Effective Management Of Topical Nosocomial Aspergillus Spp. Infections In Three Severely Burned Patients.

Nosocomial opportunistic fungal infections by Aspergillus spp. represent increasing morbidity and mortality factors for severely burned patients, who are fragile and immunocompromised. Voriconazole (VRC), a modern antifungal drug, is used as a first-line therapy against systemic mold and yeast infections. Little has been published about the place, relative importance and efficacy of voriconazole in the treatment protocols involving Aspergillus spp. in Burn Centers. The objective of the present work was to assess the place and importance of voriconazole for the treatment of burn patients presenting superficial Aspergillus spp. infections. We performed a retrospective evaluation of VRC treatment in three severely burned patients with superficial nosocomial Aspergillus spp. infections in our Burn Center. Results showed that VRC allowed for control and cure of topical nosocomial Aspergillus spp. infections. In two cases, treatment with VRC had to be discontinued because of hepatotoxicity. In two cases, following or during systemic treatment with VRC, a 1% terbinafine cream was applied to resolve the infection in order to continue standard wound management. Overall, VRC has been shown to be an effective antifungal agent and is an alternative to amphotericin B to fight Aspergillus spp. infections developing in the wounds of severely burned patients.

La survenue d'une aspergillose chez les patients gravement brûlés, dès lors immunodéprimés, est une cause de morbidité et de mortalité. Le voriconazole (VRC) est un antifongique utilisé en première intention dans le traitement des infections à moisissures. La littérature est pauvre au sujet de son utilisation dans l'aspergillose chez le brûlé. Cette étude a pour but de l 'évaluer dans le traitement de l'aspergillose cutanée chez le brûlé et a consisté en l'évaluation rétrospective de la prise en charge de trois patients de notre CTB, gravement brûlés et victimes d'une aspergillose cutanée. VRC en a permis la guérison, mais a dû être suspendu 2 fois en raison d'une toxicité hépatique. Dans 2 cas, il a été associé à de la crème de terbinafine à 1%. Le traitement habituel a pu être repris après guérison de l'aspergillose. Globalement, VRC semble efficace et représente une alternative à l'amphotéricine B dans le traitement de l'aspergillose cutanée chez les brûlés.

Acceptation of Folk Medicine and its "secrets" in a Swiss Burn Centre.

In Switzerland 'Secret' is a folk medicine called upon for burns. It has belonged to UNESCO's intangible cultural heritage since 2012. It is supposed to ease pain and accelerate the healing process of burns. As the practice is widely used in the population, this observational study investigated the opinion of caregivers and patients from the National Burn Center of Lausanne. Qualitative observational study based on a survey including ten questions aimed at identifying the professionals' perception of the phenomenon. Questions were developed from repeated encounters in the burn center. Data collection took five months. Thirty-six healthcare professionals (HP) and 12 selected patients (or parents for minors) discharged after burns were interviewed on a voluntary basis: all of the HPs knew about 'Secret' from the workplace, and 26 from home: 33 were convinced that it might be useful and reduce pain. The perceived efficiency of the practice (36 respondents) differs depending on professional category and personal experience. Only one HP considered the practice to be dangerous. The nurses and auxiliary nurses expressed that it should be used more widely. The 12 patients considered it as a complementary step, not a replacement for medical care. Health professionals globally considered this practice safe and helpful. The patients were interested in using parallel approaches and were careful about their expectations. This openness is probably an indication that HPs believe that acceptance of the culture and beliefs of patients and their families might positively affect response to treatment, whatever the burn size.

Il existe en Suisse une médecine traditionnelle dénommée « secret ¼ dédiée aux brûlures (supposée avoir des effets analgésiques et cicatrisants) inscrite au patrimoine immatériel de l'UNESCO depuis 2012. Dans la mesure où elle est très largement utilisée, nous avons conduit une étude observationnelle sur l'opinion qu'en ont les soignants et les patients du CTB national de Lausanne. Nous avons utilisé un questionnaire à dix items, développé après des entretiens plus informels. Trente six professionnels et 12 patients (ou parents quand le patient était mineur), interrogés après leur sortie, ont volontairement participé à l'étude. Tous les professionnels avaient entendu parler de « secret ¼ soit au travail soit chez eux (26). Trente trois étaient persuadés de son utilité analgésique, 1 seul le considérant comme dangereux. Cette opinion varie selon la catégorie professionnelle et l'expérience personnelle, les infirmières et aide- soignantes estimant qu'il devrait être plus largement utilisé. Les patients estimaient que « secret ¼ était un adjuvant ne devant pas remplacer la prise en charge médicalisée. Les professionnels considéraient que « secret ¼ est simple et utile. Les patients étaient intéressée par cette approche parallèle, tout en gardant une certaine retenue quant à ce qu'ils pouvaient en attendre. Cette ouverture d'esprit suggère que les professionnels pensent que la prise en compte de la culture et des croyances des patients et de leur famille peut promouvoir l'efficacité du traitement conventionnel, quelle que soit la surface brûlée.

Decellularised tissues obtained by a CO2-philic detergent and supercritical CO2.

Tissue decellularisation has gained much attention in regenerative medicine as an alternative to synthetic materials. In decellularised tissues, biological cues can be maintained and provide cellular environments still unmet by synthetic materials. Supercritical CO2 (scCO2 ) has recently emerged as a promising alternative decellularisation technique to aggressive detergents; in addition, scCO2 provides innate sterilisation. However, to date, decellularisation with scCO2 is limited to only a few tissue types with low cellular density. In the current study, a scCO2 technique to decellularise high density tissues, including articular cartilage, tendon and skin, was developed. Results showed that most of the cellular material was removed, while the sample structure and biocompatibility was preserved. The DNA content was reduced in cartilage, tendon and skin as compared to the native tissue. The treatment did not affect the initial tendon elastic modulus [reduced from 126.35 ± 9.79 MPa to 113.48 ± 8.48 MPa (p 〉 0.05)], while it reduced the cartilage one [from 12.06 ± 2.14 MPa to 1.17 ± 0.34 MPa (p 〈 0.0001)]. Interestingly, cell adhesion molecules such as fibronectin and laminin were still present in the tissues after decellularisation. Bovine chondrocytes were metabolically active and adhered to the surface of all decellularised tissues after 1 week of cell culture. The developed method has the potential to become a cost-effective, one-step procedure for the decellularisation of dense tissues.

Peptide-decorated chitosan derivatives enhance fibroblast adhesion and proliferation in wound healing.

RGD peptide sequences are known to regulate cellular activities by interacting with α5ß1, αvß5 and αvß3 integrin, which contributes to the wound healing process. In this study, RGDC peptide was immobilized onto chitosan derivative 1,6-diaminohexane-O-carboxymethyl-N,N,N-trimethyl chitosan (DAH-CMTMC) to display RGDC-promoting adhesion for enhanced wound healing. The efficiency of N-methylation, O-carboxymethylation and spacer grafting was quantitatively and qualitatively analyzed by (1)H NMR and FTIR, yielding 0.38 degree of substitution for N-methylation and >0.85 for O-carboxymethylation. The glass transition temperatures for chitosan derivatives were also studied. Peptide immobilization was achieved through sulfhydryl groups using sulfosuccinimidyl (4-iodoacetyl)amino-benzoate (sulfo-SIAB method). RGDC immobilized peptide onto DAH-CMTMC was found to be about 15.3 µg/mg of chitosan derivative by amino acid analysis (AAA). The significant increase of human dermal fibroblast (HDF) viability in vitro over 7 days suggests that RGDC-functionalized chitosan may lead to enhanced wound healing (viability >140%). Moreover, bio-adhesion and proliferation assays confirmed that coatings of RGDC-functionalized chitosan derivatives exhibit in vitro wound healing properties by enhancing fibroblast proliferation and adhesion. These results showed that RGDC peptide-functionalized chitosan provides an optimal environment for fibroblast adhesion and proliferation.

Burn patient care lost in good manufacturing practices?

Application of cell therapies in burn care started in the early 80s in specialized hospital centers world-wide. Since 2007, cell therapies have been considered as "Advanced Therapy Medicinal Products" (ATMP), so classified by European Directives along with associated Regulations by the European Parliament. Consequently, regulatory changes have transformed the standard linear clinical care pathway into a more complex one. It is important to ensure the safety of cellular therapies used for burn patients and to standardize as much as possible the cell sources and products developed using cell culture procedures. However, we can definitely affirm that concentrating the bulk of energy and resources on the implementation of Good Manufacturing Practice (GMP) alone will have a major negative impact on the care of severely burned patients world-wide. Developing fully accredited infrastructures and training personnel (required by the new directives), along with obtaining approval for clinical trials to go ahead, can be a lengthy process.We discuss whether or not these patients could benefit from cell therapies provided by standard in-hospital laboratories, thus avoiding having to meet rigid regulations concerning the use of industrial pharmaceutical products. "Hospital Exemption" could be a preferred means to offer burn patients a customized and safe product, as many adaptations may be required throughout their treatment pathway. Patients who are in need of rapid treatment will be the ones to suffer the most from regulations intended to help them.

L'utilisation de la « thérapie cellulaire ¼ au profit des patients brûlés s'est mise en place au début des années 1980 dans de nombreux centres, répartis de par le monde. Depuis 2007, les produits utilisés ont fait l'objet de directives européennes. De ce fait, la prise en charge directe du patient est devenue un parcours semé d'embûches. S'il est important d'assurer au patient l'utilisation de produits dérivés de culture cellulaire de qualité, fabriqués selon des procédés reproductibles, il est évident que la mise en place dans les unités des « Bonnes Pratiques de Fabrication ¼ entraînera des dépenses de temps et d'énergie qui auront inévitablement un impact négatif sur la prise en charge du patient très gravement brûlé. En outre, la mise à niveau de l'infrastructure et la formation du personnel (exigées par les directives actuelles) ainsi que l'obtention des essais cliniques nécessaires à l'autorisation d'utilisation de ces produits peuvent s'avérer très longues. Nous argumentons la possibilité de fabriquer ces produits de culture cellulaire dans des laboratoires hospitaliers classiques en évitant la très lourde procédure destinée principalement à l'industrie pharmaceutique. Une « exemption hospitalière ¼ pourrait être un moyen d'offrir aux brûlés une thérapeutique adaptée et sécurisée, dans la mesure où des adaptations personnalisées peuvent être nécessaires au long de leur traitement. Les patients ayant un besoin vital d'un traitement urgent seront ceux qui pâtiront le plus d'une loi sensée les protéger.

How important is hydrotherapy? Effects of dynamic action of hot spring water as a rehabilitative treatment for burn patients in Switzerland.

Burn rehabilitation using hydrotherapy can have multiple benefits for the burn patient. The therapy uses specific mineral enriched hot spring water and water jets with varied hydro-pressure to combat hypertrophy, inflammatory reaction signs, abnormal pigmentation, and, more specifically, redness and scarring. Standard operating procedures for burn rehabilitation have been developed and integrated into the Standard of Care at the CHUV hospital using localized hydro-mechanical stimulation of burn sites (20 minutes of alternating anatomical sites) followed by constant pressure large-bore and filiform showers targeting specific scarred areas. These therapeutic regimens are repeated daily for 2 to 3 weeks. Patients showed lasting effects from this regimen (up to 3-6 months), the results becoming permanent with more uniform skin structure, color and visco-elasticity in addition to a decrease in pruritus. The specifications of clinical protocols are described herein along with the virtues of hot spring hydro-pressure therapy for burn rehabilitation. The use of hydrotherapy, which has been a controversial topic among burn units across the world, is also discussed. In North America, hydrotherapy is defined only within the scope of in-patient wound cleansing and is thought to lead to microbial auto-contamination and bacterial resistance. In Switzerland and France the emphasis of hydrotherapy is on rehabilitation after the wound has closed.

L'hydrothérapie pendant la réhabilitation des patients atteints de brûlures peut avoir plusieurs avantages. Le point focal de cette thérapie est l'utilisation d'une source d'eau thermale de source chaude enrichie en minéraux et de jets d'eau avec une variation de pression afin de lutter contre l'hypertrophie, les signes de réaction inflammatoire, une pigmentation anormale et en particulière des rougeurs et des cicatrices. Pour la réhabilitation des brûlures, les procédures d'utilisation normalisées ont été développés et intégrés dans le standard des soins dans notre hôpital. Ces procedures comportent une stimulation hydro-mécanique localisée sur les sites de brûlures (20 minutes en alternant les sites atomiques), suivie par une pression constante localisée directement sur les cicatrices faite à l'aide de douches de gros diamètre et puis de douches filiformes. Ce régime thérapeutique est répétée quotidiennement pendant 2 à 3 semaines. Après le traitement, les patients ont pu observer une structure plus uniforme de leur peau ainsi qu'une amélioration de sa couleur et de sa visco-élasticité, aussi bien que la diminution du prurit, et ce durant 3 à 6 mois. Ici nous présentons les spécificités de notre protocoles cliniques et les avantages d'une traitement d'eau thermale de source sous pression pour la réhabilitation des patients brûlés. Nous parlerons également de l'utilisation de l'hydrothérapie, qui est un sujet de controverse parmi les unités de soins aux brûlures à travers le monde. En Amérique du Nord, l'hydrothérapie est définie uniquement dans le cadre du nettoyage des plaies des patients hospitalisés, et elle peut conduire à l'auto-contamination microbienne et la résistance bactérienne. En Suisse et en France, l'hydrothérapie concerne uniquement la réhabilitation des plaies une fois cellesci fermées.

Development of a cost-effective method for platelet-rich plasma (PRP) preparation for topical wound healing.

Platelet-rich plasma (PRP) is a volume of plasma fraction of autologous blood having platelet concentrations above baseline whole-blood values due to processing and concentration. PRP is used in various surgical fields to enhance soft-tissue and bone healing by delivering supra-physiological concentrations of autologous platelets at the site of tissue damage. These preparations may provide a good cellular source of various growth factors and cytokines, and modulate tissue response to injury. Common clinically available materials for blood preparations combined with a two-step centrifugation protocol at 280g each, to ensure cellular component integrity, provided platelet preparations which were concentrated 2-3 fold over total blood values. Costs were shown to be lower than those of other methods which require specific equipment and high-cost disposables, while safety and traceability can be increased. PRP can be used for the treatment of wounds of all types including burns and also of split-thickness skin graft donor sites, which are frequently used in burn management. The procedure can be standardized and is easy to adapt in clinical settings with minimal infrastructure, thus enabling large numbers of patients to benefit from a form of cellular therapy.

Le plasma riche en plaquettes (PRP) est un volume de la fraction plasmatique du sang autologue ayant des concentrations au-dessus des valeurs de base des plaquettes sang total en raison de l'élaboration et de la concentration. Le PRP est utilisé dans différents domaines de la chirurgie pour améliorer la guérison des tissus mous et des os en délivrant des concentrations supraphysiologiques de plaquettes autologues au niveau du site de lésion tissulaire. Ces préparations peuvent fournir une bonne source cellulaire de divers facteurs de croissance et, de cytokines et moduler la réponse tissulaire à une lésion. Des matériaux communs cliniquement disponibles pour des préparations hématiques en association avec un protocole de centrifugation en deux étapes à 280g chacun, afin d'assurer l'intégrité des composants cellulaires, ont fourni des préparations de plaquettes qui ont été concentrées 2-3 fois par rapport aux valeurs de sang total. Les coûts se sont avérés inférieurs à ceux des autres méthodes qui nécessitent un équipement spécifique et des jetables ayant un coût élevé, tandis que la sécurité et la traçabilité peuvent être augmentées. Le PRP peut être utilisé pour le traitement de tous les types de lésions, y compris les brûlures comme aussi pour le traitement des sites donateurs de greffes cutanées d'épaisseur variable, qui sont fréquemment utilisées dans la gestion des brûlures. La procédure peut être standardisée et facilement adaptée dans les milieux cliniques avec une infrastructure minimale, permettant ainsi à un grand nombre de patients de bénéficier de cette forme de thérapie cellulaire.

Whole-cell bioprocessing of human fetal cells for tissue engineering of skin.

Current restrictions for human cell-based therapies have been related to technological limitations with regards to cellular proliferation capacity (simple culture conditions), maintenance of differentiated phenotype for primary human cell culture and transmission of communicable diseases. Cultured primary fetal cells from one organ donation could possibly meet the exigent and stringent technical aspects for development of therapeutic products. Master and working cell banks from one fetal organ donation (skin) can be developed in short periods of time and safety tests can be performed at all stages of cell banking. For therapeutic use, fetal cells can be used up to two thirds of their life-span in an out-scaling process and consistency for several biological properties includes protein concentration, gene expression and biological activity. As it is the intention that banked primary fetal cells can profit from the prospected treatment of hundreds of thousands of patients with only one organ donation, it is imperative to show consistency, tracability and safety of the process including donor tissue selection, cell banking, cell testing and growth of cells in out-scaling for the preparation of whole-cell tissue-engineering products.

A novel highly informative polyA microsatellite on the telomeric side of the INK4a/ARF locus.

The INK4a/ARF locus encodes two cell cycle-regulatory proteins, p16(INK4a)and p14(ARF). Inactivation of the p16(INK4a)(MTS1) tumor suppressor gene by mutations, promoter methylation or gene deletions is a common event in the development of many different human tumors. The present report describes a novel polyA mononucleotide repeat situated 7.2 kb on the telomeric side of the INK4a/ARF locus. This highly polymorphic microsatellite marker (heterozygote frequency: 0.78) proved to be efficient for p16 allele loss and microsatellite instability analyses in human colon cancer.

Methylation silencing and mutations of the p14ARF and p16INK4a genes in colon cancer.

The INK4a-ARF locus encodes two tumor suppressor proteins involved in cell-cycle regulation, p16INK4a and p14ARF, whose functions are inactivated in many human cancers. The aim of this study was to evaluate p14ARF and p16INK4a gene inactivation and its association with some clinocopathological parameters in colon cancer. The mutational and methylation status of the p14ARF and p16INK4a genes was analyzed in 60 primary colon carcinomas and 8 colon cancer cell lines. We have identified the first two reported mutations affecting exon 1beta of p14ARF in the HCT116 cell line and in one of the primary colon carcinomas. Both mutations occur within the N-terminal region of p14ARF, documented as important for nucleolar localization and interaction with Mdm2. Tumor-specific methylation of the p14ARF and p16INK4a genes was found in 33% and 32% of primary colon carcinomas, respectively. Methylation of the p14ARF was inversely correlated with p53 overexpression (p = 0.02). p14ARF and p16INK4a gene methylation was significantly more frequent in right-sided than in left-sided tumors (p = 0.02). Methylation of the p14ARF gene occurred more frequently in well-differentiated adenocarcinomas (p = 0.005), whereas the p16INK4a gene was more often methylated in poorly differentiated adenocarcinomas (p = 0.002). The present results underline the role of p14ARF and p16INK4a gene inactivation in the development of colon carcinoma. They suggest that the methylation profile of specific genes, in particular p14ARF and p16INK4a, might be related to biologically distinct subsets of colon carcinomas and possibly to different tumorigenic pathways.

Frequent methylation silencing of p15(INK4b) (MTS2) and p16(INK4a) (MTS1) in B-cell and T-cell lymphomas.

The methylation status of p15(INK4b) (MTS2), p16(INK4a) (MTS1) and p14(ARF) (p16beta) was analyzed in 56 lymphomas by restriction-enzyme related polymerase chain reaction (PCR) (REP), methylation-specific PCR (MSP), and bisulfite genomic sequencing (BGS). Methylation of the p15 and p16 genes was detected, respectively, in 64% and 32% of the B-cell lymphomas, in 44% and 22% of the T-cell lymphomas, and in none of the 5 reactive lymph nodes analyzed. Both p15 and p16 genes were methylated more often in the high-grade (78% and 50%, respectively) than in the low-grade B-cell lymphomas (55% and 21%, respectively). For 5 cases, mapping of the methylated CpGs of the p16 promoter region confirmed the results of REP and MSP. In addition, a large variation in the methylation patterns of p16 exon 1 was observed, not only from one lymphoma to another, but also within a given tumor. Methylation of p15 and p16 was associated with an absence of gene expression, as assessed by reverse transcription-PCR. The p14 gene was unmethylated and normally expressed in all 56 tumors. We found no mutations of p15, p16, or p14 in any of the 56 lymphomas. Our results suggest a role for p15 and p16 gene methylation during lymphomagenesis and a possible association between p15 and p16 inactivation and aggressive transformation in B-cell and T-cell lymphomas.

Consequences of seven novel mutations on the expression and structure of keratinocyte transglutaminase.

We report the molecular characterization of seven new keratinocyte transglutaminase mutations (R315C, S358R, V379L, G473S, R687C, deletion Delta679-696, R127Stop) found in lamellar ichthyosis patients. Arg-315, Ser-358, Val-379, and Gly-473 are highly conserved residues in transglutaminases while Arg-687 and Delta679-696 are not. All mutations strongly decreased transglutaminase activity and protein levels. The mutation R127Stop diminished the amount of mRNA. Structural analysis of these mutations based on the factor XIII A-subunit crystal structure demonstrated that Arg-315, Ser-358, Val-379, and Gly-473 are located in the catalytic core domain, and Arg-687 and the deletion are in the beta-barrel domains. The side chains of amino acids Arg-315, Ser-358, and Gly-473 make ionic and hydrogen bonds important for folding and structural stability of the enzyme but are not directly involved in catalysis. Val-379 is two amino acids away from the active site cysteine, and its change into leucine disturbs the active site structure. The decreased activity and protein level after expression of the R687C and Delta679-696 TGK cDNA in TGK negative keratinocytes excluded that they are polymorphisms. These results identify important amino acids in the central core domain of transglutaminases and show that the C-terminal end influences the structural and functional integrity of TGK.