Secretory microRNA Profiles of Third- and Fourth-Stage Dirofilaria immitis Larvae with Different Macrocyclic Lactone Susceptibility: In Search of Biomarkers for Early Detection of Infection

The canine heartworm, Dirofilaria immitis, is among the most important parasites of dogs in the United States and worldwide, and may cause severe and potentially fatal disease. Current diagnostic recommendations rely on serological detection of an adult female antigen, and visualization of microfilariae in the blood. Therefore, a reliable diagnosis can be only performed approximately six months post-infection. There is a growing need to characterize novel diagnostic markers that are capable of detecting the early stages of heartworm infection, in special markers associated with third-stage larvae (L3) and fourth-stage larvae (L4). The early detection of infection would guide medical interventions that could impede the development of patent infections and further parasite transmission. We cultured D. immitis L3 and L4 of two laboratorial strains with different susceptibility statuses to macrocyclic lactone drugs in vitro. Excretory/secretory microRNAs were sequenced and analyzed. We identified two miRNA novel candidates secreted abundantly by both L3 and L4 of both strains. These candidates were previously detected in the secretions of other D. immitis stages and one of them was found in the blood of D. immitis-infected dogs. These miRNAs have not been found in the secretions of other nematodes and could be D. immitis-specific diagnostic biomarkers, which could allow for the early detection of infection.

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Cryopreservation of Dirofilaria immitis microfilariae and third-stage larvae

Journal of Helminthology ◽

10.1017/s0022149x00011019 ◽

1983 ◽

Vol 57 (4) ◽

pp. 319-324 ◽

Cited By ~ 11

Author(s):

J. B. Lok ◽

M. Mika-Grieve ◽

R. B. Grieve

Keyword(s):

Hydroxyethyl Starch ◽

Dirofilaria Immitis ◽

Fourth Stage ◽

The Third ◽

Third Stage

AbstractMicrofilariae of Dirofilaria immitis retained their infectivity for susceptible mosquitoes after cooling to −196°C in the presence of 5% dimethylsulphoxide (Me2SO) using a two-step cooling sequence. Motility and in vitro development of cryopreserved microfilariae also compared favourably with unfrozen controls. Third-stage larvae frozen by the same cooling sequence in the presence of either 5% Me2SO or 16% hydroxyethyl starch were motile upon thawing. Thawed larvae completed the third- to fourth-stage moult in vitro at a frequency approximately 5 to 10% of that seen in unfrozen controls.

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The effect of catecholamines and catecholamine antagonists on the third larval moult of Dirofilaria immitis in vitro

Journal of Helminthology ◽

10.1017/s0022149x00014711 ◽

1992 ◽

Vol 66 (4) ◽

pp. 273-278 ◽

Cited By ~ 2

Author(s):

E. V. Warbrick ◽

S. A. Ward

Keyword(s):

Dirofilaria Immitis ◽

Parasitic Nematode ◽

The Third ◽

Adrenergic Antagonist ◽

Third Stage ◽

Larval Moult

ABSTRACTVarious catecholamines and catecholamine antagonists have been examined for their effects on the third larval moult of the parasitic nematode, Dirofilaria immitis, cultured in vitro. The non-selective α and β agonist, noradrenaline, and the β agonist, isoprenaline, had no effect on the timing of the third stage moult when used at a concentration of 10−5M. The α-adrenergic antagonist. phentolamine, resulted in worm mortality at 10−5M. At 10−7M, both phentolamine and the β-antagonist, propranolol caused a significant reduction in the numbers of larvae capable of completing the third stage moult. Idazoxan, an a2-antagonist, at 10−5M did not affect worm mortality but did completely prevent ecdysis. The potential of these compounds as possible filaricides is discussed.

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Angiostrongylus cantonensis (Nematode: Metastrongiloidea): In Vitro Cultivation of Infective Third-Stage Larvae to Fourth-Stage Larvae

PLoS ONE ◽

10.1371/journal.pone.0072084 ◽

2013 ◽

Vol 8 (8) ◽

pp. e72084 ◽

Cited By ~ 3

Author(s):

Rong-Jyh Lin ◽

Jie-Wen He ◽

Li-Yu Chung ◽

June-Der Lee ◽

Jiun-Jye Wang ◽

...

Keyword(s):

Angiostrongylus Cantonensis ◽

In Vitro Cultivation ◽

Fourth Stage ◽

Third Stage

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In vitro Culture of Dirofilaria immitis Third- and Fourth-Stage Larvae under Defined Conditions

Journal of Parasitology ◽

10.2307/3282093 ◽

1987 ◽

Vol 73 (2) ◽

pp. 377 ◽

Cited By ~ 20

Author(s):

David Abraham ◽

Meisen Mok ◽

Marcia Mika-Grieve ◽

Robert B. Grieve

Keyword(s):

In Vitro Culture ◽

Dirofilaria Immitis ◽

Fourth Stage

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A scanning electron microscopy study of Anisakis physeteris molecularly identified: from third stage larvae from fish to fourth stage larvae obtained in vitro

Parasitology Research ◽

10.1007/s00436-018-5896-5 ◽

2018 ◽

Vol 117 (7) ◽

pp. 2095-2103 ◽

Cited By ~ 2

Author(s):

Dolores Molina-Fernández ◽

Francisco Javier Adroher ◽

Rocío Benítez

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Microscopy Study ◽

Electron Microscopy Study ◽

Fourth Stage ◽

Scanning Electron Microscopy Study ◽

Third Stage ◽

Anisakis Physeteris ◽

Scanning Electron

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Thermotaxis in third and fourth-stageDirofilaria immitislarvae

Journal of Helminthology ◽

10.1017/s0022149x00008257 ◽

1986 ◽

Vol 60 (1) ◽

pp. 61-64 ◽

Cited By ~ 1

Author(s):

M. Mok ◽

D. Abraham ◽

R. B. Grieve ◽

C. B. Thomas

Keyword(s):

Thermal Gradient ◽

Dirofilaria Immitis ◽

Fourth Stage ◽

Successful Transmission ◽

Larval Migration ◽

Third Stage

AbstractThird-stage and fourth-stageDirofilaria immitislarvae exhibited positive thermotaxis when placed in a thermal gradient. Negative thermotaxis was not observed. Positive thermotaxis may be important for the successful transmission and for directing third and fourth-stage larval migration toward predilection sites in the Host.

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In vitro development of cyathostomin larvae from the third stage larvae to the fourth stage: morphologic characterization, effects of refrigeration, and species-specific patterns

Veterinary Parasitology ◽

10.1016/j.vetpar.2009.04.029 ◽

2009 ◽

Vol 163 (4) ◽

pp. 348-356 ◽

Cited By ~ 7

Author(s):

Emanuele Brianti ◽

Salvatore Giannetto ◽

Donato Traversa ◽

Sharon R. Chirgwin ◽

Krishna Shakya ◽

...

Keyword(s):

Fourth Stage ◽

In Vitro Development ◽

The Third ◽

Third Stage ◽

Species Specific ◽

Vitro Development

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Establishing a Living Biobank of Patient-Derived Organoids of Intraductal Papillary Mucinous Neoplasms of the Pancreas

10.1101/2020.09.11.283168 ◽

2020 ◽

Author(s):

Francisca Beato ◽

Dayana Reverón ◽

Kaleena B. Dezsi ◽

Antonio Ortiz ◽

Joseph O. Johnson ◽

...

Keyword(s):

Early Detection ◽

Unmet Need ◽

The United States ◽

Cancer Center ◽

Pancreatic Cysts ◽

Intraductal Papillary Mucinous Neoplasms ◽

Cross Sectional ◽

Clinical Model ◽

Mucinous Neoplasms

AbstractPancreatic cancer (PaCa) is the third leading cause of cancer-related deaths in the United States. There is an unmet need to develop strategies to detect PaCa at an early, operable stage and prevent its progression. Intraductal papillary mucinous neoplasms (IPMNs) are cystic PaCa precursors that comprise nearly 50% of pancreatic cysts detected incidentally via cross-sectional imaging. Since IPMNs can progress from low- and moderate-grade dysplasia to high-grade dysplasia and invasion, the study of these lesions offers a prime opportunity to develop early detection and prevention strategies. Organoids are an ideal preclinical platform to study IPMNs, and the objective of the current investigation was to establish a living biobank of patient-derived organoids (PDO) from IPMNs. IPMN tumors and adjacent normal pancreatic tissues were successfully harvested from 15 patients with IPMNs undergoing pancreatic surgical resection at Moffitt Cancer Center & Research Institute (Tampa, FL) between May of 2017 and March of 2019. Organoid cultures were also generated from cryopreserved tissues. Organoid count and size were determined over time by both Image-Pro Premier 3D Version 9.1 digital platform and Matlab application of a Circular Hough Transform algorithm, and histologic and genomic characterization of a subset of the organoids was performed using immunohistochemistry and targeted sequencing, respectively. The success rates for organoid generation from IPMN tumor and adjacent normal pancreatic tissues were 81% and 87%, respectively. IPMN organoids derived from different epithelial subtypes showed different morphologies in vitro, and organoids recapitulated histologic and genomic characteristics of the parental IPMN tumor. In summary, this pre-clinical model has the potential to provide new opportunities to unveil mechanisms of IPMN progression to invasion and to shed insight into novel biomarkers for early detection and targets for chemoprevention.

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