Arlette De Coninck

Therapeutic vaccination with an autologous mRNA electroporated dendritic cell vaccine in patients with advanced melanoma.

The immunostimulatory capacity of dendritic cells is improved by co-electroporation with mRNA encoding CD40 ligand, constitutively active toll-like receptor 4, and CD70 (TriMix-DC). This pilot clinical trial evaluated the feasibility, safety, and immunogenicity of a therapeutic vaccination containing autologous TriMix-DC co-electroporated with mRNA encoding a human leukocyte antigen class II-targeting signal linked to 1 of 4 melanoma-associated antigens (MAGE-A3, MAGE-C2, tyrosinase, and gp100) in patients with advanced melanoma. Thirty-five American Joint Committee on Cancer stage III/IV melanoma patients received autologous TriMix-DC (4 administrations 2 weeks apart). Immune monitoring was performed by evaluating skin biopsies of delayed type IV hypersensitivity (DTH) reactions for presence of vaccinal antigen-specific DTH-infiltrating lymphocytes (DIL). Thereafter, patients could receive interferon-alpha-2b (IFN-&agr;-2b) 5 MU subcutaneously 3 times weekly and additional TriMix-DC every 8 weeks. TriMix-DC-related adverse events comprised grade 2 local injection site reactions (all patients), and grade 2 fever and lethargy (2 patients). Vaccinal antigen-specific DIL were found in 0/6 patients tested at vaccine initiation and in 12/21 (57.1%) assessed after the fourth vaccine. A positive postvaccination DTH test correlated with IL-12p70 secretion capacity of TriMix-DC. No objective responses to TriMix-DC alone were seen according to RECIST. Twenty-nine patients received IFN-&agr;-2b after the fourth vaccine without unexpected adverse events. During TriMix-DC/IFN-&agr;-2b combination therapy, 1 partial response and 5 stable disease (disease control of >6 months with regression of metastases) were observed in 17 patients with evaluable disease at baseline. In conclusion, this study demonstrated that therapeutic vaccination with autologous TriMix-DC is feasible, safe, and immunogenic and can be combined with sequential IFN-&agr;-2b.

Randomized trial comparing cryopreserved cultured epidermal allografts with hydrocolloid dressings in healing chronic venous ulcers

BACKGROUND Cultured epidermal allografts have been successfully used to treat a variety of wounds. Their postulated mechanism of action is through release of cytokines that stimulate epithelialization. On the basis of previous experience we expected ulcers treated with cryopreserved cultured allografts (CCAs) to be healed by 6 weeks. Hydrocolloid dressings (HCDs) have also been reported to be effective in the treatment of venous ulcers. OBJECTIVE Our purpose was to compare the effectiveness of CCAs with HCDs in healing chronic venous ulcers. METHODS Forty-three patients with 47 ulcers were enrolled in a randomized controlled trial. Ulcers not healed by 6 weeks were changed to the other treatment. RESULTS No difference in the number of healed ulcers between the two groups was observed at 6 weeks. Healing rate, percent reduction of initial ulcer size, and radial progression toward wound closure were significantly greater for CCAs than for HCDs. Pain relief was not significantly different. CONCLUSION CCAs achieve more rapid healing and greater reduction in ulcer size than HCDs.

Feeder layer- and animal product-free culture of neonatal foreskin keratinocytes: improved performance, usability, quality and safety

Since 1987, keratinocytes have been cultured at the Queen Astrid Military Hospital. These keratinocytes have been used routinely as auto and allografts on more than 1,000 patients, primarily to accelerate the healing of burns and chronic wounds. Initially the method of Rheinwald and Green was used to prepare cultured epithelial autografts, starting from skin samples from burn patients and using animal-derived feeder layers and media containing animal-derived products. More recently we systematically optimised our production system to accommodate scientific advances and legal changes. An important step was the removal of the mouse fibroblast feeder layer from the cell culture system. Thereafter we introduced neonatal foreskin keratinocytes (NFK) as source of cultured epithelial allografts, which significantly increased the consistency and the reliability of our cell production. NFK master and working cell banks were established, which were extensively screened and characterised. An ISO 9001 certified Quality Management System (QMS) governs all aspects of testing, validation and traceability. Finally, as far as possible, animal components were systematically removed from the cell culture environment. Today, quality controlled allograft production batches are routine and, due to efficient cryopreservation, stocks are created for off-the-shelf use. These optimisations have significantly increased the performance, usability, quality and safety of our allografts. This paper describes, in detail, our current cryopreserved allograft production process.

Healing of full-thickness wounds in pigs: effects of occlusive and non-occlusive dressings associated with a gel vehicle.

This study, based upon a pig model, was conducted to investigate the effects of moist and dry healing conditions on wound closure (epithelialization, granulation tissue, contraction) of full-thickness wounds. Thirty-two full-thickness square wounds (3 cm x 3 cm) covered with either an occlusive polyurethane dressing (Tegaderm) or a non-occlusive dressing (Melolin) were evaluated. The effect of the presence or the absence of a gel (3% Idroramnosan) was also investigated with both dressings. The dressings were renewed twice a week. The time required for wound closure was 19.2 +/- 1.6 days for Tegaderm and 26.6 +/- 3.0 days (means +/- SD) for Melolin, respectively. The healing time of the full-thickness porcine wounds was significantly (P < 0.001) reduced by the occlusive dressing. Equivalent results were found with the 3% gel, indicating that the gel can be used as a neutral vehicle. The healing rate, calculated according to Gilmans method, was also significantly (P < 0.001) enhanced by the occlusive dressing. This progression was 0.073 +/- 0.004 cm/day and 0.050 +/- 0.009 cm/day (means +/- SD) for Tegaderm and Melolin, respectively. The contribution of contraction to wound closure was similar in all wounds, indicating that the occlusive dressing did not have an effect on wound contraction. Histological evaluation was performed on full-thickness skin biopsies of whole wound harvested from the time of wound closure to 3 months after. At any time point, no significant histological variations were observed between the different treated wounds. This study demonstrates in a porcine model that for full-thickness wounds, as for split-thickness wounds, occlusive dressing enhances healing rate and shortens the time for wound repair. The shortened healing time is a function primarily of the effect of occlusive dressing on epithelialization, especially the third phase of wound resurfacing.

Allogeneic cultured epidermal grafts heal chronic ulcers although they do not remain as proved by DNA analysis

To investigate whether allogeneic cultured keratinocytes are rejected or not, and to find out how beneficial their effect on wound healing could be, patients with chronic ulcers were grafted with allogeneic cultured human keratinocytes. In order to examine the epidermal origin of the healed wound, DNA analysis was performed and compared to donor and recipient blood-cell DNA. Healing was observed in 84% of the grafted ulcers by granulation tissue stimulation and would edge effect. In little time 60% of the grafted chronic ulcers healed completely. Although no rejection was observed, DNA analysis revealed that the grafted allogeneic keratinocytes were finally replaced by the patients own epidermis. This study confirmed that cultured allogeneic keratinocytes that have been grafted on ulcers, play an important role in the wound healing process.

Glycerol treatment as recovery procedure for cryopreserved human skin allografts positive for bacteria and fungi

Human donor skin allografts are suitable and much used temporary biological (burn) wound dressings. They prepare the excised wound bed for final autografting and form an excellent substrate for revascularisation and for the formation of granulation tissue. Two preservation methods, glycerol preservation and cryopreservation, are commonly used by tissue banks for the long-term storage of skin grafts. The burn surgeons of the Queen Astrid Military Hospital preferentially use partly viable cryopreserved skin allografts. After mandatory 14-day bacterial and mycological culture, however, approximately 15% of the cryopreserved skin allografts cannot be released from quarantine because of positive culture. To maximize the use of our scarce and precious donor skin, we developed a glycerolisation-based recovery method for these culture positive cryopreserved allografts. The inactivation and preservation method, described in this paper, allowed for an efficient inactivation of the colonising bacteria and fungi, with the exception of spore-formers, and did not influence the structural and functional aspects of the skin allografts.

Potential release of aluminum and other metals by food-grade aluminum foil used for skin allograft cryo preservation

Since 1991, the skin bank of the Queen Astrid Military Hospital uses food-grade aluminum foil as a primary support for storing cryo preserved human donor skin (511 donors). The possible release of heavy metals into the cryo preservation media (30% (v/v) glycerol in physiological water) and the possible impact this release could have on the quality of the cryo preserved donor skin was evaluated. Aluminum was the principal detection target. Possible contaminants of the aluminum foil as such (arsenic, cadmium, chromium and lead) were also investigated. The evaluation was set up after a Belgian Competent Authority inspection remark. Aluminum was detected at a concentration of 1.4 mg/l, arsenic and lead were not detected, while cadmium and chromium were detected in trace element quantities. An histological analysis revealed no differences between cryo preserved and fresh donor skin. No adverse reactions in patients, related to the presence of aluminum or heavy metal traces, were reported since the introduction of the cryo preserved donor skin in our burn wound centre.

Hemorrhagic regression of melanoma metastases during therapeutic vaccination: a report of three cases.

Melanoma metastases are characterized by pronounced neo-angiogenesis and spontaneous bleeding frequently occurring within central nervous system metastases. Clinically apparent spontaneous hemorrhage within subcutaneous melanoma metastases, however, is a rare event that coincides with progression of such metastases. We report, to our knowledge the first observation, on regression of subcutaneous metastases with hemorrhage of the overlying skin in three patients with stage IV melanoma who participated in clinical trials on therapeutic vaccination. In two patients, loss of arterial flow on Doppler ultrasound imaging was documented in the metastasis at the time of hematoma formation. One patient suffered from an intracranial hemorrhage in a subcentimetric brain metastasis coincident with the hemorrhagic regression of some of his skin metastases.

Healing of full‐thickness wounds treated with lyophilized cultured keratinocyte cell lysate in genetically diabetic mice

The effect of a lyophilized cell lysate prepared from cultured human keratinocytes on the healing of full‐thickness wounds was evaluated in an impaired healing model. Full‐thickness wounds (8 mm in diameter) were made on the dorsal areas of female genetically diabetic mice C57 BL/KsJ (db/db) and their normal (db/+) littermates. Wounds were covered with an occlusive polyurethane film dressing and were treated for 5 days either with the lyophilized cell lysate from cultured human keratinocytes prepared in phosphate‐buffered saline solution or with phosphate‐buffered saline solution. In normal (db/+) mice, all wounds were closed 16 days after wounding, and more than 90% of the wound closure was due to wound contraction. Wound contraction accounted for a similar extent of wound closure in both lyophilized cell lysate‐treated and phosphate‐buffered saline solution‐treated wounds. In contrast, in the diabetic (db/db) mice, after histologic examination of the wounds 32 days after wounding, four of ten lyophilized cell lysate‐treated wounds and four of seven phosphate‐buffered saline‐treated wounds were found to be closed. Moreover, applications of lyophilized cell lysate from cultured human keratinocytes to full‐thickness wounds in diabetic db/db mice significantly decreased the contribution of contraction to wound closure. Day 32 after wounding, contraction contribution to wound closure amounted to 57.7%± 4.7% and 80.4%± 3.2% (mean ± standard error of the mean, p < 0.005) of the initial wound areas, respectively, for lyophilized cell lysate‐treated and phosphate‐buffered saline solution‐treated wounds. At this time of wound healing, the thickness of the dermis was increased 1.7‐fold by the keratinocyte cell lysate treatment, but neither epithelial migration from the wound edges nor the thickness of the regenerated epithelium were significantly affected. In conclusion, in diabetic (db/db) mice the application of lyophilized cell lysate from cultured human keratinocytes influenced the healing of the dermis and wound contraction, but had no effect on reepithelialization.

Rote Beulen am Rücken eines Neugeborenen

Ein weibliches Baby wurde per Kaiserschnitt nach fetalem Stress von einer 40-jährigen Mutter geboren. Im Fruchtwasser wurde Mekonium nachgewiesen sowie eine perinatale Asphyxie (die Apgar-Werte lagen bei 1 beziehungsweise 5 nach 1 beziehungsweise 5 Minuten; der arterielle pHWert der Nabelschnur lag bei 6,96) festgestellt. Das Baby wurde mit einem Beatmungsbeutel beatmet, zwei Minuten lang wurde eine Herzdruckmassage durchgeführt. Darüber hinaus wurde ihr Körper für 72 Stunden auf 32°C heruntergekühlt. Fünf Tage später stellte man am Rücken des Babys feste, verhärtete erythematöse Flecken und Plaques fest (Abbildung 1 ). Rote Beulen am Rücken eines Neugeborenen A red, bumpy back in a neonate Diagnosequiz