bromochloroacetic-acid and Wounds-and-Injuries

Wound dressings have experienced continuous and significant changes over the years based on the knowledge of the biochemical events associated with chronic wounds. The development goes from natural materials used to just cover and conceal the wound to interactive materials that can facilitate the healing process, addressing specific issues in non-healing wounds. These new types of dressings often relate with the proteolytic wound environment and the bacteria load to enhance the healing. Recently, the wound dressing research is focusing on the replacement of synthetic polymers by natural protein materials to delivery bioactive agents to the wounds. This article provides an overview on the novel protein-based wound dressings such as silk fibroin keratin and elastin. The improved properties of these dressings, like the release of antibiotics and growth factors, are discussed. The different types of wounds and the effective parameters of healing process will be reviewed.

Topics: Animals; Anti-Bacterial Agents; Bandages; Collagen; Drug Delivery Systems; Elastin; Fibroins; Humans; Hydrolysis; Keratins; Polymers; Wound Healing; Wounds and Injuries

Topics: Animals; Basement Membrane; Binding Sites; Cell Movement; Epidermal Cells; Epidermis; Extracellular Matrix; Humans; Keratins; Proteins; Receptors, Cell Surface; Skin; Wounds and Injuries

Severe illness or trauma alters the body's metabolic rate. After injury, host-defence protein synthesis and increased energy requirements are satisfied from available protein, usually active muscle tissue. A prolonged hypercatabolic state persists and may lead to increased morbidity and mortality in severely burned patients. Growth hormone (GH) is an anabolic agent shown to decrease some of the deleterious effects of hypermetabolism. This article will review the effects of GH on burn wound repair and gut healing. Studies on GH have shown a significant reduction in wound-healing times in burned patients given GH at a dose of 0.6 IU/kg/day (0.2 mg/kg/day). At this dose, other studies have shown no increase in mortality, and a number of beneficial effects in critically burned children have been demonstrated. Animal studies have suggested that insulin-like growth factor I (IGF-I), stimulated through the GH axis, plays an important role in the reconstitution of intestinal epithelial integrity following mucosal injury. Many encouraging papers report positive results regarding both the efficacy and safety of GH and IGF-I, therefore warranting continued investigation.

Topics: Adolescent; Burns; Child; Child, Preschool; Cicatrix; Collagen; Dose-Response Relationship, Drug; Double-Blind Method; Epithelial Cells; Female; Growth Hormone; Human Growth Hormone; Humans; Insulin-Like Growth Factor I; Intestinal Mucosa; Keratins; Laminin; Male; Wounds and Injuries

Impaired wound healing is a major medical problem in diabetes. The objective of this study was to determine the possible application of an insoluble fraction of fur-derived keratin biomaterial as a wound dressing in a full thickness surgical skin wound model in mice ( n = 20) with iatrogenically induced diabetes. The obtained keratin dressing was examined in vitro and in vivo. In vitro study showed the keratin dressing is tissue biocompatible and non-toxic for murine fibroblasts. Antimicrobial examination revealed the keratin dressing inhibited the growth of S. aureus and E. coli. In vivo studies showed the obtained dressing significantly ( p < 0.05) accelerated healing during the first week after surgery compared to control wounds. Keratin dressings were incorporated naturally into granulation and regenerating tissue without any visible signs of inflammatory response, which was confirmed by clinical and histopathological analysis. It is one of the first studies to show application of insoluble keratin proteins and its properties as a wound dressing. The obtained keratin dressing accelerated wound healing in mice with iatrogenically induced diabetes. Therefore, it can be considered as a safe and efficient wound dressing. Although future studies are needed to explain the molecular mechanism behind fur-derived keratin effect during the multilayer wound healing process, our findings may open the way for a new class of insoluble fur keratin dressings in chronic difficult to heal wounds treatment.

Topics: Animals; Anti-Bacterial Agents; Bandages; Biocompatible Materials; Cell Survival; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Escherichia coli; Fibroblasts; Keratins; Male; Mice; Mice, Inbred C57BL; NIH 3T3 Cells; Skin; Staphylococcus aureus; Streptozocin; Wound Healing; Wounds and Injuries

Keratin is an interesting protein needed for wound healing and tissue recovery. We have recently proposed a new, simple and inexpensive method to obtain fur and hair keratin-derived biomaterials suitable for medical application. The aim of the study was to evaluate the role of the fur keratin-derived protein (FKDP) dressing in the allogenic full-thickness surgical skin wound model. The data obtained using scanning electron microscopy showed that employed processed biomaterial had higher surface porosity compared with control raw material. From the MTS test, it was found keratin biomaterial is not only toxic to the NIH/3T3 cell line (p < 0.05), but also enhances cell proliferation compared with the control. In vivo studies have shown keratin dressings are tissue biocompatible, accelerate wound closure and epithelialization to the statistically significant differences on day 5 (p < 0.05) in comparison to control wounds. Histological examination revealed, that in FKDP-treated wounds the inflammatory response contained predominantly macrophages whilst their morphological untreated variants showed mixed cell infiltrates rich in neutrophils. Predominant macrophages based response creates more favorable environment for healing. In FKDP-dressed wounds the number of microhemorrhages was also significantly decreased (p < 0.05) as compared with undressed wounds. Applied keratin dressing favors reconstruction of a more regular skin structure and assures better cosmetic effect in terms of scar formation and appearance. In conclusion, fur keratin-derived protein dressings significantly accelerated wound healing in the mouse model. Further studies are needed to determine the molecular mechanisms involved in the multilayer wound healing process and to assess the possible use of these dressings for medical purposes.

Topics: Animals; Biocompatible Materials; Biological Dressings; Disease Models, Animal; Keratins; Mice; Skin; Wound Healing; Wounds and Injuries

Keratin proteins have been shown to play a key role in wound healing. Controlled keratin gene (KRT) expression promotes cell growth, migration and differentiation, and as an example of the importance of keratin proteins, absence of KRT17 has been shown to delay wound closure. In addition, downregulation of KRT6 and KRT16 in non-healing chronic venous ulcers suggests that deregulation of keratin expression contributes to non-healing phenotype. A sample of 45 chronic wounds of mixed aetiologies presenting in 31 patients were treated with keratin-based novel topical wound healing products. Thirty-seven wounds or 82% of wounds were either healed or reduced in size of >50% during treatment, with 29 (64%) healing completely and an additional 8 wounds experiencing 50% wound size reduction or greater. Of the wounds that responded, 15 required antimicrobial treatment during their course of treatment, suggesting that keratin dressing treatment should be interrupted briefly and then restarted when wound infection occur.

Topics: Adult; Aged; Aged, 80 and over; Bandages; Chronic Disease; Female; Humans; Hydrogels; Keratins; Male; Middle Aged; Prospective Studies; Wound Healing; Wounds and Injuries; Young Adult

The majority of studies on scar formation have mainly focused on the dermis and little is known of the involvement of the epidermis. Previous research has demonstrated that the scar tissue-derived keratinocytes are different from normal cells at both the genetic and cell biological levels; however, the mechanisms responsible for the fundamental abnormalities in keratinocytes during scar development remain elusive. For this purpose, in this study, we used normal, wound edge and hypertrophic scar tissue to examine the morphological changes which occur during epidermal regeneration as part of the wound healing process and found that the histological structure of hypertrophic scar tissues differed from that of normal skin, with a significant increase in epidermal thickness. Notably, staining of the basement membrane (BM) appeared to be absent in the scar tissues. Moreover, immunofluorescence staining for cytokeratin (CK)10, CK14, CK5, CK19 and integrin-β1 indicated the differential expression of cell markers in the epidermal keratinocytes among the normal, wound edge and hypertrophic scar tissues, which corresponded with the altered BM structures. By using a panel of proteins associated with BM components, we validated our hypothesis that the BM plays a significant role in regulating the cell fate decision of epidermal keratinocytes during skin wound healing. Alterations in the structure of the BM promote basal keratinocytes to adopt a proliferative phenotype both in vivo and in vitro.

Topics: Adolescent; Adult; Basement Membrane; Biomarkers; Cell Differentiation; Cicatrix, Hypertrophic; Epidermis; Female; Gene Expression Profiling; Gene Expression Regulation; Humans; Keratinocytes; Keratins; Male; Phenotype; Wound Healing; Wounds and Injuries; Young Adult

Antimicrobial peptides (AMPs) are endogenous antibiotics that directly affect microorganisms, and also have a variety of receptor-mediated functions. One such AMP, Tilapia piscidin 4 (TP4), was isolated from Nile tilapia (Oreochromis niloticus); TP4 has antibacterial effects and regulates the innate immune system. The aim of the present study was to characterize the role of TP4 in the regulation of wound closure in mice and proliferation of a keratinocyte cell line (HaCaT) and fibroblast cell line (Hs-68). In vitro, TP4 stimulated cell proliferation and activated collagen I, collagen III, and keratinocyte growth factor (KGF) gene expression in Hs-68 cells, which induces keratin production by HaCaT cells. This effect was detectable at TP4 concentrations of 6.25 µg/mL in both cell lines. In vivo, TP4 was found to be highly effective at combating peritonitis and wound infection caused by MRSA in mouse models, without inducing adverse behavioral effects or liver or kidney toxicity. Taken together, our results indicate that TP4 enhances the survival rate of mice infected with the bacterial pathogen MRSA through both antimicrobial and wound closure activities mediated by epidermal growth factor (EGF), transforming growth factor (TGF), and vascular endothelial growth factor (VEGF). The peptide is likely involved in antibacterial processes and regulation of tissue homeostasis in infected wounds in mice. Overall, these results suggest that TP4 may be suitable for development as a novel topical agent for wound dressing.

Topics: Animals; Anti-Bacterial Agents; Cell Line; Cell Proliferation; Cells, Cultured; Collagen Type I; Collagen Type III; Epidermal Growth Factor; Fibroblast Growth Factor 7; Fibroblasts; Keratinocytes; Keratins; Methicillin-Resistant Staphylococcus aureus; Mice; Tilapia; Transforming Growth Factors; Vascular Endothelial Growth Factor A; Wound Healing; Wounds and Injuries

Topics: Animals; Biotechnology; Extinction, Biological; Horns; Keratins; Perissodactyla; Printing, Three-Dimensional; Prostheses and Implants; Prosthesis Design; Wounds and Injuries

Lesions with great loss of skin and extensive burns are usually treated with heterologous skin grafts, which may lead rejection. Cell therapy with mesenchymal stem cells is arising as a new proposal to accelerate the healing process. We tested a new therapy consisting of sodium carboxymethylcellulose (CMC) as a biomaterial, in combination with adipose-derived stem cells (ADSCs), to treat skin lesions in an in vivo rat model. This biomaterial did not affect membrane viability and induced a small and transient genotoxicity, only at the highest concentration tested (40 mg/mL). In a rat wound model, CMC at 10 mg/mL associated with ADSCs increased the rate of cell proliferation of the granulation tissue and epithelium thickness when compared to untreated lesions (Sham), but did not increase collagen fibers nor alter the overall speed of wound closure. Taken together, the results show that the CMC is capable to allow the growth of ADSCs and is safe for this biological application up to the concentration of 20 mg/mL. These findings suggest that CMC is a promising biomaterial to be used in cell therapy.

Topics: Adipose Tissue; Animals; Carboxymethylcellulose Sodium; Cell Proliferation; Cell Survival; Cell- and Tissue-Based Therapy; Cells, Cultured; Collagen; Disease Models, Animal; Immunohistochemistry; Keratins; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Rats, Wistar; Skin; Tissue Engineering; Tissue Scaffolds; Wound Healing; Wounds and Injuries

Low level light therapy (LLLT) is an attractive alternative to enhance wound healing. So far most studies are performed with red or infrared irradiation. However, we recently showed that blue light (470 nm) can significantly influence biological systems, improving perfusion by release of nitric oxide from nitrosyl complexes with haemoglobin in a skin flap model in rats. Here, we compared the effects of blue and red low level light by light-emitting diodes (LEDs) on in vivo wound healing in an excision wound model in rats.. Circular excision wounds were surgically created on the dorsum of each rat. Excisions on either the left or right side were illuminated post-OP and on five consecutive days for 10 min by LED at 470 nm or 630 nm with an intensity of 50 mW/cm(2),while protecting the contralateral side from exposure. In the control group, neither side was illuminated. On day 7 post-OP, we analysed planimetric and histological parameters, as well as expression of keratin-1, keratin-10 and keratin-17 on mRNA level.. Illumination substantially influenced wound healing. Blue light significantly decreased wound size on day 7, which correlated with enhanced epithelialisation. Light also affected mRNA expression. Both wavelengths decreased keratin-1 mRNA on day 7 post-OP, while keratin-10 mRNA level was elevated in both light treated group compared to control. Keratin-17 mRNA was also elevated in the red light group, but was unchanged in the blue light group.. In contrast to previous studies, we showed that also blue light significantly influences wound healing. Furthermore, our data suggest that light therapy can play an important role in normotrophic wound healing by affecting keratin expression. Illumination would provide an easily applicable, safe and cost-effective treatment of surface wounds.

Topics: Animals; Disease Models, Animal; Keratins; Low-Level Light Therapy; Male; Phototherapy; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin; Swine; Wound Healing; Wounds and Injuries

Although chronic wounds are common, treatment for these disabling conditions remains limited and largely ineffective. In this study, we examined the benefit of bone marrow-derived mesenchymal stem cells (BM-MSCs) in wound healing. Using an excisional wound splinting model, we showed that injection around the wound and application to the wound bed of green fluorescence protein (GFP)(+) allogeneic BM-MSCs significantly enhanced wound healing in normal and diabetic mice compared with that of allogeneic neonatal dermal fibroblasts or vehicle control medium. Fluorescence-activated cell sorting analysis of cells derived from the wound for GFP-expressing BM-MSCs indicated engraftments of 27% at 7 days, 7.6% at 14 days, and 2.5% at 28 days of total BM-MSCs administered. BM-MSC-treated wounds exhibited significantly accelerated wound closure, with increased re-epithelialization, cellularity, and angiogenesis. Notably, BM-MSCs, but not CD34(+) bone marrow cells in the wound, expressed the keratinocyte-specific protein keratin and formed glandular structures, suggesting a direct contribution of BM-MSCs to cutaneous regeneration. Moreover, BM-MSC-conditioned medium promoted endothelial cell tube formation. Real-time polymerase chain reaction and Western blot analysis revealed high levels of vascular endothelial growth factor and angiopoietin-1 in BM-MSCs and significantly greater amounts of the proteins in BM-MSC-treated wounds. Thus, our data suggest that BM-MSCs promote wound healing through differentiation and release of proangiogenic factors. Disclosure of potential conflicts of interest is found at the end of this article.

Topics: Angiogenic Proteins; Animals; Cell Differentiation; Diabetes Mellitus, Type 2; Female; Fibroblasts; Genes, Reporter; Green Fluorescent Proteins; Keratins; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred Strains; Mice, Mutant Strains; Mice, Transgenic; Neovascularization, Physiologic; Random Allocation; Skin; Transplantation, Homologous; Wound Healing; Wounds and Injuries

The specific aim of this study was to measure the TS of rat skin wounds during the first week following surgical injury. Biomechanical and histological data were collected daily (days 1 to 7 following surgery) from separate groups of Sprague-Dawley rats (N = 12) each with two 3 cm long parallel skin incisions on the back. The wounds were immediately closed by four simple sutures. A control group (N = 15) was used to obtain TS measurements of unwounded skin. TS was measured by applying a ramp load until wound separation and estimated by dividing the yield strength by the wound area. The time course of biomechanical recovery followed a step-plateau pattern with the largest increase in TS observed one day after surgery (0 - 1.60 g/cm(2)). The plateau stage extended from day 1 to 5 (1.60 - 3.88 g/cm(2)). The final step (day 5-7) indicated a period of rapid rise in wound TS (3.88 - 11.57 g/cm(2)). Since even on day 7 the mean TS was only 4% of unwounded skin, the wound had to be protected from tensile loads. Histological analysis confirmed that the early changes in TS (day 1) correlated with the fibrin accumulation of the wound edges followed by a plateau stage caused by the tissue proliferation. The rapid increase in wound TS was characterized by cross-linking the incisions with collagen fibres with escalating organization. We conclude that from a biomechanical perspective, sutures can be removed during the "plateau phase", but the wound must be protected from tensile loads.

Topics: Animals; Female; Keratins; Muscle, Skeletal; Rats; Rats, Sprague-Dawley; Regeneration; Skin; Sutures; Tensile Strength; Wound Healing; Wounds and Injuries

Fetal epithelium retains the ability to re-epithelialize a wound in organotypic culture in a manner not dependent on the presence of underlying dermal substrata. This capacity is lost late in the third trimester of gestation or after embryonic day 17 (E(17)) in the rat such that embryonic day 19 (E(19)) wounds do not re-epithelialize. Moreover, wounds created in E(17) fetuses in utero heal in a regenerative, scar-free fashion. To investigate the molecular events regulating re-epithelialization in fetal skin, the wound-induced expression profile and tissue localization of activator protein 1 (AP-1) transcription factors c-Fos and c-Jun was characterised in E(17) and E(19) skin using organotypic fetal cultures. The involvement of mitogen-activated protein kinase (MAPK) signaling in mediating wound-induced transcription factor expression and wound re-epithelialization was assessed, with the effect of wounding on the expression of keratinocyte differentiation markers determined. Our results show that expression of AP-1 transcription factors was induced immediately by wounding and localized predominantly to the epidermis in E(17) and E(19) skin. c-fos and c-jun induction was transient in E(17) skin with MAPK-dependent c-fos expression necessary for the re-epithelialization of an excisional wound in organotypic culture. In E(19) skin, AP-1 expression persisted beyond 12 h post-wounding, and marked upregulation of the keratinocyte differentiation markers keratin 10 and loricrin was observed. No such changes in the expression of keratin 10 or loricrin occurred in E(17) skin. These findings indicate that re-epithelialization in fetal skin is regulated by wound-induced AP-1 transcription factor expression via MAPK and the differentiation status of keratinocytes.

Topics: Animals; Butadienes; Cell Differentiation; Enzyme Activation; Female; Fetus; Genes, fos; Keratinocytes; Keratins; Mitogen-Activated Protein Kinases; Nitriles; Rats; Rats, Sprague-Dawley; Skin; Transcription Factor AP-1; Wound Healing; Wounds and Injuries

Apoptosis of the epithelium is deemed to play a pivotal role in the pathogenesis of sepsis. A neoepitope in cytokeratin 18 (CK18), termed M30 neoantigen, becomes available at an early caspase cleavage event during apoptosis of epithelium-derived cells and is not detectable in vital or necrotic epithelial cells. A monoclonal antibody, M30, specifically recognizes a fragment of CK18 cleaved at Asp396 (M30 neoantigen). We used an enzyme-linked immunosorbent assay (ELISA) to measure M30 antigen levels in the sera of 15 septic patients. Healthy humans and critical ill patients suffering from severe trauma served as controls. Mann-Whitney U test was used to calculate significance, and a P value of <0.01 was considered to be statistically significant. Serum levels of the CK18 neoepitope M30 were significantly increased in septic patients (236.88 +/- 47.4 U/L) versus trauma (97.2 +/- 17.1 U/L) and healthy controls (66.9 +/- 9.2 U/L) (P < 0.01 and P < 0.008, respectively). The increased serum level of the CK18 neoepitope in septic patients indicates a heightened apoptotic turnover in epithelial cells as compared with trauma patients and healthy controls. Interestingly, nonsurviving trauma patients exhibited a significant increase in the M30 neoantigen as compared with survivors and healthy controls (P < 0.003 and P < 0.002, respectively). The detection of CK18 neoepitope M30 in the serum might be a useful marker in tracing apoptotic epithelium in septic patients.

Topics: Adult; Biomarkers; Epitopes; Female; Humans; Keratins; Male; Middle Aged; Reference Values; Retrospective Studies; Sepsis; Wounds and Injuries

There is evidence that neuropeptides, especially the opiate receptor agonists, are involved in wound healing. We have previously observed that beta-endorphin, the endogenous ligand for the mu-opiate receptor, stimulates the expression of cytokeratin 16 in a dose-dependent manner in human skin organ cultures. Cytokeratin 16 is expressed in hyperproliferative epidermis such as psoriasis and wound healing. Therefore we were interested to study whether epidermal mu-opiate receptor expression is changed at the wound margins in acute and chronic wounds. Using classical and confocal microscopy, we were able to compare the expression level of mu-opiate receptors and the influence of beta-endorphin on transforming growth factor beta type II receptor in organ culture. Our results show indeed a significantly decreased expression of mu-opiate receptors on keratinocytes close to the wound margin of chronic wounds compared to acute wounds. Additionally beta-endorphin upregulates the expression of transforming growth factor beta type II receptor in human skin organ cultures. These results suggest a crucial role of opioid peptides not only in pain control but also in wound healing. Opioid peptides have already been used in animal models in treatment of wounds; they induce fibroblast proliferation and growth of capillaries, and accelerate the maturation of granulation tissue and the epithelization of the defect. Furthermore opioid peptides may fine-tune pain and the inflammatory response while healing takes place. This new knowledge could potentially be used to design new locally applied drugs to improve the healing of painful chronic wounds.

Topics: Acute Disease; beta-Endorphin; Chronic Disease; Dose-Response Relationship, Drug; Gene Expression Regulation; Humans; Keratins; Organ Culture Techniques; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Opioid, mu; Receptors, Transforming Growth Factor beta; Wound Healing; Wounds and Injuries

To study the redistribution of epidermal stem cells in regenerating wound tissues, and to elucidate the role of epidermal stem cells during wound repair.. 80 circular full-thickness wounds were produced on both sides of the back in 20 male Wistar rats (4 wounds in each animal). Then the 80 wounds were randomly divided into 2 groups: group A (treated with sulfadiazine silver- sulfadiazine zinc cream, n = 40) and group B (without any treatment, n = 40). The infection and healing process of the wounds were observed with naked eyes. Five mice were killed one time 3, 7, 14, and 21 days after the wounding. Two wounds with surrounding normal skin tissues were collected from each mouse. Routine histological examination was conducted with HE staining. Beta(1) integrin and keratin19 (K19), markers of epidermal stem cells, were employed to determine the distribution of epidermal stem cells with streptavidin-peroxidase (SP) immunohistochemical method.. The healing rate of wounds was 80% (32/40) in group A, and 60% (24/40) in group B. No beta(1) integrin and K19 positive cells was found in the granulation tissue of all wounds in these 2 groups at any time point during the healing process. However, a few beta1 integrin and K19 positive cells, bearing no anatomic relation with the epidermal stem cells in the basal layer, were found scattering in the stratum spinosum and stratum granulosum of the epidermis on the wound edges. The closer to the wound the more concentrated the beta(1) integrin and keratin19 positive cells. Their numbers increased gradually along with the shrinking of the wound surface until the wound completely healed. The numbers of the beta(1) integrin and keratin19 positive cells decreased gradually after epithelialization. The numbers of the beta(1) integrin and keratin19 positive cells were obviously lower in the infected wounds than in the uninfected wounds.. Epidermal stem cells participated in the healing of wound. The function of redistribution of epidermal stem cells in wound edges seems to promote the re-epithelialization of granulating wounds.

Topics: Animals; Epithelium; Integrin beta1; Keratins; Male; Random Allocation; Rats; Rats, Wistar; Stem Cells; Wound Healing; Wounds and Injuries

Topics: Humans; Keratins; Lymphatic Metastasis; Lymphatic System; Massage; Neoplastic Cells, Circulating; Sentinel Lymph Node Biopsy; Wounds and Injuries

Cutaneous sensory nerves mediate inflammation and wound healing by the release of neuropeptides such as substance P. Neutral endopeptidase is a cell surface enzyme that degrades substance P and thereby terminates its biologic actions. The distribution of neutral endopeptidase in normal skin and wounded human skin, however, has not been examined. The objectives of this study were to evaluate neutral endopeptidase expression in wounded and unwounded skin as well as in cells derived from human skin. Neutral endopeptidase was strikingly localized in normal skin by immunohistochemistry to keratinocytes of the epidermal basal layer, to hair follicles, eccrine and sebaceous glands as well as to endothelium of blood vessels and to large nerves. Standard incisional human wounds were studied at several time points between 1 h and 28 d after wounding. Staining for neutral endopeptidase was noted in the wound bed 6 h after wounding. In contrast to normal skin, staining of all the epidermal cell layers was noted in the migrating tongue of epithelium in l d wounds. Similar full-thickness staining was noted in 3 d and 7 d wounds in all layers of the new wound epithelium and in a "transition epithelium" near the wound edge. By 28 d post wounding neutral endopeptidase staining again was detected only in the basal layer of the epidermis. Neutral endopeptidase mRNA was detected in normal skin and wounds as well as cultured keratinocytes, fibroblasts and endothelial cells. Neutral endopeptidase enzymatic bioactivity was demonstrated in cultured keratinocytes. While it is known that several metalloproteinases important to tissue repair are produced by keratinocytes, this is the first evidence that keratinocytes produce neutral endopeptidase. Neutral endopeptidase may terminate the proinflammatory and mitogenic actions of neuropeptides in normal skin and wounds.

Topics: Aged; Antibodies; Antibody Specificity; Blotting, Western; Coloring Agents; Drug Contamination; Endothelium, Vascular; Female; Fibroblasts; Humans; Immunohistochemistry; Keratinocytes; Keratins; Male; Microcirculation; Middle Aged; Neprilysin; Reverse Transcriptase Polymerase Chain Reaction; Skin; Wounds and Injuries

Besides group A (GAS), Lancefield group C beta-haemolytic streptococci (GCS) have been implicated as a causative agent in outbreaks of purulent pharyngitis. In this study we have investigated a class CI M protein of a Streptococcus dysgalactiae1:256, revealed that 26% of these sera showed serological cross-reactivity between a 68-kDa cartilage protein and the N-terminal part of MC. Only 8% of the sera of healthy patients showed this property. In additional, MC also cross-reacted with antibodies recognising epidermal keratins. The cross-reacting 68-kDa protein from cartilage was different from human serum albumin, but was recognised with anti-vimentin immune serum. The MC was cloned and the gene sequenced. By using PCR, recombinant gene fragments encoding characteristic peptide fragments of MC were expressed in Escherichia coli. The peptides were used to map the binding sites for plasma proteins and to locate the cross-reacting epitopes on the MC molecule. In consequence, sequence alignments revealed that MC shared homologous regions with vimentin and different keratins. Our data, obtained with MC, suggest that not only infections with GAS but also infections with GCS and possibly GGS (the latter species can also produce class CI M-like proteins) may be responsible for the formation of streptococcal-associated sequel diseases.

Topics: Amino Acid Sequence; Animals; Antigens, Bacterial; Bacterial Outer Membrane Proteins; Bacterial Proteins; Blood Proteins; Blotting, Western; Carrier Proteins; Cartilage; Chick Embryo; Cross Reactions; Electrophoresis, Polyacrylamide Gel; Epitopes; Humans; Immune Sera; Keratins; Molecular Sequence Data; Phagocytosis; Protein Binding; Rabbits; Sequence Homology, Amino Acid; Streptococcal Infections; Streptococcus; Virulence; Wounds and Injuries

Alterations in cell to cell adhesion are necessary to enable the type of cell movements that are associated with epithelial wound healing and malignant invasion. Several studies of transformed cells have related epithelial cell movement to changes in the cell surface expression of the carbohydrate structures represented by the ABO blood group antigens and, in particular, by Lewis antigens and their biosynthetic precursors. To study further the relationship between cell surface carbohydrates and keratinocyte cell movement, experimental wounds were created in human oral mucosa and examined by immunohistochemical methods for their expression of selected cytokeratins (K5, K16, K19), basement membrane components (laminin alpha5 and gamma2-chains, BP180, collagen IV and collagen VII), and blood group antigen precursor structures Le(x), sialosyl-Le(x), Le(y), H antigen, N-acetyllactosamine, and sialosyl-T antigen. The changes induced by wounding in the expression of collagen IV, laminin gamma2-chain (laminin-5), and laminin alpha5-chain were similar to those found in skin wounds and served to define the region of epithelial movement. This region was found to show a marked increase in staining for both Lewis antigen Y (Le(y)) and H blood group antigen, and decreased staining of Le(x), thus indicating an upregulation in wounded epithelium of the fucosyltransferases responsible for the synthesis of the H antigen. The changes in carbohydrate expression extended beyond the wound margin into the nonwounded epithelium, a pattern of expression similar to K16, which was also strongly upregulated in both the outgrowth and the adjacent nonwounded epithelium. These findings provide further support for an influence of such carbohydrate structures on the migratory behavior of epithelial cells.

Topics: Adult; Antibodies; Cell Movement; Glycoconjugates; Humans; Keratins; Laminin; Lewis Blood Group Antigens; Membrane Glycoproteins; Mouth Mucosa; Tissue Distribution; Wound Healing; Wounds and Injuries

Fibronectin, collagen type III, laminin, and cytokeratin 5 were visualized in normal skin and in skin showing early or advanced signs of autolytic decomposition to prove whether the immunohistochemical analysis of these antigens can provide useful information for an age-estimation of skin wounds obtained from putrified corpses. In cases with early signs of decomposition (visible course of veins, greenish discoloration) and without microscopic alterations like relaxation of the epidermal cell layers or destruction of the blood vessel structures, the staining pattern was identical to that found in normal, non-putrefied skin. In skin already showing microscopic alteration of the tissue structure, fibronectin and collagen type III could not be localized unambiguously. The distribution of laminin and cytokeratin 5, however, was well preserved. In advanced putrefied skin no reliable staining results could be obtained for fibronectin, collagen type III, and laminin. Even though cytokeratin 5 was still detectable in remnants of decomposition-resistant skin appendages, no information useful for an age-estimation of skin wounds can be obtained due to the autolytic detachment of the epidermal layers.

Topics: Collagen; Humans; Immunoenzyme Techniques; Keratins; Laminin; Postmortem Changes; Skin; Wound Healing; Wounds and Injuries

Transforming growth factor-alpha (TGF-alpha) is thought to be the major autocrine factor controlling growth in epidermal cells. To explore further the role of TGF-alpha in epidermal growth and differentiation, we used a human keratin K14 promoter to target expression of rat TGF-alpha cDNA to the stratified squamous epithelia of transgenic mice. Unexpectedly, the only regions of epidermis especially responsive to TGF-alpha overexpression were those that were normally thick and where hair follicle density was typically low. This included most, if not all, body skin from 2-day- to 2-week-old mice, and ear, footpad, tail, and scrotum skin in adult mice. In these regions, excess TGF-alpha resulted in thicker epidermis and more stunted hair growth. Epidermal thickening was attributed both to cell hypertrophy and to a proportional increase in the number of basal, spinous, granular, and stratum corneum cells. During both postnatal development and epidermal differentiation, responsiveness to elevated TGF-alpha seemed to correlate with existing epidermal growth factor (EGF) receptor levels, and we saw no evidence for TGF-alpha-mediated control of EGF receptor (EGFR) expression. In adults, no squamous cell carcinomas were detected, but benign papillomas were common, developing primarily in regions of mechanical irritation or wounding. In addition, adult transgenic skin that was still both sensitive to TGF-alpha and subject to mild irritation displayed localized regions of leukocytic infiltration and granular layer loss, characteristics frequently seen in psoriasis in humans. These unusual regional and developmental effects of TGF-alpha suggest a natural role for the growth factor in (1) controlling epidermal thickness during development and differentiation, (2) involvement in papilloma formation, presumably in conjunction with TGF-beta, and (3) involvement in psoriasis, in conjunction with some as yet unidentified secondary stimulus stemming from mild mechanical irritation/bacterial infection.

Topics: Animals; Animals, Newborn; Blotting, Northern; Cell Differentiation; Cell Division; Cells, Cultured; Epidermis; ErbB Receptors; Humans; Keratins; Mice; Mice, Transgenic; Papilloma; Phenotype; Psoriasis; Transforming Growth Factor alpha; Wounds and Injuries

Fifteen patients, eight with burn or scald wounds and seven with split-thickness donor sites, were treated with cultured epithelial allografts. Skin was obtained from HIV-negative donors undergoing circumcision and sheets of epithelium were cultured using the 3T3 feeder method. Multiple post-operative biopsies were performed at various time intervals and stained with a panel of monoclonal antibodies against cytokeratins, involucrin, transferrin receptor and epidermal growth factor receptor. Fresh cultured epithelial sheets, normal skin, standard treated donor sites and burns treated with autografts were also studied. Cytokeratin-10 expression was not observed at treated sites until 4 weeks post-grafting, when normal suprabasal levels were observed. Cytokeratins 13 and 16, usually observed in highly proliferative states such as psoriasis, were observed in epithelial-treated sites for up to 6 months. Other proliferation markers such as Ki67 and transferrin receptor were only expressed 2-3 weeks post-operatively. Involucrin, a marker of keratinocyte terminal differentiation, was expressed throughout newly formed epidermis until 15 weeks, when the normal pattern of granular expression was observed. These results indicate that although the cultured 'allograft' does not survive, it may modulate the proliferation and differentiation of spontaneously regenerating epithelium.

Topics: Adult; Aged; Biological Dressings; Burns; Cell Differentiation; Cell Division; Cells, Cultured; Child; Child, Preschool; Epidermal Cells; Epidermis; Epithelium; Humans; Immunohistochemistry; Keratins; Middle Aged; Skin Transplantation; Time Factors; Wounds and Injuries

The intermediate filament protein keratin 16 is expressed in hyperproliferative epidermis. The present study aims to clarify the relationship between the expression of this keratin type, hyperproliferation (percentage of cells in SG2M phases), and keratinization (keratin 10 expression). These three parameters were quantified in biopsy material taken at different time intervals following sellotape stripping--this being a dynamic in vivo model for the induction of hyperproliferation. From the biopsy specimens cell suspensions were prepared, labeled with antibodies KS8.12 (specially directed against keratin 16) and RKSE60 (directed against keratin 10), and analyzed using flow cytometry. Percentages of cells in SG2M phases were assessed by measuring the relative DNA content after propidium iodide staining. Keratin 16 expression in the suprabasal layer anticipated epidermal proliferation, suggesting a role of the suprabasal compartment in the induction of epidermal growth. Keratin 10 expression decreased about 1 day after the onset of keratin 16 expression, indicating that these processes do not depend directly upon each other.

Topics: Adult; Epidermis; Female; Flow Cytometry; Gene Expression; Humans; Keratins; Male; Time Factors; Wounds and Injuries

When confluent keratinocyte cultures were wounded by cutting with a blade, the cells rapidly retracted from the wounded site, leaving an area denuded of cells. Within 3-4 h of wounding, keratinocytes began to migrate from the edges and gradually reepithelialized the entire denuded area. Mitomycin C did not prevent the reepithelialization but did dramatically inhibit [3H]thymidine incorporation into the leading edge of cells. These results indicate that cell proliferation was not required for reepithelialization. Using a rabbit antibody against urokinase-type plasminogen activator (u-PA) and an avidin-biotin-peroxidase detection method, we localized u-PA in the keratinocytes at the leading edge of the migrating cultures. Cytochalasin B dramatically inhibited the extent of migration and also altered cell morphology; nonetheless, urokinase was detected in the limited number of cells that moved into the wounded area, even in the presence of cytochalasin B. A small but consistent enhancement (36% +/- 9) of plasminogen activator activity was observed in the supernatant of wounded cultures. These data suggest that plasminogen activator may be involved in the migration of keratinocytes that occurs during wound healing.

Topics: Cell Movement; Cells, Cultured; Cytochalasin B; Epidermal Cells; Humans; Infant, Newborn; Keratins; Male; Mitomycin; Mitomycins; Plasminogen Activators; Urokinase-Type Plasminogen Activator; Wounds and Injuries

Serosal tissues consist of a surface mesothelial layer and subsurface spindled connective tissue cells. Surface cells are decorated with antibodies to both low and high molecular weight cytokeratin whereas subserosal cells only express the intermediate filament vimentin. Serosal injury results in the proliferation of multipotential subserosal cells (MSC) which have the ultrastructural morphology of myofibroblasts and yet co-express low molecular weight cytokeratin and vimentin. These cells appear responsible for the re-establishment of surface mesothelium during which they acquire high molecular weight cytokeratin and loose vimentin. There are many parallels between reactive and neoplastic serosal tissues. Desmoplastic/sarcomatoid mesotheliomas resemble the MSC and co-express low molecular weight cytokeratin and vimentin and epithelial mesotheliomas resemble surface mesothelium and express both low and high molecular weight cytokeratin. The ability of normal serosal tissue to modulate its cell shape and intermediate filament expression helps understand the diversity of serosal tumors.

Topics: Antibodies; Antibodies, Neoplasm; Antigens, Neoplasm; Humans; Intermediate Filaments; Keratins; Mesothelioma; Microscopy, Electron; Models, Biological; Neoplasm Proteins; Serous Membrane; Vimentin; Wounds and Injuries

Topics: Animals; Chromatography, DEAE-Cellulose; Electrophoresis, Starch Gel; Hair; Keratins; Male; Rats; Skin; Wounds and Injuries

Topics: Animals; Epinephrine; Glucocorticoids; Glycoproteins; Granulation Tissue; Growth Inhibitors; Homeostasis; Humans; Keratins; Langerhans Cells; Melanocytes; Mice; Mitosis; Sebaceous Glands; Skin; Sleep; Sweat Glands; Wound Healing; Wounds and Injuries