Human Skin Microbiota: High Diversity of DNA Viruses Identified on the Human Skin by High Throughput Sequencing

The human skin is a complex ecosystem that hosts a heterogeneous flora. Until recently, the diversity of the cutaneous microbiota was mainly investigated for bacteria through culture based assays subsequently confirmed by molecular techniques. There are now many evidences that viruses represent a significant part of the cutaneous flora as demonstrated by the asymptomatic carriage of beta and gamma-human papillomaviruses on the healthy skin. Furthermore, it has been recently suggested that some representatives of the Polyomavirus genus might share a similar feature. In the present study, the cutaneous virome of the surface of the normal-appearing skin from five healthy individuals and one patient with Merkel cell carcinoma was investigated through a high throughput metagenomic sequencing approach in an attempt to provide a thorough description of the cutaneous flora, with a particular focus on its viral component. The results emphasize the high diversity of the viral cutaneous flora with multiple polyomaviruses, papillomaviruses and circoviruses being detected on normal-appearing skin. Moreover, this approach resulted in the identification of new Papillomavirus and Circovirus genomes and confirmed a very low level of genetic diversity within human polyomavirus species. Although viruses are generally considered as pathogen agents, our findings support the existence of a complex viral flora present at the surface of healthy-appearing human skin in various individuals. The dynamics and anatomical variations of this skin virome and its variations according to pathological conditions remain to be further studied. The potential involvement of these viruses, alone or in combination, in skin proliferative disorders and oncogenesis is another crucial issue to be elucidated

No evidence for viral sequences in mycosis fungoides and Sézary syndrome skin lesions: a high-throughput sequencing approach

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Human polyomavirus related to African green monkey lymphotropic polyomavirus

International audienceWhile studying the virome of the skin surface of a patient with a Merkel cell carcinoma (MCC) by using unbiased, high-throughput sequencing, we identified a human polyomavirus nearly identical to human polyomavirus 9, a virus recently reported in blood and urine of renal transplantion patients and closely related to the African green monkey lymphotropic polyomavirus. Specific PCR analysis further identified this virus in 2/8 patients with MCC, but only in 1/111 controls without MCC. This virus was shed for >20 months by the MCC index patient and was on the skin of the spouse of the index patient. These results provide information on the viral ecology of human skin and raise new questions regarding the pathology of virus-associated skin disorders

Long-term safety and tolerability of bapineuzumab in patients with Alzheimer’s disease in two phase 3 extension studies

BACKGROUND: Immunotherapy with monoclonal antibodies that target amyloid beta has been under investigation as a treatment for patients with Alzheimer's disease (AD). The 3000 and 3001 phase 3 clinical studies of intravenous bapineuzumab assessed safety and efficacy in patients with mild to moderate AD recruited in over 26 countries. This article describes the long-term safety and tolerability of bapineuzumab in the extension studies for these two protocols. METHODS: The long-term safety and tolerability of intravenous-administered bapineuzumab in patients with AD was evaluated in apolipoprotein E ε4 allele noncarriers (Study 3002, extension of Study 3000) and apolipoprotein E ε4 allele carriers (Study 3003, extension of Study 3001). Those receiving bapineuzumab in the parent study were continued at the same dose; if receiving placebo, patients began bapineuzumab. Bapineuzumab doses were 0.5 mg/kg in both studies and also 1.0 mg/kg in the noncarrier study. Clinical efficacy of bapineuzumab was also assessed in exploratory analyses. RESULTS: Because of lack of efficacy in two other phase 3 trials, the parent protocols were stopped early. As a result, Studies 3002 and 3003 were also terminated. In total, 492 and 202 patients were enrolled in Studies 3003 and 3002, respectively. In apolipoprotein E ε4 carriers (Study 3003), treatment-emergent adverse events occurred in 70.7% of the patients who originally received placebo and 66.9% of those who originally received bapineuzumab. In noncarriers, treatment-emergent adverse events occurred in 82.1% and 67.6% of patients who received placebo + bapineuzumab 0.5 mg/kg and placebo + bapineuzumab 1.0 mg/kg, respectively, and in 72.7% and 64.3% of those who received bapineuzumab + bapineuzumab 0.5 mg/kg and 1.0 mg/kg, respectively. Amyloid-related imaging abnormalities with edema or effusions were the main bapineuzumab-associated adverse events in both studies, occurring in approximately 11% of placebo + bapineuzumab and 4% of bapineuzumab + bapineuzumab groups overall. Exploratory analyses of clinical efficacy were not significantly different between groups in either study. CONCLUSIONS: In these phase 3 extension studies, intravenous bapineuzumab administered for up to approximately 3 years showed no unexpected safety signals and a safety profile consistent with previous bapineuzumab trials. TRIAL REGISTRATION: Noncarriers (Study 3002): ClinicalTrials.gov NCT00996918 . Registered 14 October 2009. Carriers (Study 3003): ClinicalTrials.gov NCT00998764 . Registered 16 October 2009

Evaluation of high-throughput sequencing for identifying knwon and unknown viruses in biological samples

High-throughput sequencing furnishes a large number of short sequence reads from uncloned DNA and hasrapidly become a major tool for identifying viruses in biological samples, and in particular when the targetsequence is undefined. In this study, we assessed the analytical sensitivity of a pipeline for detection of virusesin biological samples based on either the Roche-454 genome sequencer or Illumina genome analyzer platforms.We sequenced biological samples artificially spiked with a wide range of viruses with genomes composed ofsingle or double-stranded DNA or RNA, including linear or circular single-stranded DNA. Viruses were addedat a very low concentration most often corresponding to 3 or 0.8 times the validated level of detection ofquantitative reverse transcriptase PCRs (RT-PCRs). For the viruses represented, or resembling those represented,in public nucleotide sequence databases, we show that the higher output of Illumina is associated witha much greater sensitivity, approaching that of optimized quantitative (RT-)PCRs. In this blind study,identification of viruses was achieved without incorrect identification. Nevertheless, at these low concentrations,the number of reads generated by the Illumina platform was too small to facilitate assembly of contigswithout the use of a reference sequence, thus precluding detection of unknown viruses. When the virus load wassufficiently high, de novo assembly permitted the generation of long contigs corresponding to nearly full-lengthgenomes and thus should facilitate the identification of novel viruses

T cell clonal expansions in ileal Crohn’s disease are associated with smoking behaviour and postoperative recurrence

T cell clonal expansions are present in the inflamed mucosa of patients with Crohn's disease (CD) and may be implicated in postoperative recurrence after ileocolonic resection.Methods T cell receptor (TCR) analysis was performed in 57 patients included in a prospective multicentre cohort. Endoscopic recurrence was defined by a Rutgeerts score >iO. DNA and mRNA were extracted from biopsies collected from the surgical specimen and endoscopy, and analysed by high throughput sequencing and microarray, respectively.Results TCR repertoire in the mucosa of patients with CD displayed diverse clonal expansions. Active smokers at time of surgery had a significantly increased proportion of clonal expansions as compared with non-smokers (25.9%vs17.9%, p=0.02). The percentage of high frequency clones in the surgical specimen was significantly higher in patients with recurrence and correlated with postoperative endoscopic recurrence (area under the curve (AUC) 0.69, 95% CI 0.54 to 0.83). All patients with clonality above 26.8% (18/57) had an endoscopic recurrence. These patients with a high clonality were more frequently smokers than patients with a low clonality (61% vs 23%, p=0.005). The persistence of a similar TCR repertoire at postoperative endoscopy was associated with smoking and disease recurrence. Patients with high clonality showed increased expression of genes associated with CD8 T cells and reduced expression of inflammation-related genes. Expanded clones were found predominantly in the CD8 T cell compartment.Conclusion Clonal T cell expansions are implicated in postoperative endoscopic recurrence. CD patients with increased proportion of clonal T cell expansions in the ileal mucosa represent a subgroup associated with smoking and where pathogenesis appears as T cell driven

Human papillomavirus sequences assembly and assignment in each sample.

<p># uc denotes unclassified HPVs.</p>*<p>Whole genome sequences confirmed through Sanger sequencing and submitted into Genbank.</p><p>New HPV sequences appear in bold and sequences labeled with * are those related to complete genome submitted into Genbank with the JF966371 to JF966379 respective accession numbers (correspondences are displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038499#pone-0038499-t004" target="_blank">table 4</a>).</p

Genomic organization of 9 new Human gamma-papillomavirus sequences.

<p>For HPV's genes E6 to L1, open reading frame positions and numbering are indicated with the size of the respective predicted protein in brackets.</p><p>- $ deduced from the second start codon likely predicted for in vivo use.</p><p>- * JF966371 and JF966375 are two strains from the same new HPV species.</p><p>- **JF966378 and JF966379 are two strains from the recently described HPV148 species.</p

MCPyV genome sequence coverage versus MCPyV viral load in the six samples.

<p>Genome coverage is expressed as number of reads per nucleotide and viral load is in genome copies per ng of whole DNA. Coefficient of correlation is: <i>R²</i> = 0.894; and best fit regression equation is: [MCPyV viral load]  = 4.1 [MCPyV coverage]- 28,850.</p