Lung progenitors from lambs can differentiate into specialized alveolar or bronchiolar epithelial cells.

26International audienceBACKGROUND: Airways progenitors may be involved in embryogenesis and lung repair. The characterization of these important populations may enable development of new therapeutics to treat acute or chronic lung disease. In this study, we aimed to establish the presence of bronchioloalveolar progenitors in ovine lungs and to characterize their potential to differentiate into specialized cells. RESULTS: Lung cells were studied using immunohistochemistry on frozen sections of the lung. Immunocytochemistry and flow cytometry were conducted on ex-vivo derived pulmonary cells. The bronchioloalveolar progenitors were identified by their co-expression of CCSP, SP-C and CD34. A minor population of CD34pos/SP-Cpos/CCSPpos cells (0.33% +/- 0.31) was present ex vivo in cell suspensions from dissociated lungs. Using CD34 magnetic positive-cell sorting, undifferentiated SP-Cpos/CCSPpos cells were purified (>80%) and maintained in culture. Using synthetic media and various extracellular matrices, SP-Cpos/CCSPpos cells differentiated into either club cells (formerly named Clara cells) or alveolar epithelial type-II cells. Furthermore, these ex vivo and in vitro derived bronchioloalveolar progenitors expressed NANOG, OCT4 and BMI1, specifically described in progenitors or stem cells, and during lung development. CONCLUSIONS: We report for the first time in a large animal the existence of bronchioloalveolar progenitors with dual differentiation potential and the expression of specialized genes. These newly described cell population in sheep could be implicated in regeneration of the lung following lesions or in development of diseases such as cancers

Copy number variation and differential expression of a protective endogenous retrovirus in sheep.

The Jaagsiekte sheep retrovirus exJSRV and its endogenous counterpart enJSRV co-exist in sheep. exJSRV, a betaretrovirus, is the etiological agent of ovine pulmonary adenocarcinoma, and it has been demonstrated in vitro that an enJSRV Gag variant bearing the R-to-W amino acid change at position 21 was able to block exJSRV budding from the cells, providing a potential protective role for the host. In this work, we developed a fast mutation detection assay based on the oligo ligation assay (OLA) that permits the quantification of the relative proportions of the R21 and W21 Gag variants present in individual genomes and in cDNA obtained from normal and exJSRV-induced lung tumors. We have shown that the W21/R21 ratio is variable within and between breeds. We also describe for the first time that putative protecting enJSRV variants were expressed in alveolar type II cells (AECII), the major target of exJSRV

Interstitial lung disease associated with Equine Infectious Anemia Virus infection in horses

International audienceEIA (Equine Infectious Anemia) is a blood-borne disease primarily transmitted by haematophagous insects or needle punctures. Other routes of transmission have been poorly explored. We evaluated the potential of EIAV (Equine Infectious Anemia Virus) to induce pulmonary lesions in naturally infected equids. Lungs from 77 EIAV seropositive horses have been collected in Romania and France. Three types of lesions have been scored on paraffin-embedded lungs: lymphocyte infiltration, bronchiolar inflammation, and thickness of the alveolar septa. Expression of the p26 EIAV capsid (CA) protein has been evaluated by immunostaining. Compared to EIAV-negative horses, 52% of the EIAV-positive horses displayed a mild inflammation around the bronchioles, 22% had a moderate inflammation with inflammatory cells inside the wall and epithelial bronchiolar hyperplasia and 6.5% had a moderate to severe inflammation, with destruction of the bronchiolar epithelium and accumulation of smooth muscle cells within the pulmonary parenchyma. Changes in the thickness of the alveolar septa were also present. Expression of EIAV capsid has been evidenced in macrophages, endothelial as well as in alveolar and bronchiolar epithelial cells, as determined by their morphology and localization. To summarize, we found lesions of interstitial lung disease similar to that observed during other lentiviral infections such as FIV in cats, SRLV in sheep and goats or HIV in children. The presence of EIAV capsid in lung epithelial cells suggests that EIAV might be responsible for the broncho-interstitial damages observed

Early Steps of Jaagsiekte Sheep Retrovirus-Mediated Cell Transformation Involve the Interaction between Env and the RALBP1 Cellular Protein

International audienceOvine pulmonary adenocarcinoma is a naturally occurring lung cancer in sheep induced by the Jaagsiekte sheep retrovirus (JSRV). Its envelope glycoprotein (Env) carries oncogenic properties, and its expression is sufficient to induce in vitro cell transformation and in vivo lung adenocarcinoma. The identification of cellular partners of the JSRV envelope remains crucial for deciphering mechanisms leading to cell transformation. We initially identified RALBP1 (RalA binding protein 1; also known as RLIP76 or RIP), a cellular protein implicated in the ras pathway, as a partner of JSRV Env by yeast two-hybrid screening and confirmed formation of RALBP1/Env complexes in mammalian cells. Expression of the RALBP1 protein was repressed in tumoral lungs and in tumor-derived alveolar type II cells. Through its inhibition using specific small interfering RNA (siRNA), we showed that RALBP1 was involved in envelope-induced cell transformation and in modulation of the mTOR (mammalian target of rapamycin)/p70S6K pathway by the retroviral envelope. IMPORTANCE: JSRV-induced lung adenocarcinoma is of importance for the sheep industry. While the envelope has been reported as the oncogenic determinant of the virus, the cellular proteins directly interacting with Env are still not known. Our report on the formation of RALBP/Env complexes and the role of this interaction in cell transformation opens up a new hypothesis for the dysregulation observed upon virus infection in sheep

La Charente

24 octobre 18791879/10/24 (A8,N2092)-1879/10/24.Appartient à l’ensemble documentaire : PoitouCh

List of breeds analyzed. IL16 to 63 are inbred lines derived from the <i>Pre-Alpes du Sud</i> French breed.

<p>List of breeds analyzed. IL16 to 63 are inbred lines derived from the <i>Pre-Alpes du Sud</i> French breed.</p

A. Canonical structure of the enJSRV provirus.

<p>The W/R variant at position 21 is indicated by a star. The 470 bp amplicon is represented by a gray bar. B. Ligation products obtained from the OLA detection assay of W21 and R21 variants. The sequence of the phosphate-modified downstream oligo is indicated in italics. The upstream oligo that detected the R21 to W21 mutation is labeled with HEX, whereas the two FAM-labeled oligos detected the R21 variant. Probes that were mismatched to the target by a single nucleotide at their junctions were not ligated. The ligation products were discriminated by size (60 nt for W21 coding sequence vs. 64 nt for R21 coding sequences) and color (HEX for W21 vs. FAM for R21). The W and R codons are underlined.</p

Distribution of the W21/R21 ratios between the DNA and RNA extracts from non-tumoral (respectively n = 6 and n = 5) and tumoral samples (respectively n = 7 and n = 7).

<p>Two Wilcoxon tests were used to compare the medians of W21/R21 ratios measured in DNA and RNA extracts from non-tumoral (p-value = 0.41, non significant) and tumoral samples (p-value = 0.001, highly significant).</p

Distribution of W21 variants among 41 genomes from 10 breeds and 5 inbred lines.

<p>Individuals are numbered according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041965#pone-0041965-t001" target="_blank">Table 1</a>. The W21/R21 ratios are indicated as percentage.</p

Boxplot representation of triplicate W21/R21 ratio measurements among randomly selected 10 samples.

<p>For each sample, W21/R21 measurement was repeated three times. Each dot represents a single measurement. Numbers correspond to the samples listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041965#pone-0041965-g001" target="_blank">Figure 1</a>. NT1 and NT2 correspond to DNA extracted from two non-tumoral samples.</p