C. Gandia

Heterotopic sternum transplant in rats: A new model of a vascularized bone marrow transplantation.

We introduced the heterotopic vascularized sternum transplant as a more simple and pure alternative to allogeneic hind limb transplantation for the study of bone marrow transplantation. We report the clinical and histopathological manifestations after transplantation of syngeneic and allogeneic sternal grafts with and without immunossupression with FK‐506. Syngeneic grafts maintained normal histology, whereas allografts showed rejection, which was prevented by FK‐506. FK‐506‐treated allografts developed chimerism that was present throughout the observation period. Transplantation of the sternum may be a valuable model to study vascularized bone marrow transplantation and its effects on repopulation of bone marrow of the host, chimerism, and tolerance.

Utilization of dried blood spot citrulline level as a noninvasive method for monitoring graft function following intestinal transplantation

Background. Citrulline concentrations have been proposed as a marker for intestinal allograft rejection. We instituted dried blood spot (DBS) specimen monitoring of citrulline to simplify sample collection posttransplant. This study demonstrates the correlation between plasma and dried blood spot specimen citrulline concentrations after intestinal transplantation. Methods. Plasma and DBS samples were analyzed by hydrophilic interaction chromatography tandem mass spectrometry. Comparison of the strength of linear correlation was made according to the type of surgery, sonication time, DBS citrulline levels, and the time interval between the blood sample collection and the assay date. Results. A very strong linear correlation exists between the plasma and DBS citrulline concentrations (r2=0.87; P<0.001). The correlation between plasma and DBS citrulline concentrations was maintained when evaluating only the intestinal transplant recipients. There was no significant difference in the strength of linear correlation according to sonication time, cirtrulline concentrations, or length of time to assay date. Conclusions. DBS citrulline monitoring will ease sample collection following intestinal transplantation and improve the ability to detect intestinal dysfunction and rejection by a noninvasive means.

Host-derived enterocytes in intestinal grafts.

Replacement of donor lymphoid tissue by lymphocytes of recipient origin is an established phenomenon in small bowel transplants. However, replacement of donor epithelial cells of bowel grafts by host cells has not been demonstrated. The objective of our study was to determine whether donor enterocytes are replaced by host-derived enterocytes in the intestinal allograft. Graft biopsy specimens, obtained from five human male recipients of female intestine, were examined for the presence of male enterocytes. The biopsies dated from 90 to 770 days posttransplant. Formalin-fixed 3-microm specimen sections were stained for X and Y chromosomes by fluorescent in situ hybridization technique. Fluorescent microscopy of the stained sections identified male enterocytes in four patients, with a percentage of male cells ranging from 0.09% to 0.26% of the total enterocyte mass. Using the fluorescent in situ hybridization technique, we demonstrated the presence of host-derived male (XY) enterocytes in the female intestinal graft.

Correlation between allograft survival and chimeric state after bone marrow infusion in rat small bowel transplantation

Abstract: Methods to enhance natural microchimerism, which occurs after any successful organ transplant, are currently explored using unmodified donor bone marrow both in experimental and in clinical trials. Because of the potential immunomodulatory effects of donor bone marrow cells, we performed this study to evaluated the effect of single and multiple donor‐specific bone marrow infusions (DSBMI) on chimerism and small bowel allograft survival in a fully histoincompatible rat model. Forty‐five male DA rats and 45 female Lewis rats were used as donors and recipients, respectively, for a heterotopic small bowel transplant. Animals were separated into 10 groups according to the number of bone marrow infusions and immunosuppressive protocol used. Control groups (groups 1 and 2) did not receive any bone marrow infusion, groups 3 and 4 received one infusion at day 0 (150 × 106 cells), groups 5 and 6 received two infusions at days 0 and 4 (75 × 106 cells each), groups 7 and 8 received two infusions at days 4 and 10 (75 × 106 cells each), and groups 9 and 10 received five infusions at days 4, 10, 15, 20 and 25 (30 × 106 cells each). Animals in groups 1, 3, 5, 7 and 9 were immunosuppressed with 0.5 mg/kg FK506 while the remaining groups were immunosuppressed with 1 mg/kg FK506, from day 0 to 4 after transplant. Every 15 days, the chimeric state was determined by flow cytometry in order to detect cells expressing DA rat class I antigen, and small bowel biopsies were obtained from ileostomies. Animals in all groups showed minimal to moderate acute rejection at day 15 after transplant, however, vascular rejection (vasculitis, arteritis) was observed in only bone marrow groups (100% in 0.5 mg/kg and 42.1% in 1 mg/kg FK506 groups). On day 30, 58.3% of bone‐marrow‐infused animals and 66.6% of controls showed severe acute and early chronic rejection. The chimeric levels varied from 0 to 12% after transplant and were significantly higher in bone‐marrow‐infused groups compared with controls (p < 0.05). We conclude that modulation of immune response with short‐course immunosuppression and a single or multiple DSBMI did not improve allograft or recipient survival. The inability to achieve a stable chimeric state did not allow us to determine the effect of chimerism on graft and recipient survival after small bowel transplantation

Testing of a new prototype surgical stapler that automates the rollover sleeve technique for venous anastomoses.

The creation of successful vascular anastomoses is of primary importance in many surgical fields. Numerous attempts to automate this process have been made. These techniques have slowly gained acceptance, but their use is still limited. This report details feasibility testing of a new prototype stapler that automates the rollover sleeve technique for venous vascular anastomoses. Male and female mongrel dogs (n=7) (25-32 kg) were used. A segment of the right (n=5) or left (n=2) iliac vein was harvested for interposition grafts after the contra lateral side was transected. In each dog, two end-to-end venous anastomoses at the interposition grafts were performed. The standard anastomosis employed continuous mattress sutures. The experimental anastomosis was performed with a new prototype surgical stapler. The stapled anastomosis was proximal and the sutured was distal. In all experiments, it was possible to perform the experimental anastomosis with the stapler. Complications included two small leaks, one due to misfiring of a single pin in one experimental site. These leaks required suture reinforcement. One dog died of hemorrhage due to a slipped suture at the vein harvest site. One vein had thrombus seen at the sutured site although no technical abnormalities at either of the anastomoses could be found. After two weeks, grafts were inspected grossly and histologically. Healing appeared normal. There was a trend for less inflammatory cells infiltrating stapled sites; however, this was not statistically significant. The experiments demonstrate that this device can automate the rollover sleeve technique for venous anastomoses.