Anastas Popratiloff

Exosomes Derived from HIV-1-infected Cells Contain Trans-activation Response Element RNA

Background: Exosomes are extracellular vesicles that have been implicated in intercellular communication. Results: Exosomes that originate from human cells infected with HIV-1 contain virus-derived small noncoding RNA. Conclusion: Virus-derived small RNA present in exosomes exert functional consequences in naive recipient cells. Significance: Viral RNA molecules present in exosomes may be critical mediators of intercellular viral spread in infected hosts. Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 104–106 copies/ml TAR RNA in exosomes derived from infected culture supernatants and 103 copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS.

Human T-lymphotropic Virus Type 1-infected Cells Secrete Exosomes That Contain Tax Protein * □

Background: Extracellular exosomes contain various functional elements. Results: Exosomal Tax protein causes phenotypic changes in uninfected cells. Conclusion: Exosomes may play critical roles in extracellular delivery of oncogenic material derived from HTLV-1-infected cells. Significance: Exosomal delivery of Tax and other putative oncogenic components produced during HTLV-1 infection potentially contributes to pathogenesis of adult T-cell leukemia, myelopathy, or tropical spastic paraparesis. Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The HTLV-1 transactivator protein Tax controls many critical cellular pathways, including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Extracellular vesicles called exosomes play critical roles during pathogenic viral infections as delivery vehicles for host and viral components, including proteins, mRNA, and microRNA. We hypothesized that exosomes derived from HTLV-1-infected cells contain unique host and viral proteins that may contribute to HTLV-1-induced pathogenesis. We found exosomes derived from infected cells to contain Tax protein and proinflammatory mediators as well as viral mRNA transcripts, including Tax, HBZ, and Env. Furthermore, we observed that exosomes released from HTLV-1-infected Tax-expressing cells contributed to enhanced survival of exosome-recipient cells when treated with Fas antibody. This survival was cFLIP-dependent, with Tax showing induction of NF-κB in exosome-recipient cells. Finally, IL-2-dependent CTLL-2 cells that received Tax-containing exosomes were protected from apoptosis through activation of AKT. Similar experiments with primary cultures showed protection and survival of peripheral blood mononuclear cells even in the absence of phytohemagglutinin/IL-2. Surviving cells contained more phosphorylated Rb, consistent with the role of Tax in regulation of the cell cycle. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells.

Contralateral trigeminal nerve lesion reduces polyneuronal muscle innervation after facial nerve repair in rats

Functional recovery after facial nerve surgery is poor. Axotomized motoneurons (hyperexcitable upon intracellular current injections, but unable to discharge upon afferent stimulation) outgrow supernumerary branches which are misrouted towards improper muscles. We hypothesized that alterations in the trigeminal input to axotomized electrophysiologically silent facial motoneurons might improve specificity of reinnervation. To test this we compared, in the rat, behavioural, electrophysiological, and morphological parameters after transection and suture of the buccal facial nerve (buccal–buccal anastomosis, BBA) with those after BBA plus excision of the ipsi‐ or contralateral infraorbital nerve (ION). After BBA, the mystacial vibrissae dropped and remained motionless until 18–21 days post operation (days PO). After BBA plus ipsilateral ION excision, there was no recovery of vibrissae whisking at all. Following BBA plus contralateral ION excision, full restoration of whisking occurred at 7–10 days PO. Electromyography of whiskerpad muscles showed normal waveform and amplitude was also most rapidly restored after BBA plus contralateral ION excision. Neuron counts after retrograde tracing showed that the intact buccal nerve contained axons of the superior (91%) and inferior (9%) buccolabial nerves. After BBA, the superior nerve comprised 56%, the inferior 21%, and 23% of the motoneurons projected within both nerves. After BBA plus ipsilateral ION excision, misdirection worsened and values changed to 48, 39 and 13%, respectively. After BBA plus contralateral ION excision, portions improved to 69, 23 and 8%. We conclude that, by reducing the redundant axon branching, lesion of contralateral ION provides the best conditions for recovery of vibrissae rhythmical whisking after reconstructive surgery on the facial nerve.

Hypoglossal and reticular interneurons involved in oro-facial coordination in the rat.

Chewing, swallowing, breathing, and vocalization in mammals require precise coordination of tongue movements with concomitant activities of the mimetic muscles. The neuroanatomic basis for this oro‐facial coordination is not yet fully understood. After the stereotaxic microinjection of retrograde and anterograde neuronal tracers (biotin‐dextran, Fluoro‐Ruby, Fluoro‐Emerald, and Fluoro‐Gold) into the facial and hypoglossal nuclei of the rat, we report here a direct bilateral projection of hypoglossal internuclear interneurons onto facial motoneurons. We also confirm the existence of a small pool of neurons in the dorsal part of the brainstem reticular formation that project ipsilaterally to both facial and hypoglossal nuclei. For precise tracer injections, both motor nuclei were located and identified by the electrical antidromic activation of their constituent motoneurons. Injections of retrograde tracers into the facial nucleus consistently labeled neurons in the hypoglossal nucleus. These neurons prevalently lay in the ipsilateral side, were small in size, and, like classic intrinsic hypoglossal local‐circuit interneurons, had several thin dendrites. Reverse experiments — injections of anterograde tracers into the hypoglossal nucleus — labeled fine varicose nerve fiber terminals in the facial nucleus. These fiber terminals were concentrated in the intermediate subdivision of the facial nucleus, with a strong ipsilateral prevalence. Double injections of different tracers into the facial and the hypoglossal nuclei revealed a small, but constant, number of double‐labeled neurons located predominantly ipsilateral in the caudal brainstem reticular formation. Hypoglossal internuclear interneurons projecting to the facial nucleus, as well as those neurons of the parvocellular reticular formation that project to both facial and hypoglossal nuclei, could be involved in oro‐facial coordination. J. Comp. Neurol. 433:364–379, 2001.

Improved outcome of facial nerve repair in rats is associated with enhanced regenerative response of motoneurons and augmented neocortical plasticity

Within a recent study on the vibrissae motor performance after facial nerve repair in strains of blind (SD/RCS) and sighted (SD) rats we found that, despite persisting myotopic disorganization in the facial nucleus, the blind animals fully restored vibrissal whisking. Here we searched for morphological substrates of better recovery in the regenerating motoneurons and in the cerebral motor cortex. Expression analyses of the neurite growth‐related proteins f‐actin, neuronal class III β‐tubulin and plasticity‐related gene‐1, and stereological estimates of growth cone densities revealed a more vigorous regenerative response in the proximal nerve stump of blind SD/RCS rats compared with SD animals at 5–7 days after buccal nerve transection. Using c‐Fos immunoreactivity as a marker for neuronal activation, we found that the volume of the cortex acutely responding to nerve transection (facial muscles reactive volume, FMRV) in both hemispheres of intact sighted rats was twofold smaller than that measured in blind animals. One month after transection and suture of the right facial nerve (FFA) we found a twofold increase in the FMRV in both rat strains compared with intact animals. The FMRV in SD/RCS animals, but not in SD rats, returned to the values in intact rats 2 months after FFA. Our findings suggest that enhanced plasticity in the CNS and an augmented regenerative response of the injured motoneurons contribute to better functional recovery in blind rats.

Genetic manipulation of Schistosoma haematobium, the neglected schistosome.

Background Minimal information on the genome and proteome of Schistosoma haematobium is available, in marked contrast to the situation with the other major species of human schistosomes for which draft genome sequences have been reported. Accordingly, little is known about functional genomics in S. haematobium, including the utility or not of RNA interference techniques that, if available, promise to guide development of new interventions for schistosomiasis haematobia. Methods/Findings Here we isolated and cultured developmental stages of S. haematobium, derived from experimentally infected hamsters. Targeting different developmental stages, we investigated the utility of soaking and/or square wave electroporation in order to transfect S. haematobium with nucleic acid reporters including Cy3-labeled small RNAs, messenger RNA encoding firefly luciferase, and short interfering RNAs (siRNAs). Three hours after incubation of S. haematobium eggs in 50 ng/µl Cy3-labeled siRNA, fluorescent foci were evident indicating that labeled siRNA had penetrated into miracidia developing within the egg shell. Firefly luciferase activity was detected three hours after square wave electroporation of the schistosome eggs and adult worms in 150 ng/µl of mRNA. RNA interference knockdown (silencing) of reporter luciferase activity was seen following the introduction of dsRNA specific for luciferase mRNA in eggs, schistosomules and mixed sex adults. Moreover, introduction of an endogenous gene-specific siRNA into adult schistosomes silenced transcription of tetraspanin 2 (Sh-tsp-2), the apparent orthologue of the Schistosoma mansoni gene Sm-tsp-2 which encodes the surface localized structural and signaling protein Sm-TSP-2. Together, knockdown of reporter luciferase and Sh-tsp-2 indicated the presence of an intact RNAi pathway in S. haematobium. Also, we employed laser scanning confocal microscopy to view the adult stages of S. haematobium. Conclusions These findings and approaches should facilitate analysis of gene function in S. haematobium, which in turn could facilitate the characterization of prospective intervention targets for this neglected tropical disease pathogen.

Thymosin-β4 (Tβ4) Blunts PDGF-Dependent Phosphorylation and Binding of AKT to Actin in Hepatic Stellate Cells

Hepatic stellate cell transdifferentiation is a key event in the fibrogenic cascade. Therefore, attempts to prevent and/or revert the myofibroblastic phenotype could result in novel therapeutic approaches to treat liver cirrhosis. The expression of platelet-derived growth factor (PDGF)-β receptor and the proliferative response to platelet-derived growth factor-ββ (PDGF-ββ) are hallmarks of the transdifferentiation of hepatic stellate cells (HSC). In this communication, we investigated whether thymosin-β4 (Tβ4), a chemokine expressed by HSC could prevent PDGF-BB-mediated proliferation and migration of cultured HSC. Using early passages of human HSC, we showed that Tβ4 inhibited cell proliferation and migration and prevented the expression of PDGF-β receptor (PDGF-βr), α-smooth muscle actin and α1(I) collagen mRNAs. Tβ4 also inhibited the reappearance of PDGF-βr after its PDGF-BB-dependent degradation. These PDGF-dependent events were associated with the inhibition of AKT phosphorylation at both T308 and S473 amino acid residues. The lack of AKT phosphorylation was not due to the inhibition of PDGF-βr phosphorylation, the activation of phosphoinositide 3-kinase (PI3K), pyruvate dehydrogenase kinase isozyme 1 (PDK1), and mammalian target of rapamycin (mTOR). We found that PDGF-BB induced AKT binding to actin, and that Tβ4 prevented this effect. Tβ4 also prevented the activation of freshly isolated HSC cultured in the presence of Dulbeccos modified Eagles medium or Dulbeccos minimal essential medium containing 10% fetal bovine serum. In conclusion, overall, our findings suggest that Tβ4 by sequestering actin prevents binding of AKT, thus inhibiting its phosphorylation. Therefore, Tβ4 has the potential to be an antifibrogenic agent.

Developmental change in expression and subcellular localization of two Shaker‐related potassium channel proteins (Kv1.1 and Kv1.2) in the chick tangential vestibular nucleus

The chick tangential nucleus is a major avian vestibular nucleus whose principal cells participate in two vestibular reflexes. Intracellular recordings have shown that the principal cells acquire their mature firing pattern gradually during development. At embryonic day 16 (E16), most principal cells fire a single spike, whereas shortly after hatching (H) the vast majority fire repetitively on depolarization. The transition in firing pattern was likely due in part to a downregulation of a low‐threshold, sustained, dendrotoxin‐sensitive (DTX) potassium current, IDS. Since the DTX‐sensitive potassium channel subunits Kv1.1 and Kv1.2 generate sustained currents, in the present study we applied fluorescence immunocytochemistry and confocal microscopy to characterize their developmental expression at E16, H1, and H9. At E16, both Kv1.1 and Kv1.2 staining were confined to the principal cell bodies. Immunolabeling decreased significantly for both proteins at H1, and more so by H9. Double‐labeling with a monoclonal antibody against microtubule‐associated protein 2 (MAP2) in hatchlings showed that some Kv1.1 remained as clusters within the cell body, at the base of the dendrites, and in the axon initial segment. In hatchlings, Kv1.2 staining decreased in the cell bodies and simultaneously appeared in the neuropil, colocalized with biocytin‐labeled primary vestibular fibers and vestibular “spoon” terminals. Also, double‐labeling with synaptotagmin showed that Kv1.2 colocalized with many nonvestibular terminals surrounding the principal cell bodies. These results identified developmental decreases in the staining of these two potassium channel protein subunits and changes in their subcellular localization corresponding to the downregulation of IDS defined electrophysiologically around hatching. Accordingly, both of these protein subunits could be involved in regulating excitability of the principal cells. J. Comp. Neurol. 461:466–482, 2003.

AMPA receptor subunit expression in chick vestibular nucleus neurons

The principal cells of the chick tangential nucleus are vestibular nucleus neurons whose responses on vestibular nerve stimulation are abolished by glutamate receptor antagonists. Using confocal microscopy, we quantified immunolabeling for AMPA receptor subunits GluR1, GluR2, GluR2/3, and GluR4 in principal cells that were identified by the neuronal marker, microtubule‐associated protein 2 (MAP2). This work was focused primarily on 9 days after hatching (H9) when the principal cells have acquired some important mature electrophysiologic properties. At H9, the principal cell bodies stained strongly with GluR2/3 and GluR4, whereas GluR1 and GluR2 produced weak signals. Moreover, GluR2/3 and GluR4 receptor subunit clusters in principal cell bodies and dendrites were localized at sites contacted by biocytin‐labeled vestibular nerve terminals and synaptotagmin‐labeled terminals. Developmental expression of AMPA receptor immunolabeling was studied in the principal cell bodies at embryonic day 16 (E16) and hatching (H1). At E16, labeling for GluR4 was already strong, and continued to increase at H1 and H9. In contrast, GluR2/3 labeling was weak at E16, but increased significantly at H1, and more so by H9. GluR1 and GluR2 were present at low levels at E16 and H1. From E16 to H9, overall AMPA receptor subunit expression increased steadily, with H9 showing the strongest labeling. Ultrastructural observations at E16 and H3 confirmed the presence of immunogold labeling for AMPA receptor subunits at the vestibular nerve and non‐vestibular nerve synapses on the principal cell bodies. In summary, these results indicate that GluR3 and GluR4 are the major AMPA receptor subunits involved in excitatory synaptic transmission in principal cells during the perinatal period.

Apoptosis of cholangiocytes modulated by thioredoxin of carcinogenic liver fluke.

Chronic infection with the food-borne liver fluke, Opisthorchis viverrini, frequently induces cancer of the bile ducts, cholangiocarcinoma. Opisthorchiasis is endemic in Thailand, Lao PDR, Cambodia and Vietnam, where eating undercooked freshwater fish carrying the juvenile stage of this pathogen leads to human infection. Because inhibition of apoptosis facilitates carcinogenesis, this study investigated modulation by thioredoxin from O. viverrini of apoptosis of bile duct epithelial cells, cholangiocytes. Cells of a cholangiocyte line were incubated with the parasite enzyme after which they were exposed hydrogen peroxide. Oxidative stress-induced apoptosis was monitored using flow cytometry, growth in real time and imaging of living cells using laser confocal microscopy. Immunolocalization revealed liver fluke thioredoxin within cholangiocytes. Cells exposed to thioredoxin downregulated apoptotic genes in the mitogen activated protein kinases pathway and upregulated anti-apoptosis-related genes including apoptosis signaling kinase 1, caspase 9, caspase 8, caspase 3, survivin and others. Western blots of immunoprecipitates of cell lysates revealed binding of thioredoxin to apoptosis signaling kinase 1. Together the findings indicated that thioredoxin from O. viverrini inhibited oxidative stress-induced apoptosis of bile duct epithelial cells, which supports a role for this liver fluke oxidoreductase in opisthorchiasis-induced cholangiocarcinogenesis.