Artificial Trabecular Meshwork Structure Combining Melt Electrowriting and Solution Electrospinning.

The human trabecular meshwork (HTM) is responsible for regulating intraocular pressure (IOP) by means of gradient porosity. Changes in its physical properties, like increases in stiffness or alterations in the extracellular matrix (ECM), are associated with increases in the IOP, which is the primary cause of glaucoma. The complexity of its structure limits the engineered models to one-layered and simple approaches, which do not accurately replicate the biological and physiological cues related to glaucoma. Here, a combination of melt electrowriting (MEW) and solution electrospinning (SE) is explored as a biofabrication technique used to produce a gradient porous scaffold that mimics the multi-layered structure of the native HTM. Polycaprolactone (PCL) constructs with a height of 20-710 µm and fiber diameters of 0.7-37.5 µm were fabricated. After mechanical characterization, primary human trabecular meshwork cells (HTMCs) were seeded over the scaffolds within the subsequent 14-21 days. In order to validate the system's responsiveness, cells were treated with dexamethasone (Dex) and the rho inhibitor Netarsudil (Net). Scanning electron microscopy and immunochemistry staining were performed to evaluate the expected morphological changes caused by the drugs. Cells in the engineered membranes exhibited an HTMC-like morphology and a correct drug response. Although this work demonstrates the utility of combining MEW and SE in reconstructing complex morphological features like the HTM, new geometries and dimensions should be tested, and future works need to be directed towards perfusion studies.

Nanofibrous PCL-Based Human Trabecular Meshwork for Aqueous Humor Outflow Studies.

Primary open-angle glaucoma is characterized by the progressive degeneration of the optic nerve, with the high intraocular pressure (IOP) being one of the main risk factors. The human trabecular meshwork (HTM), specifically the juxtacanalicular tissue (JCT), is responsible for placing resistance to the aqueous humor (AH) outflow and the resulting IOP control. Currently, the lack of a proper in vitro JCT model and the complexity of three-dimensional models impede advances in understanding the relationship between AH outflow and HTM degeneration. Therefore, we design an in vitro JCT model using a polycaprolactone (PCL) nanofibrous scaffold, which supports cells to recapitulate the functional JCT morphology and allow the study of outflow physiology. Mechanical and morphological characterizations of the electrospun membranes were performed, and human trabecular meshwork cells were seeded over the scaffolds. The engineered JCT was characterized by scanning electron microscopy, quantitative real-time polymerase chain reaction, and immunochemistry assays staining HTM cell markers and proteins. A pressure-sensitive perfusion system was constructed and used for the investigation of the outflow facility of the polymeric scaffold treated with dexamethasone (a glucocorticoid) and netarsudil (a novel IOP lowering the rho inhibitor). Cells in the in vitro model exhibited an HTM-like morphology, expression of myocilin, fibronectin, and collagen IV, genetic expression, outflow characteristics, and drug responsiveness. Altogether, the present work develops an in vitro JCT model to better understand HTM cell biology and the relationship between the AH outflow and the HTM and allow further drug screening of pharmacological agents that affect the trabecular outflow facility.

Comparison of Transcriptomic Analysis of the Conjunctiva in Glaucoma-Treated Eyes with Dry Eyes and Healthy Controls.

Ocular surface disease (OSD) associated with topical glaucoma drugs is a common issue impacting treatment adherence. We aimed to identify conjunctival transcriptomic changes in glaucoma and dry eye patients, comparing them to healthy controls. Bulbar conjunctival specimens were collected via impression cytology from 33 patients treated for glaucoma, 9 patients with dry eye, and 14 healthy controls. RNA extraction and bulk RNA sequencing were performed, followed by bioinformatics analysis to detect gene dysregulation. Ingenuity pathways analysis (IPA) identified pathways and biological processes associated with these transcriptomic changes. Sequencing analysis revealed 200 modified genes in glaucoma patients compared to healthy individuals, 233 differentially expressed genes in dry eye patients versus controls, and 650 genes in treated versus dry eye samples. In glaucoma patients, 79% of altered pathways were related to host defense, while dry eye patients showed a 39% involvement of host response, 15% in cellular proliferation and integrity, and 16% of mitochondrial dysfunction. These findings were validated through qRT-PCR. Glaucoma patients showed an intensified conjunctival immune response as a potential cause of OSD, whereas in dry eye patients, in addition to the immune response, other mechanisms such as mitochondrial dysfunction or reduced cellular proliferation were observed.

Technological advances in ocular trabecular meshwork in vitro models for glaucoma research.

Glaucoma is the leading cause of irreversible blindness worldwide and is characterized by the progressive degeneration of the optic nerve. Intraocular pressure (IOP), which is considered to be the main risk factor for glaucoma development, builds up in response to the resistance (resistance to what?) provided by the trabecular meshwork (TM) to aqueous humor (AH) outflow. Although the TM and its relationship to AH outflow have remained at the forefront of scientific interest, researchers remain uncertain regarding which mechanisms drive the deterioration of the TM. Current tissue-engineering fabrication techniques have come up with promising approaches to successfully recreate the TM. Nonetheless, more accurate models are needed to understand the factors that make glaucoma arise. In this review, we provide a chronological evaluation of the technological milestones that have taken place in the field of glaucoma research, and we conduct a comprehensive comparison of available TM fabrication technologies. Additionally, we also discuss AH perfusion platforms, since they are essential for the validation of these scaffolds, as well as pressure-outflow relationship studies and the discovery of new IOP-reduction therapies.

Continuous intraocular pressure monitoring in patients with obstructive sleep apnea syndrome using a contact lens sensor.

PURPOSE: To analyse nocturnal intraocular pressure (IOP) fluctuations in patients with obstructive sleep apnea syndrome (OSAS) using a contact lens sensor (CLS) and to identify associations between the OSAS parameters determined by polysomnographic study (PSG) and IOP changes. METHOD: Prospective, observational study. Twenty participants suspected of having OSAS were recruited. During PSG study, IOP was monitored using a CLS placed in the eye of the patient. The patients were classified according to the apnea-hypopnea index (AHI) in two categories, severe (>30) or mild/moderate (<30) OSAS. We evaluated several parameters determined by the IOP curves, including nocturnal elevations (acrophase) and plateau times in acrophase (PTs) defined by mathematical and visual methods. RESULTS: The IOP curves exhibited a nocturnal acrophase followed by PTs of varying extents at which the IOP remained higher than daytime measurement with small variations. We found significant differences in the length of the PTs in patients with severe OSAS compared to those with mild/moderate disease (P = 0.032/P = 0.028). We found a positive correlation between PTs and OSAS severity measured by the total number of apneic events (r = 0.681/0.751 P = 0.004/0.001) and AHI (r = 0.674/0.710, P = 0.004/0.002). Respiratory-related arousal and oxygen saturation also were associated significantly with the IOP PT length. CONCLUSIONS: Periods of nocturnal IOP elevation lasted longer in severe OSAS patients than those with mild/moderate OSAS and correlate with the severity of the disease. The length of the nocturnal PT is also associated to respiratory parameters altered in patients with OSAS.

Ocular surface analysis and automatic non-invasive assessment of tear film breakup location, extension and progression in patients with glaucoma.

BACKGROUND: Tear film stability is the key event in ocular surface diseases. The purpose of this study is to evaluate spatial and temporal progression of the tear film breakup using an automatic non-invasive device. METHODS: Non-invasive tear breakup time (NITBUT) parameters, such as First NITBUT (F-NITBUT) and Average NITBUT (A-NITBUT), were evaluated in 132 glaucoma and 87 control eyes with the Keratograph 5 M device. Further analysis of this data was used to determine size, location and progression of tear film breakup with automatically identified breakup areas (BUA). The progression from First BUA (F-BUA) to total BUA (T-BUA) was expressed as Dry Area Growth Rate (DAGR). Differences between both groups were analysed using Student t-test for parametric data and Mann-Whitney U test for non-parametric data. Pearson's correlation coefficient was used to assess the relationship between parametric variables and Spearman in the case of non-parametric variables. RESULTS: F-NITBUT was 11.43 ± 7.83 s in the control group and 8.17 ± 5.73 in the glaucoma group (P = 0.010). A-NITBUT was 14.04 ± 7.21 and 11.82 ± 6.09 s in control and glaucoma groups, respectively (P = 0.028). F-BUA was higher in the glaucoma group than in the control group (2.73 and 2.28; P = 0.022) and was more frequently located at the centre of the cornea in the glaucoma group (P = 0.039). T-BUA was also higher in the glaucoma group than in the control group (13.24 and 9.76%; P = 0.012) and the DAGR was steeper in the glaucoma group than in the control group (34.38° and 27.15°; P = 0.009). CONCLUSIONS: Shorter NITBUT values and bigger, more central tear film breakup locations were observed in the glaucoma group than in the control group. The DAGR indicates that tear film rupture is bigger and increases faster in glaucomatous eyes than in normal eyes.

Relationship between OSDI questionnaire and ocular surface changes in glaucomatous patients.

PURPOSE: To evaluate the quality of life of glaucoma patients using the Ocular Surface Disease Index (OSDI) questionnaire and their association with dry eye clinical signs. METHODS: The study included patients into three groups. The treated group diagnosed with bilateral open-angle glaucoma and treated with one or more topical medication at least 1 year. The operated group underwent glaucoma surgery without the need for topical medications. The control group entered subjects without ocular diseases or previous surgeries. Dry eye clinical signs were evaluated; noninvasive tear break-up time, Meibomian gland depletion (MGD), and conjunctival hyperemia were measured using the Keratograph 5 M. The total-OSDI (T-OSDI) score was divided into the visual field-OSDI and discomfort-OSDI scores. RESULTS: Two hundred and nine subjects participated in this cross-sectional study, 147 using glaucoma medications, 21 patients underwent glaucoma surgery and 41 were controls. The T-OSDI and subscores were higher in glaucoma patients compared with controls (p < 0.05); we found no differences between treated and surgically groups. Correlations were observed between the T-OSDI values and Schirmer test (p = 0.016), ocular surface staining (p < 0.001) and the MGD (p = 0.006). The subscores were associated with the ocular surface staining (VF p = 0.013 and D p = 0.003). In treated patients, the number of drops per day correlates with T-OSDI and subscores (p = 0.017 and p = 0.005). CONCLUSION: OSDI scores increased in the glaucoma patients compared to controls without significant changes between treated and surgical patients. OSDI scores were associated to dry eye signs and medication in glaucoma patients.

Long noncoding RNA EGOT negatively affects the antiviral response and favors HCV replication.

The role of long noncoding RNAs (lncRNAs) in viral infection is poorly studied. We have identified hepatitis C virus (HCV)-Stimulated lncRNAs (CSRs) by transcriptome analysis. Interestingly, two of these CSRs (PVT1 and UCA1) play relevant roles in tumorigenesis, providing a novel link between HCV infection and development of liver tumors. Expression of some CSRs seems induced directly by HCV, while others are upregulated by the antiviral response against the virus. In fact, activation of pathogen sensors induces the expression of CSR32/EGOT RIG-I and the RNA-activated kinase PKR sense HCV RNA, activate NF-κB and upregulate EGOT EGOT is increased in the liver of patients infected with HCV and after infection with influenza or Semliki Forest virus (SFV). Genome-wide guilt-by-association studies predict that EGOT may function as a negative regulator of the antiviral pathway. Accordingly, EGOT depletion increases the expression of several interferon-stimulated genes and leads to decreased replication of HCV and SFV Our results suggest that EGOT is a lncRNA induced after infection that increases viral replication by antagonizing the antiviral response.

HCV infection, IFN response and the coding and non-coding host cell genome.

HCV is an ideal model to study how the infected cell is altered to allow the establishment of a chronic infection. After infection, the transcriptome of the cell changes in response to the virus or to the antiviral pathways induced by infection. The cell has evolved to sense HCV soon after infection and to activate antiviral pathways. In turn, HCV has evolved to block the antiviral pathways induced by the cell and, at the same time, to use some for its own benefit. In this review, we summarize the proviral and antiviral factors induced in HCV infected cells. These factors can be proteins and microRNAs, but also long noncoding RNAs (lncRNAs) that are induced by infection. Interestingly, several of the lncRNAs upregulated after HCV infection have oncogenic functions, suggesting that upregulation of lncRNAs could explain, at least in part, the increased rate of liver tumors observed in HCV-infected patients. Other lncRNAs induced by HCV infection may regulate the expression of coding genes required for replication or control genes involved in the cellular antiviral response. Given the evolutionary pressure imposed by viral infections and that lncRNAs are specially targeted by evolution, we believe that the study of proviral and antiviral lncRNAs may lead to unexpected discoveries that may have a strong impact on basic science and translational research.

Type I Interferon Regulates the Expression of Long Non-Coding RNAs.

Interferons (IFNs) are key players in the antiviral response. IFN sensing by the cell activates transcription of IFN-stimulated genes (ISGs) able to induce an antiviral state by affecting viral replication and release. IFN also induces the expression of ISGs that function as negative regulators to limit the strength and duration of IFN response. The ISGs identified so far belong to coding genes. However, only a small proportion of the transcriptome corresponds to coding transcripts and it has been estimated that there could be as many coding as long non-coding RNAs (lncRNAs). To address whether IFN can also regulate the expression of lncRNAs, we analyzed the transcriptome of HuH7 cells treated or not with IFNα2 by expression arrays. Analysis of the arrays showed increased levels of several well-characterized coding genes that respond to IFN both at early or late times. Furthermore, we identified several IFN-stimulated or -downregulated lncRNAs (ISRs and IDRs). Further validation showed that ISR2, 8, and 12 expression mimics that of their neighboring genes GBP1, IRF1, and IL6, respectively, all related to the IFN response. These genes are induced in response to different doses of IFNα2 in different cell lines at early (ISR2 or 8) or later (ISR12) time points. IFNß also induced the expression of these lncRNAs. ISR2 and 8 were also induced by an influenza virus unable to block the IFN response but not by other wild-type lytic viruses tested. Surprisingly, both ISR2 and 8 were significantly upregulated in cultured cells and livers from patients infected with HCV. Increased levels of ISR2 were also detected in patients chronically infected with HIV. This is relevant as genome-wide guilt-by-association studies predict that ISR2, 8, and 12 may function in viral processes, in the IFN pathway and the antiviral response. Therefore, we propose that these lncRNAs could be induced by IFN to function as positive or negative regulators of the antiviral response.

Negative regulation of the interferon response by an interferon-induced long non-coding RNA.

Long non-coding RNAs (lncRNAs) play critical roles in diverse cellular processes; however, their involvement in many critical aspects of the immune response including the interferon (IFN) response remains poorly understood. To address this gap, we compared the global gene expression pattern of primary human hepatocytes before and at three time points after treatment with IFN-α. Among ∼ 200 IFN-induced lncRNAs, one transcript showed ∼ 100-fold induction. This RNA, which we named lncRNA-CMPK2, was a spliced, polyadenylated nuclear transcript that was induced by IFN in diverse cell types from human and mouse. Similar to protein-coding IFN-stimulated genes (ISGs), its induction was dependent on JAK-STAT signaling. Intriguingly, knockdown of lncRNA-CMPK2 resulted in a marked reduction in HCV replication in IFN-stimulated hepatocytes, suggesting that it could affect the antiviral role of IFN. We could show that lncRNA-CMPK2 knockdown resulted in upregulation of several protein-coding antiviral ISGs. The observed upregulation was caused by an increase in both basal and IFN-stimulated transcription, consistent with loss of transcriptional inhibition in knockdown cells. These results indicate that the IFN response involves a lncRNA-mediated negative regulatory mechanism. lncRNA-CMPK2 was strongly upregulated in a subset of HCV-infected human livers, suggesting a role in modulation of the IFN response in vivo.

Long Non-Coding RNA BST2/BISPR is Induced by IFN and Regulates the Expression of the Antiviral Factor Tetherin.

Many long non-coding RNAs (lncRNAs) are expressed in cells but only a few have been well characterized. In these cases, lncRNAs have been shown to be key regulators of several cellular processes. Therefore, there is a great need to understand the function of more lncRNAs and their regulation in response to stimuli. Interferon (IFN) is a key molecule in the cellular antiviral response. IFN binding to its receptor activates transcription of several IFN-stimulated genes (ISGs) that function as potent antivirals. In addition, several ISGs are positive or negative regulators of the IFN pathway. This is essential to ensure a strong antiviral response and a later return of the cell to homeostasis. As the ISGs described to date are coding genes, we sought to determine whether IFN also regulates the expression of long non-coding ISGs. To this aim, we used RNA sequencing to analyze the transcriptome of control and HuH7 cells treated with IFNα2. The results show that IFN-treatment regulates the expression of several unknown non-coding transcripts. We have validated two lncRNAs upregulated after treatment with different doses of type I IFNα2 in different cells or with type III IFNλ. These lncRNAs were also induced by influenza and vesicular stomatitis virus mutants unable to block the IFN response, but not by several wild-type lytic viruses tested. These lncRNA genes were named lncISG15 and lncBST2 as they are located close to ISGs ISG15 and BST2, respectively. Interestingly, inhibition experiments showed that lncBST2 is a positive regulator of BST2. Therefore lncBST2 has been renamed BISPR, from BST2 IFN-stimulated positive regulator. Our results may have therapeutic implications as lncBST2/BISPR, but also lncISG15 and their coding neighbors, are increased in cells infected with hepatitis C virus and in the liver of infected patients. These results allow us to hypothesize that several lncRNAs could be activated by IFN to control the potency of the antiviral IFN response.

Resumen de congreso: 20 años después del primer Simposium Internacional sobre el virus de la hepatitis C y otros virus relacionados / No disponible

La primera conferencia sobre el virus de la hepatitis C (VHC) y otros virus relacionados se celebró en Venecia en 1992, poco después de su descubrimiento en 1989 por el equipo de Michael Houghton en la Chiron Corporation. Esta conferencia se planteó con el objetivo de avanzar en los conocimientos sobre el entonces misterioso virus responsable de la mayoría de las hepatitis «no A, no B». Durante estos 20 años desde la primera conferencia, su nivel científico ha ido incrementándose proporcionalmente a los avances en la investigación básica, epidemiológica y clínica. Lo que comenzó siendo un pequeño simposio sobre un nuevo virus del que se conocía bien poco, se ha convertido en el congreso de referencia: un punto de encuentro de investigadores básicos, clínicos, epidemiólogos y expertos en salud pública, y miembros de la industria donde se presentan los avances más importantes y su aplicación al tratamiento y control de la infección. El pasado mes de septiembre de 2012 se celebró la decimonovena edición, de nuevo en Venecia (AU)

The hepatitis C virus (HCV) was discovered by the team of Michael Houghton at Chiron Corporation in 1989 and the first symposium on HCV and related viruses was held in Venice,Italy, shortly after, in 1992. This conference was organized to advance knowledge on whatthen was a mysterious virus responsible for most cases of «non-A, non-B» hepatitis. During the20 years since the first conference, the scientific quality of presentations has steadily increased, together with the tremendous advances in basic and clinical research and epidemiology.What started as a small conference on a new virus, about which there were very few data,has today become a first-in-class congress: a meeting place for basic researchers, clinicians,epidemiologists, public health experts, and industry members to present the most importantadvances and their application to HCV treatment and control. The nineteenth HCV symposiumwas held in September 2012, once again in Venice (AU)

[Meeting Report: 20 years after the First International Symposium on hepatitis C virus and related viruses]. / Resumen de congreso: 20 años después del primer Simposium Internacional sobre el virus de la hepatitis C y otros virus relacionados.

The hepatitis C virus (HCV) was discovered by the team of Michael Houghton at Chiron Corporation in 1989 and the first symposium on HCV and related viruses was held in Venice, Italy, shortly after, in 1992. This conference was organized to advance knowledge on what then was a mysterious virus responsible for most cases of «non-A, non-B¼ hepatitis. During the 20 years since the first conference, the scientific quality of presentations has steadily increased, together with the tremendous advances in basic and clinical research and epidemiology. What started as a small conference on a new virus, about which there were very few data, has today become a first-in-class congress: a meeting place for basic researchers, clinicians, epidemiologists, public health experts, and industry members to present the most important advances and their application to HCV treatment and control. The nineteenth HCV symposium was held in September 2012, once again in Venice.

Comparison of Heterologous Prime-Boost Strategies against Human Immunodeficiency Virus Type 1 Gag Using Negative Stranded RNA Viruses.

This study analyzed a heterologous prime-boost vaccine approach against HIV-1 using three different antigenically unrelated negative-stranded viruses (NSV) expressing HIV-1 Gag as vaccine vectors: rabies virus (RABV), vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV). We hypothesized that this approach would result in more robust cellular immune responses than those achieved with the use of any of the vaccines alone in a homologous prime-boost regimen. To this end, we primed BALB/c mice with each of the NSV-based vectors. Primed mice were rested for thirty-five days after which we administered a second immunization with the same or heterologous NSV-Gag viruses. The magnitude and quality of the Gag-specific CD8(+) T cells in response to these vectors post boost were measured. In addition, we performed challenge experiments using vaccinia virus expressing HIV-1 Gag (VV-Gag) thirty-three days after the boost inoculation. Our results showed that the choice of the vaccine used for priming was important for the detected Gag-specific CD8(+) T cell recall responses post boost and that NDV-Gag appeared to result in a more robust recall of CD8(+) T cell responses independent of the prime vaccine used. However, the different prime-boost strategies were not distinct for the parameters studied in the challenge experiments using VV-Gag but did indicate some benefits compared to single immunizations. Taken together, our data show that NSV vectors can individually stimulate HIV-Gag specific CD8(+) T cells that are effectively recalled by other NSV vectors in a heterologous prime-boost approach. These results provide evidence that RABV, VSV and NDV can be used in combination to develop vaccines needing prime-boost regimens to stimulate effective immune responses.

Contribution of double-stranded RNA and CPSF30 binding domains of influenza virus NS1 to the inhibition of type I interferon production and activation of human dendritic cells.

The influenza virus nonstructural protein 1 (NS1) inhibits innate immunity by multiple mechanisms. We previously reported that NS1 is able to inhibit the production of type I interferon (IFN) and proinflammatory cytokines in human primary dendritic cells (DCs). Here, we used recombinant viruses expressing mutant NS1 from the A/Texas/36/91 and A/Puerto Rico/08/34 strains in order to analyze the contribution of different NS1 domains to its antagonist functions. We show that the polyadenylation stimulating factor 30 (CPSF30) binding function of the NS1 protein from A/Texas/36/91 influenza virus, which is absent in the A/Puerto Rico/08/34 strain, is essential for counteracting these innate immune events in DCs. However, the double-stranded RNA (dsRNA) binding domain, present in both strains, specifically inhibits the induction of type I IFN genes in infected DCs, while it is essential only for inhibition of type I IFN proteins and proinflammatory cytokine production in cells infected with influenza viruses lacking a functional CPSF30 binding domain, such as A/Puerto Rico/08/34.

Cytopathogenesis of vesicular stomatitis virus is regulated by the PSAP motif of M protein in a species-dependent manner.

Vesicular stomatitis virus (VSV) is an important vector-borne pathogen of bovine and equine species, causing a reportable vesicular disease. The matrix (M) protein of VSV is multifunctional and plays a key role in cytopathogenesis, apoptosis, host protein shut-off, and virion assembly/budding. Our previous findings indicated that mutations of residues flanking the (37)PSAP(40) motif within the M protein resulted in VSV recombinants having attenuated phenotypes in mice. In this report, we characterize the phenotype of VSV recombinant PS > A4 (which harbors four alanines (AAAA) in place of the PSAP motif without disruption of flanking residues) in both mice, and in Aedes albopictus C6/36 mosquito and Culicoides sonorensis KC cell lines. The PS > A4 recombinant displayed an attenuated phenotype in infected mice as judged by weight loss, mortality, and viral titers measured from lung and brain samples of infected animals. However, unexpectedly, the PS > A4 recombinant displayed a robust cytopathic phenotype in insect C6/36 cells compared to that observed with control viruses. Notably, titers of recombinant PS > A4 were approximately 10-fold greater than those of control viruses in infected C6/36 cells and in KC cells from Culicoides sonorensis, a known VSV vector species. In addition, recombinant PS > A4 induced a 25-fold increase in the level of C3 caspase activity in infected C6/36 cells. These findings indicate that the PSAP motif plays a direct role in regulating cytopathogenicity in a species-dependent manner, and suggest that the intact PSAP motif may be important for maintaining persistence of VSV in an insect host.

In Vivo Replication and Pathogenesis of Vesicular Stomatitis Virus Recombinant M40 Containing Ebola Virus L-Domain Sequences.

The M40 VSV recombinant was engineered to contain overlapping PTAP and PPxY L-domain motifs and flanking residues from the VP40 protein of Ebola virus. Replication of M40 in cell culture is virtually indistinguishable from that of control viruses. However, the presence of the Ebola PTAP motif in the M40 recombinant enabled this virus to interact with and recruit host Tsg101, which was packaged into M40 virions. In this brief report, we compared replication and the pathogenic profiles of M40 and the parental virus M51R in mice to determine whether the presence of the Ebola L-domains and flanking residues altered in vivo characteristics of the virus. Overall, the in vivo characteristics of M40 were similar to those of the parental M51R virus, indicating that the Ebola sequences did not alter pathogenesis of VSV in this small animal model of infection.

Adenovirus and miRNAs.

Adenovirus infection has a tremendous impact on the cellular silencing machinery. Adenoviruses express high amounts of non-coding virus associated (VA) RNAs able to saturate key factors of the RNA interference (RNAi) processing pathway, such as Exportin 5 and Dicer. Furthermore, a proportion of VA RNAs is cleaved by Dicer into viral microRNAs (mivaRNAs) that can saturate Argonaute, an essential protein for miRNA function. Thus, processing and function of cellular miRNAs is blocked in adenoviral-infected cells. However, viral miRNAs actively target the expression of cellular genes involved in relevant functions such as cell proliferation, DNA repair or RNA regulation. Interestingly, the cellular silencing machinery is active at early times post-infection and can be used to control the adenovirus cell cycle. This is relevant for therapeutic purposes against adenoviral infections or when recombinant adenoviruses are used as vectors for gene therapy. Manipulation of the viral genome allows the use of adenoviral vectors to express therapeutic miRNAs or to be silenced by the RNAi machinery leading to safer vectors with a specific tropism. This article is part of a "Special Issue entitled:MicroRNAs in viral gene regulation".

Quantitative and qualitative features of heterologous virus-vector-induced antigen-specific CD8+ T cells against Trypanosoma cruzi infection.

We studied some aspects of the quantitative and qualitative features of heterologous recombinant (re) virus-vector-induced, antigen-specific CD8(+) T cells against Trypanosoma cruzi. We used three different, highly attenuated re-viruses, i.e., influenza virus, adenovirus and vaccinia virus, which all expressed a single, T. cruzi antigen-derived CD8(+) T-cell epitope. The use of two out of three vectors or the triple virus-vector vaccination regimen not only confirmed that the re-vaccinia virus, which was placed last in order for sequential immunisation, was an effective booster for the CD8(+) T-cell immunity in terms of the number of antigen-specific CD8(+) T cells, but also demonstrated that (i) the majority of cells exhibit the effector memory (T(EM)) phenotype, (ii) robustly secrete IFN-γ, (iii) express higher intensity of the CD122 molecule and (iv) present protective activity against T. cruzi infection. In contrast, placing the re-influenza virus last in sequential immunisation had a detrimental effect on the quantitative and qualitative features of CD8(+) T cells. The triple virus-vector vaccination was more effective at inducing a stronger CD8(+) T-cell immunity than using two re-viruses. The different quantitative and qualitative features of CD8(+) T cells induced by different immunisation regimens support the notion that the refinement of the best choice of multiple virus-vector combinations is indispensable for the induction of a maximum number of CD8(+) T cells of high quality.