Comparative analysis of the oral mucosae from rodents and non-rodents: Application to the nonclinical evaluation of sublingual immunotherapy products

<div><p>Background</p><p>A comparative characterization of the oral mucosa in various animals is needed to identify the best animal model(s) for nonclinical evaluation of sublingual immunotherapy products. With this aim, we studied the histological characteristics and immune cell infiltrates of oral mucosae from common animal species.</p><p>Methods</p><p>Three oral regions (<i>i</i>.<i>e</i>. ventral surface of the tongue, mouth floor and cheek) obtained from eight animal species, including rodents (<i>i</i>.<i>e</i>. mice, rats, hamsters, guinea pigs) and non-rodents (<i>i</i>.<i>e</i>. rabbits, dogs, minipigs and monkeys) were characterized by histology and immunohistology in comparison with a human tongue.</p><p>Results</p><p>Rodents exhibit a thin keratinized epithelium with low epithelial extensions, whereas non-rodents, most particularly minipigs and monkeys, display a non-keratinized epithelium with larger rete ridges, similarly to humans. Glycogen-rich cells in the superficial epithelial layers are observed in samples from both minipigs, monkeys and humans. Comparable immune subpopulations detected in the 3 oral regions from rodent and non-rodent species include MHC-II⁺ antigen presenting cells, mostly CD163⁺ macrophages, located in the <i>lamina propria</i> (<i>LP)</i> and muscle tissue in the vicinity of resident CD3⁺CD4⁺ T cells. Limited numbers of mast cells are also detected in the <i>LP</i> and muscle tissue from all species.</p><p>Conclusion</p><p>The oral mucosae of minipigs and monkeys are closest to that of humans, and the immune networks are quite similar between all rodents and non-rodents. Taking into account the ethical and logistical difficulties of performing research in the latter species, rodents and especially mice, should preferentially be used for pharmacodynamics/efficacy studies. Our data also support the use of minipigs to perform biodistribution and safety studies of sublingual immunotherapy products.</p></div

Detection of glycogen in the oral mucosae of mice, minipigs, monkeys and humans.

<p>Representative photomicrographs of mucosal (<i>i</i>.<i>e</i>. ventral surface of the tongue, mouth floor or cheek as indicated in the figure) tissue sections (magnification x400) embedded in paraffin and stained with PAS and PAS diastase are shown.</p

Histology of the ventral surface of the tongue from rodents, non-rodent species and humans.

<p>Representative photomicrographs of mucosal (<i>i</i>.<i>e</i>. ventral surface of the tongue) tissue sections (magnification x400 for animal species and x200/x400 for human) embedded in paraffin and stained with Hematoxylin-Eosin (HE) are shown.</p

Collagen fibers density in the mucosa of the ventral surface of tongues from rodents, non-rodent species and humans.

<p>Representative photomicrographs of mucosal (<i>i</i>.<i>e</i>. ventral surface of the tongue) tissue sections (magnification x200) embedded in paraffin and stained with Masson trichrome are shown.</p

Mapping of immune cells in the ventral surface of tongues from rats, dogs, minipigs and monkeys.

<p>Mucosal tissues from animal species were processed for immunohistology analysis, as described in Methods. Slides were stained with the following specific antibodies: anti-MHC-II, anti-CD163, anti-CD172a, anti-CD3, and anti-CD4 to detect and quantify positive cells in tissue sections or with toluidine blue to quantify mast cells (magnification x200). Representative photomicrographs of mucosal tissue sections are shown.</p

Identification of Novel Short Ragweed Pollen Allergens Using Combined Transcriptomic and Immunoproteomic Approaches

<div><p>Background</p><p>Allergy to short ragweed (<i>Ambrosia artemisiifolia</i>) pollen is a serious and expanding health problem in North America and Europe. Whereas only 10 short ragweed pollen allergens are officially recorded, patterns of IgE reactivity observed in ragweed allergic patients suggest that other allergens contribute to allergenicity. The objective of the present study was to identify novel allergens following extensive characterization of the transcriptome and proteome of short ragweed pollen.</p><p>Methods</p><p>Following a Proteomics-Informed-by-Transcriptomics approach, a comprehensive transcriptomic data set was built up from RNA-seq analysis of short ragweed pollen. Mass spectrometry-based proteomic analyses and IgE reactivity profiling after high resolution 2D-gel electrophoresis were then combined to identify novel allergens.</p><p>Results</p><p>Short ragweed pollen transcripts were assembled after deep RNA sequencing and used to inform proteomic analyses, thus leading to the identification of 573 proteins in the short ragweed pollen. Patterns of IgE reactivity of individual sera from 22 allergic patients were assessed using an aqueous short ragweed pollen extract resolved over 2D-gels. Combined with information derived from the annotated pollen proteome, those analyses revealed the presence of multiple unreported IgE reactive proteins, including new Amb a 1 and Amb a 3 isoallergens as well as 7 novel candidate allergens reacting with IgEs from 20–70% of patients. The latter encompass members of the carbonic anhydrase, enolase, galactose oxidase, GDP dissociation inhibitor, pathogenesis related-17, polygalacturonase and UDP-glucose pyrophosphorylase families.</p><p>Conclusions</p><p>We extended the list of allergens identified in short ragweed pollen. These findings have implications for both diagnosis and allergen immunotherapy purposes.</p></div

IgE reactivity of recombinant Der f 36 and Der p 36.

<p>Recombinant non-glycosylated Der f 36 and Der p 36 were produced in <i>P</i>. <i>pastoris</i>. IgE reactivity was assessed by western blot using a pool of sera from HDM-sensitized individuals. Culture supernatant from a mock strain was used as a negative control.</p

2D-gel reference map of the short ragweed pollen proteome.

<p>Proteins from an aqueous short ragweed pollen extract were separated by 2D-gel electrophoresis and stained with Sypro Ruby. Proteins spots were picked and analyzed by LC-MS/MS after trypsin digestion. Proteins were identified using the Transcriptome-Derived Proteome collection supplemented with missing known allergens. Numbers refer to spots analyzed by mass spectrometry. Identification details are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136258#pone.0136258.t001" target="_blank">Table 1</a>.</p

Experimental workflow of HDM transcriptome, proteome and allergome analyses.

<p>Messenger RNAs from <i>D</i>. <i>farinae</i> and <i>D</i>. <i>pteronyssinus</i> were sequenced using next generation sequencing. Following <i>de novo</i> assembly, translated sequence databases were derived after coding sequences (CDS) prediction and used as references to identify proteins in aqueous extracts from mite bodies and feces by LC-MS/MS analysis. In parallel, extracts from whole cultures were submitted to 2D-gel electrophoresis to establish reference 2D maps of species-specific proteomes and assign IgE reactivity.</p

Proteome and IgE reactivity maps of <i>Dermatophagoides</i> species.

<p>Water-soluble HDM proteins were separated by 2D-gel electrophoresis and stained with Sypro Ruby or probed with a pool of serum IgEs from HDM-sensitized donors to establish 2D proteome (left panel) and IgE reactivity (right panel) maps for <i>D</i>. <i>farinae</i> (A, B) and <i>D</i>. <i>pteronyssinus</i> (C, D) species. Protein spots were picked and analyzed by LC-MS/MS after trypsin digestion, using the transcriptome-derived protein sequence collection supplemented with allergen sequences registered in the WHO/IUIS allergen database in order to facilitate identification. Spots corresponding to novel allergens (<i>i</i>. <i>e</i>. Der f 36 and Der p 36) are circled.</p