Treatment of therapy-refractive ulcera cruris of various origins with autologous keratinocytes in fibrin sealant.

BACKGROUND: Evaluation of the effects of cultivated, subconfluent, autologous keratinocytes in fibrin sealant (BioSeed-S) on the healing of therapy-refractive chronic wounds. PATIENTS AND METHODS: Open observational study in 60 patients with chronic leg ulcers and impaired wound healing of various origins. After whole-skin excision and cultivation of the autologous keratinocytes, the suspended cells were applied to the preconditioned wound in fibrin sealant. Wound epithelization and wound size were recorded at defined times. RESULTS: Fifty-two of the 60 participating patients could be evaluated. After 6 weeks, 29 ulcers (55.8%) were healed. The mean epithelization increased between the 8th and 42nd postoperative day from 23% to 62.5%. In 50.0% of the patients, global assessment of the wound showed a high degree of epithelization or healing after 42 days. In 32.6% of treated patients, improvement was observed, while no healing tendency was to be found in 17.4%. CONCLUSION: The present observational study indicates that the transplantation of autologous keratinocytes suspended in fibrin sealant could be of advantage in the treatment of refractive leg ulcers.

[Gene therapy perspectives in modulation of wound healing]. / Gentherapeutische Perspektiven zur Modulation der Wundheilung.

A variety of reasons can afflict wound healing. Current research is focussed on the acceleration of wound healing by stimulating molecular processes. Gene therapy may offer completely new ways to treat chronic wounds. Possible advantages of gene therapeutic modulation of wound healing might be a long term efficiency, systemic or local regulation of gene expression and low side-effects. Current goals comprise the improvement of transfection efficiency and specificity. In vivo applications are therefore focussed on optimized inducible or even cell-type specific promotors, as well as on improved local application techniques. Studies from our laboratory demonstrate the possibility to combine modern cell culture techniques with different types of gene transfer. This enables the simultaneous grafting of manipulated cells to the wound with the continuous delivery of specific proteins of interest. Experimentally, this lead to accelerated closure of partial and full thickness animal wounds. Clinically, gene therapy for the treatment of chronic wounds seems to be a realistic goal within the next years and might be applicable for a variety of novel indications.

[Treatment of chronic wounds with cultured autologous keratinocytes as suspension in fibrin glue]. / Behandlung chronischer Wunden mit kultivierten autologen Keratinozyten als Suspension in Fibrinkleber.

Cultivated keratinocytes have been used for treatment of chronic wounds. Our group developed a new application form, using a suspension of subconfluently cultivated keratinocytes in fibrin glue (keratinocyte-fibrin-glue-suspension = KFGS), which has successfully been used in burn patients. Altogether 8 patients (average: 57 yrs.) with complex chronic wounds of different origin were treated with KFGS. All wounds, which had been existing from 4 months to 14 years, showed good reepithelization. Up to now there has been observed stable wound closure for 4 years after grafting. This study demonstrates the wound healing potency of a keratinocyte-fibrin-glue-suspension also for chronic wounds. Fibrin glue seems to be both an ideal application vehicle as a biological matrix for the cultivated keratinocytes. The transplantation of cultivated autologous keratinocytes as suspension in fibrin glue is a promising way in the treatment of chronic wounds.

[Free microsurgical flap-plasty in reconstructive therapy of diabetic foot ulcer]. / Freie mikrochirurgische Lappenplastiken in der rekonstruktiven Therapie des diabetischen Fussulkus.

The diabetic foot ulcer is a significant clinical problem often resulting in frustranous conservative treatment or early amputation. In certain cases transfer of well vascularized tissue can improve wound healing and lead to a length-spearing therapy. In this study the concept of microsurgical tissue transfer in the treatment of diabetic foot ulcers is introduced. Following a radical debridement and, if necessary a atypical length-spearing amputation the wound is covered by a free transplanted muscle flap followed by a split-thickness skin graft. In six patients treated with this procedure the extremity could be saved. The perioperative mortality was 0%, average hospitalization was 47.6 days. One flap was lost, two vascular revision were necessary. Several necrectomies and skin grafts were performed and one donor side seroma was drained. One hernia was observed after free rectus abdominus transfer. Compared to conservative treatment or amputation this concept leads to a length-spearing therapy and can increase success rates of rehabilitation and quality of life.

[Keratinocyte transplantation and tissue engineering. New approaches in treatment of chronic wounds]. / Keratinozytentransplantation und Tissue Engineering. Neue Ansätze in der Behandlung chronischer Wunden.

Cultured keratinocytes have been used for the treatment of extensive burns since disease lethality is reduced. Consequently, the treatment of chronic wounds with keratinocytes may be promising. Cell culture technology allows to expand keratinocytes up to 6000-fold in vitro after taking a single biopsy from patient. Today the transplantation of these in vitro cultured keratinocytes in different modifications is an established clinical treatment regimen for therapy of extensive wounds. For example, keratinocyte-fibrin-glue-suspensions, mainly consisting of proliferative epidermal basal cells, were used for the treatment of burns in experimental and clinical settings to bypass the disadvantages of conventional sheet grafts. Other approaches in tissue engineering for wound healing aim at the (epi-)dermal repair by the combination of allodermis and biomaterials, i.e. collagen-sponges and microspheres. Due to most recent efforts in keratinocyte culture techniques, developments in tissue engineering, research for novel biomaterials and gene therapy, therapy of chronic wounds may prove to be more efficient. Furthermore, from the socio-economical point of view, overall costs for treatment of chronic wounds could be reduced.

Prefabrication of bilaminar-epithelialized composite flap with tissue expander and cultured keratinocytes.

This study investigated the feasibility of prefabrication of a bilaminar-epithelialized flap by using a tissue expander and cultured keratinocytes, for reconstruction of perforate defects in the oral cavity and upper aerodigestive tract. In each of six rats, a 10-ml volume expander was implanted under the inferior epigastric flap and a thin silicon catheter was introduced into periexpander space. Seven days after implantation, 10 x 10(6) cultured keratinocytes, isolated from inbred donor rats, were suspended in fibrin glue and injected into the periexpander space through the catheter (n = 4 of 6). The expansion was started immediately after cell inoculation and lasted at least 3 weeks at the speed of 2 to 3 ml every 5 to 7 days. At the end of expansion, the periexpander space was opened and the capsule around the tissue expander was found to be covered completely with a neoepithelium. Thus, a bilaminar-epithelialized flap based on femoral vessels was elevated and successfully transferred to cover the excisional perforate defect in the oral cavity with the neoepithelial side as inner lining. All flaps treated with 10 x 10(6) cultured keratinocytes survived with complete wound healing during a 1-week follow-up (n = 4 of 6). Both macroscopic and histologic findings demonstrated that a bilaminar-epithelialized composite flap can be fabricated by using a tissue expander and keratinocyte-fibrin glue suspension.

[Continuous delivery of epidermal growth factor to wounds in vivo by genetically modified fibroblasts transfected with a novel chimeric construct].

OBJECTIVE: This paper aims to explore the new method of continuous delivery of epidermal growth factor to wounds by transfected fibroblasts to promote wound repair. METHODS: It was constructed a novel chimeric expression plasmid in which the biologically active portion of the human epidermal growth factor (EGF) gene was fused in-frame to the human granulocyte colony-stimulating factor signal sequence. RESULTS: Clonally selected human fibroblasts transfected with this construct could secrete biologically active EGF. After the transplantation of irradiated gene-transfected fibroblasts suspended in fibrin glue to murine full-thickness wounds, EGF could be demonstrated for at least seven days in the wounds, slowly decreasing from initially 470 ng/L to 140 ng/L in 7 days. No EGF was found in the wound at 14 days. CONCLUSION: A single application of irradiated EGF gene transfected fibroblasts to wounds can continuously deliver the transgene in vivo and can be used to administer drugs to the wound bed during the crucial first seven days of wound-healing.

[Biological wound tissue glue systems in wound healing]. / Biologische Wundklebesysteme in der Wundheilung.

Tissue engineering relies on in vitro cell culture, biocompatible matrix materials and genetic engineering with growth and differentiation factors for guided tissue regeneration. Biogenic or semisynthetic biomaterials are an alternative as cell carriers: To circumvent the disadvantages of conventional keratinocyte sheet grafts, a keratinocyte fibrin glue suspension KFGS (H. W. Kaiser et al., Burns 20: 23, 1994), which mainly consists of epidermal stem cells, has been tested experimentally in nude mice and clinically in extensive burns and chronic wounds. In the "in vivo culture" on the wound, the non-confluent keratinocytes form a differentiated epithelium within days. Current research aims at guided dermal regeneration by a combination with allodermis or biomaterials (collagen sponges like TissueFaszie, Microspheres etc.). Fibrin glue (Tissuecol) has also been tested successfully as matrix for other cells like chondrocytes and fibroblasts transfected with growth factor genes (EGF/KGF).

Paracrine stimulation of keratinocytes in vitro and continuous delivery of epidermal growth factor to wounds in vivo by genetically modified fibroblasts transfected with a novel chimeric construct.

BACKGROUND: Growth factors play an important role in tissue repair. While the effectiveness of growth factor therapy in animal wound healing models and limited human clinical trials has been demonstrated, the ideal method for their administration to the wound remains unclear. Experimental data suggest that the continuous presence in the early stages of wound repair is beneficial. MATERIALS AND METHODS: We have constructed a novel chimeric expression plasmid in which the biologically active portion of the human epidermal growth factor (EGF) gene is fused in-frame to the human granulocyte colony-stimulating factor signal sequence. RESULTS: Clonally selected human fibroblasts transfected with this construct secrete biologically active EGF. After the transplantation of irradiated gene-transfected fibroblasts suspended in fibrin glue to murine full-thickness wounds, EGF can be demonstrated for at least seven days in the wounds, slowly decreasing from initially 470 pg/ml to 140 pg/ml on day 7. No EGF was found in the wound at day 14. CONCLUSIONS: A single application of irradiated EGF genetransfected fibroblasts to wounds can thus continuously deliver the transgene in vivo and could be used to administer drugs to the wound bed during the crucial first seven days of wound-healing.

High CD44 surface expression on primary tumours of malignant melanoma correlates with increased metastatic risk and reduced survival.

The cell surface glycoprotein CD44 has been implicated in the progression and metastasis of certain human tumours including malignant melanoma (MM). In animal models, certain MM cell lines, expressing high levels of CD44, displayed an augmented capacity for haematogenous metastasis, compared to those with low CD44 levels. To determine whether, in vivo, the level of CD44 expressed by primary tumours of MM (PMM) is related to their metastatic potential, CD44 expression on PMM was studied in 92 patients, classified by their metastatic risk based on histological measurement of vertical tumour thickness (VT): in situ PMM, low-risk PMM (VT < or = 0.7 mm), intermediate risk PMM (VT = 0.71-1.4 mm) and high-risk PMM (VT > 1.4 mm). Paraffin-embedded sections were stained immunohistochemically with a panCD44 MAb. The level of CD44 expression on PMM was analysed semiquantitatively with epidermal CD44 staining set as an internal standard. High levels of CD44 were detected in 58.3% of high-risk PMM, 40.6% of intermediate-risk PMM, 36.7% of low-risk PMM and 16.7% of in situ PMM. Seventy-four per cent (17/23) of patients who developed and/or died of MM metastasis were CD44 high, and importantly, among these were 5 patients, whose metastatic risk had been estimated low, based on the measurement of VT. Finally, Kaplan-Meier analysis revealed patients whose PMM were CD44 high to have a significantly reduced 5-year survival rate compared to those that were CD44 low (P < 0.05). We conclude that in our patient population, a high level expression of CD44 on PMM is associated with increased metastatic risk and reduced survival.

Detection of CD44 splice variants in formalin-fixed, paraffin-embedded specimens of human skin cancer.

The standard form of CD44 (CD44s) and CD44 isoforms, containing sequences encoded by one or several of 10 different variant CD44 exons (v1-v10), are thought to play a crucial role in the growth and metastasis of certain human tumors. Recently, monoclonal antibodies (mAbs) directed against all CD44 isoforms (panCD44), or against epitopes encoded by specific variant exons (CD44v) have been developed, which unfortunately only stain cryopreserved tissues. We wished to develop a technique to unmask chemically CD44s and CD44v epitopes in paraffin-embedded specimens of human skin cancers, so that they would be accessible for these mAbs. To address this issue, CD44s and CD44v expression was compared in cryopreserved and in formalin-fixed, paraffin-embedded biopsies obtained from the same basal cell carcinomas (BCC), squamous cell carcinomas (SCC), primary malignant melanomas (PMM) and metastatic malignant melanomas (MMM). Formalin-fixed tumors were deparaffinized and treated briefly with an antigen retrieval fluid (TUFTM) at 95 degrees C or left untreated. In untreated paraffin-embedded tissues, no CD44s or CD44v staining was detected. In contrast, in antigen retrieval fluid-treated biopsies CD44s and CD44v expression was identical to that in cryopreserved specimens of the same tumor with the exception of mAbs detecting v7/8 and v10. We conclude that antigen retrieval unmasks certain epitopes in formalin-fixed, paraffin-embedded tissues, thus facilitating future research on the relevance of CD44s and CD44v expression for human skin carcinogenesis.

In situ expression of B7 and CD28 receptor families in human malignant melanoma: relevance for T-cell-mediated anti-tumor immunity.

Work in animal models has suggested that interactions of members of the B7 receptor family (e.g., B7-1, B7-2) on tumor cells with their ligands CD28 and CTLA-4 on cytotoxic T cells (CTL) are important for the induction of anti-tumor immunity against malignant melanoma (MM). To determine whether these molecules are of relevance for CTL responses against human MM, we studied the expression of B7-1, B7-2, CD28 and CTLA-4 in primary tumors of MM (PMM), MM metastases (MMM) and benign melanocytic nevi (BMN) by immunohistochemistry (IH) and by reverse transcription polymerase chain reaction (RT-PCR). By RT-PCR, B7-1 and B7-2-specific mRNAs were detected in most PMM, MMM and BMN. These PCR-signals were derived from CD45(+)-infiltrating leukocytes and not from tumor cells since (I) MMM depleted of CD45+ cells contained no B7-1 or B7-2 mRNA; and (2) by IH, B7-1 and B7-2 were found on infiltrating dendritic cells, macrophages and a variable proportion of tumor-infiltrating lymphocytes (TIL) but not on melanoma cells or nevus cells. The important exceptions were 5/5 spontaneously regressing PMM, in which B7-1 and B7-2 were expressed by melanoma cells, that were surrounded by TIL expressing CD28 but not CTLA-4. We conclude that, in PMM, MMM and BMN, the majority of TIL are CD28+ and that B7-1 and B7-2 are expressed by CD45(+)-infiltrating antigen-presenting cells (APC) and TIL, but not by the tumor cells. However, in spontaneously regressing PMM, melanoma cells express B7-1, B7-2 and MHC class-I and -II antigens, particularly in areas with clinical and histological signs of an ongoing anti-tumor response. These data suggest that the absence of B7-1 and B7-2 favors the escape of MM from immunosurveillance, while B7-1, B7-2 expression enhances T-cell-mediated anti-tumor immunity.

CD44 isoforms containing exon V3 are responsible for the presentation of heparin-binding growth factor.

Glycosaminoglycan-modified isoforms of CD44 have been implicated in growth factor presentation at sites of inflammation. In the present study we show that COS cell transfectants expressing CD44 isoforms containing the alternatively spliced exon V3 are modified with heparan sulfate (HS). Binding studies with three HS-binding growth factors, basic-fibroblast growth factor (b-FGF), heparin binding-epidermal growth factor (HB-EGF), and amphiregulin, showed that the HS-modified CD44 isoforms are able to bind to b-FGF and HB-EGF, but not AR. b-FGF and HB-EGF binding to HS-modified CD44 was eliminated by pretreating the protein with heparitinase or by blocking with free heparin. HS-modified CD44 immunoprecipitated from keratinocytes, which express a CD44 isoform containing V3, also bound to b-FGF. We examined whether HS-modified CD44 isoforms were expressed by activated endothelial cells where they might present HS-binding growth factors to leukocytes during an inflammatory response. PCR and antibody-binding studies showed that activated cultured endothelial cells only express the CD44H isoform which does not contain any of the variably spliced exons including V3. Immunohistological studies with antibodies directed to CD44 extracellular domains encoded by the variably spliced exons showed that vascular endothelial cells in inflamed skin tissue sections do not express CD44 spliced variants. Keratinocytes, monocytes, and dendritic cells in the same specimens were found to express variably spliced CD44. 35SO4(-2)-labeling experiments demonstrated that activated cultured endothelial cells do not express detectable levels of chondroitin sulfate or HS-modified CD44. Our results suggest that one of the functions of CD44 isoforms expressing V3 is to bind and present a subset of HS-binding proteins. Furthermore, it is probable that HS-modified CD44 is involved in the presentation of HS-binding proteins by keratinocytes in inflamed skin. However, our data suggests that CD44 is not likely to be the proteoglycan principally involved in presenting HS-binding growth factors to leukocytes on the vascular cell wall.

Prolongation of the QTc-interval reflects the severity of autonomic neuropathy in primary biliary cirrhosis and in other non-alcoholic liver diseases.

It was the aim of our study to prove a potential correlation (a) between laboratory findings of cholestasis and autonomic neuropathy (AN) and (b) between the severity of AN and the prolongation of the corrected QT-time (QTc). The five standard tests of autonomic cardiac neuropathy were investigated. QTc was calculated according to Bazett's formula. 12 out of 14 patients with primary biliary cirrhosis, 18 out of 21 patients with HBsAg positive liver diseases and 11 out of 14 patients with cirrhosis of other origin had AN. No significant correlation between the laboratory parameters of cholestasis and AN was found. Abnormal QTc values (> 440 m/sec) were observed significantly more often (p < 0.002) in patients with AN than in patients without AN. Significant linear regression (p < 0.01) could be confirmed between the prolongation of the QTc-time and the severity of AN. Besides the non-invasive investigation of the cardiovascular reflexes the evaluation of the QT-time might be an additional diagnostic means to identify patients with an increased cardiovascular risk in chronic non-alcoholic liver diseases.