cameroonian tissues from untreated people were considered to be appropriate controls for the treated tissues from Ecuador as a comparison between these two groups, assessing for similarity in staining intensity of the standard control component (host plasma cells), showed no obvious difference in the staining of these cells between these two groups. The images of the plasma cells were converted to gray scale format using ImagePro®, permitting a bitmap analysis to be performed (Fig. 1). A bitmap is expressed in gray scale units, 0 being black and 255 being white; this form of analysis enables an easy comparison between different images by maintaining a constant scale and the ability to analyse multiple sections from multiple nodules without being biased by colour.Fig. 1


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 Anti-Nras staining of host cellular reaction associated with adult O. volvulus demonstrating the intensity of Nras staining in the plasma cells and the general absence of staining in other host cells. b 8-bit grayscale converted image of host plasma cell zone, showing the selection of positive cells (arrow) as control values for quantification purposes
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: a Anti-Nras staining of host cellular reaction associated with adult O. volvulus demonstrating the intensity of Nras staining in the plasma cells and the general absence of staining in other host cells. b 8-bit grayscale converted image of host plasma cell zone, showing the selection of positive cells (arrow) as control values for quantification purposes
Mentions: The computer programme ImagePro® (Media Cybernetics, Rockville, MD) was used to perform the image analysis. An image of each nodule was taken using an Olympus DP71 microscope mounted camera (Olympus, Center Valley, PA) captured at the highest resolution available and maintained in .tiff format to avoid degradation through file compression. Host plasma cells were selected by visual comparison to establish the most densely stained entities to act as the internal controls. No statistically significant difference was observed between the plasma cell controls of ivermectin-treated and the untreated nodules; this concurred with the subjective visual assessment; 5 cells was determined to be statistically a sufficient number to count (p > 0.05). Cameroonian tissues from untreated people were considered to be appropriate controls for the treated tissues from Ecuador as a comparison between these two groups, assessing for similarity in staining intensity of the standard control component (host plasma cells), showed no obvious difference in the staining of these cells between these two groups. The images of the plasma cells were converted to gray scale format using ImagePro®, permitting a bitmap analysis to be performed (Fig. 1). A bitmap is expressed in gray scale units, 0 being black and 255 being white; this form of analysis enables an easy comparison between different images by maintaining a constant scale and the ability to analyse multiple sections from multiple nodules without being biased by colour.Fig. 1

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