Eyebrow hairs from actinic keratosis patients harbor the highest number of cutaneous human papillomaviruses.

BACKGROUND: Cutaneous human papillomavirus (HPV) infections seem to be associated with the onset of actinic keratosis (AK). This study compares the presence of cutaneous HPV types in eyebrow hairs to those in tissues of normal skin and skin lesions of 75 immunocompetent AK patients. METHODS: Biopsies from AK lesions, normal skin and plucked eyebrow hairs were collected from each patient. DNA from these specimens was tested for the presence of 28 cutaneous HPV (betaPV and gammaPV) by a PCR based method. RESULTS: The highest number of HPV prevalence was detected in 84% of the eyebrow hairs (63/75, median 6 types) compared to 47% of AK lesions (35/75, median 3 types) (p< 0.001) and 37% of normal skin (28/75, median 4 types) (p< 0.001), respectively. A total of 228 HPV infections were found in eyebrow hairs compared to only 92 HPV infections in AK and 69 in normal skin. In all three specimens HPV20, HPV23 and/or HPV37 were the most prevalent types. The highest number of multiple types of HPV positive specimens was found in 76% of the eyebrow hairs compared to 60% in AK and 57% in normal skin. The concordance of at least one HPV type in virus positive specimens was 81% (three specimens) and 88-93% of all three combinations with two specimens. CONCLUSIONS: Thus, eyebrow hairs revealed the highest number of cutaneous HPV infections, are easy to collect and are an appropriate screening tool in order to identify a possible association of HPV and AK.

Mutations in ENPP1 are associated with 'idiopathic' infantile arterial calcification.

Idiopathic infantile arterial calcification (IIAC; OMIM 208000) is characterized by calcification of the internal elastic lamina of muscular arteries and stenosis due to myointimal proliferation. We analyzed affected individuals from 11 unrelated kindreds and found that IIAC was associated with mutations that inactivated ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). This cell surface enzyme generates inorganic pyrophosphate (PP(i)), a solute that regulates cell differentiation and serves as an essential physiologic inhibitor of calcification.

Genomic characterization of ten novel cutaneous human papillomaviruses from keratotic lesions of immunosuppressed patients.

Viral warts from immunosuppressed organ transplant recipients (OTR) persist over years and may progress into non-melanoma skin cancer. The types of human papillomaviruses (HPV) in such lesions are different from that seen in the general population. A subset of these lesions is not infected with the classical wart-associated HPV types. In order to gain a better understanding of the HPV types in those lesions, we isolated ten novel HPVs from persisting keratotic lesions of immunosuppressed OTRs by rolling circle amplification and subsequent long-template PCR. Additionally, we sequenced and characterized the whole genome of the ten novel HPV types. Phylogenetic analyses revealed that nine HPV types belonged to the genus Gammapapillomavirus (γ-PV) and one to the genus Betapapillomavirus. In a phylogenetic analysis using L1 fragments of human and non-human PV types, primate papillomaviruses and our novel HPV types nested within the genus γ-PV in a highly polyphyletic pattern. This study significantly broadens the knowledge concerning the diversity and evolution of the poorly known γ-PV types.

Extension of the typing in a general-primer-PCR reverse-line-blotting system to detect all 25 cutaneous beta human papillomaviruses.

beta-Papillomaviruses (PV) seem to be involved in the pathogenesis of cutaneous squamous cell carcinoma and its early stage actinic keratosis. In this study, typing was extended of a previously described consensus primer-mediated beta- and gamma-cutaneous HPV PCR method followed by reverse-line-blotting (BGC-PCR/RLB) to detect all 25 known beta-PV and to examine their prevalence in actinic keratosis. The typing format of the BGC-PCR assay was extended by adding hybridization probes of six beta-PV (HPV 75, 76, 80, 92, 93, and 96) to the RLB system. Subsequently, tumor and normal skin tissues were collected from 75 patients with actinic keratosis, allowing typing for a total of 25 beta- and 5 gamma-types. The analytical sensitivity was between 10 copies (HPV 75, 80, 92, 93, and 96) and 100 copies (HPV 76). Except for that of HPV 76, none of the added probes showed any cross-hybridization with other beta-HPV. HPV DNA was detected in 45% of actinic keratosis and in 33% of normal skin by BGC-PCR, and at least one of the six added beta-types was present in 19% of actinic keratoses and in 13% of normal skin. Six beta-HPV types were added successfully to the typing format of the BGC-PCR/RLB system. The potential role of these types in the development of non-melanoma skin cancer awaits further studies.

Detection and typing of cutaneous human papillomavirus types--a comparison of three different methods.

Cutaneous human papillomavirus (HPV) may play a role in the development of cutaneous squamous cell carcinoma. HPV copy numbers in cutaneous squamous cell carcinoma are very low and hence sensitive and reliable detection methods are important, particularly to examine the natural history of cutaneous HPV. In the present study, the presence of cutaneous HPV types was examined in 194 skin swabs and in a subgroup of 91 skin swabs, and compared using three different PCR based methods: (i) beta/gamma cutaneous HPV PCR reverse-line-blotting (BGC-PCR RLB), (ii) multiplex cutaneous papillomavirus genotyping (McPG) and (iii) FAP PCR. The HPV prevalence was 75% (68/91) with BGC-PCR RLB, 64% (124/194) with McPG and 72% (139/194) with FAP PCR. The agreement for the detection of HPV between the three methods in the subset of 91 samples was 73% (66/91; kappa=0.34) for BGC-PCR RLB and McPG, 75% (68/91; kappa=0.32) for BGC-PCR RLB and FAP PCR, and 69% (63/91; kappa=0.25) for McPG and FAP PCR. For McPG and FAP PCR, 194 specimens were tested in total, with an overall agreement of 66% (129/194; kappa=0.24) for the detection of HPV. The concordance between the three methods was moderate, which could be explained by different HPV types detectable with each method; the high number of multiple infections and the low viral copy number in human skin. Overall, many cutaneous HPV types were identified and multiple HPV types were found frequently in the human skin swabs.

Generation of retinal pigment epithelial cells from small molecules and OCT4 reprogrammed human induced pluripotent stem cells.

Autologous retinal pigment epithelium (RPE) grafts derived from induced pluripotent stem cells (iPSCs) may be used to cure blinding diseases in which RPE dysfunction results in photoreceptor degeneration. Four-, two-, and one-factor-derived iPSCs (4F-, 2F-, and 1F-iPSCs, respectively) were differentiated into fully functional cuboidal pigmented cells in polarized monolayers that express RPE-specific markers. 1F-iPSCs-RPE (1F-iPS-RPE) strongly resembles primary human fetal RPE (hfRPE) based on proteomic and untargeted metabolomic analyses, and using novel in vivo imaging technology coupled with electroretinography, we demonstrated that 1F-iPS-RPE mediate anatomical and functional rescue of photoreceptors after transplantation in an animal model of RPE-mediated retinal degeneration. 1F-iPS0RPE cells were injected subretinally as a suspension and formed a monolayer dispersed between host RPE cells. Furthermore, 1F-iPS-RPE do not simply provide trophic support to rescue photoreceptors as previously speculated but actually phagocytose photoreceptor outer segments in vivo and maintain visual cycling. Thus, 1f-iPS-RPE grafts may be superior to conventional iPS-RPE for clinical use because 1F-IPS-RPE closely resemble hfRPE, mediate anatomical and functional photoreceptor rescue in vivo, and are generated using a reduced number of potentially oncogenic reprogramming factors.

Lung epithelial injury by B. anthracis lethal toxin is caused by MKK-dependent loss of cytoskeletal integrity.

Bacillus anthracis lethal toxin (LT) is a key virulence factor of anthrax and contributes significantly to the in vivo pathology. The enzymatically active component is a Zn(2+)-dependent metalloprotease that cleaves most isoforms of mitogen-activated protein kinase kinases (MKKs). Using ex vivo differentiated human lung epithelium we report that LT destroys lung epithelial barrier function and wound healing responses by immobilizing the actin and microtubule network. Long-term exposure to the toxin generated a unique cellular phenotype characterized by increased actin filament assembly, microtubule stabilization, and changes in junction complexes and focal adhesions. LT-exposed cells displayed randomly oriented, highly dynamic protrusions, polarization defects and impaired cell migration. Reconstitution of MAPK pathways revealed that this LT-induced phenotype was primarily dependent on the coordinated loss of MKK1 and MKK2 signaling. Thus, MKKs control fundamental aspects of cytoskeletal dynamics and cell motility. Even though LT disabled repair mechanisms, agents such as keratinocyte growth factor or dexamethasone improved epithelial barrier integrity by reducing cell death. These results suggest that co-administration of anti-cytotoxic drugs may be of benefit when treating inhalational anthrax.

Cutaneotropic human beta-/gamma-papillomaviruses are rarely shared between family members.

Several cutaneotropic human papillomavirus (HPV) types seem to be involved in the early onset of cutaneous squamous-cell carcinoma. To test the hypothesis that cutaneotropic HPV infections are facilitated because of close and frequent skin contact (for example, between child and mother), we examined HPV prevalence in hair follicle cells from 134 volunteers (1-89 years of age, median 42 years) from 13 families. We used a high-throughput HPV-typing approach with a sensitive beta-/gamma-cutaneous PCR method, followed by reverse line blotting, to detect 30 cutaneotropic HPV types. HPV prevalence in all individuals was 42% and increased with age from 5% at < or =20 years to 27% at 21-40 years, 53% at 41-60 years, and 76% at >60 years. The effect of life age was significant, independent of couples and family members shown by regression analyses (P < or =10(-8)). A higher similarity of HPV infection patterns was observed in couples versus two randomly chosen individuals (P

Inhibitory action of NoxA1 on dual oxidase activity in airway cells.

Imbalance between pro- and antioxidant mechanisms in the lungs can compromise pulmonary functions, including blood oxygenation, host defense, and maintenance of an anti-inflammatory environment. Thus, tight regulatory control of reactive oxygen species is critical for proper lung function. Increasing evidence supports a role for the NADPH oxidase dual oxidase (Duox) as an important source for regulated H2O2 production in the respiratory tract epithelium. In this study Duox expression, function, and regulation were investigated in a fully differentiated, mucociliary airway epithelium model. Duox-mediated H2O2 generation was dependent on calcium flux, which was required for dissociation of the NADPH oxidase regulatory protein Noxa1 from plasma membrane-bound Duox. A functional Duox1-based oxidase was reconstituted in model cell lines to permit mutational analysis of Noxa1 and Duox1. Although the activation domain of Noxa1 was not required for Duox function, mutation of a proline-rich domain in the Duox C terminus, a potential interaction motif for the Noxa1 Src homology domain 3, caused up-regulation of basal and stimulated H2O2 production. Similarly, knockdown of Noxa1 in airway cells increased basal H2O2 generation. Our data indicate a novel, inhibitory function for Noxa1 in Duox regulation. This represents a new paradigm for control of NADPH oxidase activity, where second messenger-promoted conformational change of the Nox structure promotes oxidase activation by relieving constraint induced by regulatory components.

Modulation of gene expression via disruption of NF-kappaB signaling by a bacterial small molecule.

The control of innate immune responses through activation of the nuclear transcription factor NF-kappaB is essential for the elimination of invading microbial pathogens. We showed that the bacterial N-(3-oxo-dodecanoyl) homoserine lactone (C12) selectively impairs the regulation of NF-kappaB functions in activated mammalian cells. The consequence is specific repression of stimulus-mediated induction of NF-kappaB-responsive genes encoding inflammatory cytokines and other immune regulators. These findings uncover a strategy by which C12-producing opportunistic pathogens, such as Pseudomonas aeruginosa, attenuate the innate immune system to establish and maintain local persistent infection in humans, for example, in cystic fibrosis patients.

N-(3-oxo-acyl)homoserine lactones signal cell activation through a mechanism distinct from the canonical pathogen-associated molecular pattern recognition receptor pathways.

Innate immune system receptors function as sensors of infection and trigger the immune responses through ligand-specific signaling pathways. These ligands are pathogen-associated products, such as components of bacterial walls and viral nuclear acids. A common response to such ligands is the activation of mitogen-activated protein kinase p38, whereas double-stranded viral RNA additionally induces the phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha). Here we have shown that p38 and eIF2alpha phosphorylation represent two biochemical markers of the effects induced by N-(3-oxo-acyl)homoserine lactones, the secreted products of a number of Gram-negative bacteria, including the human opportunistic pathogen Pseudomonas aeruginosa. Furthermore, N-(3-oxo-dodecanoyl)homoserine lactone induced distension of mitochondria and the endoplasmic reticulum as well as c-jun gene transcription. These effects occurred in a wide variety of cell types including alveolar macrophages and bronchial epithelial cells, requiring the structural integrity of the lactone ring motif and its natural stereochemistry. These findings suggest that N-(3-oxo-acyl)homoserine lactones might be recognized by receptors of the innate immune system. However, we provide evidence that N-(3-oxo-dodecanoyl)homoserine lactone-mediated signaling does not require the presence of the canonical innate immune system receptors, Toll-like receptors, or two members of the NLR/Nod/Caterpillar family, Nod1 and Nod2. These data offer a new understanding of the effects of N-(3-oxo-dodecanoyl)homoserine lactone on host cells and its role in persistent airway infections caused by P. aeruginosa.