Ghép Da: Chỉ Định, Ứng Dụng Và Các Nghiên Cứu Hiện Tại (2011)

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Full thickness skin grafts are composed of epidermis and the full thickness of dermis,
including adnexial structures such as hair follicles and sweat glands. Full thickness skin
grafts are most commonly used to repair defects in face and fingers as an alternative to local
flaps. FTSGs can provide excellent color, texture and thickness matches for facial defects,
and may be especially useful for the repair of defects of the nasal tip, dorsum, ala, and side
wall as well as the lower eyelid and ear [1-3].
Selection of a donor site for a FTSG depends on the color, texture, thickness and sebaceous
qualities of the skin surrounding the defect [1, 3]. Most FTSGs are taken above the
shoulders, whose color, vascular pattern, texture, thickness and density and distribution of
adnexial structures best match the tissue surrounding facial defects [1].
The most frequently used grafts, especially for the dermocosmetic purposes, are thick or
intermediate split thickness grafts for the repair of areas greater than 3 cm diameter and full
thickness grafts for the repair of smaller areas [1].
3. Preputial skin grafts
In last decade, an extraordinary type of FTSG; PSG has been used as an alternative graft
source and has promising results. The use of PSG is not a new idea. Its usage is well
described in hypospadias surgery till now [5]. In last two decades choice of PSG as a graft
source has been reported for many clinical conditions such as burn, release of contractures,
syndactly repair, eyelid and anal canal reconstruction, intraoral burn reconstruction, closure
of defects after nevus excision and penile skin defect repair [6-12]. Most of these reports are
single case reports. Its usage in burned patients is described in literature in relatively larger
series of patients [13-15]. But PSG is still not used as a routine full thickness graft side
among surgeons [13, 14].
Modern treatment of deep partial-thickness and full-thickness burns are operative
debridement with subsequent skin graft coverage. Currently, nearly 95 % success rate is the
standard of care for skin grafting. For this success, adequate wound bed preparation, careful
selection of donor sites, and appropriate perioperative care are critical. From these factors,
careful selection of donor sites is important as donor site problems may occur.
In patients with extensive burns, all unburned areas can be used as donor sites. However, in
small burns, selection of donor site can be problematic. Since all donor sites scar to some
degree, it is advised to take skin from an area that will be otherwise hidden under most
circumstances [16].
Preputial skin graft is a full thickness and expandable graft that can be easily harvested and
used [13]. The prepuce is very thin, pliable skin. Its major advantages are its relatively large
size and, as a full thickness graft, its very low tendency to contract [14]. Also, like the split
thickness graft, it has high viability. A thinning procedure, which is required for the harvest
of other donor sites, is not required. Additional advantages of PSG are; they do not need
donor site care, do not causes scar, donor site is hidden and can be harvested with simple
surgical instruments. In small burns, it has the advantage of not to disrupt any healthy skin
area for graft harvesting [13, 14] (Figure 1).
In one series of the patients of which all were burned children, PSG has been used liberally.
In this report all of the patients were burned children with an average age of 3,6. Preputial
skin graft was the only donor site for 25% of patients and average size of PSG was 6 x 4 cm.

Preputial Skin Grafts

143



Fig. 1. Circumcision and preparation of skin graft

Skin Grafts – Indications, Applications and Current Research

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In 2 patients, PSG was used for periareolar grafting where hyperpigmented healing was
preferred (Figure 2). General anesthesia was required in 91% of patients because of need for
additional donor sites. Overall graft survival was 100% without any complication of donor
site [13] (Table-1).In another series of patients, PSG has been successfully used for burn
contracture release, acute burn treatment and for defects in scalp and defects from acute
trauma. Mean defect size was 3.5x5.5 cm in this study in which PSG was enough to cover
these defects in 90% of the patients. All the recipient area was opened on postoperative day
5. Graft survival was 100 % and no complication on donor site [14] (Table-1).


Fig. 2. Periareolar grafting with preputial skin


Number
of
Patients
Mean
age of
Patients
PSG was
the only
donor Site
(%)
Average
Defect
Size
Mean
PSG
Size
Graft
Survival
(%)
Indication
for PSG use
Donor
site
morbidity
(%)
Dogrul
et al.
12
3.6
25
N/A
6x4 cm
100
Burn
0
Yıldrım
et al.
11
7.9
90,9
3.5x5.5
cm
N/A
100
Burn,
contracture
release, scalp
defect and
defects from
trauma
0
Table 1. Two series of patients in which PSG was liberally used.

Preputial Skin Grafts

145
Recently in a study performed by Mcheik and et al, keratinocytes isolated from preputial
skin after double enzymatic digestion. They cultured keratinocytes and obtained an average
of 8.8 million cells per foreskin. And they concluded that keratinocyte resulting from
foreskin have a high capacity of division. These cells can divide a long time before
differentiation and enabled them to propose with their paitents the keratinocytes from
foreskin for wound healing especially for burns in children [17].
Only limitation of PSG is its healing with hyperpigmentation which limits its usage in face
and neck region which are the most common recipient side of FTSGs [13, 14]. But it can be
used in extremity and scalp defects [14]. But this color mismatch may be an advantage if it is
used in special areas like periareolar area [13]. Other limitation of PSG may be potential
complications of circumcision.
Circumcision is the surgical procedure for harvesting PSG. As with any surgical procedure,
bleeding and infection are probably the most common complications of circumcision [18].
Other complications include hematoma formation, diffuse swelling and pain from
inadequate anesthesia and tearing of the sutures due to erection before healing is complete
[18]. Urethral injury and penile necrosis are exceptional but reported complications [18].
These complications decrease with experience and can be as low as 0.034 % where
circumcision is routinely performed [14] .
In some countries, circumcision is the most common surgical procedure in boys because of
religious and cultural reasons. It is routinely performed to all male children with a very low
complication rates [13]. There are also some absolute medical indications for circumcision,
such as phimosis secondary to balanitis xerotica, obliterans and recurrent balanophosthitis
[19, 20]. Relative indications of circumcision are paraphimosis, phimosis, preputial pearls,
reduntant foreskin, hypospadias surgery [19]. Besides, it may have some medical benefits
such as improved hygiene, reduced risks of urinary and sexually transmitted infections, and
of penile and cervical cancer [20]. However, opponents deny or minimize these benefits and
put forward complications of circumcision and loss of penile sensation [20]. But we think
that; in burn patients, benefits of PSG outweigh from potential complications of
circumcision.
Although PSG is still not in routine use in the era of reconstructive surgery, reported
series have promising results [13, 14]. Especially in burned children with small sized
defects, PSG may be the only graft that adequately used for closure of defects with
almost nil donor site complication and 100 % graft survival rate. Only limitation for its
usage is hyperpigmentation. Every surgeon must keep PSG in mind as an alternative
donor site.
4. References
[1] Ratner, D., Skin grafting. Semin Cutan Med Surg, 2003. 22(4): p. 295-305.
[2] Andreassi, A., et al., Classification and pathophysiology of skin grafts. Clin Dermatol, 2005.
23(4): p. 332-7.
[3] Ratner, D., Skin grafting. From here to there. Dermatol Clin, 1998. 16(1): p. 75-90.
[4] Petruzzelli, G.J. and J.T. Johnson, Skin grafts. Otolaryngol Clin North Am, 1994. 27(1): p.
25-37.

Skin Grafts – Indications, Applications and Current Research

146
[5] Baran, C.N., et al., What is new in the treatment of hypospadias? Plast Reconstr Surg, 2004.
114(3): p. 743-52.
[6] Fontenot, C., J. Ortenberg, and D. Faust, Hypospadiac or intact foreskin graft for syndactyly
repair. J Pediatr Surg, 1999. 34(12): p. 1826-8.
[7] Emory, R.E. and C.H. Chester, Prepuce pollicization: a reminder of an alternate donor. Plast
Reconstr Surg, 2000. 105(6): p. 2100-1.
[8] D'Alessio, A., et al., Use of preputial skin as cutaneous graft after nevus excision. Int J Pediatr.
2010: p. 951270.
[9] Radopoulos, D., I. Vakalopoulos, and P. Thanos, Preputial graft in penile curvature
correction: preliminary results. Int J Impot Res, 2009. 21(1): p. 82-7.
[10] Silfen, R., D.A. Hudson, and P.J. Skoll, The use of the prepuce for reconstruction of an
intraoral burn. Ann Plast Surg, 2000. 44(3): p. 317-9.
[11] Oates, S.D. and A.K. Gosain, Syndactyly repair performed simultaneously with circumcision:
use of foreskin as a skin-graft donor site. J Pediatr Surg, 1997. 32(10): p. 1482-4.
[12] Mak, A.S., A.M. Poon, and M.K. Tung, Use of preputial skin for the release of burn
contractures in children. Burns, 1995. 21(4): p. 301-2.
[13] Dogrul, A.B., et al., Preputial skin can be used in all boys with burns requiring grafting. Ulus
Travma Acil Cerrahi Derg, 2009. 15(1): p. 58-61.
[14] Yildirim, S., et al., The preputium: an overlooked skin graft donor site. Ann Plast Surg, 2001.
46(6): p. 630-4.
[15] Chlihi, A., et al., [The use of preputial skin as cutaneous graft after circumcision. Report of 30
clinical cases]. Ann Chir Plast Esthet, 2002. 47(3): p. 214-8.
[16] Helmbach DM, F.L., ed. Principles of Burn Surgery. Principles and Practice of Burn
Surgery, ed. H.D. Barrett-Nerrin JP. 2005, Mercel Dekker: New York. 135 - 62.
[17] McHeik, J.N., et al., [Cultured keratinocyte cells from foreskin and future application for
burns in children]. Ann Chir Plast Esthet, 2009. 54(6): p. 528-32.
[18] Akoz, T., et al., Unusual complications of circumcision. Plast Reconstr Surg, 1998. 101(7):
p. 1915-8.
[19] Malone, P. and H. Steinbrecher, Medical aspects of male circumcision. BMJ, 2007.
335(7631): p. 1206-90.
[20] Holman, J.R. and K.A. Stuessi, Adult circumcision. Am Fam Physician, 1999. 59(6): p.
1514-8.

12
Treatment of Adult-Acquired Buried Penis
W. Britt Zimmerman1,2 and Richard A. Santucci1,2
1Michigan State University – College
of Osteopathic Medicine
2Detroit Receiving Hospital
Detroit Medical Center
United States of America
1. Introduction
The first know description of buried penis was by Keyes in 1919. Buried penis was
described as the “absence of the penis exists when the penis, lacking its proper sheath of
skin, lies buried beneath the integument of the abdomen, thigh or scrotum (Keyes, 1919).”
Classically, the entity of buried penis has been primarily discussed in the pediatric
population (Boemers & De Jong, 1995, Crawford, 1977, Donatucci & Ritter, 1998, Lim et al.,
1995, Maizels et al., 1986, McCahill et al., 1995, Wollin et al., 1990). However, a buried penis
afflicts many adults leaving them with the inability to have sexual relations, practice
personal hygiene, or stand to void since they are unable to direct their penis during
micturition (Cromie et al., 1998). Unlike the pediatric patient population, adults with buried
penis have acquired this condition as a result of obesity, over exuberant circumcision,
scarring due to lichen sclerosis et atrophicus (previously as known as balanitis xerotica
obliterans) or lymphedema (Alici et al., 1998, Chopra et al., 2002, Donatucci & Ritter, 1998,
Sivakumar et al., 2004, Tang et al., 2008). This chapter discusses the pathophysiology of a
buried penis, patient selection for operative management, and surgical management with
associated complications.
2. History of buried penis management
Since the initial description by Keyes and colleagues, various names have been associated
with “buried penis” which describes the pathology in both the pediatric and adult
population. These names range from buried penis (Keyes, 1919), concealed penis (Wollin et
al., 1990) webbed penis (Crawford, 1977), and inconspicuous penis (Maizels et al., 1986). In
1951, Campbell and colleagues described the buried penis as being underneath the
perineum, hypogastrium, thigh, or subcutaneous fat of scrotum (Campbell, 1951). Usually,
buried penis and concealed penis are used interchangeably (Glanz, 1968).
Two authors have offered classification systems in identifying buried penis. In 1977,
Crawford proposed three categories: concealed penis, buried penis (partial or complete) and
penoscrotal webs (Crawford, 1977). Later in 1986, Maizels proposed a classification system
in four categories: buried penis, webbed penis, trapped penis, and micropenis (Maizels et
al., 1986) (Table 1).

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Buried Penis Classification

Crawford’s Classification
Maizel’s Classification
1
Concealed penis
Buried penis
2
Buried penis (partial or complete)
Webbed penis
3
Penoscrotal webs
Trapped penis
4

Micropenis
Table 1. Buried penis classification systems
3. Etiology
The underlying pathological process of a buried penis includes an abnormal hypermobility
of the ventral skin on dartos fascia and inadequate attachments to the underlying Buck’s
fascia. The corpora remains stationery and the surrounding tissues (skin and dartos) migrate
distally (Alter & Ehrlich, 1999, Casale et al., 1999, Frenkl et al., 2004). Hence, the overlying
penile skin acts as a redundant drape, or “tent pole” over which skin is draped. The process
may advance because the corporal bodies remain fixed to the pubic bone by the suspensory
ligament and because of this fixation, the obese patient’s abdomen descends and envelopes
the penis. As the penis becomes covered or “buried,” excessive moisture begins to develop
which then promotes bacterial overgrowth, tissue maceration, and infection. As chronic
infections continue and a cycle is established, there is a perpetuation of skin breakdown
followed by more inflammation and scarification. In addition, the patient is unable to direct
their penis for voiding resulting in urine soilage. Urine is trapped and the cycle continues.
Most patients are obese and have associated comorbidities such as diabetes mellitus, which
may worsen the patient’s prognosis (Donatucci & Ritter, 1998).
4. Patient presentation / selection
Many patients present to their primary care physician or Urologist with in the inability to
participate in sexual activity secondary to painful erections or the inability to achieve
adequate penile length (Chopra et al., 2002, Cromie et al., 1998) (Figure 1). Additionally,
patients may also suffer from social embarrassment, recurrent balanitis, and psychological
stress (Chopra et al., 2002, Frenkl et al., 2004).
When selecting patients for surgical correction, many indications exist. Frankl et al (2004)
describes; in the pediatric population indications for surgery which include recurrent
balanitis, secondary phimosis, difficulty holding the penis during voiding, urinary stream
spraying, social embarrassment, and parent’s concern for future social embarrassment
(Frenkl et al, 2004). These are widely accepted (Adham et al., 2000, Alici et al, 1998, Chuang
et al., 2001, Donatucci & Ritter, 1998, Frenkl et al., 2004). However, in dealing with the adult
population, the primary indications are related to sexuality. Other indications include
complete or partial penile concealment with the inability to stand to void, chronic urinary

Treatment of Adult-Acquired Buried Penis

149
soilage, dysuria, inability to participate in penetrative intercourse, and erectile pain
(Donatucci & Ritter, 1998, Tang et al., 2008).

.


A)



B)


C)



D)
Fig. 1. Buried penis etiologies vary: widely A: Large abdominal pannus, escutcheon and
normal size scrotum. B: Large abdominal pannus and severe scrotal lymphedema. C: Buried
penis following abdominoplasty, note normal abdomen and scrotum. D: Buried penis as a
result of scrotal lymphedema.
The ideal candidates for operative correction of a buried penis are patients who are
motivated and prepared for postoperative wound care. Wound complications are reported
post-operatively, and these can be significant (Anaya & Dellinger, 2006, Rogliani et al.,
2006). Additionally, the patient must be medically and cardiovascular cleared for general
anesthesia.

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5. Patient preparation
Patient education is paramount during pre-operative management. Many of these patients
suffer from depression and some have even considered suicide in the past (Tang et al., 2008).
Hence, the patient should be made aware of the extensive postoperative course that will
ensue. Obesity and their abdominal girth often have a negative impact on wound healing
and may limit their dexterity and ability to care for the groin wounds (Anaya & Dellinger,
2006, Rogliani et al., 2006).
6. Surgical technique
Major reconstructive urologic surgery is required to treat adults with a buried penis. Many
techniques have been described. The objectives are all primarily the same: restore the
normal anatomy from the underlying pathological process.
Surgical approaches vary and should correlate to the etiology of the buried penis. Some
patients present with a normal sized scrotum and abdomen without a pannus (Figure 2). In
corollary, many patients suffer from a lack of penile skin as in an over exuberant
circumcision. In simple situations like this, the penile skin is released and approximated to
the base of the penile shaft, and the defect is covered by split thickness skin grafts. In more
complicated scenarios, as in a patient with scrotal lymphedema or an abnormally large
abdominal pannus, a more aggressive reconstructive surgical approach is mandated.


Fig. 2. Buried penis with normal abdomen and scrotal skin and poor penile shaft skin
Various surgical techniques for buried penis have been reported. Maizels et al. (1986)
performed a lipectomy of the suprapubic fat pad and suspensory ligament incision. This
was accompanied by pubic bone anchoring of the penopubic tissue (Maizels et al, 1986).
Similarly, Alter and colleagues (1999) advocated securing the subdermis of the penoscrotal
junction to the tunica albugenia ventrally to stabilize the penile and scrotal skin and prevent
migration of the penis within the scrotum. Additionally, these authors advocate securing the
subdermal penopubic junction to the rectus abdominal fascia (Alter & Ehrlich, 1999).

Treatment of Adult-Acquired Buried Penis

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Wollin et al. (1990) described a complete degloving of penis with lysis of improper ventral
dartos attachments. This is followed by an island pedicle flap of preputial skin and
transposed to the ventral aspect of the penis (Wollin et al., 1990).
Donatucci et al. (1998) described a treatment algorithm ranging from release of scar
contracture and primary closure. If insufficient release of the phallus occurs, then procession to
panniculectomy is warranted. Depending on the adequacy of skin or soft tissue in for closure,
the next step would be initially to use primary skin closure versus Z-plasty. If native skin is not
available and/or of poor quality, then split thickness skin grafts or flaps may be necessary.
Skin flaps should only be used when an inadequate graft bed exists (Donatucci & Ritter, 1998).
Chopra et al. (2002) described a technique using a large elliptical panniculectomy with scar
excision and penile skin removal. Division of the suspensory ligament of the penis, release
of the cicatrix and reattachment of the suspensory ligament of the penis should be
undertaken if necessary. Finally, a standard abdominoplasty and full thickness skin grafting
to the penis is performed. Skin grafting should be performed in a spiral fashion to diminish
any linear scar contraction (Chopra et al., 2002).
Many authors have also addressed the obese patient’s overlying suprapubic fat pad by
performing dermatolipectomy or suction lipectomy with abdominoplasty (Adham et al.,
2000, Alter & Ehrlich, 1999, Horton et al., 1987).
Six principles for success have been summarized in the buried penis patient population by
Chopra et al. (2002) (Table 2). First, release of the underlying cicatrix or scar which has
anchored the penile tissue preventing it from being mobilized, should occur. Secondly, the
majority of the pannus should be removed in the obese patient. Third, with the reduction of
the abdominal fat pad, there is a need to reestablish the normal male escutcheon
(suprapubic fat pad). The next step, in patients with a normal scrotum, is to reapproximate
the median raphe of the scrotum to its’ normal anatomic position at the base of the penis.
Then, skin resurfacing must be carried out depending on the condition of the penile shaft.
This can be achieved with either skin grafts or flaps. However, in the adult patient, non-hair
bearing coverage is preferred. Finally, an appropriate dressing needs to be applied as the
fragility of the skin graft must be respected (Chopra et al., 2002).

Six Principles for Successful Buried Penis Management
1
Release/excision of cicatrix
2
Removal of as much pannus as possible
3
Reestablishment of normal male escutcheon
4
Reattachment of median raphe to the base of the penis
5
Resurfacing the denuded penis with a full-thickness skin graft, sutured in spiral
fashion to avoid linear contracted scarring
6
Circumferential generic foam dressings applied snugly around the graft for
immobilization and splinting
Table 2. Six principle for successful surgical management of buried penis by Chopra et al.

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152
A different surgical approach is described by Tang et al. (2008). This technique in the obese
patient includes escutcheonectomy, scrotoplasty, and penile split-thickness grafting with
fibrin glue fixation (Tang et al., 2008). This begins with a circumcision incision two to three
millimeters proximal to the coronal sulcus. This allows for complete exposure of the glans
penis and penile shaft. The penoscrotal webs and aberrant scrotal tissues are released if
necessary. If a scrotoplasty is performed, the wound is closed in a two layers and no drain is
placed. The escutcheonectomy is made using an elliptical incision and juxtaposition to the
base of the penis. We trim any redundant suprapubic fat and secure it as a flap to the
superficial suspensory ligaments of the penis using 2-O interrupted polyglactin sutures
(Figure 3). We make every effort to close all potential spaces to avoid seroma formation.
Closed suction drains are used under the escutcheon flap wherever necessary.




A)




B)
Fig. 3. A: An elliptical incision is made on pannus. B: dermatolipectomy is performed.

C.

Fig. 3. C: The male escutcheon is reestablished.

Treatment of Adult-Acquired Buried Penis

153
In our experience, the remaining penile shaft skin is either non-existent or of poor quality.
Hence, we use full thickness skin grafts from the discarded escutcheon or split skin grafts
(STSG) from a predetermined location, typically anterior left thigh. The STSG is harvested at
a depth of 0.012 to 0.015 inches, using an electric dermatome. The proximal aspect of the
skin graft is tacked down using multiple simple interrupted absorbable sutures. To assist in
graft success, we use a diluted fibrin sealant (freeze-dried Tissel VH, Baxter, Deerfield, IL) as
per manufacturer specifications. We spray the sealant to the entire surface of the penile shaft
using the Tissomat fibrin glue spray device (Baxter). By using the dilute fibrin glue
concentration (about 10% of the normal calcium concentration of Tissel), we lengthen the
time to hardening from seconds to minutes which allows more time for the final graft
placement (Figure 4).




A)



B)
Fig. 4. A: Excision of the pannus and skin grafting. B: Pannus excision, scrotoplasty and skin
grafting.

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154
The skin graft harvest site is covered with a transparent adhesive dressing (Opsite, Smith &
Nephew, London UK) and a small closed suction drain (TLS, Porex Surgical, Newman, GA).
This type dressing removes excessive drainage and facilitates the dressing staying longer.
The grafted penis is dressed in a 6 inch elastic antimicrobial gauze (Kerlix, Tyco/Kendall,
Manfield, MA). Local anesthesia is used with 0.5% bupivacaine containing epinephrine
1:200,000 to lessen any postoperative pain. The skin graft dressing is removed after 24 hours
and any seromas (blebs) are incised as necessary.
7. Postoperative care
Immediate postoperative care begins in the hospital. Our patients are admitted to the
hospital and are on bed rest for the first three days. On postoperative day four, the patient
returns to normal activity and is educated on wound care. Discharge typically takes place on
postoperative day five. The patient is continued with oral antibiotics such as
amoxicillin/clavulanate for two weeks. The patient is instructed to shower and dress the
wounds daily. We recommend 4-inch gauze sponges (eg, Johnson & Johnson “Topper”
dressing sponges, or other nonadherent absorbent pads). We recommend against “open
weave” gauze sponges as these have a tendency to debride and can result in surgical site
breakdown.
8. Complications
In the postoperative period, complications can occur. These can range from surgical site
infections to the development of a pulmonary embolism (Tang et al., 2008). In patients
suffering from obesity and metabolic syndromes such as diabetes, an increased risk for
Surgical Site Infection exists (Anaya & Dellinger, 2006, Rogliani et al., 2006). Patients may
also experience erectile dysfunction (ED) postoperatively. The erectile dysfunction may be
due to an underlying disease process that initially propagated the buried penis. Hence, ED
may only be recognized after the penis is once again revealed. It is also common for the
patient to experience decreased sensation over the skin graft site (Donatucci & Ritter, 1998).
This will typically improve over time as nerve re-growth occurs.
The most common defense against the aforementioned complications beyond preoperative
medical optimization and stringent perioperative care is education. The buried penis patient
has often been described as being at the “end of their rope (Tang et al., 2008).” Hence,
informed consent and patient expectations need to be framed appropriately.
9. Conclusion
Acquired adult buried penis management is a challenging reconstructive surgery. However,
with the proper use of escutcheonectomy, reduction of the suprapubic fat pad, +/-
scrotoplasty, and with use of full or split thickness skin grafting, excellent results are
possible. With diligent post-operative wound care to manage minor infections and wound
dehiscence, the patient can return to sexual activity, have profound improvement in urinary
function, and improved self-esteem as a result of improved cosmesis.
10. Acknowledgement
The author would like to acknowledge Dr. Lisa Hall Zimmerman for her dedicated
assistance of manuscript editing.

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155
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buried penis: description of a classification system and a technique to correct the
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the concealed penis. Urology, 45, 158-60.
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complications of abdominoplasty. Annals of Plastic Surgery, 57, 336-8.
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Tang SH, Kamat D, Santucci RA. (2008). Modern management of adult-acquired buried
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Journal of Urology, 65, 97-100.

Part 3
Current Research

(Trang không có chữ)

13
Skin Graft Preservation
Liangpeng Ge1, Zhenggen Huang2 and Hong Wei3
1Chongqing Academy of Animal Sciences
2Northern Hospital
3Third Military Medical University
China
1. Introduction
Skin transplants have long been of interest to plastic surgeons for the treatment of burns and
other wounds. Skin transplants provide a better micro-enviromental for wound healing and
provide physiological barrier which greatly decreases water, electrolyte, protein and heat
loss through the wound. However, skin grafts are not always available in adequate
quantities for the complete duration of treatment, making skin graft preservation important
in meeting the need for skin transplantation.
This chapter highlights progress in the field of skin graft preservation including basic
knowledge of skin preservation, harvesting, processing and preservation, and quality
control methods and is intended to provide a reference for skin preservation techniques for
doctors and researchers.
2. Thermal physics of skin low-temperature preservation
The low-temperature preservation with viable cells and tissues is a complicated procedure,
which needs to undergo multiple processes including pretreatment, freezing, preservation
under low temperature and thawing in proper order. In these processes, there is
unavoidably evident change in the functions and morphology of the tissues. An ideal
preservation procedure should be that viable cells and tissues can restore to the original
functions and morphology prior to preservation after undergoing all of these processes and
then be used for grafting and other purposes like fresh cells and tissue. So far, the effect of
low-temperature preservation does not meet the above ideal conditions. Also, doctors and
researchers are not fully aware of the change in viable tissue in the preservation processes,
especially in the processes of freezing and thawing.
Water occupies a considerable proportion ofviable cells and tissues. The moisture content of
skin is about 70%. The quality of skin is closely related to the phase transition of water
during low-temperature preservation, which including the solidification in freezing,
ebullition in freezing of viable tissue using liquid nitrogen, thawing during freezing and so
on.
Water or water vapor can generate more than 10 kinds of solid phase structure,
nevertheless, most of them need to be formed under high pressure environment (at least 200
million Pascal（Mpa）), and only hexagonal ice, cube ice or amorphous ice can be

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160
generated under low pressure. Water vapor deposits on a cold surface within the
temperature range from 0Ԩ slightly below to about -80Ԩ, or forms a hexagonal symmetric
ice crystal in a suspended state in the air, snow is a typical case in point, and cubic crystal
with diamond structure will be generated under lower temperature (about -80Ԩ~-130Ԩ)
and amorphous solid (also called glassy, vitrification) will be formed below -140Ԩ. Liquid
water usually only forms a hexagonal symmetric ice crystal upon freezing, however,
solutes-bearing water solution more easily realizes vitrification upon freezing compared
with pure water. Therefore, some components shall be added before freezing, in order to
promote the suspension of cells or tissues to more easily reach vitrification (Zhu et al.,
2002).
It is generally believed that there are two kinds of cell damaged in the freezing process: one
is intracellular ice crystals damage and the other is solution damage. Ice crystals cause the
change of cell wall and ultra-structures in the cells. Even death can occur by intracellular ice
crystals growing to a certain degree during freezing. This damage is more serious in the case
of quicker freezing. Solution damage causes cell damage by extracellular high-concentration
solution because water freezes outside of the cells. The longer cells stay in high-
concentration solution, the more serious the solution damage is. For certain types of cells
and freezing processes, ice crystals and solution damage exist simultaneously; nevertheless,
solution damage dominates in the case of slow cooling and intracellular ice damage
dominates in the case of quick cooling. And the solution damage and intracellular ice
damage shall be avoided or reduce if ice crystals are avoided (e.g. amorphous or glassy solid
are obtained), or ice crystals are formed but fail to grow to change the structure of cells (e.g.
partly vitrification) in case of snap freezing.
There are two different protocols used in skin low-temperature preservation: slow freezing
and quick freezing/snap frozen. Slow freezing reduces the temperature of the skin treated
with antifreeze fluid from about 4 to -60Ԩ at preset rate (e.g. 1-3Ԩ/min) and balanced for 4h
using a controlled-rate cooling apparatus, which adopts liquid nitrogen gas to absorb the
heat of specimen mainly by convective heat transfer, and then put skin into liquid nitrogen
for preservation. It is worthy of note that, the moisture contained in those tissues with
greater mass will releases a large amount of latent heat in a cold room at near 0Ԩ, however,
liquid nitrogen gas can’t rapidly absorb the latent heat of water phase transition due to
lower convective heat transfer coefficient, so it makes a probable great difference between
the displayed temperature by a controlled-rate cooling apparatus and the actual
temperature in the skin tissues, and it is difficult to reduce this temperature difference by
increasing cold gas volume. In case of snap frozen, the skin is incubated with antifreeze
solution and then transferred directly to liquid nitrogen, and the temperature can be cooled
from >0Ԩ to liquid nitrogen temperature within 30s. Being simple, snap frozen eliminates
special instrument and reduces cost. However, liquid nitrogen may generate grave
ebullition during snap freezing, and the big temperature difference between liquid nitrogen
and skin can not be fully utilized at the initial stage of freezing, thus, “thermo-zone
platform” (as shown in Fig.1) will be form near zero degree. To avoid this defect, on one
hand, antifreeze fluid needs to be optimized, on the other hand, a copper plate pre-cooled
with liquid nitrogen shall be used for directly contacting skin graft for snap frozen using
heat conduction style，in order to obtain better cooling effect (as shown in Fig.2).
At present, there is inadequate researche on the thawing process of frozen skin. Most
standards advocate rapid warming of the skin to optimise viability. This can be achieved by

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161

Fig. 1. Temperature (ºC) curve of skin immersing in liquid nitrogen (Available from: Basic
and application of skin preservation, people’s military medical publisher;2002.)


Fig. 2. Temperature (ºC) curve of skin in a copper plate pre-cooled with liquid nitrogen
(Available from: Basic and application of skin preservation, people’s military medical
publisher;2002.)
immersion of the packs in a 37°C waterbath. The thawing process of skin is closely related to
previous freezing process. In case of snap frozen, glassy solids are formed inside and
outside of the cells, and quick rewarming can avoid “devitrification” during temperature
rise process. “Devitrification”means glassy water or tiny ice crystals-bearing solid water is
recrystallized prior to thawing, or tiny ice crystals are rethawed into large ice crystals, which
destroy cell structures and cause cell death(Zieger et al., 1997).
3. Skin harvesting and processing
Processing methods used for banking of skin for subsequent therapeutic use depend on
whether the skin is to retain viability or not. For viable skin grafts, sterilisation techniques
cannot be applied, however antibiotics and antimycotics may be used to disinfect the tissue
with respect to bacteria and fungi (Kearney,2005; Li, 2001; Ninnmam et al,.1978).
Temperature (ºC)
Temperature (ºC)
Time (s)
Time (s)

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162
3.1 Screening of donor
One of the major concerns with the use of skin grafts from donors is the risk of disease
transmission. A case involving the transmission of HIV from a skin donor to a recipient has
been reported(Clark, 1987). To reduce the risk of disease transmission, skin donors should
be screened before skin harvesting. If skin grafts obtained from cadaveric donors，medical
records held by hospitals, family doctors, and others must be screened for conditions that
would exclude donation(Delmonico,1998). This may include current systemic infection,
diseases of unknown etiology, and previous infections with the possibility of current carrier
status (hepatitis, malaria, HIV, syphilis antibodies etc). It may also be prudent to exclude a
history of malignant disease particularly if the skin might be used on immunosuppressed
patients, for example, those suffering massive burn injuries. Behavioral history may also be
grounds for rejection of the donor owing to the high correlations between activities such as
intravenous drug abuse and viral infections such as HIV and hepatitis.
3.2 Reducing the population of microorganism
Skin tissue possesses a mixed commensal population of bacteria and fungi that lives
primarily in or around the hair and sebaceous follicles (Kearney et al.,1984). Even the
commensal bacteria may damage the skin if subsequently allowed to grow during the skin
storage period to reach massive population densities. Therefore, it is important to try to
eliminate or minimize the microbial population of skin before retrieval.
For viable skin allografts, it is not possible to apply a sterilization technique because
sterilization methods tend to inactivate cells at an equal or greater rate than for the bacterial
cells. The best that can be achieved is a combination of serology screening for viruses and
bacterial disinfection using antibiotic cocktails (Holder et al., 1998). This is generally
achieved using skin prepping techniques similar to those used on the skin of patients
undergoing operations in hospital. When banking skin in a viable state, it is important to
validate that the prepping agents have been adequately removed before harvesting the skin,
and that any residuals are not toxic to the skin cells. Although effective in reducing the
bioburden, skin prepping is unlikely to totally eliminate bacteria (Lomas et al., 2003). It is
therefore essential to prevent proliferation of any residual bacteria after retrieval, during
transportation, and during subsequent processing. Maintaining the skin in a refrigerator or
at wet ice temperatures prevents the proliferation of most bacteria. To eliminate residual
bacteria and fungi in viable skin, an antibiotic/antimycotic cocktail may be used. If used at
reduced temperatures (4Ԩ), the skin may be incubated for up to 24 hours in the cocktail;
however, only antibiotics that are effective at this temperature should be included. At
normothermic temperature (37Ԩ), most antibiotics can be considered; however, the
exposure period should be minimized.
For nonviable grafts, a range of sterilization techniques can be considered. Dry heat and
autoclaving damage the structure of the skin tissue, including denaturation of collagen, and
hence are not suitable. Techniques that have been successfully used include ethylene oxide
gas and γ irradiation; however, concerns and limitations of these methods are beginning to
appear. Ethylene oxide gas and its reaction product with chloride—ethylene chlorohydrin—
are very toxic. Although acceptable levels for these compounds had been proposed, more
recent data suggest that there is no safe level of ethylene oxide with respect to genotoxicity.
Questions have also been raised about the effectiveness of γ irradiation. Many small viruses
and spore-forming bacteria are fairly resistant to γ irradiation. The high doses that would be
required to inactivate HIV in a window-period donor (more than 80 kGy) would cause

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163
extensive damage to the tissue matrix including collagen denaturation. Therefore, a current
consideration is whether “sterilization” can be achieved instead by using combinations of
microbial inactivation procedures (Kearney, 1989，2005; Prolo et al.,1980).
3.3 Time control
Viable skin grafts may be obtained from living donors or, more frequently, from cadaveric
donors. In the latter case, the skin becomes ischemic immediately after circulatory arrest.
This leads to progressive cell death over a period of days, which is accelerated at higher
temperatures. Even if the cadaver is immediately placed into a refrigerator, the body cools at
a slow rate, leading to at least some warm ischemia time. Nevertheless, viable skin can still
be retrieved up to circa 24 hours after death. If the body is not quickly refrigerated, the skin
will be subjected to a much longer warm ischemia period resulting in an accelerated
deterioration in viability. Once the skin is removed from the body, it can be cooled rapidly
by immersion into a refrigerated solution, thus immediately reducing the rate of cell death.
In the other hand, tissue banking standards generally set limits on the maximum warm and
cold ischemia times permitted after the death of the donor, because it is known that bacteria
from the gut can be released into the vascular system postmortem and migrate to the
internal organs and tissues(Kearney, 2005).
3.4 Sample of preparation skin graft from cadaveric donors
3.4.1 Donor selection
Donor skin procurement must comply with local legislation. To ensure skin grafts quality,
donors were aged between 8–60 years. The exclusion criteria for harvesting skin are
basically the same as for donors of parenchymatous organs. Further exclusion criteria
comprise skin diseases, injuries in the areas from which skin may be harvested, and blood
samples were tested for HIV, HbA, HbB, HbC and syphilis antibodies.
3.4.2 Skin harvesting
Skin is cleaned with povidone-iodine 10% solution and skin with adipose layer harvested
with a knife. Areas from which skin is harvested are limited to the following regions: ventral
and dorsal trunk, left and right upper arm, and left and right thigh. Immediately after the
removal, grafts were placed in saline, refrigerated and transferred to the processing room.
The hair was removed and skin was soaked in 0.1% benzalkonium bromide for 15 min to
sterilize. Split skin of 0.2–0.3mm was harvested under sterile conditions by a dermatome.
Thereafter split skin was washed extensively and soaked for 10 min 3 times in saline with
Penicillin /Streptomycin; then in Neomycin–saline (0.5%) for 10 min, 3 times. All the
following procedures are carried out under sterile conditions.
3.5 Sample of preparation skin graft from porcine
Domestic pigs were sacrificed and the skin was washed extensively with soap and water.
The hair was removed by razor and the carcass was cleaned with povidone-iodine 10%
solution. After cleaning, 0.3 mm to 0.6 mm partial-thickness skin from the pig`s back was
harvested with a dermatome. Immediately after the removal, the harvested skins were
placed into sterile boxes in normal saline supplemented with penicillin/streptomycin and
sent to the laboratory, then soaked in 0.1% benzalkonium bromide for 15 min to sterilise and
rinsed thrice in normal saline supplemented with penicillin/streptomycin in a laminar flow
hood (Chiu&Burd, 2005).

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4. Viable skin grafts preservation
Fresh cadaver allograft is still considered the ‘gold standard’ biologic dressing for closure of
burns. Unfortunately, the use of fresh allografts is severely impeded by their inadequate
availability. Skin graft preservation for the purpose of delayed application is still a basic tool
in burn treatment and plastic and reconstructive surgery. Even if the skin is placed into an
oxygenated nutrientrich medium, ischemic necrosis of the tissue still occurs because the
diffusion path from the tissue periphery to the central cells is extensive, oxygen and
nutrients cannot diffuse fast enough to supply the cells, and toxic metabolites cannot be
removed quickly enough. Therefore, method for the viable storage of skin is to reduce the
temperature hence reduces the metabolic rate of the cells and the nutritional demands and
metabolite production (Bravo et al,. 2000; Ge et al., 2010; Robb et al., 2001; Sterne et al., 2000).
4.1 Storage in 4°C
As early as 1903, Wentscher reported the successful storage and grafting of skin autografts
at temperatures near 0Ԩ for 14 days (Wentscher, 1903). Further studies by Carrel using
normothermic and hypothermic storage conditions confirmed the utility of the latter (Carrel,
1912). Eventually, refrigerator storage for skin autografts became the norm in burn units.
The simplest technique was to fold the skin so that the cut surfaces were in apposition and
then wrap the skin sandwich in tulle gras and/or saline gauze to prevent desiccation. Using
this method, autologous skin can be used after around 2 weeks of storage, although cell
viability is very low at this point. Unlike autograft, allograft has already lost some viability
by the time it is retrieved; therefore, maximum storage times using this method are reduced
to 7 or 8 days. Many attempts have been made to extend the refrigerator storage period for
skin. The addition of homologous serum at 10% to 33% was found to be beneficial because it
provided nutrients and diluted and buffered acids produced as byproducts of metabolism
(Allgower & Blocker, 1952). Alternatively, tissue culture media as a source of nutrients and
various buffering systems have been evaluated. The buffering systems, however, only
extend slightly the period of useful storage. Different methods for storing skin grafts around
4Ԩ are shown in Table 1 and show a large variance in storage time although the storage
methods are similar. The main reason for this variance may be criteria incompatible and the
absence of reliable detection methods of early research especially before 1970. The storage of
skin grafts in a 4Ԩ refrigerator is a simple and practicable technique; however, the skin
showed a very slow viability decrease with time stored at 4Ԩ (Chang et al., 1998; DeBono et
al., 1998; DeLW, 1980; Matsuka et al., 1993; May & Wainwright, 1985). The preservation time
of fresh skin should not exceed 3 days when stored in normal saline and must be used
within two weeks even when stored in tissue culture media with 10% serum.
4.2 Storage in -20°C
Wang has success develop non-freeze complex cryoprotectants( 10% DMSO, 10% propylene
glycol，10% PEG，10% glycerin) to avoid ice crystal formation when skin stored in -
20Ԩ，the skin remain 76% activity in 14th day, higher than that of stored at liquid
nitrogen（60-70%）, then skin activity lower than that of stored at liquid nitrogen in the
following time, but still remain 50% viability(the ultimate rate to survival post-
transplantation) in 60 days, so skin grafts stored at -20Ԩ should be first consider if skin will
be used within 2 weeks.(Wang et al., 2002).

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165
Year
Author
Category
Stored method
Stored time
1903
Wentsch
Human
Exsiccatus ice-box
3-14d
1912
Carrel
chicken
0Ԩ, normal saline with serum,
paraffin oil
Several weeks
1912
Carrel
Dog
0Ԩ, normal saline，blood
Several months
1922
Filatov
Human
0Ԩ
Several days
1945
Matthews
Human
3-6Ԩ，normal saline，oxygen
deficiency
3-8 weeks
1949
Hanks，Wallace
Rabbit
0-8Ԩ，10% serum
2 weeks
1952 Allgower，Blocker
Human
5Ԩ,10% serum
14 days
1952
Hyatt
Human
4Ԩ,10% serum
185 days
1954
Skoog
Rat
3Ԩ
3 weeks
1957
Perry
Human
4Ԩ，Earle medium，10% serum
84 days
1963
Grasham
Human
4Ԩ，10% serum
6-8 weeks
1971
Bondoc，Burke
Human
4Ԩ，serum
14 days
1972
Lawrene
Human
4Ԩ，normal saline
1 week
1985 May，Wainwright
Pig
4Ԩ，Earle medium
1 week
2002
Alotto
Human
4Ԩ，RPMI 1640, 1% human serum
albumin
72 hours
2003
Castagnoli
Human
4Ԩ，RPMI 1640, 1% human serum
albumin
4 days
2010
Ge
Pig
4Ԩ，normal saline or DMEM
72/96hours
Table 1. Effect of skin stored around 4Ԩ
4.3 Deep freezing
Deep freezing is preservation of skin grafts in ultra-low temperature refrigerator, which
generally maintain temperature under -80Ԩ，and now -150Ԩ ultra-low temperature
refrigerator has been made in China. The advantage of this method is simplicity of operator,
low maintain cost and relative long preservation time (not exceed 1 year) (May et al., 1985,
1988).
To achieve high viability of skin grafts, cryoprotective agents (CPAs) should be added
before freezing. The general properties of cryoprotectants are that the molecules can pass
through the cell membrane into the cell and be relatively nontoxic at very high multimolar
concentrations. Several cryoprotective chemicals have been identified. CPAs are glycerol
and dimethyl sulfoxide. Cryoprotective agents are able to prevent solution effect injury.
CPAs mode of action is probably 2-fold. First, they act as solvents for the salt, thus reducing
the salt concentration that the cells are subjected to at the high subzero temperatures (where
solution effects are most damaging). Secondly, the presence of CPAs within the cells prevent
excessive shrinkage of the cells during this cooling phase. Therefore, in the presence of
CPAs, it is possible to use very slow cooling rates that minimize intracellular ice formation
while protecting the cells against solution effects. High viabilities of all cell types can thus be
achieved using this slow cooling rate.
Although CPAs are relatively nontoxic at low temperatures, the toxicity can become
significant at higher temperatures. In addition, the rate at which CPAs enter the cells is
temperature and CPA dependent, being faster at higher temperatures. Therefore, the

Skin Grafts – Indications, Applications and Current Research

166
optimum temperature and the exposure time need to be validated. The basal medium into
which the CPA is dissolved for incubation of the skin tissue should be a balanced salt
solution, in which the zwitterionic buffers hydroxyethyl piperazine ethanesulfonic acid
(HEPES) and trimethylamino-ethanesulfonic acid (TES) have been shown to work well
(Basaran et al., 2006). To avoid osmotic lysis of the cells, either the saline can be added
gradually or an impermeant solute such as sucrose can be added to the saline to reduce the
difference in osmolarity.
4.4 Cryopreservation
A better method for the long-term preservation of skin grafts is cryopreservation. The
cryopreservation technique facilitates the cooling of the tissues to ultralow temperatures
while protecting the viability of the cells (Aggarwal et al., 1985; Bondox & Burke, 1971; Cui
et al., 2007; Fujita et al., 2000; Marrel et al., 1986). Once the skin is at a temperature lower
than -130Ԩ, no further loss of cell viability is incurred. The boiling point of nitrogen gas is -
196Ԩ, so skins immersed into liquid nitrogen should be able to keep viabily indefinitely in
abstracto.
There are two different cryopreservation protocols used in skin preservation: slow freezing
and quick freezing/snap frozen. Slow freezing can be achieved using a controlled-rate
cooling apparatus (Blondet et al., 1982). There is an optimum cooling rate for any cell type
that produces maximum cell survival. On either side of this optimum, the survival rate falls.
In the presence of CPA, a cooling rate of -1 ºC per minute will ensure survival of most of the
cells within skin tissue. As the cooling rate is increased，cell populations are sequentially
and adversely affected. Many cells in the body derived from leukocytes or closely related
lineages are known to be exquisitely sensitive to cryogenic injury. The depletion of
immunostimulatory “passenger leukocytes” was demonstrated by increasing the cooling
rate for pancreatic islets of Langerhans while maintaining the viability of the insulin-
producing islet cells (Ingham et al., 1993). This concept of cooling rate–dependent
immunomodulation was evaluated for skin tissue. A cooling rate of -30Ԩ/min was shown
to maintain the viability of keratinocytes and fibroblasts while reducing the immunogenicity
(as assessed by the mixed epidermal cell/lymphocyte response assay) of murine allografts
by 95%. This was assumed to be due to an effect of the faster cooling rate on the major
immunostimulatory cell in the skin—the Langerhans cell.
Quick freezing/snap frozen is skin vitrification technique. Vitrification is defined as “the
instant solidification of a solution brought about by an extreme elevation in viscosity during
cooling, without ice crystal formation”. In other words, vitrification is faster and lacks some
of the typical disadvantages seen in traditional slow freezing (Mukaida, 2003). It bypasses
the ice-crystal formation phase and instantaneously solidifies into a glass-like structure,
moves the water directly into a glass-like phase. In a glass, the molecules do not rearrange
themselves into grainy ice crystals as the solution cools, but instead become locked together
while still randomly arranged as in a fluid, forming a “solid liquid” as the temperature falls
below the glass transition temperature (Silvestre et al., 2002). This technique was simple,
and no expensive equipment was needed. Initially, this method needed a 2-step procedure
because the cryoprotective agent was toxic. Kasai et al., (1990) modified this impractical to a
1-step method and incubated the skin with vitrification solution at room temperature and
was transferred directly into liquid nitrogen.

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167
Ben-Bassat et al., (1996, 2001) evaluation graft performance of cryopreserved cadaveric skin
by programmed freezing (1°C/min). The results demonstrate that graft performance of
cryopreserved skin decreased with time as seen in the lower percent of samples with high
scores of separate histologic criteria after prolonged storage (Ben-Bassat et al., 1996).
Nevertheless, paired comparison analysis between cryopreserved and fresh skin indicated
that this decrease was not significant for storage of 5 years; however, it was highly
significant for 6 years of storage (Ben-Bassat et al., 2001).
Table 2 shows the distinction of different preservation and the viability of skin frozen at -
20Ԩ attenuated to 50% (the ultimate rate to survival post-transplantation) in 60 days. Skin
grafts stored at -20Ԩ should be used within two months. To be similar, the skins should be
transplanted in one year of freezing in -80Ԩ and 5 years in liquid nitrogen. Viability declines
rapidly after thawing of the skin and further storage before use cannot be recommended.


Commom
refrigerator
Freezer
ultra-low temperature
refrigerator
Liquid
nitrogen
Temperature(Ԩ)
4
-18～-20
-80
-196
Equipment cost
Cheap
Cheap
Costly
moderately
Sustain cost
Cheap
Cheap
Cheap
Costly
Electricity effect
Great
Great
Great
None
Storage time
3～7days
60days
1 year
5 years
Viability（%）
30～80
50～60
50～60
60～70
Table 2. Comparison of different preservation methods
5. Nonviable skin grafts preservation
With non-viable skin (or dermis) there is no requirement to maintain the viability of tlhe
skin cells. Nevertheless, it is still important that degradative changes do not adversely affect
the tissue matrix and bacteria are not allowed to proliferate; therefore, many of the practices
discussed earlier will still apply. A major difference from viable skin, however, is that much
more effective disinfection or sterilisation methods can be applied to the skin, including
virucidal treatments.
5.1 Freeze drying
In the early 1950s, the US Navy Tissue Bank was the first to commence large-scale freeze
drying of human tissues for implantation. In 1955, the use of freeze-dried skin was first
reported (Brown et al., 1955). It was subsequently shown that freeze drying reduced the
immunogenicity of skin without interfering with its beneficial properties（Abbott &
Hembree , 1970）.This process involves the removal of water from skin in the frozen state
by sublimation, which is achieved by applying a vacuum to the tissue and condensing the
removed water molecules downstream. Sublimation from the frozen state helps to protect
molecules that would otherwise be adversely affected by high salt concentrations at higher
temperatures, for example, denaturation of proteins. Drying must continue until enough
water has been removed to prevent degradation reactions, which equates to less than 5%
residual water as measured gravimetrically. Although accepted in most tissue banking
standards, “residual water” is not identical to “water activity”, which is the most
appropriate measure.

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168
5.2 Glycerolization preservation
In 1980s, glycerin was applied in clinical practice as skin graft preservation fluid, and had
very good effect (Hermans, 1989; Huang et al., 2004; Richter et al., 1997; Van Baare et al.,
1994,1998; Vuola & Pipping, 2002). EuroSkin Bank has done considerable fruitful work in
the area and developed a solute preservation method using glycerol (De Backere & Mackie,
1997; Mackie, 1997). The skin was incubated in successively more concentrated glycerol
solutions (50%, 70%, and 85%) and maintained long term in 85% glycerol. More recently, the
glycerol and water flux kinetics have been characterized and a more efficient validated
protocol was proposed. Study demonstrated that the 85% concentration originally chosen by
the Euroskin Bank is optimal for minimizing degradative reactions (Ross & Kearney, 2004).
It is very important that the glycerol is removed from the skin before clinical use. Failure to
remove glycerol may lead to high systemic concentrations when used on open wounds. This
is achieved by repeatedly washing the skin in physiological saline, and at least 30 to 60
minutes are required to remove most of the glycerol.
5.3 Glutaraldehyde preservation
As early as 1975, Schetnter reported the successful grafting of glutaraldehyde- preserved skin
in clinical (Schetnter, 1975). The morphology of skin grafts has not evident change after treated
with glutaraldehyde, and skin antigenicity was greatly reduced because antigen determinant
site was closed by cross-linking of protein in the skin grafts. Glutaraldehyde-treated skin grafts
can be used for escharectomy and tangential excision wounds and granulation wound
covering, and has been shown to promote the formation of granulation tissues. However, such
skin grafts have least elasticity, that is, with the extension of preservation duration, the skin
becomes harder. Fresh allograft skin or lyophilized skin shall be immersed in 0.5%
glutaraldehyde solution for 7-20min and be washed with normal saline for three times prior to
use. There is no evident change in the elasticity and softeness of such skin, but its antignicity is
decreased, which helps to extend the adhesion duration of grafted skin.
5.4 Irradiation preservation
Irradiated porcine skin is the most commonly used burn wound covering in China. Fresh
porcine skin graft is sealed in a plastic bag and is irradiated with 60Co or an accelerator, then
put into an ordinary refrigerator for preservation. After being irradiated, porcine skin can
achieve integral asepsis, and its antignicity can be decreased. For gamma irradiation, the most
resistant microorganisms are viruses, D10-values as high as 13 KGy have been reported for
certain small viruses. Application of the commonest used dosage (25 KGy) to these viruses
would only reduce the bioburden by 2 logs10. The high doses that would be required to
inactivate HIV in a window-period donor (more than 80 kGy) would cause extensive damage
to the tissue matrix including collagen denaturation. There are two possible effects on the
macromolecular structure of skin matrix resulting from ionising irradiation. The first is
scission which results in the breaking of bonds thus weakening the matrix. In the presence of
water a second process involves crosslinking by new bond formation resulting from the
generation of free radicals which may lead to stiffening of the matrix.
6. Quality control
A variety of national/international organisations have issued standards or guidelines for
tissue banking, including the National Blood Service, British Association for Tissue Banking,

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and European Association of Tissue Banks, etc (Baxter, 1985; Ben-Bassat et al., 2000; British
Association for Tissue Banking, 2011; Chua et al., 2004; European Association of Tissue
Banks, 2011; Freedlander et al., 1998; Janezic, 1999; Kalter, 1997; Kearney, 1998; May, 1990;
Pillipp et al., 2004; Pianigiani et al., 2005). The core principle of skin banking quality control
is to avoid the risk of cross infection and provide high-quality skin grafts for clinical.
Therefore, donor selection, microbial/viral tests, skin viability detection and transplantation
performance will be discussed in this paper.
6.1 Donor selection
With respect to medical/behavioural history screening, the regulatory and standards
documents are once again very specific with a good deal of international consensus. Sources
of medical/behavioural information include the next of kin (or partner/close friend) of the
deceased who must be interviewed regarding medical history and any factors, practices or
behaviour that may have increased the risk of exposure to HIV/Hepatitis. In addition, the
family doctor, hospital records, and autopsy report should be consulted as additional
sources of medical and behavioural history. Evaluation of the risk of virus exposure must be
explored in detail with other aspects of medical and behavioural history. The exclusion
criteria for harvesting skin are basically the same as for donors of parenchymatous organs
including cancer, diseases of unknown aetiology and various viral, bacterial and parasitic
diseases. Further exclusion criteria comprise other skin diseases and injuries in the areas
from which skin may be harvested.
6.2 Microbial/viral tests
There is a widespread consensus on the testing of donors to preclude certain virological
diseases, including HIV, Hepatitis B, Hepatitis C, syphilis antibodies ,and in general these
follow the National Blood Service testing regimens for each country. Screening for CMV is
important when recipients are CMV negative and will receive immunosuppressive drugs
following organ transplantation. However, the natural immunosuppression associated with
burns does not appear to lead to CMV related problems in skin allograft recipients. Many
tissue banks do not screen for CMV. The microbial tests are generally done on allograft
samples and the viral tests on blood samples.Skin samples for microbial tests are incubated
in Thioglycollate and Roswell Park Memorial Institute (RPMI) 1640 without antibiotics, in a
37Ԩ incubator for 10 days.
6.3 Skin viability
The viability of graft skin is fundamental for surgical procedures involving graft taking, and
the American Association of Tissue Banks indicates that the viability of skin is an essential
prerequisite for good-quality grafts (American Association of Tissue Banks, 2011). It is a
necessity that a skin bank should certify, not only the sterility, but also the viability of the
skin allografts used and that routine quality controls be carried out to certify that grafts are
viable before transplantation. The of viability assays is two-fold: one is that of supplying an
‘‘experimental assay’’: used to optimize the various parts of a preservation procedure
during experimental development. The other offers a ‘‘predictive assay’’: used to predict the
‘‘quality’’ of an individual sample that has been stored by an established tested technique.
Various methods, both qualitative and quantitative, are currently used to determine skin
grafts viability such as cell culture, succinate dehydrogenase, trypan blue dye, skin oxygen

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consumption, Tetrazolium salts (MTT/WST-1), SYTO/EB dyes and so on (Alotto et al.,
2002). The MTT method is most widely used because it is simple, quick and precise
(Castagnoli et al., 2003; Klein, 1996; Pegg, 1989; Yang et al., 2000; Zieger et al., 1993).
6.4 Transplantation performance
Skin transplantation experiment is a straightforward and reliable skin graft quality
evaluation method. For any skin graft and preservation method, the final result is to identify
that the skin graft can survive in the living body. For evaluation of transplantation
performance of preserved skin, samples of skin were often grafted onto mice and rats and
the experiment animals were sacrificed after 4 or 7 days. The grafts were then assessed by
gross inspection including adherence to wound bed, color and pliability and by histologic
evaluation including epidermis integrity, epidermal–dermal junction, collagen organization,
the presence of fibroblasts and graft adherence (Cinamon et al., 1993).
7. Conclusions
Skin graft preservation for the purpose of delayed application is still a basic tool in burn
treatment and plastic and reconstructive surgery. It is therefore important to provide a
framework for selecting optimal guidelines for procurement, processing, preservation, and
quality control of skin grafts for doctors and researchers, thus ensuring high levels of safety
and efficacy of skin grafts.
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(Aug), pp.53-69, 0011-2240

14
Comparative Study of Skin Graft
Tolerance and Rejection in the
Frog Xenopus Laevis
Hristina Nedelkovska and Jacques Robert
Department of Microbiology and Immunology,
University of Rochester Medical Center, Rochester
USA
1. Introduction
Over the last few decades, the amphibian Xenopus laevis1 has been used as a unique
comparative model to study the developmental and immunological aspects of tissue
transplantation as well as self tolerance (Du Pasquier, Schwager et al. 1989; Robert and
Ohta 2009). The immune system of X. laevis, one of the best defined outside mammals and
chickens, is fundamentally similar to that of mammals. Notably, the evolutionary distance
of X. laevis from mammals permits distinguishing species-specific adaptations from more
conserved features of the immune system. In addition, advanced genetic resources
including the full genome sequence of the X. leavis sister species X. tropicalis, a large
collection of EST, cDNA and genomic (BAC, Fosmid) libraries for both species, efficient
transgenesis and genome wide mutagenesis has markedly empowered X. laevis as a
biomedical model. Furthermore, several different major histocompatability complex
(MHC) defined inbred strains of X. laevis, as well as clones sharing identical MHC
haplotypes but differing at multiple minor histocompatibility (H) loci, provide a unique
opportunity to study T cell regulation in vivo by skin graft. More information about these
resources can be found on the Xenbase website ()
(Bowes, Snyder et al. 2009) and the X. laevis research resource for immunobiology
() (Robert 2006).
The objectives of this review are first to provide short background information on the
immune system and skin graft biology in X. laevis, and then to examine the use of the minor
H-Antigen (Ag)-disparate skin grafting model system in X. laevis isogenic clones and to
investigate in vivo the immunostimulatory properties of certain heat shock proteins (hsps)
such as gp96 and hsp70 with particular emphasis on minor H-Ag specific T cell responses.
We will also consider the possible role of nonclassical MHC class Ib molecules in this
context. Furthermore, we will re-evaluate old data on induction of long term immunological
memory and immune tolerance in X. laevis larvae to skin graft Ags in the context of immune
regulation. Finally, we will discuss the possibility to use hsps and new genetic tools

1 All animals were handled under strict laboratory and UCAR regulations (Approval number 100577 /
2003-151), minimizing discomfort at all times.

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(genomic and transgenic technology) to revisit this immune regulation to skin Ags during
development of X. laevis. It is our conviction that by integrating the unique biological
features of X. laevis with the recently advanced genetic resources of this comparative model,
it will be possible to answer critical questions about the development of self-tolerance as
well as autoimmunity.
2. The immune system of Xenopus laevis
The frog X. laevis has been used to study skin graft rejection as well as tolerance for the last
few decades. The immune system of X. laevis, a genus whose common ancestors with
mammals diverged 350 million years ago (Kobel and Du Pasquier 1975; Evans 2008), is
fundamentally similar to that of mammals. Importantly, this extensive evolutionary distance
allows one to distinguish species-specific adaptation from conserved features of the immune
system. The X. laevis immune system is characterized by T and B lymphocytes with RAG-
mediated rearranging TCR and Ig genes, MHC class I- and class II-restricted T cell
recognition (Du Pasquier, Schwager et al. 1989; Robert and Ohta 2009), as well as innate
immune cells such as macrophages and NK cell (Horton, Horton et al. 1998; Horton, Minter
et al. 2000; Robert, Ramanayake et al. 2008; Morales, Abramowitz et al. 2010). Interestingly,
X. laevis lacks lymph nodes but it does have both a thymus and a spleen similarly to
mammals.
The additional developmental transition occurring during metamorphosis in X. laevis results
in two different life stages, the larvae and the adult, which provides a unique opportunity of
working with two distinct immune systems. Unlike mammals, larvae have external
development; therefore, they are amenable to experimental manipulation and there is easy
accessibility of early developmental stages free of maternal influences. For example, since
large areas of the larvae are transparent, the thymus is easily distinguishable and it is also
relatively simple to perform thymectomy on these animals which will render them free of T
cells in their larval as well as adult life. During metamorphosis the immune system
undergoes a remarkable developmental transformation during which surface major
histocompatability complex (MHC) class Ia (class Ia) expression becomes detectible for the
first time on erythrocytes and splenic leukocytes (Flajnik, Kaufman et al. 1986; Flajnik and
Du Pasquier 1988; Rollins-Smith, Flajnik et al. 1994). Interestingly, NK cells that are not
detected during larval life emerge concurrently with class Ia expression (Horton, Stewart et
al. 2003). X. laevis larvae are different from adults since they are naturally class Ia deficient,
but importantly these animals are immunocompetent and they have thymus-dependent
CD8 T cells (Flajnik, Kaufman et al. 1986). In addition, certain nonclassical MHC class Ib
(class Ib) genes have been found to be expressed in the thymic anlage very early in ontogeny
and preferentially by thymocytes (Goyos, Ohta et al. 2009; Goyos, Sowa et al. 2011). The
implication of this on the T cell repertoire of early larval stages is under investigation. MHC
class II antigen expression during larval life is restricted to the thymic epithelium centrally,
and to B lymphocytes and accessory cells in the periphery: whereas, it is constitutively
expressed on virtually all thymocytes and mature peripheral T as well as B cells in adults
(Flajnik, Kaufman et al. 1986; Du Pasquier and Flajnik 1990; Flajnik, Ferrone et al. 1990).
Thus, the larval and adult X. laevis immune systems have critical differences as well as
similarities. As such, comparisons between these two developmental stages afford unique
opportunities to investigate in vivo developmental and immunological aspects of tissue
transplantation. Also the differential expression of class Ia together with the ease of larval

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177
experimental manipulation allows us to explore questions regarding MHC-restriction,
autoimmunity, and the development of self-tolerance that can not be easily studied in other
animal models.
A major attribute of the X. laevis model is the availability of different MHC-defined strains
and clones. In addition to the MHC homozygous inbred strains, the J and F strains (Du
Pasquier and Chardonnens 1975; Tochinai and Katagiri 1975), the X. laevis model also
includes MHC-defined isogenic clones of frogs, such as the LG-15 and LG-6 that share the
same heterozygous MHC haplotypes (a/c) but differ at multiple minor histocompatability
(H) loci (Kobel and Du Pasquier 1975; Kobel and Du Pasquier 1977). These clones are
generated by gynogenesis, a procedure where diploid eggs from X. laevis/X. gilli hybrids are
only activated by UV irradiated sperm, and thus the spermatozoid DNA does not contribute
any genetic material to the offspring. This system allows us to easily pool cells from
different frogs and perform adoptive cell transfers since the clones share identical genetic
make up (Maniero and Robert 2004). In addition, we also have available a transplantable
thymic lymphoid tumor named 15/0 (originally a spontaneously growing tumor derived
from a LG-15 clone) that is tumorigenic in both LG-15 and LG-6 clones (Robert, Guiet et al.
1994; Robert, Guiet et al. 1995; Robert and Cohen 1998). Interestingly this tumor does not
have class Ia protein expression however it does express several class Ib molecules (Robert,
Guiet et al. 1994; Salter-Cid, Nonaka et al. 1998; Rau, Cohen et al. 2001). As a result, the
accessibility of different animals and reagents has provided us the unique opportunity to
study T cell regulation in vivo by skin transplantation.
3. Skin graft rejection in adult Xenopus laevis
Skin graft rejection is a well-established technique in X. laevis that has been used for
determining the segregation of both major and minor H-Ag loci (Chardonnens and Du
Pasquier 1973; Du Pasquier and Bernard 1980; Ramanayake, Simon et al. 2007).


Fig. 1. Skin graft rejection in Xenopus laevis. (A) Allograft one day post transplantation, 0%
rejection. The entire graft appears silvery due to the presence of irridophores. In (B) and (C)
the grafts are undergoing rejection 50 and 90% respectively. (*) indicates areas of the graft
that are still surviving. The rejected areas of the grafts are highly vascularized. (D)
Representative image of a fully (100%) rejected skin graft. This graft is “dull” and does not
have any more surviving irridophores.
We have recently described this technique in details (Nedelkovska, Cruz-Luna et al. 2010).
Succinctly, X. laevis adults are skin grafted by cutting a 5mm2 piece of donor ventral skin
A
B
C
D
*
*

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178
(abdominal skin which appears silvery due to the presence of irridophores) and inserting it
under the dorsal skin of the recipient (Chardonnens and Du Pasquier 1973; Nedelkovska,
Cruz-Luna et al. 2010). It is critical to handle the graft gently and avoid damage with the
forceps, which would obscure the results. Also care must be taken to avoid introducing
large air bubbles under the skin because that can cause displacement or loss of the graft.
After 24 hours, a window of overlaying host skin covering the graft is removed and then the
graft can be easily visualized using a dissecting microscope. Skin graft rejection is then
monitored by determining the percent of irridophore destruction over time. Rejection is
considered complete when the entire exposed graft has become “dull” and all the
irridophores have been entirely destroyed (Figure 1).
3.1 Conserved role of T cells in skin graft rejection
In adult X. laevis, as in mammals, the kinetics of skin graft rejection are dependent on the
number of MHC mismatches as well as minor H-Ags between the donor and the recipient
frogs. If the graft is either an autograft, syngenic or an isograft, it will never be rejected by
the host. However, if the donor skin displays one or two MHC haplotype mismatches, then
there will be an acute skin graft rejection that will take 18 to 22 days at 21oC to complete
(Table 1). Furthermore, if the frogs share the same MHC haplotype but only differ by minor
H-Ags then the graft will undergo chronic rejection, which will take between 30 to more
than 100 days at 21oC for full rejection depending of the genetic combinations (Table 1).
With X. laevis being ectothermic, temperature has a profound effect on skin graft rejection.
For instance, at 27oC minor H-Ag-disparate grafts between individuals of the partially
inbred F strain are rejected in 23-37 days, which is significantly faster than the rejection at
21oC that takes 60-100 days (Robert, Guiet et al. 1995). In vitro, X. laevis immune functions
are also affected by temperature (Hsu 1998). For example, in vitro T-cell proliferation in MLR
(Mixed Lymphocyte Reaction) or induced by mitogen (Meier 2003) and proliferation of
lymphoid thymic tumor cell lines (Robert, Guiet et al. 1994), occurs faster at 27°C (optimum)
than at lower temperature (18–25°C). Therefore, temperature has to be taken in account
when comparing in vivo and in vitro responses against alloantigens.
The strict T cell dependency of skin rejection has been clearly established in X. laevis by
thymectomy at early developmental stages before the full differentiation of T cell precursors
(5-6 days post-fertilization; st. 47/48 (Horton, Horton et al. 1998) stages based on
(Nieuwkoop and Faber 1967)). Adult frogs that have been thymectomized at early
developmental stage fail to reject both MHC as well as minor H-Ag-disparate allografts
(Barlow and Cohen 1983; Arnall and Horton 1987; Robert, Guiet et al. 1997). In contrast,
adults that have been thymectomized either during mid larval (st. 53-55) or late larval (st.
56-58) development remain capable to reject MHC-disparate grafts. This most likely is due
to the fact that mature T cells are able to migrate out of the thymus before it is removed;
therefore, they can have effector functions in the periphery. These data demonstrated that
skin graft rejection in adult X. laevis is thymus dependent and suggested that like in
mammals allograft rejection is mediated by CD8 as well as CD4 T cells. Additionally, there
is immunological memory against minor H-Ag because second set skin grafts are rejected in
an accelerated fashion while third party grafts are not, suggesting that these responses are
specific to the immunizing alloantigens (Table 1) (Nagata and Cohen 1983).
In order to assess more directly the role that CD8 T cells play in these responses in vivo,
depletion by antibody treatment was used. CD8 T cells were depleted using the AM22

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179
mouse anti-X. laevis CD8 monoclonal antibody (Flajnik, Ferrone et al. 1990). AM22 is specific
for CD8 T cells because cells stained with this antibody express a pan-T cell marker (XT-1) as
well as high levels of CD5 and CD45 (Gravenor, Horton et al. 1995; Robert, Sung et al. 2001).
Moreover, cells stained by AM22 are not detected in frogs that are thymectomized during
early larval life and lack T cells (Gravenor, Horton et al. 1995; Robert, Guiet et al. 1997).
AM22 depletion experiments showed that in the absence of CD8 T cells MHC-disparate
allografts did not undergo the typical acute rejection pattern but underwent slower rejection
that took 7 days longer to complete (Rau, Cohen et al. 2001). Interestingly, these grafts were
indeed completely rejected even if more mAb AM22 injections were used to prolong the
CD8 T cell depletion effect. This suggests that other cells like CD4 T cells may also be
involved in the response against skin Ags. To date this possibility has not been investigated
due to the unavailability of antibodies against X. laevis CD4. However, recently our lab has
generated and is currently characterizing a single chain CD4 antibody which will allow
exploration of the importance of CD4 T cells in allograft rejection.
To further characterize the CD8 T cells involved in graft rejection, our lab adapted a whole-
mount immunohistology technique. This procedure allows us to visualize lymphocytic
infiltration into unfixed transplanted skin tissues using fluorescent antibodies (Ramanayake,
Simon et al. 2007). Additionally, this method preserves the tissue structure and we can use
several antibodies conjugated to different fluoropores to see exactly where cells are located
and distinguish what kind of cells are present in the grafts. Therefore, this technique is a
powerful tool which we can use to characterize and monitor immune effector cells
mediating the immune responses of X. laevis against skin rejection Ags.
Using whole-mount immunohistology we found that, unlike isograft controls, MHC-
disparate grafts that were undergoing rejection were infiltrated with a large number of CD8
T cells. These CD8 T cells were mainly distributed in areas where the graft was not yet
rejected and the silvery irridophores were still persisting. Moreover, there was an inverse
correlation between the percent rejection and number of infiltrating cells. For example the
most prominent CD8 T cell infiltration occurred at day 7 when there was only 50% rejection.
Additionally, these grafts also had significant infiltration of class II positive cells which were
more numerous than the CD8 T cells. As mentioned before all adult leukocytes have class II
expression in adult X. laevis, therefore, the majority of the class II positive cells in MHC-
disparate grafts were actually CD8 T cells (~80%). This was also seen by cell morphology
since most of the cells were small round lymphocytes although this does not exclude CD4 T
cells. Besides lymphocytes, other cells morphologically similar to macrophages and
dendritic cells (such as Langerhan cells) were seen in both allografts and isografts. These
cells were already present in the grafts before transplantation, which suggests that they
could potentially serve as antigen presenting cells (APCs). However, more of these cells
from the recipient animal can infiltrate the graft and then migrate out to the spleen to prime
more CD8 T cells.
As previously discussed, the main difference between MHC-disparate and minor H-Ag-
disparate grafts is the time it takes for complete graft rejection, which is either acute or
chronic, respectively. Therefore, one might assume that this is due to the lesser number of
infiltrating effector T cells since only minor H-Ags are involved in these responses. On the
contrary, however, we found that minor H-Ag-disparate grafts were infiltrated by similar
numbers of both CD8 and class II positive cells, but with delayed kinetics (Ramanayake,
Simon et al. 2007). In these minor H-Ag-disparate allografts the peak of immune cell

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180
infiltration was also observed when the graft was about 50% rejected which in this case
occurred 15 days after transplantation, rather than 7 days as in the case of MHC mismatched
grafts (Table 1).
Whole-mount immunohistology is a very powerful technique to study infiltration of
immune cells; therefore, we are currently using different antibodies such as X. laevis CD4
and HAM56 (a human macrophage marker which cross-reacts with X. laevis macrophages)
which will allow us to visualize both effector cells as well as APCs, respectively.
3.2 Characterization of the immunological properties of heat shock proteins (HSPs)
using skin graft rejection
Our X. laevis skin grafting model has been instrumental to get better insight into the
immunological properties of certain hsps such as gp96 and hsp70. Hsps are evolutionarily
ancient and highly conserved intracellular molecular chaperones that help with intracellular
transport, folding of newly synthesized proteins, and prevent protein aggregation. In
addition, hsps have been implicated in a variety of innate as well as adaptive immune
responses. Notably, hsps have the intrinsic property to carry exogenous antigenic peptides
from the tissues which are purified from and interact with endocytic receptors expressed by
APCs. Once the hsp-Ags complexes enter the APCs the peptides are shuttled into the MHC
class Ia cross-presentation pathway where they are processed and presented by class Ia
molecules to CD8 T cells. Therefore, hsps have the ability to elicit potent CD8 T cell
responses against the chaperoned Ags. Gp96 and hsp70 bind Ags differently. While the
peptide binding of gp96 is unclear, hsp70 has defined peptide binding which is ATP
dependent (Blachere, Li et al. 1997). Peptides can be loaded in vitro onto hsp70 by simply
adding ADP to the reaction, whereas ATP addition results in peptide disassociation. Hsp70
preferentially binds peptides that contain 4-5 hydrophobic residues flanked by two basic
residues (Castellino, Boucher et al. 2000), but it has been shown that 30 amino acid long
synthetic peptides can also be complexed (Calderwood, Theriault et al. 2005).
As mentioned, the LG-6 and LG-15 clones share the same MHC haplotypes but differ by
minor H-Ags, and frogs primed with a first set of minor H-Ag-disparate skin graft reject a
second set skin significantly faster. Furthermore, this accelerated rejection is thymus-
dependent and Ag specific (e.g., a third party skin graft rejection is not accelerated). This
system has revealed to be ideal in investigating whether the ability of hsps to generate CD8
T cell responses is conserved between mammals and amphibians.
We found that if we first immunize LG-6 clones with either gp96 or hsp70 purified from LG-
15 liver (meaning that those hsps would carry LG-15 minor H-Ags) and then graft them
with an LG-15 allograft, the graft undergoes accelerated rejection in comparison to control
unimmunized animals or animals that were immunized with LG-6 derived hsps (carrying
self-Ags) (Robert, Gantress et al. 2002). Additionally, syngenic grafts were never rejected
regardless of the immunization status of the animal, which rules out possible Ag-
independent pro-inflammatory effect induced by the hsps. Furthermore, these in vivo
responses are specific to the Ags chaperoned by the hsps because if an LG-6 animal is
immunized with LG-46 derived hsp and is grafted with both LG-15 as well as LG-46 skin
(these three LG clones share the same MHC haplotype (a/c) but differ in multiple minor H-
Ags) only the LG-46 graft will have accelerated rejection, while the LG-15 graft will undergo
a typical chronic rejection (Table 1). Moreover, immunization with hsp70-peptide complexes
results in accelerated skin graft rejection, while immunization with hsp70 free of Ags (i.e.,

Comparative Study of Skin Graft Tolerance and Rejection in the Frog Xenopus Laevis

181
Ags eluted by ATP-agarose chromatography) does not change the rejection kinetics in
comparison to unimmunized animals. These data indeed show that both gp96 and hsp70 are
able to generate potent specific T cell responses in vivo against minor H-alloantigens.

Type of skin grafts¶
Minor H-Ag
disparate
1 MHC haplotype
disparate
2 MHC haplotype
disparate
Rejection at 21oC
30 - 100
18 - 22
18 - 22
Peak CD8 T cell
infiltration
15
7
7
Rejection after
priming with same
first set graft
16 – 30
ND
ND
Rejection after gp96
or hsp70
immunization

20 - 30
ND
ND
Table 1. Summary of allograft rejection and immunological memory in adult X. laevis. Data
is represented in days. ND, Not done or data not found in literature.
In mammals it is known that immune responses generated by hsps are mediated by CD8
cytotoxic T lymphocytes (CTLs). Using an in vitro killing assay, our lab confirmed that as in
mammals, immunization with gp96 and hsp70 results in the generation of anti-minor H-Ag
CTLs. Specifically, CD8 T cells purified from LG-6 animals primed with LG-15 tissue-
derived hsp70 were able to kill class Ia positive LG-15 lymphoblast but not class Ia positive
LG-6 blast targets nor class Ia negative 15/0 tumor targets. In contrast, CD8 T cells from
animals immunized with Ag free hsp70 or unimmunized animals did not kill any of the
blast targets. The same results were obtained using gp96 and different combinations of
cloned frogs. Besides showing that the ability of hsps to generate CTL responses is
conserved in amphibians, this study has provided definitive evidence of MHC class Ia
restriction and Ag specificity of cytotoxic CD8 T cell effectors in X. laevis.
To explore in vivo the anti-minor H-Ag CD8 T cell effector capacity generated by gp96, our
lab adapted a carboxyfluorescein diacetate succinimidly ester (CFSE) proliferation assay for
X. laevis (Robert, Gantress et al. 2004). CFSE is a fluorescent dye that is incorporated into
viable cells and upon cell division the CFSE content gets diluted in half, meaning that each
daughter cell will have half the CFSE fluorescent intensity in comparison to undivided cells.
This technique allows us to label, follow and calculate the percent of dividing cells as well as
to determine the number of divisions that occurred. Using this approach, splenocytes from
LG-6 frogs immunized with LG-15 gp96 were first labeled with CFSE and then were
adoptively transferred into naïve LG-6 recipients that were previously grafted with a LG-15
skin allograft or a LG-6 syngenic graft. These experiments showed that the transferred CFSE
labeled splenocytes accumulated in the spleens of animals that carried minor H-Ag-
disparate skin grafts, while no such accumulation was detected in animals with syngenic
grafts (Maniero and Robert 2004). Furthermore, by flow cytometry staining it was shown
that the majority of CFSE+ dividing cells in these animals were CD8 T cells, which
underwent several rounds of proliferation (3-4 cycles). This proliferation is not as robust as
the one seen in mammals where up to 8 divisions can be seen. This can be due to several
reasons including the difference in temperature as mentioned earlier or the strength of the
TCR signal. Currently we do not know the status of other subsets of gp96 primed T cells to

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182
minor H-Ags; however, since we know that CD8 depletion alone is not enough to
completely abrogate skin graft rejection, this would suggest that other immune effector cells
are also involved.
To test the effector function of adoptively transferred CFSE+ cells we monitored skin graft
rejection. We found that unimmunized frogs carrying a minor H-Ag-disparate graft had
accelerated skin graft rejection which reached 45-90% by day 10 after adoptive transfer of
splenocytes (Maniero and Robert 2004). On the other hand, animals that carried isogenic
grafts did not show signs of rejection. These rejection kinetics are reminiscent of a secondary
T cell mediated rejection that occurs with animals primed with gp96 carrying minor H-Ag
complexes. This means that gp96 is able to prime CD8 effector T cells that when adoptively
transferred can recognize minor H-Ags presented in vivo by an allograft and kill the
transplanted skin.
3.3 Possible role of nonclassical MHC class Ib genes in skin transplantation
As discussed above, CD8 T cells are critically involved in skin graft rejection in X. laevis and
these responses are MHC class Ia restricted. However, the involvement of nonclassical MHC
class Ib (class Ib) molecules in allorecognition has not been addressed. Class Ib genes in
comparison to class Ia are heterogeneous genes with a limited tissue distribution and low
polymorphism (Hofstetter, Sullivan et al. 2011). Both class Ia and most class Ib molecules
have similar structure and need to associate with β2-m in order to be presented at the cells
surface (Goyos, Guselnikov et al. 2007). Class Ib molecules, unlike class Ia, usually present
Ags of a more limited variability or PAMPs including peptides as well as lipids and
glycolipids. In X. laevis there are as many as 20 X. laevis class Ib (XNC) genes divided into 11
subfamilies (Flajnik, Kasahara et al. 1993; Goyos, Ohta et al. 2009). We have demonstrated
that X. laevis has the ability to generate unconventional class Ib restricted anti-15/0 CTLs
after hsp immunization (Goyos, Cohen et al. 2004). There is a possibility that after allograft
or hsp immunization a population of class Ib mediated T cell effectors can arise. These
effector cells can be both CD8 positive as well as CD8 negative as in the case in human and
mice. Interestingly, we recently found expression of the XNC11 gene, in the skin of adult
frogs. The XNC11 gene has a very unique expression pattern; it is almost exclusively
expressed at low levels in the thymus, and at high levels by several thymic lymphoid
tumors including 15/0 (Goyos, Ohta et al. 2009). We now have evidence of faint but
consistent expression of XNC11 in the skin. So far attempts to modify the expression pattern
of XNC11 by any inflammatory stimuli such as LPS and heat killed bacteria or by viral
infection has been unsuccessful. Currently, we are working under the hypothesis that
XNC11 is mainly found in the skin macrophages, Langerhan cells or unconventional T cells.
If this hypothesis is correct, it is possible that XNC11 can present Ags to effector cells during
allograft recognition or is involved in the regulation of specialized or unconventional skin
resident T cells. Our lab is in the process of generating monoclonal antibodies against
several different class Ib molecules including XNC11. These tools will allow us to directly
address if XNC11 or other class Ib genes are involved in immune responses against skin
minor H-Ags.
4. Immune responses and tolerance to skin antigens during larval and
metamorphic stages
Of particular relevance for the immunological aspects of tissue transplantation, is the fact that
unlike mammals, the X. laevis immune system undergoes striking developmental remodeling

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183
twice during its life: first during embryogenesis, and then again during the transition from larva
to adult. The thymus, first colonized by embryonic stem cells a few days after fertilization,
undergoes a second wave of stem cell immigration after losing most of its lymphocytes during
metamorphosis. The embryonic and larval periods of thymocyte differentiation take place in
different environments since during metamorphosis the whole organism is remodeled and
many new proteins are expressed that could be considered antigenic by the larval immune
system. The emerging adult lymphocytes, therefore, are likely to be subjected to a new wave of
negative selection by the adult "self," resulting in a new balance of self-tolerance. In addition,
MHC class I and class II genes are differentially regulated during metamorphosis. Although,
larvae like adults have CD8 T cells, there is no consistent expression of MHC class Ia until
metamorphosis, especially in the thymus.
In this section we will discuss the ability of X. laevis larvae to become tolerant to skin
alloantigens. The induction of allotolerance is a very complex process governed by several
different variables; therefore some conflicting data have been reported concerning its
ontogeny as will be discussed later in detail. One of the differences that may affect tolerance
induction is technical variability. Skin grafting in larvae is similar to grafting in adults but
with several important differences. A first notable difference is that due to its fragility larval
skin does not support transplantation onto a larval hosts and immune rejection is difficult to
distinguish from tissue degeneration (Horton, Horton et al. 1993). For this reason, the use of
ventral adult, instead of larval skin, graft onto larval recipients has been and is still favored.
The adult skin is introduced under the larval skin on the head between the ears of the
recipient (Chardonnens and Du Pasquier 1973). Another difference is that compared to
adults, larvae are transparent. The adult skin is usually firmly fixed one day after
transplantation and the transparent host skin retracts from the graft therefore removing the
overlaying skin is unnecessary (Figure 2). Graft rejection is scored similarly to adults where
the percent of irridophore destruction is determined. Several factors have been shown to
affect the outcome of adult skin transplantation in tadpoles, including the size of the
transplanted skin, as well as the genetic background and the developmental stage. Changes
in temperature do not affect the percent of skin graft rejection; however they do influence
the kinetics of rejection at all developmental stages. At 21oC grafts differing by 1 MHC
haplotype are rejected in 40-55 days in comparison to 25-35 days at 24oC (Cohen, DiMarzo et
al. 1985). Furthermore, size of the graft is very important for tolerance induction and in
general larger grafts have an increased chance to be tolerized. Experiments that use outbred
animals, which are genetically heterogeneous, should be considered with caution when
compared to those that use MHC defined frogs differing by minor H-Ag loci only or by
minor H-Ag loci plus one or two MHC haplotypes.
Another possible source of conflicting results concerns development, especially
metamorphosis, which may play the most important role in generating tolerance.
Metamorphosis in frogs, both initiation and completion, is under the control of the thyroid
hormone (Furlow and Neff 2006; Tata 2006). The thyroid hormone starts being produced
around stage 50, peaks at stage 60, and the levels are back to normal at the end of
metamorphosis (Figure 3). Also there are two different thyroid receptors- α and β. Thyroid
receptor-α is expressed early on after hatching while β comes up concomitantly with the
thyroid hormone (Figure 3). In addition, the action of the thyroid hormone during
metamorphosis is local and not systemic. This means that in certain tissues the changes
associated with metamorphosis may start earlier than in other tissues and this may directly
impact the ontogeny of tolerance. The complexity of the metamorphic transition is likely to
result in marked individual variation even in clonal animals, including the differences in

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184

Fig. 2. Larval skin graft. A. Minor H-Ag-disparate adult skin graft inserted on the head
region of a tadpole. B. Enlarged image of the graft shows the presence of silvery
irridophores, 0% rejection.


*Figure adapted from (Du Pasquier, Schwager et al. 1989) and (Furlow and Neff 2006)
Fig. 3. Overview of the changes occurring during metamorphosis. The different
developmental stages of X. laevis are illustrated including the period of metamorphosis
along with the morphological criteria used. Additionally, the relative expression of the
thyroid hormone as well as the thyroid hormone receptors TR-α and TR-β is depicted. Also,
total thymocyte number, which significantly decreases during metamorphosis, is shown to
correlate with impaired T cell function (MLR activity).
Thyroid Hormone
TR-α
TR-β
TH and TR
Relative
Expression
1x10
3 3x10
4 3x10
5 9x10
5 9x10
5 2x10
6 1x10
7
1.5x10
6 1x10
5
Thymocyte
Numbers
MLR
IMPAIRED
A
B

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185
the immune system. This is indeed observed by the individual variation in morphological
changes (e.g. there are easily several day differences in the time of complete tails loss in cloned
progeny).
In order to address some discrepancies in the literature we will first discuss the induction of
tolerance to minor H-Ag followed by MHC-disparate grafts. The mechanism(s) as well as
possible effector cells involved in allotolerance will also be considered. Finally, we will
provide some perspective on using new tools and methodologies to more conclusively
answer questions associated with tolerance.
4.1 Immune response and allotolerance in X. laevis larvae to minor H skin antigens
X. laevis tadpoles develop allorecognition at stage 49 (12 days post fertilization) which is
accompanied by lymphocytic infiltration of the grafts (Horton 1969). In X. laevis there are
three distinct periods throughout development during which there is a difference in the
immune responses against skin allografts (Chardonnens and Du Pasquier 1973). Those
include the periods before, during and after metamorphosis. Sibling studies showed that at
stage 53 (period before metamorphosis or premetamorphosis) allografts from siblings were
completely rejected (Table 2). Furthermore, larvae at the same stage were also grafted with
allografts from unrelated donors and again the majority of grafts underwent complete
rejection. Moreover, these tadpoles primed by a first skin graft, rejected a second set skin
grafted from the same donor at stage 58 (beginning of metamorphosis period) with
accelerated kinetics suggesting immunological memory similar to second set adult grafts.
Therefore, X. laevis larvae are immunocompetent and can indeed be sensitized against the
grafts. Also young adults (2 months post metamorphosis, postmetamorphic animals)
similarly to larvae were able to completely and acutely reject sibling allografts in 100% of the
cases with mean rejection time of 20 days (Table 2).
In contrast, during metamorphosis (which actually includes the time spanning 15 days
before (st. 58) and about a month after metamorphosis, perimetamorphic animals) up to 50%
of the sibling grafts were actually tolerized (i.e., not rejected), and those that were rejected
followed a very slow rejection kinetics (20 - 80 days for complete rejection) in comparison to
grafts on young larvae or adults (10 - 20 days). This suggests that metamorphosis is a special
developmental time during which immune tolerance can be induced. This could be due to
the fact that the immune system is undergoing complete remodeling and there is reduced
number of lymphocytes. For example, more than 50% thymocytes die during
metamorphosis (Du Pasquier and Weiss 1973). However, several pieces of evidence do not
support the hypothesis that tolerance induction at metamorphosis is due to an insufficient
number of lymphocytes. For instance, regardless whether the grafts are rejected or tolerized
they are infiltrated by lymphocytes (Horton 1969; Bernardini, Chardonnens et al. 1970),
while autografts are not. This implies that the grafts are recognized as non-self although
they are not rejected. Also if tolerance is induced due to the lack of lymphocytes one would
assume that once metamorphosis is completed and the number of lymphocytes is recovered,
these grafts would be rejected. However, that is not the case since these grafts survive for
more than two years. Interestingly, this tolerance can be broken by a third party graft
(Bernardini, Chardonnens et al. 1970), which suggests that some H-Ags may be shared
between the two different grafts. Therefore, once a T cell response is initiated against the
third party graft that response can also be cross-reactive to the tolerized graft. Thus, it

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186
appears that during metamorphosis X. laevis larvae are capable of inducing active
allotolerance against certain H loci shared by the different outbred X. laevis used
(Chardonnens and Du Pasquier 1973; Chardonnens 1975).
On the other hand, allograft tolerance capacity was found to occur also at premetamorphic
stages (Table 2) when more genetically defined and homogeneous animals were used
(DiMarzo 1980; DiMarzo and Cohen 1982a; DiMarzo and Cohen 1982b). Remarkably, both
pre and perimetamorphic larvae of inbred strains were able to induce tolerance against
allografts that were either minor H locus or even MHC-disparate, whereas all
postmetamorphic froglets rejected 100% of the grafts (Table 2). In addition, differences in
rejection depending on the developmental stage were noted; in general younger larvae (st.
47/48) had a higher propensity for becoming tolerant while older larvae (st. 57/58) had the
ability to reject more grafts. In either case, the grafts were rejected with delayed kinetics in
comparison to postmetamorphic animals. Despite the discrepancy on the ontogeny of
allotolerance in X. laevis, it is clear that perimetamorphic larvae are indeed able to become
tolerant to allografts in all cases. The inconsistency may be in part due to individual
variation of thyroid hormone levels and the impact it has on initiation of metamorphosis. It
is also possible that different tolerance mechanisms or regulatory cells are involved in
premetamorphic and metamorphic stages.
4.2 Immune response and allotolerance in X. laevis larvae to MHC-disparate grafts
Although early experiments have indicated that during metamorphosis allotolerance is
induced to minor H-Ags (Chardonnens and Du Pasquier 1973; Chardonnens 1975; DiMarzo
1980; DiMarzo and Cohen 1982a; DiMarzo and Cohen 1982b), other work has revealed that the
immunogenetics of tolerance are complex. Family studies using field-collected outbred adult
X. laevis showed that when grafted during metamorphosis skin that differed by one-MHC
haplotype had prolonged survival (more than 75 days) and the majority of those grafts were
never rejected suggesting long lasting tolerance (Barlow, DiMarzo et al. 1981). In contrast, all
post metamorphic frogs rejected their grafts. Notably, grafts that were rejected by animals
grafted during metamorphosis took at least twice as long for complete rejection. When two-
MHC haplotype-disparate grafts were transplanted during metamorphosis the majority of
grafts were rejected. However, even in this case, grafts did enjoy prolonged survival (more
than 42 days for complete rejection in comparison to 18 days in postmetamorphic hosts, Table
2). In this study even though similar results were obtained using six different families, there
was also variability from one family to another which suggests that genetics play a large role
in the induction of tolerance (Barlow, DiMarzo et al. 1981).
In order to bypass undefined genetic variation, Barlow et al. (1981) used MHC defined
inbred strains of X. laevis. In two different MHC combination when the donor and the
recipient differed by one-MHC haplotype there was 93% graft survival in perimetamorphic
hosts, while 100% grafts were rejected by postmetamorphic recipients (Table 2). When the
graft differed by two-MHC haplotypes only 30% of grafts on perimetamorphic hosts
survived more than 50 days. These data clearly show that during metamorphosis
allotolerance can easily be induced to minor H-Ags as well as one-MHC haplotype-
disparate grafts but rarely to grafts differing by two MHC haplotypes.
In addition to the genetic background of the grafts, as mentioned before, graft size plays an
important role which can tip the balance between rejection and tolerance (Bernardini,
Chardonnens et al. 1970; Chardonnens and Du Pasquier 1973; Barlow and Cohen 1983). In

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general smaller grafts (1-2 mm2) are more readily rejected (can reach up to 90% rejection) in
perimetamorphic animals when there is a one MHC haplotype difference between donor
and host. On the other hand, larger grafts (4-9 mm2) almost always induce tolerance. The
same trend holds true for grafts differing by two MHC haplotypes except the capacity for
rejection is greater since tolerance is not easily induced in this case.
4.3 Mechanisms of allotolerance
Cell transfer approaches were developed to investigate the cellular mechanism responsible
for tolerance induction and maintenance of skin Ags in X. laevis. In a set of experiments,
isogenic frogs carrying minor H-Ag-disparate grafts were injected with thymocytes and
splenocytes from isogenic metamorphosing larvae. This cell transfer induced significantly
delayed skin graft rejection in comparison to control animals that received either adult cells
or were not adoptively transferred with any cells (Du Pasquier and Bernard 1980).
Importantly, multiple injections of metamorphosing immune cells were necessary to achieve
suppression of graft rejection since a single injection did not result in the specific delay.
Additionally, this effect was not permanent; once the injections stopped the grafts were
rejected. Similarly, splenocytes from larvally tolerant frogs that were adoptively transferred
into normal frogs drastically suppressed the rejection of semi-allogeneic grafts (Nakamura,
Maeno et al. 1987). This indicates that the thymus and spleen of metamorphosing animals
contain cells, presumably lymphocytes that have tolerogenic or suppressive activity against
skin minor H-Ags. The need for repeated transfers of these cells to delay graft rejection may
be due to their low frequency and/or short life span in the spleen and thymus of the
metamorphic donor or as a result of the cell transfer. Additionally, the fact that the effect
does not last suggests that tolerance induction to skin grafts during metamorphosis is
regulatory (requiring the presence of regulatory cells) rather than deletional (absence or
deletion of potentially reactive T cells). This possibility is further supported by experiments
showing that maintenance of tolerance to minor H-Ag-disparate skin grafts can be broken
by cyclosphosphamide treatment (Horton, Horton et al. 1989).
In a complementary set of experiments, adoptive cell transfer was used to determine if
tolerance to skin minor H-Ags can be broken (Du Pasquier and Bernard 1980). Splenocytes
from isogenic adults primed by minor H-Ag-disparate skin grafts were adoptively
transferred into isogenic metamorphic recipients with tolerized skin graft genetically
identical to the one used to prime the adults. This adoptive transfer of primed anti-minor H-
Ag lymphocytes was not able to break tolerance in animals that tolerated the grafts for at
least six months. However, if a second graft, identical to the tolerized graft, was placed at
the time of the adoptive transfer, an acute rejection of this graft was initiated but stopped
within 10 days. The result was a graft that was half rejected and half healthy. This suggests
that even though the animal is tolerant to the allograft, the primed transferred cells are able
to cause graft destruction although the number and/or survival of these reactive cells is not
sufficient to complete the rejection. Presumably, if another transfer was done the graft may
be fully rejected. Another possibility is that a subset of regulatory cells home to the skin at
the time of transplantation to induce and maintain tolerance or suppression, and that the
presence of adoptively transferred anti-minor H-Ag lymphocytes prevent or delay the
migration of these regulatory cell in the transplanted skin. Taken together, these data
strongly suggest that tolerance in larvae does not depend upon deletion of alloreactive cells
but rather is maintained by suppressor or regulatory cells that can be adoptively transferred.

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The preponderant role of T cells in larvally-induced allotolerance has been established by
thymectomy. When larvae were thymectomized during early larval life before the migration
of T cell precursors into the thymic anlage, graft rejection of premetamorphic larvae as well
as postmetamorphic froglets was severely impaired (Table 2) demonstrating that without
the thymus alloreactive cells can not develop (Horton and Manning 1972; Barlow and Cohen
1983; Kaye and Tompkins 1983; Nagata and Cohen 1983). Furthermore, the impaired
rejection capacity of premetamorphic larvae that were thymectomized at early
developmental stage can be rescued by implantation of an intact larval or adult thymus
(Arnall and Horton 1986). When these T cell deficient thymectomized larvae are implanted
with an isogenic thymus their capacity to reject both minor H-Ags and MHC-disparate skin
grafts was fully restored. However, when thymectomized larvae were reconstituted with a
thymus that was either MHC-disparate or differed even by minor H-Ags from the host,
rejection was restored only to grafts that were MHC incompatible both to the host and the
thymus donor. Interestingly, these reconstituted animals had an impaired ability to reject
minor H-Ag-disparate grafts. Notably, when MHC incompatible thymi were transplanted,
skin grafts from the same donor were tolerated even though they were able to elicit
proliferative responses against the thymus donor cells in an in vitro MLR (Arnall and Horton
1986). This split tolerance was further substantiated in vivo by injecting irradiated
splenocytes from the thymus donor into reconstituted animals (Arnall and Horton 1987).
Again there was an in vivo response against the transferred splenocytes, although the grafts
were still not rejected. In addition, if live MHC-disparate splenocytes from the thymus
donor were injected, animals developed graft versus host disease and died. Based on these
results it has been concluded that the cytotoxic rather than the proliferative alloresponse is
suppressed in thymus reconstituted thymectomized animals.
In contrast to early thymectomy, late larval thymectomy did not have an effect on rejection
(Table 2). Interestingly, however, thymectomy performed during mid-larval stages
significantly impaired the ability of animals to become tolerant to allografts (Table 2)
(Barlow and Cohen 1983). Moreover, this impairment was dependent on the number of
different MHC haplotypes as well the size of the grafts.
In conclusion, it is clear that X. laevis larvae can become tolerant to allografts although the
developmental stage at which this tolerance can be established, the types and amount of
skin Ags that can be tolerized, and the cellular mechanisms involved still remain unclear.
During metamorphosis the larval immune system is drastically remodeled and larvae
develop tolerance against newly expressed adult self-antigens; therefore, allotolerance may
be a consequence of this period. This does not mean that there are not enough effector cells
or CTLs to reject the grafts since it was clearly demonstrated that MLR in these animals was
positive meaning that T cells can proliferate in the presence of alloantigens. A most likely
scenario is that during metamorphosis there is a general suppressive state where putative T
regulatory cells suppress immune responses against adult tissues in order to avoid auto-
immunity. The thyroid hormone signaling which initiates metamorphosis may be one
plausible factor of individual variability for premetamorphic larvae to become tolerant to
allografts. As explained earlier, thyroid hormone acts locally and it is possible that in some
cases animals start producing larger quantities of this hormone in certain tissues such as the
skin, which would mean that even if the animal is not showing general changes associated
with metamorphosis certain organs or tissues may be. It is possible that particular organs or
tissues will be associated with a more suppressive environment suited for induction of

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tolerance. Although, the tolerance ability of premetamorphic stages may result from the
establishment of this regulatory process that would peak during the metamorphic climax, it
is also possible that it results from a different mechanism such as insufficient or immature T
cell response. This can especially be the case at earlier larval stages (eg. st. 53) when
peripheral T cells are still few (~10,000 to 100,000).


Premetamorphic
Perimetamorphic
Postmetamorphic
Developmental Stage
47 - 58
58 – 1 month post
metamorphosis
2 months post
metamorphosis
Minor-H-Ag
disparate grafts
Tolerance induction
+*
Tolerance induction
+++
Rejection
100%
1 MHC haplotype
disparatre grafts
Tolerance induction
+*
Tolerance induction
+++
Rejection 100%
2 MHC haplotypes
disparate grafts
Rejection
Rejection
MST 42
Rejection
MST 18
Early larval life
thymectomy
Impaired graft
rejection
No effect
Impaired graft
rejection
Mid larval life
thymectomy
No effect
Impaired tolerance
No effect
Late larval life
thymectomy
No effect
No effect
No effect
Table 2. Summary of tolerance induction in X. laevis larvae and the effects of thymectomy.
*Discrepancy between different studies. Chardonnens and Du Pasquier (1973) reported that
premetamorphic larvae were capable of rejecting grafts similary to postmetamophic froglets
while several reports from the Cohen lab reported that premetamoprhic larvae (as early as
stage 47) had the capacity to induce tolerance.

+, low incidence of tolerance induction

+++, high incidence of tolerance induction
MST: Mean Survival Time (in days)
4.4 New tools to study tolerance
During metamorphosis, as described before, X. laevis larvae have to become tolerant to
newly emerging adult self-antigens, which can be recognized as foreign by the larval
immune system, and lead to autoimmunity. It is highly plausible that the same mechanisms
that are involved in allotolerance are also involved in tolerance to self; therefore, X. laevis
provides a unique model to study autoimmunity. This is illustrated by the observation that
certain animals that are thymectomized at stage 56 and than transplanted with MHC-
disparate graft have a high incidence of death due to “red disease” (Barlow and Cohen
1983). This is a disease that is autoimmune in nature and is characterized by cutaneous

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hemorrhaging around the eyes and legs and by internal hemorrhaging in the liver and
kidneys.
There is still much unknown regarding the mechanism(s) associated with suppression
during allotolerance. For instance, we still don’t know which effector cells are involved,
even though we speculate that they might be T regulatory cells. The mode and site of action
of these regulatory cells is also not defined. Using some newly generated tools and
techniques we can start to answer some of these questions. Initially, it will be of great
interest to immunize perimetamorphic larvae with hsps carrying either MHC or minor H-
Ags and then look for immunological memory toward skin grafts (same genetic background
as the Ags carried by the hsp) transplanted post metamorphosis. These experiments will
show if the Ags alone are capable of generating tolerance by possibly negatively selecting
reactive thymocytes in the thymus or by generating specific T regulatory cells toward those
particular Ags.
To further investigate the effector cells involved in these responses, we will be able to use
transgenic animals. Transgenesis in X. laevis has been widely used over the last several years
and the most common technique used is REMI (Restriction Enzyme Mediated Integration)
which requires integration of the transgene into the sperm nuclei which are than
transplanted into unfertilized eggs (Kroll and Amaya 1996). However, for generating
transgenics with our isogenic clones this technique is not useful since they are maintained
by gynogenesis. Therefore, our laboratory adapted a new transgenic approach using the I-
SceI Meganuclease (Ogino, McConnell et al. 2006; Pan, Chen et al. 2006). This method
requires a plasmid that carries the transgene of interested flanked by I-SceI recognition sites
which is digested by the meganuclease and the entire digest is injected into activated eggs.
This causes stable integration of the transgene into one to two different sites in the genome.
An added advantage of working with the clones is that all of the progeny of a given founder
will be transgenic which will give us enough larvae for experimentation.
As mentioned in the introduction the genome of the X. laevis sister species, X. tropicalis has
been fully sequenced and annotated. This has provided new possibilities to identify immune
genes using, for example, gene synteny (Robert and Ohta 2009). Furthermore, the full
sequencing of the X. laevis genome using the homozygous inbred strain J from our resource
center is now ongoing and the first assembly is already available (R. Harland, personal
communication). With these new available resources, it will become possible to identify and
isolate regulatory regions of immunologically relevant genes and produce transgenic
reporter animals expressing fluorescent reporter genes (such as GFP) under the control of
these regulatory regions as in mouse and zebra fish (Smith, Ataliotis et al. 2005; Doherty,
Johnson Hamlet et al. 2007; Hall, Flores et al. 2009). X. laevis transgenic lines expressing, for
example, GFP under the transcriptional control of the CD4 (CD4 T cells) or the Foxp3 (T
regulatory cells) promoter regions (i.e., homologs of these genes have already been
identified in the X. tropicalis genome), would permit to localize and follow the fate of these
cells in transplanted skin tissues during rejection and tolerance induction. Since tadpoles are
transparent this will allow us to easily visualize the trafficking pattern of these cells and see
if those are the effector cells that are found infiltrating tolerated grafts. Also we can sort
these cells based on their GFP expression and than adoptively transfer them into
postmetamorphic frogs to check if indeed these cells are involved in the induction of
tolerance. Additionally, by using cloned animals we will be able to pool enough larval cells