Adherence issues related to sublingual immunotherapy as perceived by allergists

Objectives: Sublingual immunotherapy (SLIT) is a viable alternative to subcutaneous immunotherapy to treat allergic rhinitis and asthma, and is widely used in clinical practice in many European countries. The clinical efficacy of SLIT has been established in a number of clinical trials and meta-analyses. However, because SLIT is self-administered by patients without medical supervision, the degree of patient adherence with treatment is still a concern. The objective of this study was to evaluate the perception by allergists of issues related to SLIT adherence. Methods: We performed a questionnaire-based survey of 296 Italian allergists, based on the adherence issues known from previous studies. The perception of importance of each item was assessed by a VAS scale ranging from 0 to 10. Results: Patient perception of clinical efficacy was considered the most important factor (ranked 1 by 54% of allergists), followed by the possibility of reimbursement (ranked 1 by 34%), and by the absence of side effects (ranked 1 by 21%). Patient education, regular follow-up, and ease of use of SLIT were ranked first by less than 20% of allergists. Conclusion: These findings indicate that clinical efficacy, cost, and side effects are perceived as the major issues influencing patient adherence to SLIT, and that further improvement of adherence is likely to be achieved by improving the patient information provided by prescribers. © 2010 Scurati et al, publisher and licensee Dove Medical Press Ltd

Simultaneous in vitro characterisation of DNA deaminase function and associated DNA repair pathways.

During immunoglobulin (Ig) diversification, activation-induced deaminase (AID) initiates somatic hypermutation and class switch recombination by catalysing the conversion of cytosine to uracil. The synergy between AID and DNA repair pathways is fundamental for the introduction of mutations, however the molecular and biochemical mechanisms underlying this process are not fully elucidated. We describe a novel method to efficiently decipher the composition and activity of DNA repair pathways that are activated by AID-induced lesions. The in vitro resolution (IVR) assay combines AID based deamination and DNA repair activities from a cellular milieu in a single assay, thus avoiding synthetically created DNA-lesions or genetic-based readouts. Recombinant GAL4-AID fusion protein is targeted to a plasmid containing GAL4 binding sites, allowing for controlled cytosine deamination within a substrate plasmid. Subsequently, the Xenopus laevis egg extract provides a source of DNA repair proteins and functional repair pathways. Our results demonstrated that DNA repair pathways which are in vitro activated by AID-induced lesions are reminiscent of those found during AID-induced in vivo Ig diversification. The comparative ease of manipulation of this in vitro systems provides a new approach to dissect the complex DNA repair pathways acting on defined physiologically lesions, can be adapted to use with other DNA damaging proteins (e.g. APOBECs), and provide a means to develop and characterise pharmacological agents to inhibit these potentially oncogenic processes

Hypersensitivity reactions due to black henna tattoos and their components: are the clinical pictures related to the immune pathomechanism?

Hypersensitivity to para-phenylenediamine (PPD) and related compounds induced by temporary black henna tattoos has become a serious health problem worldwide. Different patterns of sensitization with various clinical aspects are described in literature due to PPD associated to henna tattoo and these manifestations are likely correlated with the immunological and dermatological pathomechanisms involved. Henna is the Persian name of the plant Lawsonia inermis, Fam. Lythraceae. It is a woody shrub that grow in regions of North Africa, South Asia, India and Sri Lanka. Nowadays it is rather frequent to see temporary "tattoos" performed with henna. To make tattoos darker and long-lasting PPD has been associated to henna in tattoo drawings mixtures, so obtaining "black henna". In these years there has been a rise of contact sensitization to PPD and in medical literature an increased number of cases have been reported on temporary henna tattoo application. Here we review the various clinical patterns related to PPD and henna tattoo, to investigate the possible link between clinic-morphological pictures and the immunological response to PPD and henna. The literature underlines that different clinical manifestations are related to black henna containing PPD, and its derivative products may cause delayed-type as well as immediate-type reactions. Further studies are needed to investigate the relationship between clinical and morphological aspects of PPD contact dermatitis and the T cell subsets predominance

Hypersensitivity reactions due to black henna tattoos and their components: are the clinical pictures related to the immune pathomechanism?

Abstract Hypersensitivity to para-phenylenediamine (PPD) and related compounds induced by temporary black henna tattoos has become a serious health problem worldwide. Different patterns of sensitization with various clinical aspects are described in literature due to PPD associated to henna tattoo and these manifestations are likely correlated with the immunological and dermatological pathomechanisms involved. Henna is the Persian name of the plant Lawsonia inermis, Fam. Lythraceae. It is a woody shrub that grow in regions of North Africa, South Asia, India and Sri Lanka. Nowadays it is rather frequent to see temporary “tattoos” performed with henna. To make tattoos darker and long-lasting PPD has been associated to henna in tattoo drawings mixtures, so obtaining “black henna”. In these years there has been a rise of contact sensitization to PPD and in medical literature an increased number of cases have been reported on temporary henna tattoo application. Here we review the various clinical patterns related to PPD and henna tattoo, to investigate the possible link between clinic-morphological pictures and the immunological response to PPD and henna. The literature underlines that different clinical manifestations are related to black henna containing PPD, and its derivative products may cause delayed-type as well as immediate-type reactions. Further studies are needed to investigate the relationship between clinical and morphological aspects of PPD contact dermatitis and the T cell subsets predominance

AID-induced damage repair in the absence of replicative polymerases.

<p>(<b>A</b>) Replicative DNA polymerases were inhibited with aphidicolin (aph) during Xenopus laevis egg extracts (FE) incubation. Supercoiled DNA (scDNA) was added to FE (with bio-dC) in the presence or absence of aph, and biotinylated DNA isolated and quantitated by qPCR. The bars represent fold change as the difference between the qPCR C_t value of each sample normalised to the treated (+ aph) sample, which was set to 1. Error bars indicate ± SD (n = 3). Time line of experiment shown above the graph indicates the order of addition of substrates/proteins/nucleotides/extract/etc. or treatments. (<b>B</b>) AID-induced lesions are repaired in Xenopus laevis egg extracts. scDNA plasmids were treated (or not - bar 1) with the indicated proteins and then incubated in FE (or not - bar 2), isolated, and quantified by qPCR. The blue bars represent the ratio (fold change) of the amount of recovered plasmids from reactions carried out in the presence of G-AID (bar 3), untagged AID (AID; bar 4), GAL4 DNA binding domain (G-DBD; bar 5), or mutant GAL4-AID C87R (G-AIDmt; bar 6) versus levels of plasmids recovered from reactions that did not contain G-AID (FE alone, set to 1; bar 1). Open pink bars represent the absolute recovery of each treated sample in relation to its input. Error bars indicate ± SD (n = 3). Statistical analysis (t-test) was performed on differences of indicated fold change (brackets), with p values shown. Time line of the experiment is shown above the graph.</p

Correlation between lesion size, biotin incorporation, and plasmid recovery.

<p>(<b>A</b>) Incorporation of 1 or 2 biotinylated dCTP molecules is sufficient to recover the targeted plasmid by streptavidin purification. Schematic of the experiment is depicted at the top of the figure. Plasmids were untreated (scDNA) or nicked at one position with Nt.BsmAI (nicked DNA), followed by treatment with Klenow for 30 min at 25°C in the presence or absence of dTTP (dT) and varying ratios of biotinylated (bio-dC) and normal dCTP (dC). As indicated in the schematic, the lack of dGTP and dATP only allowed for the incorporation of 1 or 2 bio-dC molecules per plasmid. The bars (fold change) represent the difference between the qPCR C_t value for each sample before and after the streptavidin purification, normalised to the sample (scDNA or nicked DNA) treated with the Klenow and bio-dC (no dT and set to 1). Error bars indicate ± SD (n = 3). Time line of the experiment is shown above the graph. (<b>B</b>) Patch length of incorporated bio-dC does not bias the recovery of plasmids from IVR. The amount of bio-dC incorporation depending on the presence of other dNTPs was monitored by an AID-induced IVR assay. The fold change represents the difference between the qPCR C_t value of each sample normalised to the FE-treated sample (no G-AID) that was set to 1.</p

AID-induced lesions repair in vitro by the IVR system.

<p>Schematic description of the IVR assay. A supercoiled DNA plasmid (pGL4.31) containing 5 x GAL4 binding sites (UAS) is incubated with a recombinant fusion GAL4-AID protein (G-AID, represented by a yellow triangle and a red circle). Incubation at 37°C deaminates dC to create dU lesions in single stranded DNA (green star). The repair phase (yellow box) - relaxation and lesion repair - is carried out by the addition of frog egg extract (FE) in the presence of biotinylated dCTP (bio-dC) or biotinylated dATP (bio-dA) - (blue arrow), along with normal dNTPs. After DNA repair of the dU lesion the biotinylated-tagged DNA is isolated via magnetic streptavidin beads. Eluted products are subject to quantitative real-time PCR (red bar), and compared to input values of the same reaction prior to streptavidin isolation.</p

Quality control of AID used for the IVR assay.

<p>(<b>A</b>) G-AID deaminates cytosine to uracil during in vitro oligonucleotide deamination assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082097#pone.0082097-Coker1" target="_blank">[9]</a>. ssDNA oligonucleotide deamination assay was performed using an oligonucleotide SPM163 containing a single cytosine. Two concentrations (0.05 µg and 0.5 µg) of G-AID and G-AIDmt, as well as untagged AID (0.05 µg) and BSA (1 µg) were incubated with oligonucleotide for 30 min at 37°C, followed treatment with UNG and NaOH, and separated on a 17.5% PAGE gel. (<b>B</b>) Time course of G-AID activity during an IVR. G-AID was incubated with substrate for 5 to 90 min (37°C) before addition of the FE (30 min 23°C). Analysis was done as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082097#pone-0082097-g002" target="_blank">Figure 2</a>. Error bars indicate ± SD (n = 3). (<b>C</b>) G-AID does not induce topological changes on the supercoiled plasmid over time. Samples were processed as in (B), but prior to FE addition they were treated with SDS and proteinase K for 2 h at 56°C and analysed for changes in DNA topology as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082097#pone-0082097-g003" target="_blank">Figure 3A</a>. (<b>D & E</b>) G-AID and G-AIDmt were incubated with scDNA (as in C) and analysed for topological changes (D) or subjected to an IVR reaction (E). Quantitations of the topological forms of the substrate are shown in green in (E), while IVR results are shown in blue as % of input recovery. IVR analysis was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082097#pone-0082097-g002" target="_blank">Figure 2</a>. Error bars indicate ± SD (n = 3).</p

Multiple DNA repair pathways resolve AID-induced lesions.

<p>(<b>A</b>) Inhibiting the UNG activity of FE with UGI. Top - Increasing amounts of UGI were added to the FE during oligonucleotide based deglycosylation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082097#pone.0082097-Rangam1" target="_blank">[13]</a>. After NaOH treatment the fragments were separated using PAGE. Bottom - Quantification of deglycosylation activity of UNG2. Activities of lane 2 and 10 were set to 100% (no inhibition) and 0% (maximum inhibition), respectively. (<b>B</b>) UNG2 activity in FE is near saturation. 5 or 10 units of recombinant UNG2 were added to the FE reaction during the IVR reaction. Analysis was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082097#pone-0082097-g002" target="_blank">Figure 2</a> with untreated (no G-AID - not shown) set to 1. Error bars indicate ± SD (n = 3). (<b>C</b>) DNA repair pathway inhibitors alter IVR of AID lesions. AID-induced damaged plasmids were subject to FE IVR in the presence of bio-dC (bars 1–4) or bio-dA (bars 5–8). BER inhibitor UGI was added to the FE either alone (bars 2 & 6) or in combination with PCNA inhibitor T2AA (bars 4 & 8), while T2AA was also added alone (bars 3 & 7). Analysis was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082097#pone-0082097-g002" target="_blank">Figure 2</a> with untreated FE (bars 1 & 5) set to 100% (blue bar). For absolute comparisons, analysis was also shown as % of input (pink bars). Error bars indicate ± SD (n = 3). Statistical analysis (t-test) was performed on differences of indicated fold change (brackets), with p values shown.</p