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Purpose Chronic lung allograft dysfunction (CLAD) is the main obstacle to long-term survival after lung transplantation . Predictive biomarkers for CLAD are required for future studies and clinical interventions. Transbronchial biopsy (TBBx) tissue in patients with asymptomatic minimal (ISHLT grade A1) acute cellular rejection , which is not treated, affords an opportunity to study the content of rejection lesions as a determinant of subsequent clinical events. We examined the ratio of regulatory T cells (Tregs, which express Foxp3) to T effector cells (Teff) in TBBx as a predictor of outcome. We hypothesized that higher Treg content in A1 lesions would indicate a lower risk of subsequent acute rejection and CLAD. Methods We identified 10 patients who developed early CLAD ( formalin fixed paraffin embedded sections from each TBBx were stained via immunofluorescence for CD3 to detect T cells, and FoxP3 to detect Tregs. The proportion of Foxp3+ nuclei in 10 random clusters of CD3+ cells was determined using ImageJ software and analyzed with GraphPad Prism. Results Contrary to our hypothesis, we observed a higher content of FoxP3+ cells in those with early onset CLAD relative to those who remained CLAD-free for 5 years (Fig 1). Additionally, those with fewer than 3.12% FoxP3+ cells tended to have longer CLAD-free survival (Fig 2). Subsequent acute rejection did not differ between the groups. Conclusion A higher content of Foxp3+ cells in first asymptomatic A1 lesions may predict increased CLAD risk. Additionally, a similar rate of subsequent acute rejection episodes in the two groups suggests Treg infiltration has a greater influence on chronic rather than acute alloimmune responses. Our findings may reflect increased T cell activity in those with early CLAD relative to those who remained CLAD-free.

Background The prognosis for esophageal cancer is poor but may be improved by neoadjuvant therapy. A complete pathologic response (pCR) is associated with improved survival. We conducted a study to profile the expression of microRNAs (miRNAs) in esophageal cancer before and after induction therapy. Our aims were to identify those miRNAs that are differentially regulated after induction therapy and attempt to describe a miRNA pattern that could predict pCR. Methods Total RNA was extracted from pretreatment and posttreatment specimens from 25 patients who underwent trimodal therapy using concurrent irinotecan/cisplatin and radiotherapy followed by surgical treatment. miRNAs were labeled and hybridized to the Illumina miRNA BeadChip microarray (Illumina, Inc, San Diego, CA). Expression data was quantified using BeadStudio software (Illumina), using a cutoff for significant gene differences of p less than 0.05 with a 2-fold difference in expression. Survival analysis was performed using SPSS, version 18 (SPSS, Inc, Chicago, IL). Results Using pretreatment biopsy specimens, 71 miRNAs were significantly different between pCR and non-pCR groups. Of these, 5 miRNAs were greater than 2-fold differentially regulated, including miR-296, recently shown to be of prognostic significance in esophageal carcinoma. After induction therapy, 568 miRNAs were found to be significantly upregulated or downregulated, 111 of which had a 2-fold difference. Patients with high levels of miR-135b or miR-145 in the posttreatment biopsy specimens had significantly shorter median disease-free survival (DFS) than did those with low levels (11.5 versus 5.1 months; p = 0.04; 11.5 versus 2.8 months; p = 0.03). Conclusions miRNA expression profiling of pretreatment biopsy specimens revealed 5 miRNAs differentially expressed in patients with pCR compared with patients without pCR. We have also identified 111 miRNAs significantly upregulated or downregulated after induction therapy, some of which may be predictive of outcome. Further study of these miRNAs may elucidate a novel understanding of mechanisms of resistance to chemotherapy or radiotherapy.

Purpose Lymphoid neogenesis, also known as tertiary lymphoid tissues, has been demonstrated as an important role in development of chronic rejection represented by obliterative bronchiolitis (OB) in human and mouse studies. Spleen tyrosine kinase (Syk) is an immunoregulatory protein regarding proliferation and receptor signaling in B cells. The study purpose was to examine the role of Syk in alloimmune-related lymphoid neogenesis in OB. Methods and Materials Using a mouse intrapulmonary tracheal transplantation (IPTT) model, BALB/c tracheae were transplanted into the left lung of C57BL/6 mice or conditional Syk knockout mice on day 0, and then C57BL/6 mice were received the treatment with Syk inhibitor (30mg/kg) or vehicle control orally once a day. Mice were sacrificed on day 28 after IPTT. Results H&E staining revealed the lumen of transplanted trachea to be open and the lymphoid aggregates smaller in Syk knock-out mice and mice treated with Syk inhibitor compared to control. Quantification of the graft occlusion (mean±SEM) revealed significant attenuation in mice treated with Syk inhibitor compared to control (19.1±4.6 vs 63.2±7.0 vs 97.4±0.8(%), p Conclusions The treatment with Syk inhibitor and Syk deletion not only abrogated development of OB but also attenuated formation of lymphoid neogenesis through impairment of B cell maturation and proliferation. Inhibition of Syk is a novel potential strategy to control chronic rejection associated with lymphoid neogenesis after organ transplantation.

and 10.8-fold in average respectively in allograft-implanted lungs compared to isograft-implanted lungs at day 14 after IPTT. At day 28, control allografts showed near-complete obliteration while anti-CCL21 Ab treated allografts had significantly attenuated lumen obliteration (95.9 2.6% (mean SEM) vs. 73.6 3.9%, n 5, p 0.05). Immunohistochemically, T-cell and B-cell zones in lymphoid neogenesis were organized in control allografts, but disorganized in allografts treated by anti-CCL21 Ab. Disorganization of lymphoid neogenesis in the T cell zone was also observed in plt mice after allograft IPTT and graft obliteration was significantly attenuated by day 28 compared with control allografts (66.6 15.2%, n 3, p 0.05). Conclusions: Ab-mediated blockade or genetic deletion of CCL21 attenuates graft obliteration and induces disorganization of lymphoid neogenesis. CCL21 plays an important role in the formation of lymphoid-like stroma and contributes to lymphoid neogenesis, which is associated with chronic graft dysfunction after lung transplantation.