RNA interference uses small RNAs (sRNA), which target genes for sequence-specific silencing. The parasite Entamoeba histolytica contains an abundant repertoire of 27 nt antisense (AS) sRNA with 5′-polyphosphate termini, but their roles in regulating gene expression have not been well established. We demonstrate that a gene-coding region to which large numbers of AS sRNAs map can serve as a ‘trigger’ and silence the gene fused to it. Silencing is mediated by generation of AS sRNAs with 5′-polyphosphate termini that have sequence specificity to the fused gene. The mechanism of silencing is independent of the placement of the trigger relative to the silenced gene but is dependent on the sRNA concentration to the trigger. Silencing requires transcription of the trigger-gene fusion and is maintained despite loss of the trigger plasmid. We used this approach to silence multiple amebic genes, including an E. histolytica Myb gene, which is upregulated during oxidative stress response. Silencing of the EhMyb gene decreased parasite viability under oxidative stress conditions. Thus, we have developed a new tool for genetic manipulation in E. histolytica with many advantages over currently available technologies. Additionally, these data shed mechanistic insights into a eukaryotic RNA interference pathway with many novel aspects.

Telomere length (TL) decreases with cellular ageing and biological stressors. As advanced donor and recipient ages are risk factors for chronic lung allograft dysfunction (CLAD), we hypothesised that decreased age-adjusted donor TL would predict earlier onset of CLAD. Shorter donor TL was associated with increased risk of CLAD or death (HR 1.26 per 1 kb TL decrease, 95% CI 1.03 to 1.54), particularly for young donors. Recipient TL was associated with cytopenias but not CLAD. Shorter TL was also seen in airway epithelium for subjects progressing to CLAD (p=0.02). Allograft TL may contribute to CLAD pathogenesis and facilitate risk stratification.

Background Acute cellular rejection is a major cause of morbidity following lung transplantation. Because regulatory T cells (Treg) limit rejection of solid organs, we hypothesized that donor-reactive Treg increase after transplantation with development of partial tolerance and decrease relative to conventional CD4+ (Tconv) and CD8+ T cells during acute cellular rejection. Methods To test these hypotheses, we prospectively collected 177 peripheral blood mononuclear cell (PBMC) specimens from 39 lung transplant recipients at the time of transplantation and during bronchoscopic assessments for acute cellular rejection. We quantified the proportion of Treg, CD4+ Tconv, and CD8+ T cells proliferating in response to donor-derived, stimulated B cells. We used generalized estimating equation-adjusted regression to compare donor-reactive T cell frequencies with acute cellular rejection pathology. Results An average of 16.5±9.0% of pretransplantation PBMC Treg were donor-reactive, compared with 3.8%±2.9% of CD4+ Tconv and 3.4±2.6% of CD8+ T cells. These values were largely unchanged following transplantation. Donor-reactive CD4+ Tconv and CD8+ T cell frequencies both increased 1.5-fold (95% CI 1.3-1.6, P <0.001 and 95% CI 1.2-1.6, P = 0.007, respectively) during A2-grade rejection compared with no rejection. Surprisingly, donor-reactive Treg frequencies increased by 1.7-fold (95% CI 1.4-1.8, P <0.001). Conclusions Contrary to prediction, overall proportions of donor-reactive Treg are similar before and after transplantation and increase during A2-grade rejection. This suggests how A2 rejection can be self-limiting. The observed increases over high baseline proportions in donor-reactive Treg were insufficient to prevent acute lung allograft rejection.