Lymphopenia With Clinical and Laboratory Features of Combined Immune Deficiency in an 11-Year-Old Female With FANCD2 Variants and Fanconi Anemia

Fanconi anemia (FA) is an inherited bone marrow failure and cancer predisposition disorder due to mutations in DNA repair pathways proteins (FANC). The dysfunctional proteins are unable to repair DNA breaks and cause genomic instability. Mutations in many of the 19 FANC genes are well characterized biochemically and clinically. Little is known about the FANCD2 gene which acts downstream of the FA-core proteins. Here we report a 11-year-old female previously diagnosed with FA and bone marrow failure. Gene sequencing demonstrated deletion of exons 2-18 and a pathologic missense mutation (c. 2444G>A, p. Arg815Gln) in FANCD2 (Chr3). Her medical history is significant for an episode of pneumococcal sepsis despite adequate vaccination. Repeated blood samples and immunophenotyping demonstrated severe lymphopenia. There were markedly low CD4+ T-cell counts with a low CD4:CD8 ratio. Changes in the composition of the B-cell population included significantly diminished absolute total B-cells, and decreased mature cells. There was no immunogenic response to vaccination against S. pneumoniae. The NK-cell count was unaffected and demonstrated normal spontaneous and stimulated cytotoxic response. Bone marrow analysis demonstrated hypocellularity without dysplasia. The clinical and laboratory features are suggestive of combined immune deficiency. FANCD2 may be involved in the transition of immature B and T cells to mature cells, a process that requires substantial DNA recombination not observed in NK cells. Additional genetic and biochemical evaluation is needed to further characterize the novel genetic and clinical findings

Transfer of MicroRNAs by Embryonic Stem Cell Microvesicles

Microvesicles are plasma membrane-derived vesicles released into the extracellular environment by a variety of cell types. Originally characterized from platelets, microvesicles are a normal constituent of human plasma, where they play an important role in maintaining hematostasis. Microvesicles have been shown to transfer proteins and RNA from cell to cell and they are also believed to play a role in intercellular communication. We characterized the RNA and protein content of embryonic stem cell microvesicles and show that they can be engineered to carry exogenously expressed mRNA and protein such as green fluorescent protein (GFP). We demonstrate that these engineered microvesicles dock and fuse with other embryonic stem cells, transferring their GFP. Additionally, we show that embryonic stem cells microvesicles contain abundant microRNA and that they can transfer a subset of microRNAs to mouse embryonic fibroblasts in vitro. Since microRNAs are short (21–24 nt), naturally occurring RNAs that regulate protein translation, our findings open up the intriguing possibility that stem cells can alter the expression of genes in neighboring cells by transferring microRNAs contained in microvesicles. Embryonic stem cell microvesicles may be useful therapeutic tools for transferring mRNA, microRNAs, protein, and siRNA to cells and may be important mediators of signaling within stem cell niches

Targeting the Plasmodium vivax equilibrative nucleoside transporter 1 (PvENT1) for antimalarial drug development

Infection with Plasmodium falciparum and vivax cause most cases of malaria. Emerging resistance to current antimalarial medications makes new drug development imperative. Ideally a new antimalarial drug should treat both falciparum and vivax malaria. Because malaria parasites are purine auxotrophic, they rely on purines imported from the host erythrocyte via Equilibrative Nucleoside Transporters (ENTs). Thus, the purine import transporters represent a potential target for antimalarial drug development. For falciparum parasites the primary purine transporter is the P. falciparum Equilibrative Nucleoside Transporter Type 1 (PfENT1). Recently we identified potent PfENT1 inhibitors with nanomolar IC50 values using a robust, yeast-based high throughput screening assay. In the current work we characterized the Plasmodium vivax ENT1 (PvENT1) homologue and its sensitivity to the PfENT1 inhibitors. We expressed a yeast codon-optimized PvENT1 gene in Saccharomyces cerevisiae. PvENT1-expressing yeast imported both purines ([3H]adenosine) and pyrimidines ([3H]uridine), whereas wild type (fui1Δ) yeast did not. Based on radiolabel substrate uptake inhibition experiments, inosine had the lowest IC50 (3.8 μM), compared to guanosine (14.9 μM) and adenosine (142 μM). For pyrimidines, thymidine had an IC50 of 183 μM (vs. cytidine and uridine; mM range). IC50 values were higher for nucleobases compared to the corresponding nucleosides; hypoxanthine had a 25-fold higher IC50 than inosine. The archetypal human ENT1 inhibitor 4-nitrobenzylthioinosine (NBMPR) had no effect on PvENT1, whereas dipyridamole inhibited PvENT1, albeit with a 40 μM IC50, a 1000-fold less sensitive than human ENT1 (hENT1). The PfENT1 inhibitors blocked transport activity of PvENT1 and the five known naturally occurring non-synonymous single nucleotide polymorphisms (SNPs) with similar IC50 values. Thus, the PfENT1 inhibitors also target PvENT1. This implies that development of novel antimalarial drugs that target both falciparum and vivax ENT1 may be feasible. Keywords: Purines, Transporter, Malaria, Drug development, Nucleoside/nucleobase transport, Parasite, Plasmodium vivax, Single-nucleotide polymorphism (SNP

Transfer of microRNAs by embryonic stem cell microvesicles.

Microvesicles are plasma membrane-derived vesicles released into the extracellular environment by a variety of cell types. Originally characterized from platelets, microvesicles are a normal constituent of human plasma, where they play an important role in maintaining hematostasis. Microvesicles have been shown to transfer proteins and RNA from cell to cell and they are also believed to play a role in intercellular communication. We characterized the RNA and protein content of embryonic stem cell microvesicles and show that they can be engineered to carry exogenously expressed mRNA and protein such as green fluorescent protein (GFP). We demonstrate that these engineered microvesicles dock and fuse with other embryonic stem cells, transferring their GFP. Additionally, we show that embryonic stem cells microvesicles contain abundant microRNA and that they can transfer a subset of microRNAs to mouse embryonic fibroblasts in vitro. Since microRNAs are short (21-24 nt), naturally occurring RNAs that regulate protein translation, our findings open up the intriguing possibility that stem cells can alter the expression of genes in neighboring cells by transferring microRNAs contained in microvesicles. Embryonic stem cell microvesicles may be useful therapeutic tools for transferring mRNA, microRNAs, protein, and siRNA to cells and may be important mediators of signaling within stem cell niches

ESMVs contain miRNAs.

<p>The relative abundance of several miRNAs in ESMVs compared with ESCs was determined by real time quantitative RT-PCR. The miRNAs tested include <i>miR-16</i> (lane 1), <i>miR-21</i> (lane 2), <i>miR-22</i> (lane 3), <i>miR-290</i> (lane 4), <i>miR-291-3p</i> (lane 5), <i>miR-292-3p</i> (lane 6), <i>miR-294</i> (lane 7), <i>miR-295</i> (lane 8), and the small nuclear RNA, <i>RNU6b</i> (lane 9). (A) Box plots of relative abundance in ESMVs compared with ESCs (n = 9). The boxed area represents the mean±quartile and the whiskers extend out to the minimum and maximum values. Bootstrap ANOVA was performed and a significant difference was detected between all groups (<i>p</i> = 0.008). (B) The 95^th percentile confidence interval for each miRNA was determined and plotted on a bar graph. Non-overlapping groups are significantly different from each other. RNU6b is significantly less abundant than all miRNAs tested except <i>for miR-22, miR-290,</i> and <i>miR-291.</i> The majority of miRNAs tested do not differ significantly from one another except for <i>miR-295</i>, which is significantly more abundant than <i>miR-290</i> and <i>miR-291</i>.</p

ESMVs transfer miRNAs.

<p>MEFs were incubated with ESMVs for 1, 12, 36, or 54 hours and transfer of miRNAs was determined by real time quantitative RT-PCR (n = 5). Time point 0 represents MEFs without ESMVs. The difference in Ct values between the negative control (MEFs alone) and each experimental group (miR-290, miR-291-3p, miR-292-3p, miR-294, miR-295, miR-16, and RNU6b) is shown. Positive values indicate transfer of miRNA.</p

Primers used for RT-PCR.

<p>Primers used for RT-PCR.</p

ESMVs transfer GFP.

<p>ESCs without the GFP transgene were labeled with DiD and then incubated with ESMVs containing GFP. All confocal images were taken using a 100×, 1.4 NA objective with the pinhole set to 1 airy unit. (A) DiD signal from ESCs incubated with ESMVs. (B) GFP signal from ESCs incubated with ESMVs. Arrows indicate punctate signal, likely representing docked vesicles. Arrowheads indicate diffuse signal, likely from the diffusion of GFP inside the cell or from the production of newly translated GFP. (C) Overlay of A+B. (D) DiD signal from control ESCs without ESMVs. (E) No GFP signal can be detected in the absence of ESMVs. (F) Overlay of D+E. All scale bars are 5 μm.</p

ESMVs contain GFP mRNA and protein expressed from a GFP transgene in ESCs.

<p>(A) 300 ng of ESMV RNA from an ESC line expressing GFP were used for RT, and 35 cycles of PCR amplification were performed with the GFP primers shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004722#pone-0004722-t001" target="_blank">Table 1</a>. A 2% agarose gel was loaded with the RT-PCR products from ESMVs (lane 1), ESCs (lane 2), and a “no RT” control of ESMVs (lane 3). A 406bp band corresponding to the GFP amplicon is observed in both the ESMV and ESC lanes. (B) (Left) Box plot of relative abundance of GFP in ESMVs compared with ESCs (n = 8). (Right) Comparison of amplification curves for <i>GFP</i> (top) and <i>β-actin</i> (bottom) in ESCs (1) and ESMVs (2). Note that while quantitative RT-PCR was performed in the linear range of amplification, (panel B), the end-point PCR products shown in panel (A) are only qualitative and well outside of the linear range. (C) Immunoblot of an 8% urea-SDS polyacrylamide/Tris-glycine buffered gel loaded with 20 μg total protein/lane, using polyclonal anti-GFP antibody (1∶1000) and horse anti-rabbit secondary antibody (1∶5000). The secondary antibody was conjugated to alkaline phosphatase and visualized with BCIP/NBT. A single ∼35kD immunoreactive band corresponding to GFP in ESCs (lane 1) and ESMVs (lane 2) was detected.</p