34 research outputs found
Human herpesvirus 8 cellular gene modulation
No full textP0663 Human herpesvirus 8 cellular gene modulation
Barbara Matteoli*
1
, Rita Romano
2
,FrancescaTabacchi
2
, Moreno Ferroni
1
1 LAMM,SRL,Lucca, Italy,
2 Department of Public Health and Infectious Diseases,Sapienza University of Rome,
Rome, Italy
Background:Sinceinflammation is the basis of viral pathogenesis and may beinduced either directly by thevirus
or by thecell in responseto infection, theexpression pattern of cellular genes involved in this process was
investigated in human epithelial and lymphoid cells infected with Human herpes virus 8 (HHV8) .
Materials/methods:Theexpression of 297 human genes involved in 65 pathways associated with inflammation
have been studied in HEK293 and PBMC cells 24h after infection with HHV8 (A1 and C3 strains) by DualChip
Microarray.Signal quantification was performed by SilverQuant Image Viewer and analysis software(Eppendorf,
Germany) and SPSS 16.0 for Windows (SPSS, USA) with two way ANOVA followed by Scheffecontrast.
Results:Expression ratios of most of the genes werecloseto 1 or changed only <2-fold cutoff (in positive or
negativevalue), suggesting that they were not significantly changed in consequenceto HHV8 infection. Overall,a
higher number of genes resulted modulated in HHV8-infected HEK293 cells with respect to PBMCs. A robust
increasein theexpression was found in genes involved in inflammation (i.e., AGT, CCR8, CXCR4, IL-6, IL-9, IL10RA,
IL-17, IL-23A), signal transduction (i.e.,FGF5,FGF7, MAP3K1, MAPk9m TGFBR1,TRAF2),cellular cycle(IGFBP1),
immuneresponse(i.e., IL-16,TLR4),apoptosis (TNFSF4,TNFSF11),and others genes belonging to different
categories (i.e., BMP4, BMPR2,EPHA3, HGFBP1, RUNX1, RUNX2,SP1).With a >2-fold threshold filter gene
modulation as significant,a high numbers of genes resulted significantly modulated both in HEK293 cell lineand
PBMCs infected both with the A1 (169 and 104 genes, respectively) and C3 subtypes (131 and 93 genes,
respectively). Next, to increasethesignificance, only those genes with >4-fold changes in their expression were
considered. A total of 24 genes resulted strongly induced,especially after A1 subtypeinfection.Thecomparison of
transcriptional profiling between both viral subtypes showed that only 4 genes arestrongly induced by different
viral subtypes (i.e., BMP4, BMPR2, RUNX2 and TNFSF11).
Conclusions: New human genes have been involved in HHV8 infection;commonly modulated genes may be
used as target for antiviral/chemiotherapic treatmen
“Modulazione dell'espressione genica in cellule linfoidi ed epitelioidi infettate con il Virus Erpetico Umano 8 (HHV8) ed analisi dell’attività chemioterapica/antivirale della Distamicina A (DA)”
No full textPurpose of this PhD study have been the analysis of the modulation of cellular gene by Human Herpes virus 8 (HHV8) infection and of the potential antiviral effect of Distamycin A (DA), an oligopeptidic antibiotic. This study describes a comprehensive picture of global gene changes soon after HHV-8 active infection of two susceptible cell types. We found that host cell expression changes after only 24 hours of infection, and it is quite different from previous gene induction studies that were carried out at later time points of virus infection. In fact, Poole et al. have used a different primary effusion lymphoma cell line (JSC1) to produce the virus that was used to infect primary endothelial cells for a period of 3–5 weeks, and gene array experiments were done when almost all of the cells changed from typical cobblestone to spindle-shaped morphology and were positive for LANA. Several features were apparent from these analysis: a) there was virtually non-overlapping list of genes in all of two viral subtypes analyzed; b) HHV-8-induced transcriptional profiles in the epithelial and PBMC were closely different. This is probably because the criteria of > 4-fold gene induction as significant is a threshold filter very tight. Nevertheless, the genes that emerge from such comparison will be of greater interest for studying their role in the unique biology of HHV-8. Furthermore, the replicative capacity of the two different strains in the two cell types and the modulation of cell involved in the inflammatory process would seem to indicate that the genotypic difference could be translated into a different virus-host relationship, also attributable to a different pathogenetic mechanism. In addition to identifying specific host response genes within each functional pathway that are already known to be important for HHV-8 infection, our findings identify novel candidate genes that are not yet known in HHV-8 biology.
Obtained results show a lack of cytotoxicity of DAlipo on cultured HEK293, RCE and PBMC cells. It might be noted that the CC50 value found in the present study falls at the low extreme of the range of CC50 values reported in the literature. In fact, available data show CC50 values ranging from 35 μM in a Hep-2 cell line to 154 μM in a HeLa cell line and 385 μM in rabbit primary kidney cells. It has been reported that DA inhibits the activity of all DNA polymerases. This mechanism of action could explain its antiviral activity. Because of the lack of information concerning the relevant genetic definition of HHV8 A1 and C3 subtypes from BCBL1 and BC3 cell lines, whether the observed behavior be the result of the expression of specific mutated genes or a non specific consequence of viral strain virulence has to be established. Clearly, further studies are needed to explain the different degree of sensitivity of HHV8 subtypes to DA and Dalipo. The significant antiviral activity of DAlipo and the lack of citotoxicity in epithelial and lymphoid cell lines shown in the present study indicates the compound as an antiherpetic drug potentially useful in both topic and systemic treatments. It could be used either alone or in association with other antiviral treatment, the latter to be employed at lower dosages and for shorter periods of time, thus limiting side effects. The antiviral activity of DAlipo formulation also in lymphoid cell lines may be due to the hydrophobic or amphiphilic drug penetration efficacy increased through inclusion of the drug in liposomes. DAlipo showed, when compared with the free drug, higher antiviral activity against HHV8 A1 and C3 subtypes in PBMCs and might therefore represent a promising DA formulation that could be used also for systemic treatments
Liposomes as a potential ocular delivery system of distamycin A
No full textLiposomes containing Distamycin A (DA) may be clinically useful in the treatment of ocular HSV infections, especially in acyclovir-resistant HSV keratitis. This study evaluated the in vitro and in vivo performance of a topical controlled release liposomal formulation containing DA (DA-Lipo) aimed at reducing the toxicity of the encapsulated active agent and improving drug uptake by ocular tissues. The bioavailability of DA in the tear fluid and the DA uptake into the cornea were increased after instillation of DA-Lipo in rabbits, reaching the DA corneal concentration corresponding to IC50 values against HSV without any sign of transcorneal permeation of drug. DA-Lipo was definitely less cytotoxic then plain DA in rabbit corneal epithelial cells. These results provide new insights into the correlation between the in vitro data and the drug kinetics following ocular applications of liposomal vesicle
Comparison of oncogenic HPV type-specific viral DNA load and E6/E7 mRNA detection in cervical samples: Results from a multicenter study
No full textMeasuring pH in insulin secretory granules by phasor-based fluorescence lifetime imaging of a genetically encoded sensor
No full textIt is widely accepted that the pH of insulin granules is acidic, and that its active regulation during granule maturation plays a role in the process of insulin secretion by β-cells. Yet, a calibrated measurement of the absolute granule pH with organelle specificity is still lacking. To tackle this issue, we use the genetically encoded E1GFP pH reporter inserted into the C-peptide of proinsulin and expressed in Insulinoma 1E cells. Following verification of correct targeting of the E1GFP reporter in the insulin granules, phasor-based Fluorescence Lifetime Imaging Microscopy (FLIM) is applied to obtain a calibrated and probe-concentration-independent measurement of insulin-granule pH. Our results confirm the acidic nature of insulin granules under maintenance cell culture conditions, with an average luminal pH of ~5.8, and show that acidity is actively maintained, as evidenced by its near-neutralization upon treatment with the vacuolar H+-ATPase inhibitor concanamycin. Additionally, by ..
In vivo and in vitro evidence for an association between the route-specific transmission of HHV-8 and the virus genotype
No full textThe study was performed to determine if there is an association between the genotype and transmission of HHV-8 types A and C. These HHV-8 subtypes are prevalent in the area of North of Sardinia, which is an island off west Italy's mainland that has a high HHV-8 seroprevalence (35%). Blood and saliva samples from 30 patients with classic Kaposi's sarcoma who were lifetime residents of North Sardinia were analyzed to identify the HHV-8 genotype and quantitate the viral load. Genotype A, especially A1 subtype, was found more frequently (9/30 patients) and had a significantly higher viral load in saliva compared to blood (P=0.029), where type C was found more frequently but with a viral load lower than 10 3copies/ml. To determine if there is a correlation between the viral genotype and cellular tropism, type A1 and C3 HHV-8 viral particles were obtained from cell lines BCBL1 and BC3 infected chronically with HHV-8 A1 and C3 genotypes respectively and used to infect HEK293 epithelial origin cells and PBMCs in vitro. The data indicate that the A1 HHV-8 genotype is tropic and replicates at higher levels in the epithelial cell lines. © 2012 Wiley Periodicals, Inc
Modelling and Control of HIV Dynamics
No full textVarious models of HIV infection and evolution have been considered in the literature. This paper considers a variant of the Wodarz and Nowak mathematical model, adding "aggressiveness" as a new state variable in order to quantify the strength of the virus and its response to drugs. Although the model proposed is relatively simple, simulation results suggest that it may be useful in predicting the impact of the effectiveness of therapy on HIV dynamics
Tolleranza all'insulto ischemico e invecchiamento mitocondriale: ruolo dei canali mito-KATP.
No full textLe patologie cardiovascolari, in particolare l’infarto del miocardio, risultano incrementate con l’invecchiamento, in più si osserva una minore tolleranza all’insulto ischemico da parte di cuori senescenti, e una perdita di efficacia delle strategie cardioprotettive. Come conseguenza si osserva anche un’aumentata vulnerabilità cellulare, riconducibile a una disfunzione dei mitocondri. In più sappiamo che i canali mitocondriali del potassio ATP-sensibili (mito-KATP) svolgono un ruolo fondamentale nella cardioprotezione da danno ischemico, tanto che la ricerca si è indirizzata verso lo sviluppo di attivatori sempre più selettivi verso questo target.
Recenti studi dimostrano che l’attivatore di riferimento dei canali mito-KATP, diazossido, perde la sua efficacia protettiva quando somministrato a cuori ottenuti da animali senescenti (Schulman et al., 2001), senza tuttavia fare alcuna speculazione sulle alterazioni fisiologiche responsabili di tale mancato effetto.
Pertanto, lo scopo della mia ricerca, è stato quello di valutare gli effetti sui parametri funzionali di mitocondri cardiaci ottenuti da animali giovani e senescenti, da parte di due attivatori dei canali mito-KATP, diazossido e F163.
Attraverso il primo protocollo sperimentale è stato valutato il potenziale di membrana mitocondriale (Δψm), in mitocondri isolati da ratto giovane e senescente in seguito all’aggiunta degli attivatori dei mito-KATP. L’analisi è stata condotta potenziometricamente con l’utilizzo di un mini-elettrodo sensibile al Tetrafenilfosfonio (TPP+), ione liposolubile che si distribuisce fra interno ed esterno del mitocondrio in relazione al potenziale di membrana, come descritto dall’equazione di Nernst.
Nella seconda parte della ricerca sono stati valutati i movimenti del K+ utilizzando una sonda fluorescente tallio-sensibile (catione K+-mimetico). Mediante questo approccio sperimentale sono stati osservati, attraverso l’analisi spettrofluorimetrica, i flussi di ioni Tl+ indotti dagli attivatori dei mito-KATP, diazossido e F163, e indotti dalla valinomicina (ionoforo del K+, usato come riferimento) nel ratto giovane e nel ratto senescente.
Nell’ultima parte della tesi, sono stati analizzati i movimenti del Ca++ attraverso la membrana mitocondriale nel ratto giovane e nel ratto senescente con l’utilizzo degli attivatori dei canali mito-KATP, diazossido e F163. L’analisi è stata condotta potenziometricamente con l’utilizzo di un mini-elettrodo sensibile al Ca++.
Il lavoro ha permesso di identificare che la maggiore vulnerabilità al danno da ischemia/riperfusione tipica negli animali senescenti può risiedere in un cattivo funzionamento del mitocondrio, dovuto ad una alterata gestione dei flussi di K+ tra citosol e matrice mitocondriale.
Tale alterazione sembra avere pesanti ripercussioni sul corretto funzionamento dei canali mito-KATP, che rappresentano un fondamentale meccanismo endogeno di protezione da danno da ischemia/riperfusione
