Comparative growth and development of Trichinella spiralis in vitro and in vivo, with a redescription of the life cycle

Abstract Our work aimed to evaluate the possible effect of Annona muricata (Graviola) leaf extract on Trichinella spiralis in in vitro and in vivo studies. Trichinella spiralis worms were isolated from infected mice and transferred to three culture media – group I (with no drugs), group II (contained Graviola) and group III (contained albendazole) – then they were examined using the electron microscope. In the in vivo study, mice were divided into five groups: GI (infected untreated), GII (prophylactically treated with Graviola for seven days before infection), GIII (infected and treated with Graviola), GIV (infected and treated with albendazole) and GV (infected and treated with a combination of Graviola plus albendazole in half doses). Drug effects were assessed by adults and larvae load beside the histopathological small intestinal and muscular changes. A significant reduction of adult and larval counts occurred in treated groups in comparison to the control group. Histopathologically, marked improvement in the small intestinal and muscular changes was observed in treated groups. Also, massive destruction of the cultured adults’ cuticle was detected in both drugs. This study revealed that Graviola leaves have potential activity against trichinellosis, especially in combination with albendazole, and could serve as an adjuvant to anti-trichinellosis drug therapy.

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In vitro life cycle of pentamidine-resistant amastigotes: stability of the chemoresistant phenotypes is dependent on the level of resistance induced.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.9.1898 ◽

1997 ◽

Vol 41 (9) ◽

pp. 1898-1903 ◽

Cited By ~ 30

Author(s):

D Sereno ◽

J L Lemesre

Keyword(s):

Life Cycle ◽

Culture Conditions ◽

Cross Resistance ◽

Diminazene Aceturate ◽

Drug Pressure ◽

Mouse Macrophages ◽

Wild Type Clone ◽

Different Levels

Using a continuous drug pressure protocol, we induced pentamidine resistance in an active and dividing population of amastigote forms of Leishmania mexicana. We selected in vitro two clones with different levels of resistance to pentamidine, with clone LmPENT5 being resistant to 5 microM pentamidine, while clone LmPENT20 was resistant to 20 microM pentamidine. Resistance indexes (50% inhibitory concentration [IC50] after drug presure/IC50 before drug pressure) of 2 (LmPENT5) and 6 (LmPENT20) were determined after drug selection. Both resistant clones expressed significant cross-resistance to diminazene aceturate and primaquine. Pentamidine resistance was not reversed by verapamil, a calcium channel blocker known to reverse multidrug resistance (A. J. Bitonti, et al., Science 242:1301-1303, 1988; A. R. C. Safa et al., J. Biol. Chem. 262:7884-7888, 1987). No difference in the in vitro infectivity for resident mouse macrophages was observed between the wild-type clone (clone LmWT) and pentamidine-resistant clones. During in vitro infectivity experiments, when the life cycle was performed starting from the intramacrophagic amastigote stage, the drug resistance of the resulting LmPENT20 amastigotes was preserved even if the intermediate promastigote stage could not be considered resistant to 20 microM pentamidine. In the same way, when a complete developmental sequence of L. mexicana was achieved axenically by manipulation of appropriate culture conditions, the resulting axenically grown LmPENT20 amastigotes remained pentamidine resistant, whereas LmPENT5 amastigotes lost their ability to resist pentamidine, with IC50s and index of resistance values close to those for the LmWT clone. These results strongly indicate that the level of pentamidine tolerated by resistant amastigotes after the life cycle was dependent on the induced level of resistance. This fact could be significant in the in vivo transmission of drug-resistant parasites by Phlebotominae. Particular attention should be given to the finding that the emergence of parasite resistance is a potential risk of the use of inadequate doses as therapy in humans.

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Transcriptomic analysis reveals a role for the nervous system in regulating growth and development of Fasciola hepatica juveniles

10.1101/2022.01.13.476286 ◽

2022 ◽

Author(s):

Emily Robb ◽

Erin McCammick ◽

Duncan Wells ◽

Paul McVeigh ◽

Erica Gardiner ◽

...

Keyword(s):

Nervous System ◽

Rapid Growth ◽

Growth And Development ◽

Liver Fluke ◽

Fasciola Hepatica ◽

Expression Profiles ◽

Neuronal Signalling ◽

Growth Development

Fasciola spp. liver fluke have significant impacts in veterinary and human medicine. The absence of a vaccine and increasing anthelmintic resistance threaten sustainable control and underscore the need for novel flukicides. Functional genomic approaches underpinned by in vitro culture of juvenile Fasciola hepatica facilitate control target validation in the most pathogenic life stage. Comparative transcriptomics of in vitro and in vivo maintained 21 day old F. hepatica finds that 86% of genes are expressed at similar levels across maintenance treatments suggesting commonality in core biological functioning within these juveniles. Phenotypic comparisons revealed higher cell proliferation and growth rates in the in vivo juveniles compared to their in vitro counterparts. These phenotypic differences were consistent with the upregulation of neoblast-like stem cell and cell-cycle associated genes in in vivo maintained worms. The more rapid growth/development of in vivo juveniles was further evidenced by a switch in cathepsin protease expression profiles, dominated by cathepsin B in in vitro juveniles and then by cathepsin L in in vivo juveniles. Coincident with more rapid growth/development was the marked downregulation of both classical and peptidergic neuronal signalling components in in vivo maintained juveniles, supporting a role for the nervous system in regulating liver fluke growth and development. Differences in the miRNA complements of in vivo and in vitro juveniles identified 31 differentially expressed miRNAs, notably fhe-let-7a-5p , fhe-mir-124-3p and, miRNAs predicted to target Wnt-signalling, supporting a key role for miRNAs in driving the growth/developmental differences in the in vitro and in vivo maintained juvenile liver fluke. Widespread differences in the expression of neuronal genes in juvenile fluke grown in vitro and in vivo expose significant interplay between neuronal signalling and the rate of growth/development, encouraging consideration of neuronal targets in efforts to dysregulate growth/development for parasite control.

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Organoids and Bioengineered Intestinal Models: Potential Solutions to the Cryptosporidium Culturing Dilemma

Microorganisms ◽

10.3390/microorganisms8050715 ◽

2020 ◽

Vol 8 (5) ◽

pp. 715 ◽

Cited By ~ 6

Author(s):

Samantha Gunasekera ◽

Alireza Zahedi ◽

Mark O’Dea ◽

Brendon King ◽

Paul Monis ◽

...

Keyword(s):

Life Cycle ◽

Host Cells ◽

Protozoan Parasites ◽

Future Directions ◽

In Vivo Models ◽

Severe Diarrhea ◽

Transformed Cell Lines ◽

Gnotobiotic Piglets

Cryptosporidium is a major cause of severe diarrhea-related disease in children in developing countries, but currently no vaccine or effective treatment exists for those who are most at risk of serious illness. This is partly due to the lack of in vitro culturing methods that are able to support the entire Cryptosporidium life cycle, which has led to research in Cryptosporidium biology lagging behind other protozoan parasites. In vivo models such as gnotobiotic piglets are complex, and standard in vitro culturing methods in transformed cell lines, such as HCT-8 cells, have not been able to fully support fertilization occurring in vitro. Additionally, the Cryptosporidium life cycle has also been reported to occur in the absence of host cells. Recently developed bioengineered intestinal models, however, have shown more promising results and are able to reproduce a whole cycle of infectivity in one model system. This review evaluates the recent advances in Cryptosporidium culturing techniques and proposes future directions for research that may build upon these successes.

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Recombinant human megakaryocyte growth and development factor stimulates thrombocytopoiesis in normal nonhuman primates

Blood ◽

10.1182/blood.v86.1.54.bloodjournal86154 ◽

1995 ◽

Vol 86 (1) ◽

pp. 54-59 ◽

Cited By ~ 145

Author(s):

AM Farese ◽

P Hunt ◽

T Boone ◽

TJ MacVittie

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Growth And Development ◽

Nonhuman Primates ◽

Normal Male ◽

Platelet Counts ◽

Development Factor ◽

Development In Vitro

Megakaryocyte growth and development factor (MGDF) is a novel cytokine that binds to the c-mpl receptor and stimulates megakaryocyte development in vitro and in vivo. This report describes the ability of recombinant human (r-Hu) MGDF to affect megakaryocytopoiesis in normal nonhuman primates. r-HuMGDF was administered subcutaneously to normal, male rhesus monkeys once per day for 10 consecutive days at dosages of 2.5, 25, or 250 micrograms/kg of body weight. Bone marrow and peripheral blood were assayed for clonogenic activity and peripheral blood counts were monitored. Circulating platelet counts increased significantly (P < .05) for all doses within 6 days of r-HuMGDF administration and reached maximal levels between day 12 and day 14 postcytokine administration. The 2.5, 25.0, and 250.0 micrograms/kg/d doses elicited peak mean platelet counts that were 592%, 670%, and 449% of baseline, respectively. Bone marrow-derived clonogenic data showed significant increases in the concentration of megakaryocyte (MEG)- colony-forming unit (CFU) and granulocyte-erythroid-macrophage- megakaryocyte (GEMM)-CFU, whereas that of granulocyte-macrophage (GM)- CFU and burst-forming unit-erythroid (BFU-e) remained unchanged during the administration of r-HuMGDF. These data show that r-HuMGDF is a potent stimulator of thrombocytopoiesis in the normal nonhuman primate.

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Platelets Exposed to Elevated Levels of Endogenous Thrombopoietin In Vivo Have a Reduced Response to Megakaryocyte Growth and Development Factor In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0037-1612918 ◽

2001 ◽

Vol 85 (01) ◽

pp. 152-159 ◽

Cited By ~ 5

Author(s):

Uichi Nishiyama ◽

Haruhiko Morita ◽

Yoshifumi Torii ◽

Tomoaki Kuwaki ◽

Eiko Shimizu ◽

...

Keyword(s):

Growth And Development ◽

Platelet Reactivity ◽

Biochemical Properties ◽

Human Platelets ◽

Janus Kinase ◽

Intravenous Injections ◽

Development Factor ◽

Lower Reactivity

SummaryThrombopoietin (TPO), or megakaryocyte growth and development factor (MGDF), has been shown to potentiate the sensitivity of normal human platelets to various agonists in vitro. The present study investigated the functional and biochemical properties of platelets from mice rendered thrombocytopenic by sublethal irradiation with regard to the reactivity to recombinant murine MGDF (rmMGDF) in vitro. During the course of reversible thrombocytopenia following irradiation, platelets from irradiated mice which had lower platelet counts and reciprocally higher plasma TPO levels showed lower reactivity to rmMGDF in agonist-induced platelet aggregation. Intravenous injections of recombinant soluble murine c-Mpl (sMpl), which has the ability to capture TPO, after irradiation restored the reactivity of platelets at the platelet nadir to rmMGDF. On the other hand, platelets prepared from normal mice 3 h after a single intravenous injection of pegylated rmMGDF did not respond to rmMGDF. There was a marked decrease in c-Mpl and Janus kinase 2 (JAK2) in platelets from irradiated mice at the platelet nadir. Similar results were observed with platelets from mice administered pegylated rmMGDF. JAK2 was only moderately decreased, however, in platelets from mice given sMpl after irradiation. These results indicate that exposure of platelets to increased endogenous TPO levels in vivo in thrombocytopenic mice leads to a reduction in the platelet reactivity to rmMGDF in vitro. Further, these results suggest that the c-Mpl-mediated signaling pathway, which is essential for the priming effect of rmMGDF, is defective in thrombocytopenic murine platelets.

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Pegylated megakaryocyte growth and development factor abrogates the lethal thrombocytopenia associated with carboplatin and irradiation in mice

Blood ◽

10.1182/blood.v86.12.4486.bloodjournal86124486 ◽

1995 ◽

Vol 86 (12) ◽

pp. 4486-4492 ◽

Cited By ~ 132

Author(s):

MM Hokom ◽

D Lacey ◽

OB Kinstler ◽

E Choi ◽

S Kaufman ◽

...

Keyword(s):

Growth And Development ◽

Granulocyte Colony Stimulating Factor ◽

Potent Inducer ◽

Development Factor ◽

Internal Bleeding ◽

Life Threatening ◽

Stimulating Factor ◽

Sublethal Irradiation

Megakaryocyte growth and development factor (MGDF) is a potent inducer of megakaryopoiesis in vitro and thrombopoiesis in vivo. The effects of MGDF appear to be lineage-selective, making this cytokine an ideal candidate for use in alleviating clinically relevant thrombocytopenias. This report describes a murine model of life-threatening thrombocytopenia that results from the combination treatment of carboplatin and sublethal irradiation. Mortality of this regimen is 94% and is associated with widespread internal bleeding. The daily administration of pegylated recombinant human MGDF (PEG-rMGDF) significantly reduced mortality (to < 15%) and ameliorated the depth and duration of thrombocytopenia. The severity of leucopenia and anemia was also reduced, although it was not clear whether these effects were direct. Platelets generated in response to PEG-rMGDF were morphologically indistinguishable from normal platelets. PEG-rMGDF administered in combination with murine granulocyte colony-stimulating factor completely prevented mortality and further reduced leukopenia and thrombocytopenia. These data support the concept that PEG-rMGDF may be useful to treat iatrogenic thrombocytopenias.

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In vitro infection of megakaryocytes and their precursors by human cytomegalovirus

Blood ◽

10.1182/blood.v95.2.487 ◽

2000 ◽

Vol 95 (2) ◽

pp. 487-493 ◽

Cited By ~ 55

Author(s):

Kirsten Crapnell ◽

Esmail D. Zanjani ◽

Aniruddho Chaudhuri ◽

Joao L. Ascensao ◽

Stephen St. Jeor ◽

...

Keyword(s):

Life Cycle ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Allogeneic Bone Marrow Transplantation ◽

Laboratory Strain ◽

Allogeneic Bone ◽

Cd34 Cells ◽

Hcmv Disease

Apart from congenital human cytomegalovirus (HCMV) infection, manifest HCMV disease occurs primarily in immunocompromised patients. In allogeneic bone marrow transplantation, HCMV is frequently associated with graft failure and cytopenias involving all hematopoietic lineages, but thrombocytopenia is the most commonly reported hematologic complication. The authors hypothesized that megakaryocytes (MK) may be a specific target for HCMV. Although the susceptibility of immature hematopoietic progenitors cells to HCMV has been established, a productive viral life cycle has only been linked to myelomonocytic maturation. The authors investigated whether HCMV can also infect MK and impair their function. They demonstrated that HCMV did not affect the thrombopoietin (TPO)-driven proliferation of CD34+ cells until MK maturation occurred. MK challenged with HCMV showed a 50% more rapid loss of viability than mock-infected cells. MK and their early precursors were clearly shown to be susceptible to HCMV in vitro, as evidenced by the presence of HCMV in magnetic column-purified CD42+ MK and 2-color fluorescent staining with antibodies directed against CD42a and HCMV pp65 antigen. These findings were confirmed by the infection of MK with a laboratory strain of HCMV containing the β-galactosidase (β-gal) gene. Using chromogenic β-gal substrates, HCMV was detected during MK differentiation of infected CD34+ cells and after infection of fully differentiated MK. Production of infectious virus was observed in cultures infected MK, suggesting that HCMV can complete its life cycle. These results demonstrate that MK are susceptible to HCMV infection and that direct infection of these cells in vivo may contribute to the thrombocytopenia observed in patients infected with HCMV.

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