The genetic manipulation of antigen-presenting dendritic cells (DC) offers promise for stimulating the immune response, in particular for anticancer and antiviral protocols. As adeno-associated virus (AAV) has shown promise as a gene delivery vector for transducing a variety of hematopoietic cell types, we have investigated AAV's ability to genetically alter DC. In this analysis, we modified the standard granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) treatment of adherent monocytes to generate DC. In our protocol, adherent monocytes were first infected with an AAV/GM-CSF/Neo vector, and the addition of IL-4 was delayed for 2 days to allow for a brief period of monocyte proliferation. AAV-mediated transduction of the GM-CSF and Neo genes into monocytes/DC precursors was demonstrated by G418 selection, GM-CSF secretion, GM-CSF RNA expression (reverse transcriptase-polymerase chain reaction amplification [RT-PCR]), and cell proliferation. Cells resulting from infection with AAV/GM-CSF/Neo virus, and subsequent IL-4 and tumor necrosis factor-alpha (TNF-alpha) treatment, displayed multiple classic markers consistent with mature DC. Finally, chromosomal integration of the AAV vector was also demonstrated in sorted CD83+ DC. These data strongly suggest that AAV vectors will be useful for the genetic manipulation of DC and suggest that the transduction of the GM-CSF gene was able to fully replace the need for exogenous GM-CSF in the production of mature DC.
Since its discovery in 1966, adeno-associated virus type 2 (AAV) has been described as a helper-dependent parvovirus. However, in this study we demonstrate that AAV undergoes its complete life cycle, devoid of helper viruses or genotoxic agents, in the organotypic epithelial raft tissue culture system, a model of normal skin. AAV progeny production directly correlated with epithelial differentiation, as nondifferentiating keratinocytes were defective for this activity. Large nuclear virus arrays of particles of approximately 26 nm (parvovirus size) were observed in the granular layers of the raft epithelium by electron microscopy. Additionally, dosage-dependent histologic changes, some of which might be interpreted as cytopathology, were induced in the AAV-infected epithelial tissues. These data suggest a new biological model for AAV; that is, AAV is an epithelial-tropic autonomous parvovirus that can alter normal squamous differentiation.
Adeno-associated virus (AAV), a common genital virus, may have a "protective" role against human papillomavirus (HPV)-associated cervical cancer. Epidemiological studies indicate a negative correlation between AAV infection and the incidence of cervical cancer. In contrast, HPV is positively associated with cervical cancer. To investigate interactions between these two viruses we used the organotypic "raft" culture system. The raft culture system is capable of supporting the complete HPV life cycle. Raft tissues that were actively replicating HPV were superinfected with AAV type 2 (AAV-2). We observed a multiplicity of infection (m.o.i.)-dependent enhancement and inhibition of HPV DNA replication, concomitant with AAV-2 replication. The data suggest that at low m.o.i. of AAV-2 infection, HPV DNA replication was slightly increased compared to controls and AAV-2 replicated poorly. At high AAV-2 m.o.i., HPV DNA replication was reduced and AAV-2 replicated to high levels. AAV-2 replication was increased in the presence of HPV compared to primary human keratinocyte, squamous cell carcinoma, and HaCat raft cultures infected with AAV-2 alone. These data suggest that HPV may provide types of "enhancer/helper" functions for AAV-2 replication and progeny formation. Infection with AAV-2 had significant effects on epithelial morphology. During infection with low m.o.i. of AAV-2 the epithelium stratified to a greater extent than in controls. With high m.o.i. of AAV-2 infections, tissue cytopathic effects were observed, indicating an additional factor responsible for the effect of AAV-2 on HPV replication and infection. Our results demonstrate a complex interaction between AAV-2, HPV, and skin during dual infection.
BackgroundMyxovirus resistance protein A (MxA) is a molecule induced after interferon-beta injection, mostly used to evaluate its bioactivity. There is little available data on clinical utility of baseline MxA mRNA status. The objective of the study is to investigate whether baseline MxA mRNA expression can predict relapse and disease progression in multiple sclerosis patients treated with interferon-beta.MethodsBaseline blood samples were obtained before the first interferon-beta dose was administered to evaluate MxA mRNA expression using real-time polymerase chain reaction (PCR). Demographic and clinical variables were prospectively recorded to define treatment responder and non responder groups.Results104 patients were included in the study. Baseline MxA mRNA expression was significantly lower in the group of patients who met the definition of responders (1.07 vs 1.95, Student t test, p<0.0001). A threshold of 1.096 was established using Receiver Operating Characteristic analysis to differentiate between responders and non-responders (sensitivity 73.9%, specificity 69.0%). Survival analysis using this threshold showed that time to next relapse (p<0.0001) and to EDSS progression (p = 0.01) were significantly higher in patients with lower MxA titers.ConclusionThe results suggest that baseline MxA mRNA levels may be useful for predicting whether multiple sclerosis patients will respond or not to interferon-beta treatment.
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