EURURO Vol. 71 No. 4

585 692

1. Introduction
Penile transplantation is becoming an option for penile
tissue loss . Two human penile transplantations have
been performed to date The first transplant, which
was reported in 2006, was surgically removed 14 d
following transplantation due to skin necrosis and psycho-
logical distress of the recipient and his wife . The second
penile transplant was performed in 2015. Although it has
not been reported in the scientific literature, press briefings
suggest that it is a successful transplant and that the
recipient has erectile function .
The goal of penile transplantation is not only cosmetic
reconstruction and urinary transport but attainment of
natural erectile function . How rejection episodes and
antirejection therapies will affect erection physiology is
unknown. Rejection affects organ microvasculature, and
erectile function is sensitive to microvascular disease
. Consequently, new-onset loss of function in the
setting of penile transplantation might be a harbinger of
rejection. Although similar immunosuppression treatments
are used for solid organs, extrapolation of erection
physiology results is difficult, given that organ failure
comorbidities are also significant risk factors for erectile
dysfunction (ED).
Current penile transplant models are inadequate to
assess erectile function; therefore, we used an ex vivo
mixed lymphocyte reaction (MLR) with human cavernous
tissue obtained during penile prosthesis implantation and
cocultured it with allogenic peripheral blood mononuclear
cells (PBMCs) to better understand how erectile tissue
physiology and penile tissue architecture are affected by
allograft rejection and immunosuppression regimens. MLR
is an established model used for >30 yr to study PBMC
activation in response to allogenic tissue Historically,
MLR is performed by mixing donor and recipient PBMCs. In
this study, we mixed ‘‘donor’’ penile tissues with ‘‘recipient’’
PBMCs to investigate how rejection affects cavernous tissue.
By performing tissue myography and molecular studies, we
showed that smooth muscle relaxation is impaired in the
setting of experimental penile transplant rejection and that
cyclosporin A (CsA), although effective at preventing
rejection, did not improve smooth muscle relaxation in
this model of transplantation. Moreover, compared with
control, smooth muscle function was not impaired by
FK506 treatment. These data provide important insight into
how penile tissues and erectile tissue physiology are
affected by rejection and immunosuppression treatment
and highlight the importance of understanding this process
to optimize clinical results in vascularized composite
allotransplantation.
2. Methods
2.1. Experimental design
All studies were approved by and conducted in accordance
with the Johns Hopkins Hospital institutional review board.
Following informed consent, cavernous tissue was collected
from 10 men (‘‘donors’’) with a median age of 59 yr (range
53–73 yr) undergoing penile prosthesis implantation for ED.
Indications for surgery included Peyronie’s disease (four
patients), postprostatectomy ED (three patients), and
vasculogenic ED (three patients). Peripheral blood was
collected from donors and from a healthy Blood Group B
volunteer (‘‘recipient’’) via venipuncture. PBMCs were
isolated using BD Vacutainer Cell Preparation Tubes
(Beckton, Dickinson and Company, Franklin Lakes, NJ,
USA), according to the manufacturer’s instructions, and
seeded at a concentration of 1.0 106 cells/ml. Cavernous
tissues were cultured in 24-well plates at 37 8C with 5% CO 2
in air in 2 mL of media composed of RPMI 1640 (Corning,
Corning, NY, USA), 10% fetal bovine serum (Corning), and 1%
antibiotic antimycotic (Thermo-Fisher Scientific, Waltham,
MA, USA), unless otherwise stated. Ex vivo MLRs were
prepared by culturing cavernous tissues for 48 h withmedia
(control), autologous (donor) PBMCs, allogenic (recipient)
PBMCs, and 1 m M CsA (Sigma-Aldrich, St. Louis, MO, USA)
. At 1 mM, CsA has been shown to prevent PBMC
activation in MLRs In addition to the media control,
cocultured PBMCs and cavernous tissue from the same
person represented an autologous transplant (eg, kidney
autotransplantation) or penile replantation (following trau-
matic injury) in that an immune rejection response is not
expected. Additional tissues for myography were cultured
for 24 h without PBMCs in media alone or with 1 mM CsA or
20 nM FK506 (LC Laboratories, Woburn, MA, USA)
2.2. Characterization of peripheral blood mononuclear cell
activation by flow cytometry
To establish PBMC activation and efficacy of CsA, flow
cytometry was used to measure PBMC proliferation
. Following PBMC isolation as described, donor PBMCs
were labeled with 5,6-carboxyfluorescein diacetate succi-
nimidyl ester, and recipient PBMCs were labeled with
CellTrace Violet (Thermo Fisher Scientific), according to the
manufacturer’s instructions. Ex vivo MLR was prepared as
described, and PBMCs were collected after 48 h of culture.
Cells were then labeled for viability using a membrane-
impermeable dye (Live/Dead Blue; Thermo Fisher Scientif-
ic). Cells were analyzed using an LSRII Flow Cytometer
System (Beckton Dickinson and Company), and prolifera-
tion rates and proliferative indices were calculated using
FlowJo Software (FlowJo LLC, Ashland, OR, USA).
2.3. Tissue fluorescent and histochemical staining
Live confocal laser microscopy was performed on cavernous
tissue following ex vivo MLR using a Zeiss AxioObserver
with Laser Scanning Microscope 700 Confocal Module (Carl
Zeiss Microscopy, Jena, Germany). Immediately following
collection, cavernous tissues were labeled with cell-
permeant red fluorescent marker carboxy-SNARF-1, acet-
oxymethyl ester, acetate (Life Technologies), according to
the manufacturer’s instructions . Tissues were then
labeled with fluorescent Image-iT LIVE Green Caspase-3
and -7 Detection Kit (Life Technologies), according to the
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