Ex Vivo Plasmodium malariae Culture Method for Antimalarial Drugs Screen in the Field

Abstract Bone is one of the most common sites for metastasis across cancers. Cancer cells that travel through the vasculature and invade new tissues can remain in a non-proliferative dormant state for years before colonizing the metastatic site. Switching from dormancy to colonization is the rate-limiting step of bone metastasis. Here we develop an ex vivo co-culture method to grow cancer cells in mouse bones to assess cancer cell proliferation using healthy or cancer-primed bones. Profiling soluble factors from conditioned media identifies the chemokine CXCL5 as a candidate to induce metastatic colonization. Additional studies using CXCL5 recombinant protein suggest that CXCL5 is sufficient to promote breast cancer cell proliferation and colonization in bone, while inhibition of its receptor CXCR2 with an antagonist blocks proliferation of metastatic cancer cells. This study suggests that CXCL5 and CXCR2 inhibitors may have efficacy in treating metastatic bone tumors dependent on the CXCL5/CXCR2 axis.

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Analysis of Plasmodium vivax schizont transcriptomes from field isolates reveals heterogeneity of expression of genes involved in host-parasite interactions

Scientific Reports ◽

10.1038/s41598-020-73562-7 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Sasha V. Siegel ◽

Lia Chappell ◽

Jessica B. Hostetler ◽

Chanaki Amaratunga ◽

Seila Suon ◽

...

Keyword(s):

Plasmodium Vivax ◽

Ex Vivo ◽

Culture Method ◽

Surface Proteins ◽

Parasite Development ◽

Field Isolates ◽

Host Parasite Interactions ◽

Short Period ◽

Host Parasite

Abstract Plasmodium vivax gene regulation remains difficult to study due to the lack of a robust in vitro culture method, low parasite densities in peripheral circulation and asynchronous parasite development. We adapted an RNA-seq protocol “DAFT-seq” to sequence the transcriptome of four P. vivax field isolates that were cultured for a short period ex vivo before using a density gradient for schizont enrichment. Transcription was detected from 78% of the PvP01 reference genome, despite being schizont-enriched samples. This extensive data was used to define thousands of 5′ and 3′ untranslated regions, some of which overlapped with neighbouring transcripts, and to improve the gene models of 352 genes, including identifying 20 novel gene transcripts. This dataset has also significantly increased the known amount of heterogeneity between P. vivax schizont transcriptomes from individual patients. The majority of genes found to be differentially expressed between the isolates lack Plasmodium falciparum homologs and are predicted to be involved in host-parasite interactions, with an enrichment in reticulocyte binding proteins, merozoite surface proteins and exported proteins with unknown function. An improved understanding of the diversity within P. vivax transcriptomes will be essential for the prioritisation of novel vaccine targets.

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A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Journal of Visualized Experiments ◽

10.3791/50359 ◽

2013 ◽

Cited By ~ 1

Author(s):

Laura A. Dyer ◽

Cam Patterson

Keyword(s):

Ex Vivo ◽

Culture Method ◽

Mouse Heart ◽

Embryonic Mouse

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Optimization of Ex Vivo Murine Bone Marrow Derived Immature Dendritic Cells: A Comparative Analysis of Flask Culture Method and Mouse CD11c Positive Selection Kit Method

Bone Marrow Research ◽

10.1155/2018/3495086 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 2

Author(s):

Rahul Ashok Gosavi ◽

Sukeshani Salwe ◽

Sandeepan Mukherjee ◽

Ritwik Dahake ◽

Sweta Kothari ◽

...

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

Positive Selection ◽

Mhc Class Ii ◽

Cell Population ◽

Ex Vivo ◽

Culture Method ◽

Flask Culture ◽

Suspended Cell ◽

Significant Difference

12–14 days of culturing of bone marrow (BM) cells containing various growth factors is widely used method for generating dendritic cells (DCs) from suspended cell population. Here we compared flask culture method and commercially available CD11c Positive Selection kit method. Immature BMDCs’ purity of adherent as well as suspended cell population was generated in the decreasing concentration of recombinant-murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) in nontreated tissue culture flasks. The expression of CD11c, MHCII, CD40, and CD86 was measured by flow cytometry. We found significant difference (P<0.05) between the two methods in the adherent cells population but no significant difference was observed between the suspended cell populations with respect to CD11c+ count. However, CD11c+ was significantly higher in both adhered and suspended cell population by culture method but kit method gave more CD11c+ from suspended cells population only. On the other hand, using both methods, immature DC expressed moderate level of MHC class II molecules as well as low levels of CD40 and CD86. Our findings suggest that widely used culture method gives the best results in terms of yield, viability, and purity of BMDCs from both adherent and suspended cell population whereas kit method works well for suspended cell population.

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Ex Vivo Culture Models of Hidradenitis Suppurativa for defining molecular pathogenesis and treatment efficacy of novel drugs

10.1101/2021.10.11.464007 ◽

2021 ◽

Author(s):

Kayla Goliwas ◽

Mahendra P Kashyap ◽

Jasim Khan ◽

Rajesh Sinha ◽

Zhiping Weng ◽

...

Keyword(s):

Hidradenitis Suppurativa ◽

Ex Vivo ◽

Drug Efficacy ◽

Culture Method ◽

Gene Clusters ◽

Dynamic Expression ◽

Novel Drugs ◽

Culture Models ◽

Ex Vivo Culture ◽

Gene Status

Hidradenitis suppurativa (HS) is a complex inflammatory and debilitating skin disease for which no effective treatment is available. This is partly because of the unavailability of suitable human or animal models with which exact pathobiology of the disease can be defined. Here, we describe the development of air-liquid (A-L) interface, liquid-liquid/liquid-submersion (L-S) and bioreactor (Bio) ex vivo skin culture models. All three ex vivo platforms were effective for culturing skin samples up to day-14, with the tissue architecture and integrity remaining intact for at least 3 days for healthy skin while for 14 days for HS skin. Up to day-3, no significant differences were observed in % early apoptotic cells among all three platforms. However, an increase was observed in late apoptotic/necrotic cells in HS skin at day-3 in A-L and Bio culture of HS skin. These cultures efficiently support the growth of various cells populations, including keratinocytes and immune cells. Profiling of the inflammatory genes using HS skin from these ex vivo cultures showed dynamic expression changes at day-3 and day-14. All of these cultures are necessary to represent the inflammatory gene status of HS skin at day-0 suggesting that not all gene clusters are identically altered in each culture method. Similarly, cytokine/chemokine profiling of the supernatant from vehicle- and drug-treated ex vivo HS cultures again showed better prediction of drug efficacy against HS. Overall, development of these three systems collectively provide a powerful tool to uncover the pathobiology of HS progression and screen various drugs against HS.

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Signaling Modulations of miR-206-3p in Tooth Morphogenesis

International Journal of Molecular Sciences ◽

10.3390/ijms21155251 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5251

Author(s):

Sanjiv Neupane ◽

Yam Prasad Aryal ◽

Tae-Young Kim ◽

Chang-Yeol Yeon ◽

Chang-Hyeon An ◽

...

Keyword(s):

Gene Expression ◽

Expression Pattern ◽

Ex Vivo ◽

Developmental Regulation ◽

Expression Patterns ◽

Tooth Germ ◽

Culture Method ◽

Tooth Pulp ◽

Altered Expression ◽

Tooth Morphogenesis

MicroRNAs (miRNAs) are a class of naturally occurring small non-coding RNAs that post-transcriptionally regulate gene expression in organisms. Most mammalian miRNAs influence biological processes, including developmental changes, tissue morphogenesis and the maintenance of tissue identity, cell growth, differentiation, apoptosis, and metabolism. The miR-206-3p has been correlated with cancer; however, developmental roles of this miRNA are unclear. In this study, we examined the expression pattern and evaluated the developmental regulation of miR-206-3p during tooth morphogenesis using ex-vivo culture method. The expression pattern of miR-206-3p was examined in the epithelium and mesenchyme of developing tooth germ with stage-specific manners. Perturbation of the expression of miR-206-3p clearly altered expression patterns of dental-development–related signaling molecules, including Axin2, Bmp2, Fgf4, Lef1 and Shh. The gene expression complemented with change in cellular events including, apoptosis and proliferation which caused altered crown and pulp morphogenesis in renal-capsule–calcified teeth. Especially, mislocalization of β-Catenin and SMAD1/5/8 were observed alongside dramatic alterations in the expression patterns of Fgf4 and Shh. Overall, our data suggest that the miR-206-3p regulate the cellular physiology during tooth morphogenesis through modulation of the Wnt, Bmp, Fgf, and Shh signaling pathways to form proper tooth pulp and crown.

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ABDOMINAL AORTIC ANEURYSMS: A NOVEL EX VIVO CELL CULTURE METHOD TO ANALYSE INFLAMMATORY AND ANGIOGENIC PATHWAYS

Cardiovascular Pathology ◽

10.1016/j.carpath.2004.03.047 ◽

2004 ◽

Vol 13 (3) ◽

pp. 18

Author(s):

Alexandra Kovalic ◽

Claudia Monaco ◽

Roger M Greenhalgh ◽

Ian J Franklin ◽

Ewa M Paleolog

Keyword(s):

Cell Culture ◽

Ex Vivo ◽

Culture Method ◽

Abdominal Aortic Aneurysms ◽

Aortic Aneurysms ◽

Abdominal Aortic

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Molecular Surveillance and Ex Vivo Drug Susceptibilities of Plasmodium vivax Isolates From the China–Myanmar Border

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.738075 ◽

2021 ◽

Vol 11 ◽

Author(s):

Weilin Zeng ◽

Hui Zhao ◽

Wei Zhao ◽

Qi Yang ◽

Xinxin Li ◽

...

Keyword(s):

Drug Resistance ◽

Multidrug Resistance ◽

Plasmodium Vivax ◽

Ex Vivo ◽

Antimalarial Drugs ◽

Multidrug Resistance Protein 1 ◽

Molecular Surveillance ◽

Dihydropteroate Synthase ◽

Synonymous Substitutions ◽

Resistance Protein

Drug resistance in Plasmodium vivax may pose a challenge to malaria elimination. Previous studies have found that P. vivax has a decreased sensitivity to antimalarial drugs in some areas of the Greater Mekong Sub-region. This study aims to investigate the ex vivo drug susceptibilities of P. vivax isolates from the China–Myanmar border and genetic variations of resistance-related genes. A total of 46 P. vivax clinical isolates were assessed for ex vivo susceptibility to seven antimalarial drugs using the schizont maturation assay. The medians of IC50 (half-maximum inhibitory concentrations) for chloroquine, artesunate, and dihydroartemisinin from 46 parasite isolates were 96.48, 1.95, and 1.63 nM, respectively, while the medians of IC50 values for piperaquine, pyronaridine, mefloquine, and quinine from 39 parasite isolates were 19.60, 15.53, 16.38, and 26.04 nM, respectively. Sequence polymorphisms in pvmdr1 (P. vivax multidrug resistance-1), pvmrp1 (P. vivax multidrug resistance protein 1), pvdhfr (P. vivax dihydrofolate reductase), and pvdhps (P. vivax dihydropteroate synthase) were determined by PCR and sequencing. Pvmdr1 had 13 non-synonymous substitutions, of which, T908S and T958M were fixed, G698S (97.8%) and F1076L (93.5%) were highly prevalent, and other substitutions had relatively low prevalences. Pvmrp1 had three non-synonymous substitutions, with Y1393D being fixed, G1419A approaching fixation (97.8%), and V1478I being rare (2.2%). Several pvdhfr and pvdhps mutations were relatively frequent in the studied parasite population. The pvmdr1 G698S substitution was associated with a reduced sensitivity to chloroquine, artesunate, and dihydroartemisinin. This study suggests the possible emergence of P. vivax isolates resistant to certain antimalarial drugs at the China–Myanmar border, which demands continuous surveillance for drug resistance.

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