Purpose: Primary cutaneous T-cell lymphomas (CTCL) are neoplastic disorders of skin-homing T cells. Affected skin areas show morphologic similarities with alterations in other T-cell-mediated dermatoses. Furthermore, as in atopic dermatitis but in contrast with psoriasis, patients with CTCL are frequently afflicted by cutaneous bacterial infections that support the survival of lymphoma cells. Our aim was to investigate the mechanisms of elevated susceptibility to cutaneous infections in patients with CTCL.Experimental Design: Skin samples from CTCL, psoriasis, and atopic dermatitis patients were used to illuminate the antibacterial competence status and the presence of its modulating cytokines. For substantiation of findings, 3-dimensional epidermis models, isolated and in vitro generated Th-subpopulations, were applied. Parameters were analyzed via qPCR and IHC.Results: CTCL lesions compared with psoriatic lesions presented an impaired upregulation of antibacterial proteins (ABPs), with levels even below those in atopic dermatitis. This was associated with a relative deficiency of the ABP-inducing cytokine IL-17 and a strong presence of the ABP-downregulating cytokine IL-13. The simultaneous presence of the Th17-cell cytokine IL-26 indicated that IL-17 deficiency in CTCL lesions results from functional deviation of Th17 cells. Accordingly, IL-17 but not IL-26 production by Th17 cells in vitro was inhibited by IL-4Ra ligand. Levels of other ABP inducers were comparable between CTCL and psoriasis lesions. The same was true about IL-22/TNF-a targets, including the keratinocyte hyperregeneration marker K16 and the matrix-degrading enzyme MMP1.Conclusion: Our results suggest that the cutaneous bacterial infections in CTCL are caused by impaired ABP induction as consequence of Th2-mediated biased Th17-cell function. Clin Cancer Res; 20(21); 5507-16. Ó2014 AACR.
Adeno-associated virus (AAV) has long been known to inhibit helper adenovirus (Ad) replication independently of AAV Rep protein expression. More recently, replication of Ad serotype 5 (Ad5)/AAV serotype 2 (AAV-2) hybrid vectors was shown to be inhibited in cis by a sequence near the 3= end of AAV rep, termed the Rep inhibition sequence for adenoviral replication (RISAd). RIS-Ad functions independently of Rep protein expression. Here we demonstrate that inhibition of adenoviral replication by RIS-Ad requires an active AAV p40 promoter and the 5= half of the intron. In addition, Ad inhibition is critically dependent on the integrity of the p40 transcription start site (TSS) leading to short p40-associated transcripts. These do not give rise to effector molecules capable of inhibiting adenoviral replication in trans, like small polypeptides or microRNAs. Our data point to an inhibitory mechanism in which RNA polymerase II (Pol II) pauses directly downstream of the p40 promoter, leading to interference of the stalled Pol II transcription complex with the adenoviral replication machinery. Whereas inhibition by RIS-Ad is mediated exclusively in cis, it can be overcome by providing a replication-competent adenoviral genome in trans. Moreover, the inhibitory effect of RIS-Ad is not limited to AAV-2 but could also be shown for the corresponding regions of other AAV serotypes, including AAV-5. These findings have important implications for the future generation of Ad5/AAV hybrid vectors. IMPORTANCEInsertion of sequences from the 3= part of the rep gene of adeno-associated virus (AAV) into the genome of its helper adenovirus strongly reduces adenoviral genome replication. We could show that this inhibition is mediated exclusively in cis without the involvement of trans-acting regulatory RNAs or polypeptides but nevertheless requires an active AAV-2 p40 promoter and p40-associated short transcripts. Our results suggest a novel inhibitory mechanism that has so far not been described for AAV and that involves stalled RNA polymerase II complexes and their interference with adenoviral DNA replication. Such a mechanism would have important implications both for the generation of adenoviral vectors expressing the AAV rep and cap genes and for the regulation of AAV gene expression in the absence and presence of helper virus.A deno-associated virus (AAV) is characterized by a bipartite replication cycle, with productive infection being critically dependent upon the presence of a helper virus, such as adenovirus (Ad) (1) or herpes simplex virus (HSV) (2). In the absence of helper virus, AAV can establish latent infection by integration into the host genome (3, 4). During the productive replication cycle, AAV is able to inhibit the propagation of the coinfecting helper virus, especially evident in the case of adenovirus (5, 6). This inhibition has been attributed to the expression of the large regulatory (Rep) proteins Rep78 and Rep68. These promote the replication of the AAV genome and induce the expression of the AAV seroty...
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