Signaling through C5a receptor and C3a receptor diminishes function of murine natural regulatory T cells.

Thymus-derived (natural) CD4(+) FoxP3(+) regulatory T cells (nT reg cells) are required for immune homeostasis and self-tolerance, but must be stringently controlled to permit expansion of protective immunity. Previous findings linking signals transmitted through T cell-expressed C5a receptor (C5aR) and C3a receptor (C3aR) to activation, differentiation, and expansion of conventional CD4(+)CD25(-) T cells (T conv cells), raised the possibility that C3aR/C5aR signaling on nT reg cells could physiologically modulate nT reg cell function and thereby further impact the induced strength of T cell immune responses. In this study, we demonstrate that nT reg cells express C3aR and C5aR, and that signaling through these receptors inhibits nT reg cell function. Genetic and pharmacological blockade of C3aR/C5aR signal transduction in nT reg cells augments in vitro and in vivo suppression, abrogates autoimmune colitis, and prolongs allogeneic skin graft survival. Mechanisms involve C3a/C5a-induced phosphorylation of AKT and, as a consequence, phosphorylation of the transcription factor Foxo1, which results in lowered nT reg cell Foxp3 expression. The documentation that C3a/C3aR and C5a/C5aR modulate nT reg cell function via controlling Foxp3 expression suggests targeting this pathway could be exploited to manipulate pathogenic or protective T cell responses.

sCD200 present in mice receiving cardiac and skin allografts causes immunosuppression in vitro and induces Tregs.

BACKGROUND: CD200 overexpression in transgenic mice increases skin, cardiac, and renal allograft survival. Elevated levels of soluble CD200 (sCD200) are found in the serum of cancer individuals. We investigated whether sCD200 levels increase in mice with prolonged graft survival. METHODS: Control or CD200 BL/6 recipients of BALB/c cardiac or skin grafts received low-dose rapamycin (0.5 mg/kg) at 36-hr intervals, a dose shown previously not to augment the survival of control grafts or alter the host antigraft immunity or graft gene expression profiles. Separate groups received high-dose rapamycin (1.5 mg/kg). Serum was obtained at 8, 15, and 80 days after grafting and assayed for sCD200 (enzyme-linked immunosorbent assay), for the suppression of immunity in mixed leukocyte cultures (MLCs), for the induction of regulatory T cells able to suppress cytotoxic T lymphocyte induction in MLCs, and for the ability to transfer graft survival to naïve recipients. RESULTS: Both CD200 and conventional mice with early enhanced graft survival had increased levels of sCD200 in serum, which induced Tr1 able to suppress MLCs. Suppression was abolished after passage of serum over a CD200 immunoadsorbent column. Dendritic cells maturing in the presence of sCD200 serum could induce populations of Foxp3 regulatory T cells able to suppress MLCs in vitro. In CD200 mice with long-term surviving cardiac (skin) allografts in the absence of continued transgene induction (>80 days [>35 days]), sCD200 levels returned to baseline, with no loss of grafts, but sera were unable to suppress MLCs in vitro. sCD200 serum adoptively transferred increased graft survival to naïve mice. CONCLUSION: We conclude that monitoring sCD200 at early times after engraftment may predict allograft survival.

Perforator flap from proximal lateral leg for head and neck reconstruction.

INTRODUCTION: Perforator flap transfer has the benefits of minimal donor site morbidity and customization in design. This study reports on the proximal lateral leg perforator (PLLP) flap transfer for head and neck reconstruction. MATERIALS AND METHODS: From January 2000 through December 2009, 18 patients underwent head and neck reconstruction with the PLLP flap, including 16 males and 2 females ranging in age between 32 and 80 years old. The mean follow-up time was 9.8 months. RESULTS: Mother vessels of PLLP flaps can arise 77.8% (14/18) from the peroneal system, 11.1% (2/18) from the posterior tibial system, or 11.1% (2/18) from the common popliteal system. The skin dimension ranges from 4 cm × 7 cm to 6.5 cm × 18 cm. The mean of the perforator size is 1.96 mm (1.5 mm to 2.3 mm). The mean of the pedicle length is 6.96 cm (4.5 cm to 10 cm). All flaps survived completely except one flap that had partial necrosis. CONCLUSIONS: The PLLP flap for head and neck reconstruction offers versatility in design, diverse tissues for composition, a two-team approach, and negligible donor site morbidity, and it spares major vessels. The unpredictable pedicle length and the feasibility for microvascular anastomosis in the perforator level can be adapted with further experience and refining techniques.

Using the pedicle flap for taking pressure off the pedicle in distally based skin flaps.

This report describes our experience using the pedicle flap for taking pressure off the pedicle in distally based skin flaps. Between July 2007 and March 2010, distally based skin flaps were used to treat 34 patients with soft tissue defects in the lower leg, ankle, and foot. A pedicle flap was used to take pressure off the pedicle in all of these flaps. There were 21 men and 13 women with a mean age at surgery of 47 years (range, 21 to 67 years). The sizes of the soft tissue defects varied from 5.0 cm × 2.0 cm to 17.0 cm × 10.0 cm. The pedicle flap sizes varied from 1.0 cm × 1.0 cm to 5.0 cm × 3.5 cm. All flaps survived completely except three, which suffered a marginal partial flap necrosis due to venous congestion. These flaps healed by secondary intention after changing the dressing. Using the pedicle flap takes pressure off the pedicle and facilitates primary closure after transposition of the distally based skin flaps, which is a simple and effective method to prevent complications of these flaps.

Enhancement of venous drainage with vein stripper for reversed pedicled neurocutaneous flaps.

The flaps based on the vascular axis of superficial sensitive cutaneous nerves had gained increased popularity in reconstructive surgery because of such major advantages as preservation of major extremity arteries and avoidance of microsurgical procedures. However, postoperative venous congestion resulting in partial or total necrosis is still a common problem for these flaps. The aim of the current study is to introduce a new method for reducing the postoperative venous congestion of neural island flap with the results of reconstruction of the soft tissue defects of foot and ankle. This method was used to treat 19 patients with various chronic soft tissue defects of the foot and ankle between 2011 and 2012. We observed that the novel method presented in this report enables effective venous drainage, solving the postoperative venous congestion problem of these flaps.

Microdialysis: characterisation of haematomas in myocutaneous flaps by use of biochemical agents.

Metabolic markers are measured by microdialysis to detect postoperative ischaemia after reconstructive surgery with myocutaneous flaps. If a haematoma develops around the microdialysis catheter, it can result in misinterpretation of the measurements. The aim of the present study was to investigate whether a haematoma in a flap can be identified and dissociated from ischaemia, or a well-perfused flap, by a characteristic chemical profile. In 7 pigs, the pedicled rectus abdominal muscle flap was mobilised on both sides. A haematoma was made in each flap and two microdialysis catheters were placed, one in the haematoma, and the other in normal tissue. One flap was made ischaemic by ligation of the pedicle. For 6 hours, the metabolism was monitored by measurement every half-an-hour of the concentrations of glucose, lactate, pyruvate, and glycerol from all 4 catheters. After 3 hours of monitoring, intravenous glucose was given as a challenge test to identify ischaemia. The non-ischaemic flap could be differentiated from the ischaemic flap by low glucose, and high lactate, concentrations. It was possible to identify a catheter surrounded by a haematoma in ischaemic as well as non-ischaemic muscle from a low or decreasing concentration of glucose together with a low concentration of lactate. All four sites could be completely dissociated when the concentrations of glucose and lactate were evaluated and combined with the lactate:glucose ratio and a flow chart. The challenge test was useful for differentiating between haematomas in ischaemic and non-ischaemic tissue.

Unlinked memory helper responses promote long-lasting humoral alloimmunity.

Essential help for long-lived alloantibody responses is theoretically provided only by CD4 T cells that recognize target alloantigen, processed and presented by the allospecific B cell. We demonstrate that in an alloresponse to multiple MHC disparities, cognate help for class-switched alloantibody may also be provided by CD4 T cells specific for a second "helper" alloantigen. This response was much shorter-lived than when help was provided conventionally, by Th cell recognition of target alloantigen. Nevertheless, long-lasting humoral alloimmunity developed when T cell memory against the helper alloantigen was first generated. Costimulatory blockade abrogated alloantibody produced through naive Th cell recognition of target alloantigen but, crucially, blockade was ineffective when help was provided by memory responses to the accessory helper alloantigen. These results suggest that memory Th cell responses against previously encountered graft alloantigen may be the dominant mechanism for providing help to generate new specificities of alloantibody in transplant patients receiving immunosuppression.

A combination of local inflammation and central memory T cells potentiates immunotherapy in the skin.

Adoptive T cell therapy uses the specificity of the adaptive immune system to target cancer and virally infected cells. Yet the mechanism and means by which to enhance T cell function are incompletely described, especially in the skin. In this study, we use a murine model of immunotherapy to optimize cell-mediated immunity in the skin. We show that in vitro-derived central but not effector memory-like T cells bring about rapid regression of skin-expressing cognate Ag as a transgene in keratinocytes. Local inflammation induced by the TLR7 receptor agonist imiquimod subtly yet reproducibly decreases time to skin graft rejection elicited by central but not effector memory T cells in an immunodeficient mouse model. Local CCL4, a chemokine liberated by TLR7 agonism, similarly enhances central memory T cell function. In this model, IL-2 facilitates the development in vivo of effector function from central memory but not effector memory T cells. In a model of T cell tolerogenesis, we further show that adoptively transferred central but not effector memory T cells can give rise to successful cutaneous immunity, which is dependent on a local inflammatory cue in the target tissue at the time of adoptive T cell transfer. Thus, adoptive T cell therapy efficacy can be enhanced if CD8(+) T cells with a central memory T cell phenotype are transferred, and IL-2 is present with contemporaneous local inflammation.

A study of the imbalance in B cell-expressed nucleoside triphosphate diphosphohydrolase 1-induced ADP degradation in graft injury during acute antibody-mediated rejection.

OBJECTIVE: To study the effects and mechanisms of the imbalance in B cell-expressed nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1)-induced ADP degradation on graft injury during acute antibody-mediated rejection (AMR). METHODS: The acute AMR animal model was established in male NTPDase 1-wild-type Balb/c nude mice. The levels of NTPDase 1 in B cells and NTPDase1 mRNA in grafted skin, changes in platelet activation markers and average platelet velocities were determined by luciferin/luciferase enzymatic, real-time fluorescent quantitative PCR, flow cytometry and inverted microscope. The pathological changes in grafted skin were observed by electron microscopy. The effects of pretreatment with different doses of exogenous NTPDase 1 on platelet activation and graft injury were studied. RESULTS: The expression of B-cell NTPDase 1 was significantly increased at 30 min after the induction of acute AMR and restored to baseline levels after 7 days. The levels of NTPDase 1 mRNA in grafted skin were decreased at 30 min after the induction of acute AMR. After the induction of acute AMR, the levels of platelet activation markers increased significantly, whereas the average platelet velocity significantly decreased. After pretreatment with exogenous NTPDase 1, the expression of platelet activation markers significantly decreased, the average velocity of platelets increased significantly, and the necrosis of grafted skin and inflammatory reaction significantly reduced. CONCLUSIONS: An imbalance in the NTPDase 1-induced degradation of extracellular ADP may be a major cause of graft injury in acute AMR. Pretreatment with exogenous NTPDase 1 may effectively inhibit platelet activation and protect grafted skin.

Allograft rejection is restrained by short-lived TIM-3+PD-1+Foxp3+ Tregs.

Tregs play a pivotal role in inducing and maintaining donor-specific transplant tolerance. The T cell immunoglobulin and mucin domain-3 protein (TIM-3) is expressed on many fully activated effector T cells. Along with program death 1 (PD-1), TIM-3 is used as a marker for exhausted effector T cells, and interaction with its ligand, galectin-9, leads to selective death of TIM-3+ cells. We report herein the presence of a galectin-9-sensitive CD4+FoxP3+TIM-3+ population of T cells, which arose from CD4+FoxP3+TIM-3- proliferating T cells in vitro and in vivo and were often PD-1+. These cells became very prominent among graft-infiltrating Tregs during allograft response. The frequency and number of TIM-3+ Tregs peaked at the time of graft rejection and declined thereafter. Moreover, these cells also arise in a tolerance-promoting donor-specific transfusion model, representing a pool of proliferating, donor-specific Tregs. Compared with TIM-3- Tregs, TIM-3+ Tregs, which are often PD-1+ as well, exhibited higher in vitro effector function and more robust expression of CD25, CD39, CD73, CTLA-4, IL-10, and TGF-ß but not galectin-9. However, these TIM-3+ Tregs did not flourish when passively transferred to newly transplanted hosts. These data suggest that a heretofore unrecognized graft-infiltrating, short-lived subset of Tregs can restrain rejection.

Pulmonary CCL18 recruits human regulatory T cells.

CCL18 is both a constitutively expressed and an inducible chemokine, whose role in the inflammatory reaction is poorly known. The aim of this study was to evaluate whether CCL18 has the capacity to attract human T cells with a regulatory function (regulatory T cells [Treg]). Results from chemotaxis assays performed on different types of Treg showed that CD4(+)CD25(+)CD127(low) cells, but neither T regulatory type 1 clones nor Treg differentiated in vitro with anti-CD3/CD46 mAbs, were recruited by CCL18 in a dose-dependent manner. CCL18-recruited memory CD4(+) T cells were enriched in CD25(high), CD25(+)CD127(low), latency-associated peptide/TGF-ß1, and CCR4-expressing T cells, whereas there was no enrichment in Foxp3(+) cells as compared with controls. Stimulated CCL18-recruited memory T cells produced significantly increased amounts of the regulatory cytokines IL-10 and TGF-ß1, as well as IL-4, but not IFN-γ and IL-17. Cell surface CCL18 binding was found predominantly on IL-10(+) (26.3 ± 5.8%) and on a few latency-associated peptide/TGF-ß1(+) (18.1 ± 1.9%) and IL-4(+) (14.5 ± 2.9%) memory T cells. In an in vivo model of SCID mice grafted with human skin and reconstituted with autologous PBMCs, the intradermal injection of CCL18 led to the cutaneous recruitment of CD4(+), CD25(+), and IL-10(+) cells, but not Foxp3(+) cells. Furthermore, CCL18-recruited memory T cells inhibited the proliferation of CD4(+)CD25(-) effector T cells through an IL-10-dependent mechanism. These data suggest that CCL18 may contribute to maintaining tolerance and/or suppressing deleterious inflammation by attracting memory Tregs into tissues, particularly in the lung, where it is highly and constitutively expressed.

Targeting of the hNC16A collagen domain to dendritic cells induces tolerance to human type XVII collagen.

Antibodies, specific to murine DEC205, can be used to target antigens to dendritic cells. The immunodominant domain of human type XVII collagen, hNC16A, was fused to this antibody (DEC-hNC16A) and was administered as expression plasmid by gene gun transfection with the aim of inducing tolerance to human type XVII collagen in a skin transplantation model. Mice transfected with DEC-hNC16A were challenged with skin grafts from transgenic mice engineered to express human type XVII collagen. Graft survival was either prolonged or grafts were accepted infinitely (33% and 16%, respectively) upon treatment with DEC-hNC16A while 100% of grafts were rejected in untreated controls. Graft acceptance was associated with the absence of a CD4+ infiltrate and a dense CD8+ T-cell infiltrate and was not strictly dependent on antibody production. Our results show that DEC-hNC16A targets dendritic cells in vivo leading to prolonged survival of transgenic skin grafts. This indicates that DEC205-targeting may be used for the induction of tolerance to skin antigens, which would increase the chances of successful skin gene therapy of epidermolysis bullosa patients.

Impact of portable duplex ultrasonography in head and neck reconstruction.

We have reviewed the use of portable duplex ultrasonography (PDU) in 12 patients who underwent soft tissue/bone head and neck reconstruction, aiming to determine its role in the design and management of such complex cases. According to our data, there were modifications either of the surgical plan or of patient's management, based on PDU findings, in 9 (75%) of 12 patients. The use of ultrasound directed to subtle modifications in 3 patients (25%) but to significant changes of the surgical plan in the other 3 patients (25%). Also, the use of duplex ultrasound impacted significantly the postoperative management in 4 patients (33.33%). Thus, significant impact of PDU in patient's treatment was recorded in 58.33% of cases. Portable ultrasound represents generally available method for preoperative, intraoperative, and postoperative diagnosis and decision making in free tissue transfer, hence could replace in the future the unidirectional Doppler in the hands of head and neck surgeons.

Effects of thermal water on skin regeneration.

An experimental study was carried out in an animal (New Zealand white rabbit) wound model to evaluate any effects of a hypotonic, bicarbonate-calcium-magnesium mineral water (Comano thermal water) on skin regeneration, comparing the healing rate of split-thickness skin graft donor sites treated with the thermal water wet dressing versus a standard petrolatum gauze dressing versus a saline solution wet dressing. The study was performed in two steps; an overall of 22 animals were enrolled in the study. The wound healing progress was evaluated both by the surgeons and by the histologists. Sixty-four punch biopsies were examined in all. The histological samples were examined after staining with haematoxylin and eosin, Masson's and orcein staining and under a transmission electron microscope. The data were statistically analysed. The Comano thermal water proved to improve skin regeneration, not only by increasing keratinocyte proliferation and migration but also favourably modulating the regenerated collagen and elastic fibres in the dermis. We propose that the results of the topical treatment with the thermal water could be due to the favourable combination of a local wet environment with an anti-inflammatory action and that the regenerative properties of Comano thermal water observed in rabbits could also be applied for human use.

Use of autologous skin equivalents with artificial dermal matrix (integra) in donor site coverage in radial forearm free flaps: preliminary cases.

PURPOSE: The radial forearm flap is one of the most commonly used methods for intraoral reconstruction in oral carcinoma surgery. One of its disadvantages is the residual functional and unaesthetic defect in the donor site. The objective of this report is to describe preliminary cases of a novel method to cover such donor sites based on the use of autologous skin equivalents (ASEs) and an artificial dermal matrix (Integra, Prim, Barcelona, Spain). MATERIALS AND METHODS: The donor sites of 2 patients were treated with the artificial dermal matrix after raising a radial forearm flap. A skin biopsy and a blood sample were taken to construct an ASE. After 3 weeks, the ASE was applied over the dermal template and left to heal. The functional and esthetic results were recorded. RESULTS: Good functional and esthetic results were achieved, with correct wrist motility, although a natural skin color could not be achieved. Neither the Integra nor the ASE was rejected. Total wound coverage was achieved at 4 months, and completely normal skin was observed at 6 months. CONCLUSIONS: This technique of combining an artificial dermal matrix with an ASE could be an alternative method to cover the donor sites of radial forearm flaps.

Haptoglobin activates innate immunity to enhance acute transplant rejection in mice.

Immune tolerance to transplanted organs is impaired when the innate immune system is activated in response to the tissue necrosis that occurs during harvesting and implantation procedures. A key molecule in this immune pathway is the intracellular TLR signal adaptor known as myeloid differentiation primary response gene 88 (MyD88). After transplantation, MyD88 induces DC maturation as well as the production of inflammatory mediators, such as IL-6 and TNF-α. However, upstream activators of MyD88 function in response to transplantation have not been identified. Here, we show that haptoglobin, an acute phase protein, is an initiator of this MyD88-dependent inflammatory process in a mouse model of skin transplantation. Necrotic lysates from transplanted skin elicited higher inflammatory responses in DCs than did nontransplanted lysates, suggesting DC-mediated responses are triggered by factors released during transplantation. Analysis of transplanted lysates identified haptoglobin as one of the proteins upregulated during transplantation. Expression of donor haptoglobin enhanced the onset of acute skin transplant rejection, whereas haptoglobin-deficient skin grafts showed delayed acute rejection and antidonor T cell priming in a MyD88-dependent graft rejection model. Thus, our results show that haptoglobin release following skin necrosis contributes to accelerated transplant rejection, with potential implications for the development of localized immunosuppressive therapies.

Comparison of sensory recovery at the subfascial and suprafascial donor sites of the free radial flap.

The radial flap may be raised using a subfascial or suprafascial approach. The latter donor site is associated with fewer healing complications. We retrospectively evaluated the quality of sensory recovery within two comparable groups of 30 patients with subfascial and suprafascial donor sites. When considering the two groups, two-point discrimination was the modality most commonly reduced, with 97% of patients in both groups having reduced sensation in at least one anatomical zone. Sensation of sharp touch was most often lost; 90% in the subfascial and 83% in the suprafascial groups lost sensation in at least one anatomical zone. Roughly half the patients had reduced perception of light touch (43% and 50%), whilst perception of heat (27% and 17%) and cold (33% and 27%) were lost least often. At least one modality in at least one anatomical zone was lost or reduced in all patients, and roughly two-thirds (73% and 63%) had a reduction in 3 or more. The only significant difference between the donor and non-donor arms was reduced perception of sharp touch in the anterior forearm in both groups (p<0.001). Perception at the two sites (including the anatomical snuff box) was similar except for superior thenar palmar light touch (p=0.015) in the suprafascial group, which may indicate injury to the thenar cutaneous sensory branches during subfascial dissection.