Toxoplasma gondii infection of fibroblasts causes the production of exosome-like vesicles containing a unique array of mRNA and miRNA transcripts compared to serum starvation

Abstract

Background: Until recently thought to be of little significance unless occurring during pregnancy, Toxoplasma gondii infection of human hosts is now known to play a larger role in mental health and is a growing concern in the health care community. We sought to elucidate a possible mechanism by which Toxoplasma infection may cause some of the behavioural pathology now associated with infection. We hypothesized that exosomes may be playing a role.

Methods: We utilized electron microscopy to detect the presence and size of extracellular vesicles in the supernatants of Toxoplasma-infected human foreskin fibroblasts (HFF). We then utilized microarray analysis to discern mRNA and miRNA content of the vesicles isolated from supernatants of Toxoplasma-infected (Toxo) and serum-starved (SS) HFF.

Results: We recovered extracellular vesicles with a size consistent with exosomes that we called exosome-like vesicles (ELVs) from the supernatants of SS and Toxo cultures. The mRNA and miRNA content of these ELVs was highly regulated creating specific and unique expression profiles comparing Toxo ELVs, SS ELVs and RNA isolated from whole cell homogenates. Interestingly, among the most enriched mRNA isolated from ELVs of Toxo cells are 4 specific mRNA species that have been described in the literature as having neurologic activity: Rab-13, eukaryotic translation elongation factor 1 alpha 1, thymosin beta 4 and LLP homolog. In addition, miRNA species uniquely expressed in Toxo ELVs include miR-23b, a well-known regulator of IL-17.

Conclusion: While the production of ELVs containing mRNAs that modify behaviour are consistent with reported Toxoplasma pathology, the mechanism of enrichment and ultimate in vivo effect of these mRNA and miRNA containing ELVs remains to be investigated.

SHORT COMMUNICATION

Toxoplasma gondii infection of fibroblasts causes the production of exosome-like vesicles containing a unique array of mRNA and miRNA transcripts compared to serum starvation

Samuel M. Pope1* and Cecilia Lässer2

1Department of Biomedical Sciences, Marian University College of Osteopathic Medicine, Indianapolis IN, USA; 2Department of Internal Medicine and Clinical Nutrition, Krefting Research Centre, University of Gothenburg, Gothenburg, Sweden

Abstract

Background : Until recently thought to be of little significance unless occurring during pregnancy, Toxoplasma gondii infection of human hosts is now known to play a larger role in mental health and is a growing concern in the health care community. We sought to elucidate a possible mechanism by which Toxoplasma infection may cause some of the behavioural pathology now associated with infection. We hypothesized that exosomes may be playing a role.

Methods : We utilized electron microscopy to detect the presence and size of extracellular vesicles in the supernatants of Toxoplasma-infected human foreskin fibroblasts (HFF). We then utilized microarray analysis to discern mRNA and miRNA content of the vesicles isolated from supernatants of Toxoplasma-infected (Toxo) and serum-starved (SS) HFF.

Results : We recovered extracellular vesicles with a size consistent with exosomes that we called exosome-like vesicles (ELVs) from the supernatants of SS and Toxo cultures. The mRNA and miRNA content of these ELVs was highly regulated creating specific and unique expression profiles comparing Toxo ELVs, SS ELVs and RNA isolated from whole cell homogenates. Interestingly, among the most enriched mRNA isolated from ELVs of Toxo cells are 4 specific mRNA species that have been described in the literature as having neurologic activity: Rab-13, eukaryotic translation elongation factor 1 alpha 1, thymosin beta 4 and LLP homolog. In addition, miRNA species uniquely expressed in Toxo ELVs include miR-23b, a well-known regulator of IL-17.

Conclusion : While the production of ELVs containing mRNAs that modify behaviour are consistent with reported Toxoplasma pathology, the mechanism of enrichment and ultimate in vivo effect of these mRNA and miRNA containing ELVs remains to be investigated.

Keywords: Toxoplasma; mRNA; miRNA; exosomes; extracellular vesicles; microarray

To access the supplementary material to this article, please see Supplementary files under Article Tools online.

Received: 29 July 2013; Revised: 30 September 2013; Accepted: 11 October 2013; Published: 11 December 2013

Journal of Extracellular Vesicles 2013. © 2013 Samuel M. Pope and Cecilia Lässer. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 3.0 Unported License (http://creativecommons.org/licenses/by-nc/3.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Citation: Journal of Extracellular Vesicles 2013, 2: 22484 - http://dx.doi.org/10.3402/jev.v2i0.22484

Toxoplasma gondii (Toxoplasma) is a ubiquitous protozoan coccidian parasite of global public health concern. Members of the Felidae (cat) family are the primary hosts, where the parasite undergoes sexual reproduction. All other mammals - including the infection of nearly 2 billion humans worldwide - serve as a secondary host, where the parasite is tropic for the brain. In the brain, the Toxoplasma tachyzoite multiplies via binary fission before entering a metabolically inactive state, bradyzoite, in a cyst-like lesion. This tropism for neurologic tissue is considered by some to be the cause of well-described Toxoplasma-induced behavioural effects in secondary hosts. The cause remains debated; however, recent studies have shown cyst location is random and behavioural effects are not (1,2). In rodents, Toxoplasma infection causes a reduced fear response to feline odours (3-7). While Toxoplasma infection in humans used to be considered transitory, it is now clear there are long-lasting effects of infection. In humans, Toxoplasma infection is positively correlated with the development of schizophrenia and Alzheimer's disease (8-11). It is known that other protozoans make functional exosomes as reported in the literature for Plasmodium falciparum, Trichomonas sp. and Leishmania (12-14). It has also been reported that Toxoplasma-infected cells make proinflammatory exosomes that increase Tumour necrosis factor-? (TNF-?) production. However, there was no further characterization of those extracellular vesicles (15). It is also known that viral infection causes a cell to change the protein content of the exosomes it secretes (16). Therefore, we hypothesize that Toxoplasma and/or toxoplasma-infected cells produce exosomes that could be a contributing factor to pathology and perhaps behavioural changes in secondary hosts. Exosomes, a sub-population of extracellular vesicles, are small vesicles formed in the endosomal pathway and loaded with various RNAs and proteins. Exosomes are released from the parent cells constitutively, with increases in times of cellular stress. They are then internalized by or bind to neighbouring cells where they release their contents. The contents of the exosomes then exert effects on the recipient cell based on the species of RNA and protein contained in the exosome.

Since the mechanism of Toxoplasma infection associated behavioural pathology is yet to be elucidated, and other protozoa produce exosomes, we hypothesized that exosomes from Toxoplasma or host cells produce exosomes that play a role in pathogenesis. To investigate our hypothesis, we examined supernatants of infected HFF and found extracellular vesicles of the correct size for exosomes that we called exosome-like vesicles (ELVs) by electron microscopy. Furthermore, when we explored the RNA contents using microarrays, we found human mRNA content to be unique when compared to ELVs from uninfected cells.

Methods

Cell culture and Toxoplasma infection

Human foreskin fibroblasts (HFF) were grown to confluence in tissue culture DMEM 10% foetal bovine serum (FBS) and infected with approximately 2.0 MOI, multiplicity of infection, of fresh Prugniaud strain of Toxoplasma gondii or deprived of FBS. Serum starvation was used as a way to induce exosome production from the HFF at the time points analyzed, as normal levels of exosome production in unstressed HFF did not yield enough RNA for our study. Serum starvation ultimately induces apoptosis but is known to increase the production of extracellular vesicles as well (17). After an incubation period of 24- or 72-hours, the supernatant was collected from the cultures. Each experiment consisted of 20 T-75 flasks per condition that were pooled for vesicles isolation. We conducted 3 experiments at the 72-hour time period and 1 at the 24-hour time period.

Extracellular vesicle isolation

Extracellular vesicles were isolated by a method outlined in Lässer et al. 2012 (18). In brief, supernatants were harvested from SS and Toxo groups and centrifuged at 300×g for 10 minutes at 4°C. The supernatant was transferred to new centrifuge tubes where it was centrifuged at 16,500×g for 20 minutes at 4°C. The supernatant was then transferred to ultra-centrifuge tubes and centrifuged at 140,000×g for 45 minutes at 4°C. The pellet containing extracellular vesicles was resuspended in 200 µl PBS. A sample was sent for electron microscopy to determine the presence and size of extracellular vesicles.

RNA isolation

The ELVs-samples were treated with 1,000 U RNAse and DNAse for 20 minutes at 37°C. After nuclease treatment, RNA was purified from the ELVs utilizing the Trizol© RNA purification method as per manufacturer's recommendations. RNA was quantified utilizing UV spectrophotometry.

RT-PCR

Purified RNA was used to create Template DNA with Invitrogen's First-Strand cDNA Synthesis Kit with Superscript III Reverse Transcriptase© and oligo (dt) as per manufacturer's protocol.

Microarray procedure

Purified RNA from whole cell lysates, ELVs from Toxoplasma-infected HFF, and ELVs from serum-starved (SS) HFF uninfected with Toxoplasma were submitted for microarray analysis to the University of Pennsylvania Microarray core facility as per their protocols (19). We used the Affymetrix human gene 1.0 st mRNA chip, covering a RefSeq (Entrez) gene count of 21,014 genes, to identify mRNA species in the RNA isolated from our exosome preparations. For microRNA analysis, the Affymetrix microRNA Genechip with Genisphere FlashTag© was used. For Toxoplasma gene product analysis, a proprietary University of Pennsylvania microarray core Toxoplasma expression chip was used. Data analysis providing differential expression values was performed by the University of Pennsylvania's microarray facility utilizing GeneSpring© software. Expression data for mRNA were confirmed by use of RT-PCR amplification of specific mRNA of interest.

Electron microscopy

Samples were placed as a drop on to Parafilm. Formvar/carbon-coated nickel grids (Ted Pella Inc., Redding, USA), which had been pre-UV-treated for 5 minutes were placed on top of the drop. The samples were fixed in 2% paraformaldehyde and washed with PBS. Next, the samples were immunostained with anti-CD63 antibody (BD Bioscience, Erembodegem, Belgium) or isotype control (Sigma-Aldrich, St. Louis, MO, USA), followed by staining with a 10 nm gold-labelled secondary antibody (Sigma-Aldrich). The samples were then fixed in 2.5% glutaraldehyde, washed in ddH2O and contrasted in 2% uranyl acetate. The preparations were examined in a LEO 912AB Omega electron microscope (Carl Zeiss NTS, Jena, Germany).

Results

Electron microscopy revealed extracellular vesicles in the preparations from Toxoplasma-infected HFF (Toxo) and SS HFF, being the correct size of exosomes (<100 nm), but no CD63 was detected (Fig. 1). We believe they are, in fact, exosomes, but we are calling them ELVs until they are further characterized. As our methodology was novel in utilizing microarray analysis of contents of ELVs to examine the effects of Toxoplasma infection on cell culture, it is important to note observations made from initial analysis of the microarray data included as acceptable microarray RNA quality control data (Supplementary File). Furthermore, we observed unique expression profiles for mRNA and miRNA data for ELVs from Toxo HFF compared to ELVs from SS HFF, which differed greatly from samples of ELVs not treated with nuclease and those from whole cell lysates, indicating our methodology of extracellular vesicle purification, RNA isolation and utilization of microarray analysis was successful (Supplementary File). While our methodology was successful and merited further analysis of the mRNA and miRNA data, not all of our data were easily interpreted. We did not see evidence of Toxoplasma RNA in ELVs, but we do not know if that is due to low levels of Toxo gene expression relative to humans or whether they are included at all (Supplementary File). Upon further analysis of the raw comparative human mRNA expression data (Supplementary File), a strategy was developed for picking salient genes from the multitude of those uniquely represented in the ELVs from Toxo compared to SS. First, we ranked the expression of Toxo/SS in descending order revealing 222 mRNA at the 24-hour time point that were more than 5-fold greater in Toxo ELVs compared to SS ELVs. We then screened out RNA with descriptions indicating they were of ribosomal, mitochondrial and microRNA origin, leaving a manageable list of genes that are highly represented in Toxo ELVs compared to SS ELVs. We chose the 11 highest represented mRNA at the 72-hour time point to further investigate (Table I).

Fig 1

Fig. 1. Electron microscopy of vesicle preparations. SS and Toxo vesicle-enriched samples were negative stained and revealed extracellular vesicles of approximately 80-90 nm, the correct dimensions for exosomes.

[Table omitted -see PDF.]

Values are reported as fold-expression Toxo/SS.

Discussion

The data we present are important because they show that a new mechanism for Toxoplasma pathogenesis is possible. ELVs from infected cells could attach and deliver their contents to uninfected cells, thereby causing the function of uninfected cells to be altered by the addition of the vesicle contents. It is clear from RT-PCR that full-length transcripts of mRNA are present, protected inside of ELVs even after strong nuclease treatment. Full-length mRNA transcripts of neurologically active proteins we report here could alter brain chemistry to the point of behavioural changes. This proposed mechanism would make the exact location of the infected cells less important, as the effect would be spread by the secretion of ELVs from infected cells throughout the brain. If this mechanism is experimentally proven, Toxoplasma could join the ranks of protozoan pathogens that manipulate the host using exosomes, such as Trichomonas vaginalis that produces exosomes that deliver their contents to host cells and modulate host cell immune responses and the parasite's adherence to host cells (13); Leishmania sp. producing its own exosomes that influence macrophages to secrete Interleukin-8, necessary to promote infection (14); and Plasmodium falciparum which has recently been shown to produce extracellular vesicles to exchange genetic material such as drug resistance genes (12). However, unlike the above listed Protozoans, Toxoplasma does not appear to be releasing ELVs containing its own genetic material.

Here, we have shown that ELVs are produced by Toxoplasma-infected HFF, full length mRNA transcript is present and that ELVs' RNA contents are not just reflecting a sample of cytoplasmic RNA. Furthermore, RNA from Toxoplasma-infected cell ELVs are distinct from RNA from ELVs produced by SS cells. Indeed, the mRNA microarray data from 24- and 72-hour post-infection time points produced similar results, but not identical, providing further support for the validity of the method and experimental findings. Among the most highly represented mRNA in ELVs from Toxoplasma-infected cells were 4 mRNA for proteins that have been previously reported to have neurologic activity - Eukaryotic translation elongation factor 1 alpha 1, Thymosin beta 4, LPP-homolog and Rab-13. Perhaps adding to the importance of the data is the fact that these ELVs were created by fibroblasts and not neurons, which might be expected to more readily produce significant amounts of neurologically relevant mRNA.

Expression data for miRNA is more difficult to interpret without direct physical experimentation to determine the specific effects, due to many miRNA species having seemingly opposite expression profiles in similar pathologic states: miR-125b reported to cause apoptosis of cancer cells on one hand and conferred a chemoresistant profile to cancer cells on the other (38-40). However, the role of one miRNA seems resolute. miR-23b is an anti-inflammatory mediator that suppresses IL-17 production and response. While it may be intended to ameliorate immune-mediated pathology, suppression of the inflammatory IL-17 response is also likely to aid in the infection process, which would be a similar function reported for Leishmania sp. (14).

The exact impact ELVs from Toxoplasma-infected cells may have on the pathogenesis of Toxoplasma cannot be certain without direct experimentation. However, these data certainly raise the possibility that neurologic effects of Toxoplasma may be, in part, impacted by production of RNA-laden ELVs we described here. It becomes attractive to conjecture that the Toxoplasma parasite could be exerting influence over the exact content of ELVs to promote its own survival. However, much more study needs to be done before such a conclusion could be made. Indeed, little is known about many of the fundamental mechanisms of ELVs production. Among the fundamental questions that beckon for further study are: How is the production of ELVs regulated? How is RNA loaded into ELVs? How does the cell specify which RNA is loaded? Our future studies will focus on these fundamental aspects of ELVs production.

Acknowledgements

Special thanks to Jan Lötval, MD, PhD, for gracious collaboration and to Daniel Beiting, PhD, for invaluable technical support and for supplying the HFF and Prugniaud strain of Toxoplasma gondii stocks. Kentucky College of Osteopathic Medicine was the sole provider of funding for these studies. Thanks to Garred R. Cline OMSIV for assistance in the laboratory.

Conflict of interest and funding

Authors have no conflicts of interest to declare. Experiments were funded with intramural institutional funds.

References

1. Berenreiterova M, Flegr J, Kubena AA, Nemec P. The distribution of Toxoplasma gondii cysts in the brain of a mouse with latent toxoplasmosis: implications for the behavioral manipulation hypothesis. PLoS One. 2011;6:e28925. PubMed Abstract | PubMed Central Full Text | Publisher Full Text

2. Di Cristina M, Marocco D, Galizi R, Proietti C, Spaccapelo R, Crisanti A. Temporal and spatial distribution of Toxoplasma gondii differentiation into Bradyzoites and tissue cyst formation in vivo. Infect Immun. 2008;76:3491-501. PubMed Abstract | PubMed Central Full Text | Publisher Full Text

3. Kannan G, Moldovan K, Xiao J-C, Yolken RH, Jones-Brando L, Pletnikov MV. Toxoplasma gondii strain-dependent effects on mouse behaviour. Folia Parasitol. 2010;57:151-5. PubMed Abstract

4. da Silva RC, Langoni H. Toxoplasma gondii: host-parasite interaction and behavior manipulation. Parasitol Res. 2009;105:893-8. PubMed Abstract | Publisher Full Text

5. Webster JP. The effect of Toxoplasma gondii on animal behavior: playing cat and mouse. Schizophr Bull. 2007;33:752-6. PubMed Abstract | PubMed Central Full Text | Publisher Full Text

6. Vyas A, Kim S-K, Giacomini N, Boothroyd JC, Sapolsky RM. Behavioral changes induced by Toxoplasma infection of rodents are highly specific to aversion of cat odors. Proc Natl Acad Sci U S A. 2007;104:6442-7. PubMed Abstract | PubMed Central Full Text | Publisher Full Text

7. Vyas A, Kim SK, Sapolsky RM. The effects of toxoplasma infection on rodent behavior are dependent on dose of the stimulus. Neuroscience. 2007;148:342-8. PubMed Abstract | PubMed Central Full Text | Publisher Full Text

8. Flegr J. Influence of latent Toxoplasma infection on human personality, physiology and morphology: pros and cons of the Toxoplasma-human model in studying the manipulation hypothesis. J Exp Biol. 2013;216:127-33. PubMed Abstract | Publisher Full Text

9. Arias I, Sorlozano A, Villegas E, de Dios Luna J, McKenney K, Cervilla J, et al. Infectious agents associated with schizophrenia: a meta-analysis. Schizophr Res. 2012;136:128-36. PubMed Abstract | Publisher Full Text

10. Torrey EF, Bartko JJ, Lun ZR, Yolken RH. Antibodies to Toxoplasma gondii in patients with schizophrenia: a meta-analysis. Schizophr Bull. 2007;33:729-36. PubMed Abstract | PubMed Central Full Text | Publisher Full Text

11. El-Sahn AA, Shatat HZ, Ghitany EM. Seropositivity of toxoplasmosis in patients with schizophrenia. J Egypt Public Health Assoc. 2005;80:509-24. PubMed Abstract

12. Regev-Rudzki N, Wilson Danny W, Carvalho Teresa G, Sisquella X, Coleman Bradley M, Rug M, et al. Cell-Cell Communication between Malaria-Infected Red Blood Cells via Exosome-like Vesicles. Cell. 2013;153:1120-33. PubMed Abstract | Publisher Full Text

13. Twu O, de Miguel N, Lustig G, Stevens GC, Vashisht AA, Wohlschlegel JA, et al. Trichomonas vaginalis exosomes deliver Cargo to host cells and mediate Hostratio Parasite interactions. PLoS Pathog. 2013;9:e1003482. PubMed Abstract | PubMed Central Full Text | Publisher Full Text

14. Silverman JM, Clos J, de'Oliveira CC, Shirvani O, Fang Y, Wang C, et al. An exosome-based secretion pathway is responsible for protein export from Leishmania and communication with macrophages. J Cell Sci. 2010;123:842-52. PubMed Abstract | Publisher Full Text

15. Bhatnagar S, Shinagawa K, Castellino FJ, Schorey JS. Exosomes released from macrophages infected with intracellular pathogens stimulate a proinflammatory response in vitro and in vivo. Blood. 2007;110:3234-44. PubMed Abstract | PubMed Central Full Text | Publisher Full Text

16. Meckes DG, Jr., Gunawardena HP, Dekroon RM, Heaton PR, Edwards RH, Ozgur S, et al. Modulation of B-cell exosome proteins by gamma herpesvirus infection. Proc Natl Acad Sci U S A. 2013;110(31):E2925-33. PubMed Abstract | Publisher Full Text

17. Sirois I, Raymond MA, Brassard N, Cailhier JF, Fedjaev M, Hamelin K, et al. Caspase-3-dependent export of TCTP: a novel pathway for antiapoptotic intercellular communication. Cell Death Differ. 2011;18:549-62. PubMed Abstract | PubMed Central Full Text | Publisher Full Text

18. Lässer C, Eldh M, Lotvall J. Isolation and characterization of RNA-containing exosomes. J Vis Exp. 2012;(59):e3037. doi: 10.3791/3037. Publisher Full Text

19. Scearce LM, Brestelli JE, McWeeney SK, Lee CS, Mazzarelli J, Pinney DF, et al. Functional genomics of the endocrine pancreas: the pancreas clone set and PancChip, new resources for diabetes research. Diabetes. 2002;51:1997-2004. PubMed Abstract | Publisher Full Text

20. Jin R, Chow VT, Tan PH, Dheen ST, Duan W, Bay BH. Metallothionein 2A expression is associated with cell proliferation in breast cancer. Carcinogenesis. 2002;23:81-6. PubMed Abstract | Publisher Full Text

21. Laghaei R, Evans DG, Coalson RD. Metal binding sites of human H-chain ferritin and iron transport mechanism to the ferroxidase sites: a molecular dynamics simulation study. Proteins. 2013;81:1042-50. PubMed Abstract | Publisher Full Text

22. Blanch A, Robinson F, Watson IR, Cheng LS, Irwin MS. Eukaryotic translation elongation factor 1-alpha 1 inhibits p53 and p73 dependent apoptosis and chemotherapy sensitivity. PLoS One. 2013;8:E66436. PubMed Abstract | PubMed Central Full Text | Publisher Full Text

23. Tsai NP, Wilkerson JR, Guo W, Maksimova MA, DeMartino GN, Cowan CW, et al. Multiple autism-linked genes mediate synapse elimination via proteasomal degradation of a synaptic scaffold PSD-95. Cell. 2012;151:1581-94. PubMed Abstract | Publisher Full Text

24. Kelkar A, Dobberstein B. Sec61beta, a subunit of the Sec61 protein translocation channel at the endoplasmic reticulum, is involved in the transport of Gurken to the plasma membrane. BMC Cell Biol. 2009;10:11. PubMed Abstract | PubMed Central Full Text | Publisher Full Text

25. Sakane A, Abdallah AA, Nakano K, Honda K, Ikeda W, Nishikawa Y, et al. Rab13 small G protein and junctional Rab13-binding protein (JRAB) orchestrate actin cytoskeletal organization during epithelial junctional development. J Biol Chem. 2012;287:42455-68. PubMed Abstract | Publisher Full Text

26. Sakane A, Honda K, Sasaki T. Rab13 regulates neurite outgrowth in PC12 cells through its effector protein, JRAB/MICAL-L2. Mol Cell Biol. 2010;30:1077-87. PubMed Abstract | PubMed Central Full Text | Publisher Full Text

27. Adachi Y, Pavlakis GN, Copeland TD. Identification and characterization of SET, a nuclear phosphoprotein encoded by the translocation break point in acute undifferentiated leukemia. J Biol Chem. 1994;269:2258-62. PubMed Abstract

28. Lam BD, Anthony EC, Hordijk PL. Cytoplasmic targeting of the proto-oncogene SET promotes cell spreading and migration. FEBS Lett. 2013;587:111-19. PubMed Abstract | Publisher Full Text

29. Jeon BJ, Yang Y, Kyung Shim S, Yang HM, Cho D, Ik Bang S. Thymosin beta-4 promotes mesenchymal stem cell proliferation via an interleukin-8-dependent mechanism. Exp Cell Res. 2013;319:2526-34.

30. Xiong Y, Zhang Y, Mahmood A, Meng Y, Zhang ZG, Morris DC, et al. Neuroprotective and neurorestorative effects of thymosin beta4 treatment initiated 6 hours after traumatic brain injury in rats. J Neurosurg. 2012;116:1081-92. PubMed Abstract | PubMed Central Full Text | Publisher Full Text

31. Xiong Y, Mahmood A, Meng Y, Zhang Y, Zhang ZG, Morris DC, et al. Neuroprotective and neurorestorative effects of thymosin beta4 treatment following experimental traumatic brain injury. Ann N Y Acad Sci. 2012;1270:51-8. PubMed Abstract | PubMed Central Full Text | Publisher Full Text

32. Eckel-Mahan KL, Storm DR. Rookie snail protein LAPS veteran C/EBP: net transcriptional proceeds for long-term facilitation. Neuron. 2006;49:645-6. PubMed Abstract | Publisher Full Text

33. Kim H, Chang DJ, Lee JA, Lee YS, Kaang BK. Identification of nuclear/nucleolar localization signal in Aplysia learning associated protein of slug with a molecular mass of 18 kDa homologous protein. Neurosci Lett. 2003;343:134-8. PubMed Abstract | Publisher Full Text

34. Ong YC, Boyle JP, Boothroyd JC. Strain-dependent host transcriptional responses to Toxoplasma infection are largely conserved in mammalian and avian hosts. PLoS One. 2011;6:e26369. PubMed Abstract | PubMed Central Full Text | Publisher Full Text

35. Zeiner GM, Norman KL, Thomson JM, Hammond SM, Boothroyd JC. Toxoplasma gondii infection specifically increases the levels of key host microRNAs. PLoS One. 2010;5:e8742. PubMed Abstract | PubMed Central Full Text | Publisher Full Text

36. Chaussabel D, Semnani RT, McDowell MA, Sacks D, Sher A, Nutman TB. Unique gene expression profiles of human macrophages and dendritic cells to phylogenetically distinct parasites. Blood. 2003;102:672-81. PubMed Abstract | Publisher Full Text

37. Ma X, Fei E, Fu C, Ren H, Wang G. Dysbindin-1, a schizophrenia-related protein, facilitates neurite outgrowth by promoting the transcriptional activity of p53. Mol Psychiatry. 2011;16:1105-16. PubMed Abstract | Publisher Full Text

38. Wang HJ, Guo YQ, Tan G, Dong L, Cheng L, Li KJ, et al. MiR-125b regulates side population in breast cancer and confers a chemoresistant phenotype. J Cell Biochem. 2013;114:2248-57.

39. Cui F, Li X, Zhu X, Huang L, Huang Y, Mao C, et al. MiR-125b inhibits tumor growth and promotes apoptosis of cervical cancer cells by targeting phosphoinositide 3-kinase catalytic subunit delta. Cell Physiol Biochem. 2012;30:1310-18. PubMed Abstract | Publisher Full Text

40. Glud M, Manfe V, Biskup E, Holst L, Dirksen AM, Hastrup N, et al. MicroRNA miR-125b induces senescence in human melanoma cells. Melanoma Res. 2011;21:253-6. PubMed Abstract | Publisher Full Text

41. Zhu S, Pan W, Song X, Liu Y, Shao X, Tang Y, et al. The microRNA miR-23b suppresses IL-17-associated autoimmune inflammation by targeting TAB2, TAB3 and IKK-alpha. Nat Med. 2012;18:1077-86. PubMed Abstract | Publisher Full Text

42. Pellegrino L, Stebbing J, Braga VM, Frampton AE, Jacob J, Buluwela L, et al. miR-23b regulates cytoskeletal remodeling, motility and metastasis by directly targeting multiple transcripts. Nucleic Acids Res. 2013;41:5400-12. PubMed Abstract | PubMed Central Full Text | Publisher Full Text

43. Torres A, Torres K, Pesci A, Ceccaroni M, Paszkowski T, Cassandrini P, et al. Diagnostic and prognostic significance of miRNA signatures in tissues and plasma of endometrioid endometrial carcinoma patients. Int J Cancer. 2013;132:1633-45. PubMed Abstract | Publisher Full Text

44. Ohyagi-Hara C, Sawada K, Kamiura S, Tomita Y, Isobe A, Hashimoto K, et al. miR-92a inhibits peritoneal dissemination of ovarian cancer cells by inhibiting integrin alpha5 expression. Am J Pathol. 2013;182:1876-89. PubMed Abstract | Publisher Full Text

45. Lai L, Song Y, Liu Y, Chen Q, Han Q, Chen W, et al. MicroRNA-92a negatively regulates Toll-like receptor (TLR)-triggered inflammatory response in macrophages by targeting MKK4 kinase. J Biol Chem. 2013;288:7956-67. PubMed Abstract | Publisher Full Text

46. Yoshizawa S, Ohyashiki JH, Ohyashiki M, Umezu T, Suzuki K, Inagaki A, et al. Downregulated plasma miR-92a levels have clinical impact on multiple myeloma and related disorders. Blood Cancer J. 2012;2:e53. PubMed Abstract | PubMed Central Full Text | Publisher Full Text

47. Niu H, Wang K, Zhang A, Yang S, Song Z, Wang W, et al. miR-92a is a critical regulator of the apoptosis pathway in glioblastoma with inverse expression of BCL2L11. Oncol Rep. 2012;28:1771-7. PubMed Abstract

48. Gu Y, Zhang Y, Zhao C, Pan Y, Smales R, Wang H, et al. Serum microRNAs as potential biomarkers of mandibular prognathism. Oral Dis. 2013. doi: 10.1111/odi.12073. [Epub ahead of print]. Publisher Full Text

49. Maes OC, Sarojini H, Wang E. Stepwise up-regulation of microRNA expression levels from replicating to reversible and irreversible growth arrest states in WI-38 human fibroblasts. J Cell Physiol. 2009;221:109-19. PubMed Abstract | Publisher Full Text

50. Kappelmann M, Kuphal S, Meister G, Vardimon L, Bosserhoff AK. MicroRNA miR-125b controls melanoma progression by direct regulation of c-Jun protein expression. Oncogene. 2013;32:2984-91. PubMed Abstract | Publisher Full Text

51. Nishida N, Mimori K, Fabbri M, Yokobori T, Sudo T, Tanaka F, et al. MicroRNA-125a-5p is an independent prognostic factor in gastric cancer and inhibits the proliferation of human gastric cancer cells in combination with trastuzumab. Clin Cancer Res. 2011;17:2725-33. PubMed Abstract | Publisher Full Text

52. Jiang L, Huang Q, Chang J, Wang E, Qiu X. MicroRNA HSA-miR-125a-5p induces apoptosis by activating p53 in lung cancer cells. Exp Lung Res. 2011;37:387-98. PubMed Abstract | Publisher Full Text

53. Zhou B, Ma R, Si W, Li S, Xu Y, Tu X, et al. MicroRNA-503 targets FGF2 and VEGFA and inhibits tumor angiogenesis and growth. Cancer Lett. 2013;333:159-69. PubMed Abstract | Publisher Full Text

54. Yan Q, Li W, Tang Q, Yao S, Lv Z, Feng N, et al. Cellular microRNAs 498 and 320d regulate herpes simplex virus 1 induction of Kaposi's sarcoma-associated herpes virus lytic replication by targeting RTA. PLoS One. 2013;8:e55832. PubMed Abstract | PubMed Central Full Text | Publisher Full Text

55. Sun D, Lee YS, Malhotra A, Kim HK, Matecic M, Evans C, et al. miR-99 family of MicroRNAs suppresses the expression of prostate-specific antigen and prostate cancer cell proliferation. Cancer Res. 2011;71:1313-s24. PubMed Abstract | PubMed Central Full Text | Publisher Full Text

56. Li D, Liu X, Lin L, Hou J, Li N, Wang C, et al. MicroRNA-99a inhibits hepatocellular carcinoma growth and correlates with prognosis of patients with hepatocellular carcinoma. J Biol Chem. 2011;286:36677-85. PubMed Abstract | PubMed Central Full Text | Publisher Full Text

*Samuel M. Pope

Department of Biomedical Sciences

Marian University College of Osteopathic Medicine

3200 Cold Spring Road

Indianapolis, IN 46222

USA

Tel: [phone]

Email: [email protected]

Refbacks

* There are currently no refbacks.

Journal of Extracellular Vesicles eISSN 2001-3078

This journal is published under the terms of the Creative Commons Attribution-Noncommercial 3.0 Unported License. Responsible editor: Clotilde Thery