Protease-modulating polyacrylate-based hydrogel stimulates wound bed preparation in venous leg ulcers--a randomized controlled trial.

BACKGROUND: Stringent control of proteolytic activity represents a major therapeutic approach for wound-bed preparation. OBJECTIVES: We tested whether a protease-modulating polyacrylate- (PA-) containing hydrogel resulted in a more efficient wound-bed preparation of venous leg ulcers when compared to an amorphous hydrogel without known protease-modulating properties. METHODS: Patients were randomized to the polyacrylate-based hydrogel (n = 34) or to an amorphous hydrogel (n = 41). Wound beds were evaluated by three blinded experts using photographs taken on days 0, 7 and 14. RESULTS: After 14 days of treatment there was an absolute decrease in fibrin and necrotic tissue of 37.6 ± 29.9 percentage points in the PA-based hydrogel group and by 16.8 ± 23.0 percentage points in the amorphous hydrogel group. The absolute increase in the proportion of ulcer area covered by granulation tissue was 36.0 ± 27.4 percentage points in the PA-based hydrogel group and 14.5 ± 22.0 percentage points in the control group. The differences between the groups were significant (decrease in fibrin and necrotic tissue P = 0.004 and increase in granulation tissue P = 0.0005, respectively). CONCLUSION: In particular, long-standing wounds profited from the treatment with the PA-based hydrogel. These data suggest that PA-based hydrogel dressings can stimulate normalization of the wound environment, particularly in hard-to-heal ulcers.

A prospective observational study of the efficacy of a novel hydroactive impregnated dressing.

OBJECTIVE: To assess the clinical efficacy, tolerance and acceptance of a novel, hydroactive-impregnated dressing (Hydrotul) in the local treatment of acute and chronic wounds. METHOD: In a prospective observational study 24 centres in France, Belgium, Germany and Austria recruited 74 patients. At each dressing change the investigators evaluated the condition of the wound, perilesional skin and patient-reported pain. Overall, five dressing changes were documented, or until complete healing occurred. The hydroactive properties of the dressing were assessed in laboratory tests by measuring fluid absorption capacity and kinetics. RESULTS: Patients were treated for an average of 17 days. The wound condition improved markedly during the observation period. The wound area covered with fibrinous slough decreased from 29% to 14%, epithelialisation increased from 19% to 54% and 22 wounds were completely healed by the end of the study. The number of patients reporting severe and moderate wound pain decreased from 35% to 19% and the proportion of patients without wound pain doubled from 27% to 60%. In laboratory tests, Hydrotul absorbed two to three times the amount of fluid compared with other impregnated wound dressings and the kinetics of absorption was much faster. CONCLUSION: The novel hydroactive impregnated dressing supports the healing process in patients with acute and chronic wounds and reduces wound pain. The dressing absorbs excess wound exudate while keeping the wound surface moist and protecting perilesional skin.

Clinical performance of a hydrogel dressing in chronic wounds: a prospective observational study.

OBJECTIVE: This prospective, multicentre application study was conducted to assess the clinical performance of Hydrosorb comfort hydrogel dressing. METHOD: Eighty-one patients (average age 67 years) with acute or chronic wounds received three dressing changes. The condition of the wound and patient-reported pain were assessed at the beginning and end of the study period. RESULTS: The mean proportion of the wound surface covered with slough fell from 63% to 34%, and the mean area of new granulation and epithelial tissue increased from 25% to 37% and 13% to 28%, respectively. The average wound size decreased from 4.7 x 2.9cm to 3.7 x 2.3cm; 29.6% of the patients reported no pain at baseline, increasing to 56.3% at the final assessment. CONCLUSION: Both acute and chronic wounds can be effectively treated with the Hydrosorb comfort hydrogel dressing.

E7 proteins from high- and low-risk human papillomaviruses bind to TGF-beta-regulated Smad proteins and inhibit their transcriptional activity.

Human papillomaviruses (HPV) infect keratinocytes of skin and mucosa. Persistent infection can lead to the formation of benign tumors. In cases of high-risk HPV, such as HPV16 or 18, these may further progress to cancer. In order to support viral replication in suprabasal keratinocytes, the HPV E7 protein employs various strategies to keep keratinocytes in cycle and counteracts anti-proliferative signals from outside. HPV16 E7 can directly interfere with transforming growth factor-beta (TGF-beta) signalling by binding to Smad proteins mediating growth arrest. It has been speculated that this property of HPV16 E7 contributes to HPV-associated carcinogenesis. Here, we show that E7 proteins from different low- and high-risk HPV types bind to Smad 1 to 4. The E7 protein from HPV1, a low-risk HPV causing plantar warts, efficiently inhibited Smad 3-induced transcription. Our data strongly indicate that the Smad-binding capacity of E7 proteins from different HPVs may preserve keratinocyte proliferation required for the productive viral life cycle rather than promoting carcinogenesis.

Reduced cellular toxicity of a new silver-containing antimicrobial dressing and clinical performance in non-healing wounds.

Bacterial colonisation of wounds may delay wound healing. Modern silver-containing dressings are antimicrobial, yet cellular toxicity is a serious side-effect. We provide data for a newly formulated silver-containing ointment dressing, Atrauman Ag, for antimicrobial activity and cytotoxicity. Atrauman Ag effectively killed a panel of commensal skin as well as pathogenic bacterial strains while cytotoxicity for HaCaT keratinocytes was only around 10%. With these favourable in vitro tests, Atrauman Ag was analysed in 86 patients with traumatic and non-healing wounds of different aetiologies. The wound state was evaluated for 3 subsequent dressing changes. The slough score was reduced from 59.2 to 35.8%, granulation tissue increased from 27 to 40% and epithelialisation went up from 12.1 to 24%. We conclude that Atrauman Ag has a superior profile of antimicrobial activity over cellular toxicity and the low silver ion release rate may prevent interference with wound-healing mechanisms.

CD40L induces matrix-metalloproteinase-9 but not tissue inhibitor of metalloproteinases-1 in cervical carcinoma cells: imbalance between NF-kappaB and STAT3 activation.

Matrix-metalloproteinases (MMPs) are essentially required for tumor cell invasion and metastasis. Production of precursor enzymes is regulated on transcriptional level, while activation of the pro-enzymes is tightly controlled by posttranscriptional mechanisms. The enzyme activity can be blocked by specific tissue inhibitors of MMPs (TIMPs). In cervical carcinomas strong up-regulation of the type IV collagenase MMP-9 had been demonstrated. We show that activation of CD40, a receptor highly expressed on cervical carcinomas, induces MMP-9 in cervical carcinoma cells, whereas TIMP-1 production inhibiting MMP-9 activity was not affected. This gene induction pattern corresponded to the differential activation of the transcription factor nuclear factor kappaB (NF-kappaB) regulating MMP-9, but not signal transducer and activator of transcription 3 (STAT3), which is involved in TIMP-1 gene regulation. Transient expression of the CD40-inducible NF-kappaB subunit p65 was sufficient for MMP-9 induction. Agents that suppressed CD40-mediated NF-kappaB activation also reduced MMP-9 induction, further supporting an important role of NF-kappaB in CD40-mediated MMP-9 induction. Our data suggest that CD40 expression in carcinoma cells might convert a CD40L-dependent immunological defense signal into a tumor-promoting signal. Selective CD40-mediated signaling through NF-kappaB but not STAT3 correlates to a shift of the balance between MMP-9 and TIMP-1 toward the protease.

Paracrine regulation of keratinocyte proliferation and differentiation.

The histoarchitecture and function of the epidermis depend on a well-controlled balance between keratinocyte proliferation and differentiation. This balance is perturbed after skin injury, and imbalance is a characteristic feature of major human skin diseases such as psoriasis and epidermal cancers. Recent studies have highlighted the importance of fibroblast-derived soluble factors for the regulation of keratinocyte proliferation and differentiation. Therefore, identification of these paracrine-acting factors and the elucidation of their mechanisms of action are necessary for understanding epidermal homeostasis, repair and disease, and these approaches will offer new potential targets for drug therapy. Here, we review exciting recent findings on the identification, regulation and function of paracrine-acting cytokines in the skin. In particular, we describe the role of fibroblast-derived mitogens as regulators of keratinocyte proliferation and differentiation, and we summarize the regulation of these factors by keratinocyte-derived interleukin 1 that involves the transcription factors c-Jun and JunB.

Loss of IL-6 receptor expression in cervical carcinoma cells inhibits autocrine IL-6 stimulation: abrogation of constitutive monocyte chemoattractant protein-1 production.

IL-6 is synthesized in human papilloma virus (HPV)-transformed cervical carcinoma cell lines and is supposed to stimulate these cells in an autocrine manner. We studied IL-6 production and responsiveness in nonmalignant HPV-transformed keratinocytes and cervical carcinoma cells in detail. IL-6 was detected in cervical carcinomas in situ. Correspondingly, HPV-positive carcinoma cell lines expressed high IL-6 levels. However, these carcinoma cell lines showed low responsiveness to IL-6 as revealed by low constitutive STAT3 binding activity, which was not further enhanced by exogenous IL-6. In contrast, in vitro-transformed nonmalignant keratinocytes without endogenous IL-6 production strongly responded to exogenous IL-6 with activation of STAT3. STAT3 protein expression levels were comparable in both responsive and nonresponsive cell lines. Also, gp130, the upstream signal-transducing receptor subunit conveying IL-6 signals into the cell, was expressed in all tested cell lines. However, the IL-6 binding subunit gp80 was lost in the malignant cells. Addition of soluble gp80 was sufficient to restore IL-6 responsiveness in carcinoma cells as shown by enhanced activation of STAT3 binding activity. As a consequence of the restored IL-6 responsiveness, carcinoma cells strongly produced the chemokine monocyte chemoattractant protein-1 (MCP-1). Our data demonstrate that cervical carcinoma cells producing high amounts of IL-6 only weakly respond to IL-6 in an autocrine manner due to limited gp80 expression. While production of IL-6 might contribute to a local immunosuppressive effect, silencing an autocrine IL-6 response prevents constitutive production of the mononuclear cell-attracting chemokine MCP-1. Both mechanisms might help the tumor to escape the immune system.

Impaired wound healing in embryonic and adult mice lacking vimentin.

It is generally assumed that the vimentin intermediate filament network present in most mesenchymally-derived cells is in part responsible for the strength and integrity of these cells, and necessary for any tissue movements that require the generation of significant tractional forces. Surprisingly, we have shown that transgenic KO mice deficient for vimentin are apparently able to undergo embryonic development absolutely normally and go onto develop into adulthood and breed without showing any obvious phenotype. However, fibroblasts derived from these mice are mechanically weak and severely disabled in their capacity to migrate and to contract a 3-D collagen network. To assess whether these functions are necessary for more challenging tissue movements such as those driving in vivo tissue repair processes, we have analysed wound healing ability in wild-type versus vimentin-deficient embryos and adult mice. Wounds in vimentin-deficient adult animals showed delayed migration of fibroblasts into the wound site and subsequently retarded contraction that correlated with a delayed appearance of myofibroblasts at the wound site. Wounds made to vimentin-deficient embryos also failed to heal during the 24 hour culture period it takes for wild-type embryos to fully heal an equivalent wound. By DiI marking the wound mesenchyme and following its fate during the healing process we showed that this impaired healing is almost entirely due to a failure of mesenchymal contraction at the embryonic wound site. These observations reveal an in vivo phenotype for the vimentin-deficient mouse, and challenge the dogma that key morphogenetic events occurring during development require generation of significant tractional forces by mesenchymal cells.

Overexpression of activin A in the skin of transgenic mice reveals new activities of activin in epidermal morphogenesis, dermal fibrosis and wound repair.

Recently we demonstrated a strong induction of activin expression after skin injury, suggesting a function of this transforming growth factor-beta family member in wound repair. To test this possibility, we generated transgenic mice that overexpress the activin betaA chain in the epidermis under the control of a keratin 14 promoter. The transgenic mice were significantly smaller than control littermates, and they had smaller ears and shorter tails. In their skin, the fatty tissue was replaced by connective tissue and a severe thickening of the epidermis was found. The spinous cell layer was significantly increased, and the epidermal architecture was highly disorganized. These histological abnormalities seem to result from increased proliferation of the basal keratinocytes and abnormalities in the program of keratinocyte differentiation. After skin injury, a significant enhancement of granulation tissue formation was detected in the activin-overexpressing mice, possibly as a result of premature induction of fibronectin and tenascin-C expression. These data reveal novel activities of activin in the regulation of keratinocyte proliferation and differentiation as well as in dermal fibrosis and cutaneous wound repair.

Efficient gene transfer into human keratinocytes with recombinant adeno-associated virus vectors.

Gene transfer into the skin is a promising approach to treat inherited or acquired dermatological diseases and systemic monogenic deficiencies. For this purpose, the efficient and sustained gene delivery into keratinocytes is of critical importance. Recombinant adeno-associated virus (rAAV) vectors hold the potential to achieve a long-term gene transfer into various human organs. In order to evaluate this potential for skin gene therapy, human keratinocytes were transduced in vitro with rAAV vectors encoding the reporter genes beta-galactosidase (rAAV/LacZ) or green fluorescent protein (rAAV/GFP). Using rAAV/LacZ at a multiplicity of infection (MOI) of five transducing particles per cell, up to 70% of human keratinocytes were transduced within 48 h. This effect was independent of individual skin donors and different body areas serving as the source for keratinocyte isolation. rAAV had no significant influence on cell viability, but induced a growth arrest in transduced keratinocytes. This growth arrest was overcome by replating cells in fresh media. rAAV/GFP-transduced keratinocytes could be passaged several times, expressed GFP for up to 50 days, and passed the transgene to their daughter cells, suggesting that keratinocyto precursor cells were also transduced. Taken together, the results suggest that rAAV is a promising gene transfer vehicle for skin gene therapy.

High prevalence of a variety of epidermodysplasia verruciformis-associated human papillomaviruses in psoriatic skin of patients treated or not treated with PUVA.

Epidermodysplasia verruciformis-associated human papillomaviruses and in particular human papillomavirus type 5 were recently shown to be highly prevalent in psoriatic skin. We have analyzed lesional skin from 54 psoriasis patients for infections with genital-specific and epidermodysplasia verruciformis-specific human papillomaviruses to define the spectrum of involved human papillomavirus types and to test if it is influenced by psoralen ultraviolet A therapy. Using polymerase chain reaction analysis we could detect human papillomavirus sequences in skin lesions of 83% of the tested patients. In contrast, human papillomavirus-DNA was only demonstrated in 19% of skin samples from 42 dermatologically healthy, immunocompetent individuals. Sequence analysis of the polymerase chain reaction amplimers revealed 14 human papillomavirus types, all belonging to the epidermodysplasia verruciformis or epidermodysplasia verruciformis-related papillomaviruses. Only in one case we identified sequences related to those of genital viruses, which, however, represented a putatively new human papillomavirus type. The most prevalent human papillomavirus type in our patient series was human papillomavirus type 36, found in 62% of the patients positive for human papillomavirus-DNA, followed by human papillomavirus type 5 (38%) and human papillomavirus type 38 (24%). Multiple infections with two to five different human papillomavirus types could be detected in skin samples of 63% of the analyzed patients. The overall human papillomavirus detection rate did not differ significantly between patients which have been subjected to psoralen ultraviolet A photochemotherapy or solely treated with topical preparations (77 vs 89%). Human papillomavirus type 5, however, could be detected significantly more frequent in lesions of psoralen ultraviolet A-treated patients (p < 0.001). Our data strongly argue for infections with epidermodysplasia verruciformis-specific papillomaviruses being an almost consistent feature of the lesional psoriatic skin and substantiate the importance of further studies to elucidate a possible involvement of human papillomaviruses in psoriasis pathology.

CD40 ligand-CD40 interaction induces chemokines in cervical carcinoma cells in synergism with IFN-gamma.

Cellular immunity plays a major role in controlling human papilloma virus infection and development of cervical carcinoma. Mononuclear cell infiltration possibly due to the action of chemokines becomes prominent in the tumor tissue. In fact, the macrophage chemoattractant protein-1, MCP-1, was detected in cervical squamous cell carcinoma in situ, whereas absent in cultured cells. From this, unknown environmental factors were postulated regulating chemokine expression in vivo. In this study, we show high CD40 expression on cervical carcinoma cells and CD40 ligand (CD40L) staining on attracted T cells in tumor tissue, suggesting a paracrine stimulation mechanism via CD40L-CD40 interactions. We therefore investigated chemokine synthesis in nonmalignant and malignant human papilloma virus-positive cell lines after CD40L exposure. Constitutive expression of MCP-1, MCP-3, RANTES, and IFN-gamma-inducible protein-10 was almost undetectable in all cell lines tested. CD40L was able to induce MCP-1 production; however, despite much higher CD40 expression in malignant cells, MCP-1 induction was significantly lower compared with nontumorigenic cells. After sensitization with IFN-gamma, another T cell-derived cytokine showing minimal effects on CD40 expression levels, CD40 ligation led to a more than 20-fold MCP-1 induction in carcinoma cell lines. An even stronger effect was observed for IFN-gamma-inducible protein-10. Our study highlights the synergism of T cell-derived mediators such as CD40L and IFN-gamma for chemokine responses in cervical carcinoma cells, helping to understand the chemokine expression patterns observed in vivo.

CD40 induces resistance to TNF-mediated apoptosis in a fibroblast cell line.

CD40, a member of the TNF receptor family, has been characterized as an important T-B cell interaction molecule. In B cells it co-stimulates isotype switching, proliferation, adhesion and is involved in cell death regulation. In addition to B cells, CD40 expression was found on transformed cells and carcinomas. However, little is known about its functions in these cell types. Recent studies show that CD40 mediates the production of pro-inflammatory cytokines in non-hematopoietic cells, inhibits proliferation or induces cell death. In some cell types the apoptotic program triggered by CD40 is only executed when protein synthesis is blocked, suggesting the existence of constitutively expressed resistance proteins. Here we demonstrate that CD40, similar to the 55-kDa TNF receptor (p55TNFR), has a dual role in the regulation of apoptosis in such cells. In the fibroblast cell line SV80 both CD40 and the p55TNFR trigger apoptosis when protein synthesis is blocked with cycloheximide (CHX). Simultaneous activation of both receptors results in markedly enhanced cell death. However, CD40 activation more than 4 h prior to a challenge with TNF/CHX paradoxically conferred resistance to TNF-induced cell death. Protection correlated with NF-kappaB induction and up-regulation of the anti-apoptotic zinc finger protein A20. Overexpression of A20 in turn rendered SV80 cells resistant to TNF cytotoxicity. In conclusion, our data provide evidence that CD40 may regulate cell death in non-hematopoietic cells in a dual fashion: the decision upon apoptosis or survival of a CD40-activated cell seems to depend on its ability to up-regulate resistance factors.

Dynamics of basement membrane formation by keratinocyte-fibroblast interactions in organotypic skin culture.

The cutaneous basement membrane zone, composed of numerous macromolecules, plays a multifunctional role in tissue regeneration and maintenance. To elucidate the cellular origin and dynamics of basement membrane formation, de novo synthesis, deposition, and ultrastructural assembly of its components were analyzed in organotypic cultures of adult skin keratinocytes on collagen gels with or without collagen-embedded dermal cells. Collagen IV and laminin-1 deposition occurred only in the presence of mesenchymal cells: patchy at day 4 and continuous after 1 week. Chain-specific mRNA expression started at day 2 in both keratinocytes and fibroblasts. It steadily increased up to day 10, however, with a reciprocal induction pattern, mRNA abundance shifting from keratinocytes to fibroblasts. On the other hand, laminin-5 staining was first observed at day 4, but in keratinocyte both mono- and cocultures. This was followed by nidogen, which was detected in cocultures but also in dermal monocultures. Laminin-5 protein persisted throughout day 21, whereas nidogen steadily increased in intensity. Expression kinetics revealed high levels of laminin-5 transcripts early and in keratinocytes only, whereas nidogen was expressed later and predominantly in fibroblasts. Although basement membrane protein deposition was continuous at day 14, the ultrastructural organization was still fragmentary, eventually normalizing at 3 weeks. These data demonstrate a dynamic interaction and cooperation of epithelial and mesenchymal skin cells in basement membrane formation. This interaction is supposedly mediated via diffusible factors. Our findings further extend the scope of epithelial-mesenchymal interactions stressing that both cell compartments are essential to constitute a tissue-specific extracellular matrix structure.

Limited T-cell repertoire in renal allograft and allogeneic melanoma transmitted by the graft.

In a patient with metastatic melanoma transmitted by the renal allograft, HLA serves as an alloantigen per se and is associated with tumor antigens at the same time. The influence of this antigeneic pattern on the Vbeta T-cell repertoire in an allogeneic melanoma, allograft, and peripheral blood mononuclear cells (PBMC) was assessed by polymerase chain reaction. Vbeta13.1 and 19 were found in both the melanoma and the graft. Vbeta14 was detected only in the melanoma and Vbeta6 was detected only in the kidney. PBMC revealed an unrestricted Vbeta pattern. Markers for cytotoxic activity of T cells--granzyme B and perforin--were not expressed during immunosuppressive therapy as clinically reflected in a nonrejecting allograft and in a progressing melanoma. In vitro PBMC proliferated to recombinant interleukin-2, whereas recombinant interferon-gamma did not augment this response. Initiation of immune therapy, in addition to discontinuation of immunosuppression, might support the rejection of the allogeneic tumor by dominant Vbeta T cells.

Differential regulation of pro-inflammatory cytokines during wound healing in normal and glucocorticoid-treated mice.

It has long been speculated that pro-inflammatory cytokines play an important role in wound repair. However, little is known about the temporal and spatial expression pattern of these cytokines during normal and impaired wound healing. In this study we show a strong and early induction of interleukins 1 alpha and beta (IL-alpha and beta) and of tumour necrosis factor alpha (TNF-alpha) expression after cutaneous injury. Highest levels of these cytokines were seen as early as 12-24 h after wounding. After completion of the proliferative phase of wound healing, mRNA levels of these cytokines returned to the basal level. During the early phase of wound repair, proinflammatory cytokines were predominantly expressed in polymorphonuclear leukocytes, suggesting a novel function of these cells in the initiation of wound healing. At later stages of the repair process, expression of IL-1 alpha, IL-1 beta and TNF-alpha was also seen in macrophages. Furthermore, TNF-alpha was detected in the hyperproliferative epithelium at the wound edge and IL-1 alpha was found in keratinocytes of the hair follicles. Induction of these cytokines after injury was significantly reduced during wound repair in healing-impaired glucocorticoid-treated mice. This finding demonstrates that wound healing defects are associated with impaired cytokine expression and suggests that the early induction of these genes is important for normal repair.