Early subretinal allograft rejection is characterized by innate immune activity

Successful subretinal transplantation is limited by considerable early graft loss, despite pharmacological suppression of adaptive immunity. We postulated that early innate immune activity is a dominant factor in determining graft survival and chose a non-immunosuppressed mouse model of retinal pigment epithelial (RPE) cell transplantation to explore this. Expression of almost all measured cytokines by DH01 RPE cells increased significantly following graft preparation and the neutrophil chemoattractant, KC/GRO/CINC, was most significantly increased. Subretinal allografts of DH01 cells (C57BL/10 origin) into healthy, non-immunosuppressed C57BL/6 murine eyes were harvested and fixed at 1, 3, 7 and 28 days post-operatively and subsequently cryosectioned and stained. Graft cells were detected using SV40 large T antigen (SV40T) immunolabeling and apoptosis/necrosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Sections were also immunolabeled for macrophage (CD11b & F4/80), neutrophil (Gr1 Ly-6G), and T-lymphocyte (CD3-ε) infiltration. Images captured with an Olympus FV1000 confocal microscope were analyzed using Imaris software. The proportion of the subretinal bolus comprising graft cells (SV40T+) was significantly (p<0.001) reduced between post-operative day (POD) 3 (90% ± 4%) and POD 7 (20% ± 7%). CD11b+, F4/80+ and Gr1 Ly-6G+ cells increased significantly (p<0.05) from POD 1 and predominated over SV40T+ cells by POD 7. Co-labeling confocal microscopic analysis demonstrated graft engulfment by neutrophils and macrophages at POD 7 and reconstruction of z-stacked confocal images confirmed SV40T inside Gr1 Ly-6G+ cells. Expression of CD3-ε was low and did not differ significantly between time-points. By POD 28, no graft cells were detectable and few inflammatory cells remained. These studies reveal for the first time a critical role for innate immune mechanisms early in subretinal graft rejection. The future success of subretinal transplantation will require more emphasis on techniques to limit innate immune-mediated graft loss, rather than focusing exclusively on suppression of the adaptive immune response

Obliteration of the fistulous tract with BioGlue® adversely affects the outcome of transanal advancement flap repair

Background: Transanal advancement flap repair (TAFR) is useful in the treatment of high transsphincteric fistulas. Initially, promising results were reported. More recent studies have indicated that TAFR fails in one out of three patients. In almost all of our patients with a failure, we have observed healing of the flap except at the site of the original internal opening. A possible explanation for this remarkable finding might be persistent inflammation in the fistulous tract, finding a way out through the original internal opening. The question is whether obliteration of the fistulous tract by local installation at a surgical adhesive, can prevent persistent inflammation to break through the original opening. The aim of this pilot study was to investigate whether concomitant instillation of BioGlue could improve the healing rate following TAFR for high transsphincteric fistulas. Methods: Between March 2006 and April 2006 a consecutive series of eight patients (four men, four women; median age 46 years) with a high transsphincteric fistula underwent TAFR after instillation of BioGlue in the fistulous tract. All patients were seen in the outpatient department for postoperative evaluation. Results: Fistula healing was observed in only one patient (12.5%). All other patients experienced one or more of the following complications: prolonged severe pain (n=5), discharge of great amounts of purulent liquid from the external opening (n=3) and abscess formation (n=2), necessitating incision and drainage. Because of this unexpected outcome we decided to terminate the study prematurely. Conclusions: Our findings indicate that obliteration of the fistulous tract with BioGlue adversely affects the outcome of TAFR for high transsphincteric fistulas