Release of pig leukocytes and reduced human NK cell recruitment during ex vivo perfusion of HLA‐E/human CD46 double‐transgenic pig limbs with human blood

Background

In pig‐to‐human xenotransplantation, interactions between human natural killer (NK) cells and porcine endothelial cells (pEC) are characterized by recruitment and cytotoxicity. Protection from xenogeneic NK cytotoxicity can be achieved in vitro by the expression of the non‐classical human leukocyte antigen‐E (HLA‐E) on pEC. Thus, the aim of this study was to analyze NK cell responses to vascularized xenografts using an ex vivo perfusion system of pig limbs with human blood.

Methods

Six pig forelimbs per group, respectively, stemming from either wild‐type (wt) or HLA‐E/hCD46 double‐transgenic (tg) animals, were perfused ex vivo with heparinized human blood for 12 hours. Blood samples were collected at defined time intervals, cell numbers counted, and peripheral blood mononuclear cells analyzed for phenotype by flow cytometry. Muscle biopsies were analyzed for NK cell infiltration. In vitro NK cytotoxicity assays were performed using pEC derived from wt and tg animals as target cells.

Results

Ex vivo, a strong reduction in circulating human CD45 leukocytes was observed after 60 minutes of xenoperfusion in both wt and tg limb groups. NK cell numbers dropped significantly. Within the first 10 minutes, the decrease in NK cells was more significant in the wt limb perfusions as compared to tg limbs. Immunohistology of biopsies taken after 12 hours showed less NK cell tissue infiltration in the tg limbs. In vitro, NK cytotoxicity against hCD46 single tg pEC and wt pEC was similar, while lysis of double tg HLA‐E/hCD46 pEC was significantly reduced. Finally, circulating cells of pig origin were observed during the ex vivo xenoperfusions. These cells expressed phenotypes mainly of monocytes, B and T lymphocytes, NK cells, as well as some activated endothelial cells.

Conclusions

Ex vivo perfusion of pig forelimbs using whole human blood represents a powerful tool to study humoral and early cell‐mediated rejection mechanisms of vascularized pig‐to‐human xenotransplantation, although there are several limitations of the model. Here, we show that (i) transgenic expression of HLA‐E/hCD46 in pig limbs provides partial protection from human NK cell‐mediated xeno responses and (ii) the emergence of a pig cell population during xenoperfusions with implications for the immunogenicity of xenografts.     

The periosteal-cutaneous chimeric medial femoral condyle free flap for subtotal ear reconstruction: A case report

When costal graft is contraindicated or refused by the patient, autologous total/subtotal auricular reconstruction represent a real challenge as limited surgical options has been described. Aim of present report is to offer a novel possible autologous reconstruction of the ear frame using a chimeric free medial femoral condyle (MFC) flap. We present a case of a 29 years old patient who had total loss of the upper 2/3 of the right ear after bombing in Somalia and secondary infected condritis (considered a relative contraindication for costal cartilage graft). The MFC flap was harvested with a chimeric skin paddle (7 × 5 cm), a thin sheet of femoral cortex (6.5 × 8 cm) was used as basal ear frame, while part of the contralateral concha was trimmed as support for the helix, with the periosteal component of the flap wrapping around the whole framework. The chimeric skin paddle assured the retroauricular skin coverage, while the anterior part of the construct was covered by a thinned dermal flap. Postoperative course was uneventful. A defatting procedure of the posterior skin paddle was performed at 2 months post-op. At 6 months post-op, the patient was satisfied with the result, could wear glasses and was socially integrated. This new application of the free chimeric MFC flap, despite being not the primary choice for ear reconstruction, guaranteed satisfactory results in terms of ear shape and infection prevention and may be considered when ordinary cartilage rib reconstruction is refused, contraindicated, or failed.     

Low Rate of Intrahospital Deep Venous Thrombosis in Acutely Ill Medical Patients: Results From the AURELIO Study

Objective

To evaluate the effect of hospitalization on deep venous thrombosis (DVT) rate by the cumulative incidence of DVT in the proximal venous tract of the lower limbs at admission and discharge.