Choosing the optimal dose in sublingual immunotherapy: Rationale for the 300 index of reactivity dose

Sublingual immunotherapy (SLIT) is an effective and well-tolerated method of treating allergic respiratory diseases associated with seasonal and perennial allergens. In contrast to the subcutaneous route, SLIT requires a much greater amount of antigen to achieve a clinical effect. Many studies have shown that SLIT involves a dose-response relationship, and therefore it is important to use a proven clinically effective dose from the onset of treatment, because low doses are ineffective and very high doses may increase the risk of side effects. A well-defined standardization of allergen content is also crucial to ensure consistent quality, potency and appropriate immunomodulatory action of the SLIT product. Several methods of measuring antigenicity are used by manufacturers of SLIT products, including the index of reactivity (IR), standardized quality tablet unit, and bioequivalent allergy unit. A large body of evidence has established the 300 IR dose of SLIT as offering optimal efficacy and tolerability for allergic rhinitis due to grass and birch pollen and HDM, and HDM-induced moderate, persistent allergic asthma. The 300 IR dose also offers consistency of dosing across a variety of different allergens, and is associated with higher rates of adherence and patient satisfaction. Studies in patients with grass pollen allergies showed that the 300 IR dose has a rapid onset of action, is effective in both adults and children in the short term and, when administered pre-coseasonally in the long term, and maintains the clinical benefit, even after cessation of treatment. In patients with HDM-associated AR and/or asthma, the 300 IR dose also demonstrated significant improvements in symptoms and quality of life, and significantly decreased use of symptomatic medication. The 300 IR dose is well tolerated, with adverse events generally being of mild or moderate severity, declining in frequency and severity over time and in the subsequent courses. We discuss herein the most important factors that affect the selection of the optimal dose of SLIT with natural allergens, and review the rationale and evidence supporting the use of the 300 IR dose

Preliminary results of the project A.I.D.A. (Auto Immunity: Diagnosis Assisted by computer)

In this paper, are presented the preliminary results of the A.I.D.A. (Auto Immunity: Diagnosis Assisted by computer) project which is developed in the frame of the cross-border cooperation Italy-Tunisia. According to the main objectives of this project, a database of interpreted Indirect ImmunoFluorescence (IIF) images on HEp 2 cells is being collected thanks to the contribution of Italian and Tunisian experts involved in routine diagnosis of autoimmune diseases. Through exchanging images and double reporting; a Gold Standard database, containing around 1000 double reported IIF images with different patterns including negative tests, has been settled. This Gold Standard database has been used for optimization of a computing solution (CADComputer Aided Detection) and for assessment of its added value in order to be used along with an immunologist as a second reader in detection of auto antibodies for autoimmune disease diagnosis. From the preliminary results obtained, the CAD appeared more powerful than junior immunologists used as second readers and may significantly improve their efficacy

Computer-Assisted Classification Patterns in Autoimmune Diagnostics: The AIDA Project

Antinuclear antibodies (ANAs) are significant biomarkers in the diagnosis of autoimmune diseases in humans, done by mean of Indirect ImmunoFluorescence (IIF)method, and performed by analyzing patterns and fluorescence intensity. This paper introduces the AIDA Project (autoimmunity: diagnosis assisted by computer) developed in the framework of an Italy-Tunisia cross-border cooperation and its preliminary results. A database of interpreted IIF images is being collected through the exchange of images and double reporting and a Gold Standard database, containing around 1000 double reported images, has been settled. The Gold Standard database is used for optimization of aCAD(Computer AidedDetection) solution and for the assessment of its added value, in order to be applied along with an Immunologist as a second Reader in detection of autoantibodies. This CAD system is able to identify on IIF images the fluorescence intensity and the fluorescence pattern. Preliminary results show that CAD, used as second Reader, appeared to perform better than Junior Immunologists and hence may significantly improve their efficacy; compared with two Junior Immunologists, the CAD system showed higher Intensity Accuracy (85,5% versus 66,0% and 66,0%), higher Patterns Accuracy (79,3% versus 48,0% and 66,2%), and higher Mean Class Accuracy (79,4% versus 56,7% and 64.2%)

Challenges in Using Cultured Primary Rodent Hepatocytes or Cell Lines to Study Hepatic HDL Receptor SR-BI Regulation by Its Cytoplasmic Adaptor PDZK1

Background: PDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expression of SR-BI protein requires PDZK1 - <5% of normal hepatic SR-BI is seen in the livers of PDZK1 knockout mice. Progress has been made in identifying features of PDZK1 required to control hepatic SR-BI in vivo using hepatic expression of wild-type and mutant forms of PDZK1 in wild-type and PDZK1 KO transgenic mice. Such in vivo studies are time consuming and expensive, and cannot readily be used to explore many features of the underlying molecular and cellular mechanisms. Methodology/Principal Findings: Here we have explored the potential to use either primary rodent hepatocytes in culture using 2D collagen gels with newly developed optimized conditions or PDZK1/SR-BI co-transfected cultured cell lines (COS, HEK293) for such studies. SR-BI and PDZK1 protein and mRNA expression levels fell rapidly in primary hepatocyte cultures, indicating this system does not adequately mimic hepatocytes in vivo for analysis of the PDZK1 dependence of SR-BI. Although PDZK1 did alter SR-BI protein expression in the cell lines, its influence was independent of SR-BI’s C-terminus, and thus is not likely to occur via the same mechanism as that which occurs in hepatocytes in vivo. Conclusions/Significance: Caution must be exercised in using primary hepatocytes or cultured cell lines when studying the mechanism underlying the regulation of hepatic SR-BI by PDZK1. It may be possible to use SR-BI and PDZK1 expression as sensitive markers for the in vivo-like state of hepatocytes to further improve primary hepatocyte cell culture conditions.National Institutes of Health (U.S.) (Grant HL052212)National Institutes of Health (U.S.) (Grant HL066105)National Institutes of Health (U.S.) (Grant ES015241)National Institutes of Health (U.S.) (Grant GM068762

Distinct Regions of the Large Extracellular Domain of Tetraspanin CD9 Are Involved in the Control of Human Multinucleated Giant Cell Formation

Multinucleated giant cells, formed by the fusion of monocytes/macrophages, are features of chronic granulomatous inflammation associated with infections or the persistent presence of foreign material. The tetraspanins CD9 and CD81 regulate multinucleated giant cell formation: soluble recombinant proteins corresponding to the large extracellular domain (EC2) of human but not mouse CD9 can inhibit multinucleated giant cell formation, whereas human CD81 EC2 can antagonise this effect. Tetraspanin EC2 are all likely to have a conserved three helix sub-domain and a much less well-conserved or hypervariable sub-domain formed by short helices and interconnecting loops stabilised by two or more disulfide bridges. Using CD9/CD81 EC2 chimeras and point mutants we have mapped the specific regions of the CD9 EC2 involved in multinucleated giant cell formation. These were primarily located in two helices, one in each sub-domain. The cysteine residues involved in the formation of the disulfide bridges in CD9 EC2 were all essential for inhibitory activity but a conserved glycine residue in the tetraspanin-defining ‘CCG’ motif was not. A tyrosine residue in one of the active regions that is not conserved between human and mouse CD9 EC2, predicted to be solvent-exposed, was found to be only peripherally involved in this activity. We have defined two spatially-distinct sites on the CD9 EC2 that are required for inhibitory activity. Agents that target these sites could have therapeutic applications in diseases in which multinucleated giant cells play a pathogenic role

Investigation of Different Iontophoretic Currents Profiles for Short-Term Applications in Cosmetics

[EN] This study aimed at investigating the effect of electrical current profile upon the iontophoretic transport of (i) ascorbic acid (AA) and (ii) ellagic acid (EA), into porcine skin in vitro, and the impact of the physicochemical properties of both actives on their mechanism of transport when formulated in cosmetic compositions. The experiments were performed using a proprietary iontophoretic device containing a roller to apply the formulation. Three current profiles were tested: (i) galvanic direct current (DC), (ii) square unipolar pulse current (SPC), and (iii) galvanic direct current (DC) + pulse current (PC). The skin samples were collected at different sampling points, extracted and analyzed by HPLC. Results suggested that the DC + PC mode for only 5 min was able to significantly increase the delivery of AA from o/w cosmetic compositions. The use of this current profile might improve the skin penetration of AA due to electromigration and passive diffusion, the latter being facilitated by the physical enhancement method. The SPC mode significantly improved the passage of EA in its neutral form from cosmetic o/w formulations by electroosmosis. Tailoring specific electrical current modes considering the ionization state of active ingredients would allow the design of short and personalized cosmetic treatments that significantly improve the penetration efficiency of the active ingredients and possibly reduce the doses applied.This research was entirely funded by L'Oreal, France.Cázares-Delgadillo, J.; Planard-Luong, L.; Gregoire, S.; Serna-Jiménez, CE.; Singhal, M.; Kalia, YN.; Merino Sanjuán, V.... (2018). Investigation of Different Iontophoretic Currents Profiles for Short-Term Applications in Cosmetics. Pharmaceutics. 10(4). https://doi.org/10.3390/pharmaceutics10040266266104R. Hamad, A.-W., Al-Momani, W. M., Janakat, S., & A. Oran, S. (2009). Bioavailability of Ellagic Acid After Single Dose Administration Using HPLC. Pakistan Journal of Nutrition, 8(10), 1661-1664. doi:10.3923/pjn.2009.1661.1664Kalia, Y. N., Naik, A., Garrison, J., & Guy, R. H. (2004). Iontophoretic drug delivery. Advanced Drug Delivery Reviews, 56(5), 619-658. doi:10.1016/j.addr.2003.10.026Marro, D., Kalia, Y. N., Begoña Delgado‐Charro, M., & Guy, R. H. (2001). Pharmaceutical Research, 18(12), 1701-1708. doi:10.1023/a:1013318412527Sobhi, R. M., & Sobhi, A. M. (2012). A single-blinded comparative study between the use of glycolic acid 70% peel and the use of topical nanosome vitamin C iontophoresis in the treatment of melasma. Journal of Cosmetic Dermatology, 11(1), 65-71. doi:10.1111/j.1473-2165.2011.00599.xHori, Y., Akimoto, R., Hori, A., Kato, K., Chino, D., Matsumoto, S., … Watanabe, Y. (2010). Skin collagen reproduction increased by ascorbic acid derivative iontophoresis by frequent-reversal bipolar electric stimulation. International Journal of Cosmetic Science, 32(3), 234-234. doi:10.1111/j.1468-2494.2010.00577_3.xJunyaprasert, V. B., Singhsa, P., Suksiriworapong, J., & Chantasart, D. (2012). Physicochemical properties and skin permeation of Span 60/Tween 60 niosomes of ellagic acid. International Journal of Pharmaceutics, 423(2), 303-311. doi:10.1016/j.ijpharm.2011.11.032Maia, A. M., Baby, A. R., Pinto, C. A. S. O., Yasaka, W. J., Suenaga, E., Kaneko, T. M., & Velasco, M. V. R. (2006). Influence of sodium metabisulfite and glutathione on the stability of vitamin C in O/W emulsion and extemporaneous aqueous gel. International Journal of Pharmaceutics, 322(1-2), 130-135. doi:10.1016/j.ijpharm.2006.05.03

Hepatitis C Virus Infection May Lead to Slower Emergence of P. falciparum in Blood

International audienceBACKGROUND: Areas endemic for Plasmodium falciparum, hepatitis B virus (HBV) and hepatitis C virus (HCV) overlap in many parts of sub-Saharan Africa. HBV and HCV infections develop in the liver, where takes place the first development stage of P. falciparum before its further spread in blood. The complex mechanisms involved in the development of hepatitis may potentially influence the development of the liver stage of malaria parasites. Understanding the molecular mechanisms of these interactions could provide new pathophysiological insights for treatment strategies in Malaria. METHODOLOGY: We studied a cohort of 319 individuals living in a village where the three infections are prevalent. The patients were initially given a curative antimalarial treatment and were then monitored for the emergence of asexual P. falciparum forms in blood, fortnightly for one year, by microscopy and polymerase chain reaction. PRINCIPAL FINDINGS: At inclusion, 65 (20.4%) subjects had detectable malaria parasites in blood, 36 (11.3%) were HBV chronic carriers, and 61 (18.9%) were HCV chronic carriers. During follow-up, asexual P. falciparum forms were detected in the blood of 203 patients. The median time to P. falciparum emergence in blood was respectively 140 and 120 days in HBV- and HBV+ individuals, and 135 and 224 days in HCV- and HCV+ individuals. HCV carriage was associated with delayed emergence of asexual P. falciparum forms in blood relative to patients without HCV infection. CONCLUSIONS: This pilot study represents first tentative evidence of a potential epidemiological interaction between HBV, HCV and P. falciparum infections. Age is an important confounding factor in this setting however multivariate analysis points to an interaction between P. falciparum and HCV at the hepatic level with a slower emergence of P. falciparum in HCV chronic carriers. More in depth analysis are necessary to unravel the basis of hepatic interactions between these two pathogens, which could help in identifying new therapeutic approaches against malaria

Suppression of mRNAs Encoding Tegument Tetraspanins from Schistosoma mansoni Results in Impaired Tegument Turnover

Schistosomes express a family of integral membrane proteins, called tetraspanins (TSPs), in the outer surface membranes of the tegument. Two of these tetraspanins, Sm-TSP-1 and Sm-TSP-2, confer protection as vaccines in mice, and individuals who are naturally resistant to S. mansoni infection mount a strong IgG response to Sm-TSP-2. To determine their functions in the tegument of S. mansoni we used RNA interference to silence expression of Sm-tsp-1 and Sm-tsp-2 mRNAs. Soaking of parasites in Sm-tsp dsRNAs resulted in 61% (p = 0.009) and 74% (p = 0.009) reductions in Sm-tsp-1 and Sm-tsp-2 transcription levels, respectively, in adult worms, and 67%–75% (p = 0.011) and 69%–89% (p = 0.004) reductions in Sm-tsp-1 and Sm-tsp-2 transcription levels, respectively, in schistosomula compared to worms treated with irrelevant control (luciferase) dsRNA. Ultrastructural morphology of adult worms treated in vitro with Sm-tsp-2 dsRNA displayed a distinctly vacuolated and thinner tegument compared with controls. Schistosomula exposed in vitro to Sm-tsp-2 dsRNA had a significantly thinner and more vacuolated tegument, and morphology consistent with a failure of tegumentary invaginations to close. Injection of mice with schistosomula that had been electroporated with Sm-tsp-1 and Sm-tsp-2 dsRNAs resulted in 61% (p = 0.005) and 83% (p = 0.002) reductions in the numbers of parasites recovered from the mesenteries four weeks later when compared to dsRNA-treated controls. These results imply that tetraspanins play important structural roles impacting tegument development, maturation or stability

Hepatocyte Permissiveness to Plasmodium Infection Is Conveyed by a Short and Structurally Conserved Region of the CD81 Large Extracellular Domain

Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a malaria infection, and thus represents an attractive target for anti-malarial interventions. Still, the molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that the tetraspanin CD81, a known receptor for the hepatitis C virus (HCV), is required on hepatocytes for infection by sporozoites of several Plasmodium species. Here we have characterized CD81 molecular determinants required for infection of hepatocytic cells by P. yoelii sporozoites. Using CD9/CD81 chimeras, we have identified in CD81 a 21 amino acid stretch located in a domain structurally conserved in the large extracellular loop of tetraspanins, which is sufficient in an otherwise CD9 background to confer susceptibility to P. yoelii infection. By site-directed mutagenesis, we have demonstrated the key role of a solvent-exposed region around residue D137 within this domain. A mAb that requires this region for optimal binding did not block infection, in contrast to other CD81 mAbs. This study has uncovered a new functionally important region of CD81, independent of HCV E2 envelope protein binding domain, and further suggests that CD81 may not interact directly with a parasite ligand during Plasmodium infection, but instead may regulate the function of a yet unknown partner protein

Temperature Shift and Host Cell Contact Up-Regulate Sporozoite Expression of Plasmodium falciparum Genes Involved in Hepatocyte Infection

Plasmodium sporozoites are deposited in the skin by Anopheles mosquitoes. They then find their way to the liver, where they specifically invade hepatocytes in which they develop to yield merozoites infective to red blood cells. Relatively little is known of the molecular interactions during these initial obligatory phases of the infection. Recent data suggested that many of the inoculated sporozoites invade hepatocytes an hour or more after the infective bite. We hypothesised that this pre-invasive period in the mammalian host prepares sporozoites for successful hepatocyte infection. Therefore, the genes whose expression becomes modified prior to hepatocyte invasion would be those likely to code for proteins implicated in the subsequent events of invasion and development. We have used P. falciparum sporozoites and their natural host cells, primary human hepatocytes, in in vitro co-culture system as a model for the pre-invasive period. We first established that under co-culture conditions, sporozoites maintain infectivity for an hour or more, in contrast to a drastic loss in infectivity when hepatocytes were not included. Thus, a differential transcriptome of salivary gland sporozoites versus sporozoites co-cultured with hepatocytes was established using a pan-genomic P. falciparum microarray. The expression of 532 genes was found to have been up-regulated following co-culture. A fifth of these genes had no orthologues in the genomes of Plasmodium species used in rodent models of malaria. Quantitative RT-PCR analysis of a selection of 21 genes confirmed the reliability of the microarray data. Time-course analysis further indicated two patterns of up-regulation following sporozoite co-culture, one transient and the other sustained, suggesting roles in hepatocyte invasion and liver stage development, respectively. This was supported by functional studies of four hitherto uncharacterized proteins of which two were shown to be sporozoite surface proteins involved in hepatocyte invasion, while the other two were predominantly expressed during hepatic parasite development. The genome-wide up-regulation of expression observed supports the hypothesis that the shift from the mosquito to the mammalian host contributes to activate quiescent salivary gland sporozoites into a state of readiness for the hepatic stages. Functional studies on four of the up-regulated genes validated our approach as one means to determine the repertoire of proteins implicated during the early events of the Plasmodium infection, and in this case that of P. falciparum, the species responsible for the severest forms of malaria