bromochloroacetic-acid and Burns--Chemical

Thermal burns typically display an injury pattern dictated by the transfer of the thermal energy into the skin and underlying tissues and creation of three zones of injury represented by a necrotic zone of disrupted cells and tissue, an intermediate zone of injured and dying cells, and a distant zone of stressed cells that will recover with proper treatment. The wound healing capabilities of a keratin biomaterial hydrogel were studied in two pilot studies, one using a chemical burn model in mice and the other a thermal burn model in swine. In both studies, keratin was shown to prevent enlargement of the initial wound area and promote faster wound closure. Interestingly, treating thermally stressed dermal fibroblast in culture demonstrated that soluble keratin was able to maintain cell viability and promote proliferation. Separation of so-called alpha and gamma fractions of the keratin biomaterial had differential effects, with the gamma fraction producing more pronounced cell survival and recovery. These results suggest that the gamma fraction, composed essentially of degraded alpha keratin proteins, may facilitate cell rescue after thermal injury. Treatment of burns with gamma keratin may therefore represent a potential therapy for wounds with an intermediate zone of damaged tissue that has the potential to contribute to spontaneous healing.

Topics: Animals; Biocompatible Materials; Burns; Burns, Chemical; Cells, Cultured; Humans; Hydrogels; Keratins; Materials Testing; Mice; Pilot Projects; Regeneration; Skin Physiological Phenomena; Sus scrofa; Wound Healing

To access the feasibility of using cultivated oral mucosal epithelial cell transplantation (COMET) for the management of severe corneal burn.. COMET was performed to promote re-epithelialization in two eyes with acute alkaline burn and one eye with chronic alkaline burn, and to reconstruct the ocular surface in two eyes with chronic thermal burn. Autologous oral mucosal epithelial cells obtained from biopsy were cultivated on amniotic membrane. Immunoconfocal microscopy for keratins and progenitor cell markers was performed to characterize the cultivated epithelial sheet. Following transplantation, the clinical outcome and possible complications were documented. The patients were followed for an averaged 29.6+/-3.6 (range: 26-34) months.. Cultivated oral mucosal epithelial sheet expressed keratin 3, 13, and progenitor cell markers p63, p75, and ABCG2. After COMET, all the corneas became less inflamed, and the corneal surface was completely re-epithelialized in 6.0+/-3.2 (range: 3-10) days in all but one patients. Microperforation occurred in one patient, and a small persistent epithelial defect developed in another. Both were solved uneventfully. In all patients, superficial corneal blood vessels invariably developed, and to further improve vision, conjunctivo-limbal autografting (N=3) and/or penetrating keratoplasty (N=3) were performed subsequently. The vision of all patients showed substantial improvement after additional surgeries.. This study showed the potential of COMET to promote re-epithelialization and reduce inflammation in acute corneal burn, and to reconstruct the corneal surface in chronic burn. COMET may, therefore, be considered an alternative treatment for severe corneal burn.

Topics: Adolescent; Adult; Amnion; Biomarkers; Burns, Chemical; Cell Culture Techniques; Cell Transplantation; Cells, Cultured; Corneal Injuries; Epithelial Cells; Epithelium, Corneal; Eye Burns; Feasibility Studies; Humans; Keratins; Male; Middle Aged; Mouth Mucosa; Tissue Engineering; Transplantation, Autologous; Young Adult

To investigate the expression of p63 and cytokeratins throughout the course of producing a cultivated autograft of limbal epithelial cells.. A 75 year old male with a severe alkali burn to his right eye received two cultivated autografts of limbal epithelial cells on amniotic membrane followed by a corneal allograft. Immunostaining for p63 and cytokeratins was performed during ex vivo expansion with 3T3 fibroblasts, following subcultivation on amniotic membrane, and on the excised corneal button.. Cultures grown in the presence of 3T3 fibroblasts or on amniotic membrane displayed positive staining for keratins 14 and 19, and p63, but poor staining for keratin 3 (K3). The excised corneal button possessed a stratified epithelium of K3 positive cells residing on amniotic membrane.. Our results document for the first time the co-expression of cytokeratins 14 and 19 with p63 in a cultivated limbal graft. These data support the conclusion that cultivated grafts of limbal epithelium contain predominantly undifferentiated cells with the potential to regenerate a normal corneal epithelium.

Topics: Aged; Burns, Chemical; Cells, Cultured; Corneal Transplantation; DNA-Binding Proteins; Epithelial Cells; Epithelium, Corneal; Eye Burns; Genes, Tumor Suppressor; Humans; Keratin-14; Keratin-3; Keratins; Limbus Corneae; Male; Phenotype; Phosphoproteins; Stem Cell Transplantation; Trans-Activators; Transcription Factors; Transplantation, Autologous; Tumor Suppressor Proteins

Cannon's disease or white sponge naevus is a relatively rare genetically determined skin disorder. It is inherited as an autosomal dominant trait that displays a high degree of penetrance and expressivity. This article describes cases of Cannon's disease in a mother and her son.

Topics: Adult; Burns, Chemical; Candidiasis, Oral; Child, Preschool; Diagnosis, Differential; Female; Genetic Diseases, Inborn; Humans; Keratins; Keratosis; Leukoplakia, Oral; Male; Mouth Diseases; Mouth Mucosa; Mutation; Pedigree

To report the expression pattern of key molecules by the reconstructed corneal epithelium after a keratolimbal allograft (KLAL) and amniotic membrane transplantation (AMT) for total limbal stem cell deficiency.. Interventional case report.. A 50-year-old woman with severe chemical burns in both eyes received an AMT as a temporary patch at the acute stage, and a KLAL with AMT as a graft at the chronic stage for total limbal stem cell deficiency. The corneal button removed during subsequent corneal transplantation was submitted for immunofluorescence staining with monoclonal antibodies against keratin K3, MUC5AC, connexin 43, integrins alpha3beta1 and alpha6beta4, and laminin 5 for comparison with a normal cornea.. Histologically, a normal stratified corneal epithelium has five to six cell layers that lay on the thick amniotic membrane basement membrane. The phenotype was of a corneal origin, based on expression of positive keratin K3, negative MUC5AC, and positive connexin 43. Furthermore, intact basement membrane complexes were present, evidenced by positive staining to integrins alpha3beta1 and alpha6beta4 and to laminin 5.. A normal corneal epithelial phenotype with normal basement membrane complexes was restored after a KLAL and AMT in a case with total limbal stem cell deficiency.

Topics: Amnion; Biomarkers; Burns, Chemical; Connexin 43; Corneal Diseases; Epithelial Cells; Epithelium, Corneal; Eye Burns; Female; Fluorescent Antibody Technique, Indirect; Humans; Immunophenotyping; Integrin alpha3beta1; Integrin alpha6beta4; Keratins; Keratoplasty, Penetrating; Laminin; Limbus Corneae; Middle Aged; Mucin 5AC; Mucins; Stem Cell Transplantation; Stem Cells; Tissue Preservation; Visual Acuity

The impaired function of corneal epithelial stem cells, located in the limbus, is responsible for corneal surface damage and is clinically characterized by recurrent epithelial defects, conjunctivalization, neovascularization, and corneal opacity. The aim of this study was to investigate corneal limbal stem cell deficiency (LSCD) by means of the impression cytology (IC) technique, using antibodies against cytokeratin 19 (CK19) and cytokeratin 3 (CK3), and to evaluate the diagnostic potential of this approach.. Over a 3-year period (October 1998-June 2001), we collected 113 pairs of IC samples from the eyes of 85 patients with a range of ocular surface diseases and performed an immunocytochemical analysis of CK19 and CK3. Samples with more than 50% cellularity were considered suitable for diagnostic purposes, while samples with less than 50% cellularity were considered with caution. CK19-positive cells in corneal IC were considered an expression of LSCD. We arbitrarily scored LSCD as mild (<25% of CK19-positive cells), moderate (25-50%), and severe (>50%).. One hundred thirteen pairs of IC specimens were obtained from 85 patients; 32 patients (37.6%) had alkaline burns, 18 (21.2%) had other chemical or physical corneal injuries, 13 (15.3%) had complications from wearing contact lenses, 8 (9.4%) had severe microbial keratitis, and 14 (16.5%) had suspicious limbal deficit due to other causes. Nine patients underwent bilateral sampling and 12 had to be resampled. Thirteen pairs of IC specimens were obtained during the follow-up of 8 patients who had undergone limbal stem cell transplantation. In 3 of these patients, IC confirmed reversion to corneal immunophenotype (CK3+/CK19-), whereas in 4, residual limbal damage was still evident; 1 patient relapsed. In the remaining 100 pairs of impressions, we found 77 cases of LSCD, whereas in 16 pairs, we did not find LSCD. Seven pairs were defined as "not valuable" because of the poor quality of both CK samples. Diffuse LSCD, moderate or severe in degree, was found in 26 of 32 patients (81.2%) with alkali burns, whereas mild diffuse LSCD or sectoral LSCD was found in 13 of 18 patients (72.2%) with other chemical-physical injuries, in 10 of 13 patients (76.9%) wearing contact lenses, in 7 of 8 patients (87.5%) with severe microbial keratitis, and in 12 of 14 patients (85.7%) with other corneal pathologies. The quality of impressions was assessed in 77 cases and found to be good or discrete for both CKs in 32 cases (41.5%) and poor in 45 (58.5%): in 46.7% of these cases, the IC was poor only for CK19 and in 45.4% only for CK3.. Immunocytochemistry for seeking out CK19- and CK3-positive cells on corneal IC is a simple and practical method to investigate LSCD. We believe that this technique could have an important role in evaluating patients undergoing therapeutic penetrating keratoplasty to select those who would benefit from limbal stem cell transplantation. Since sampling has been shown to be a critical point, we believe that any improvement in this area will also help to improve the methodology and will contribute to its wider utilization.

Topics: Burns, Chemical; Corneal Injuries; Eye Diseases; Humans; Immunohistochemistry; Keratin-3; Keratins; Limbus Corneae; Stem Cells

Keratocytes synthesize keratan-sulfate proteoglycans (KSPG), lumican and keratocan, to develop and maintain proper collagen interfibrillar spacing and fibril diameter characteristics of the transparent cornea. The purposes of this study are to compare the expression patterns of KSPGs and keratin 12 (K12) respectively by corneal keratocytes and epithelial cells after three different types of injuries; partial and total epithelial debridement and alkali burn.. Corneas of 8-12 week old C57Bl/6J or FVBN mice were wounded by partial epithelial (2 mm in diameter) and total epithelial debridement, and alkali burn (0.1 M NaOH, 30 s) and were allowed to heal for various periods of time, from 1 to 84 days. The corneas were then subjected to light microscopy, in situ and Northern hybridization and RT-PCR for examining the expression of K12 and KSPG in the corneal epithelium and stroma, respectively. Immunohistochemistry with anti-alpha-smooth muscle actin (alpha-SMA) was used to identify myofibroblasts in the stroma of injured cornea.. In 2-3 days, partial epithelial denuded corneas were resurfaced by corneal epithelium positive for K12, and stromal edema caused by debridement disappeared. Total epithelial debridement wounded corneas were resurfaced by conjunctival epithelial cells in 2 weeks. Stromal edema in the total epithelial debridement corneas began to subside after 6 weeks. Corneal epithelial cells resurfaced alkali burned corneas within 3-5 days. In situ and Northern hybridization showed a decrease in keratocan and lumican expression at 6 weeks and increased at 12 weeks post-injury in all wound types. Alpha-SMA positive myofibroblasts in the cornea were detected via immunostaining at the time point when KSPG expression was lowest, 6 weeks post-injury.. The results suggest keratocan and lumican are down-regulated during wound healing at 6 weeks and returned to higher levels at 12 weeks post-injury; implicating that the cells repopulating the injured corneal stroma regained the characteristic function of keratocytes independent of the wound types. However, complete epithelial removal results in irreversible loss of K12 expression.

Topics: Animals; Blotting, Northern; Burns, Chemical; Chondroitin Sulfate Proteoglycans; Cornea; Corneal Injuries; Corneal Stroma; Down-Regulation; Epithelium, Corneal; Eye Burns; Eye Injuries; Eye Proteins; Fibroblasts; Immunoenzyme Techniques; In Situ Hybridization; Keratan Sulfate; Keratin-12; Keratins; Lumican; Mice; Mice, Inbred C57BL; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium Hydroxide; Wound Healing

To investigate the proliferation and differentiation of cultured corneal stem cells and determine the effect of corneal stem cells cultured on amniotic membranes on the limbal area for treating corneal burns.. The proliferation and differentiation of corneal stem cells in vitro had been examined using colony-forming efficiency and immunohistochemistry. The stem cells had been cultured on amniotic membranes and transplanted to the limbal area for treating corneal burns.. Corneal stem cells had a high proliferation capacity in primary and first passage, cytokeratin 3 was not expressed in primary culture but partly in first passage. The stem cells could proliferate to form cell layer on an amniotic membrane. When transplanted, stem cells could survive on limbus. After transplantation, ocular inflammation resolved, the cornea re-epithelialized, the stromal opacity reduced, the superficial neovascularity was lessened and the conjunctival fornix re-established.. Ocular surface conditions could be improved by allograft of corneal stem cells cultured on amniotic membranes.

Topics: Alkalies; Amnion; Animals; Burns, Chemical; Cell Culture Techniques; Cell Differentiation; Cell Division; Cell Transplantation; Cells, Cultured; Epithelium, Corneal; Eye Burns; Graft Survival; Humans; Keratins; Limbus Corneae; Rabbits; Stem Cells; Treatment Outcome

The healing of alkali-injured corneas is characterized by the persistence of polymorphonuclear leukocytes (PMN) in tissues and recurrent corneal epithelial defects. It has been suggested that the proteolytic enzymes secreted by PMN may account in part for the recurrent epithelial defects in the alkali-burned corneas. Cytoplasmic keratins, which form intracellular intermediate filaments, participate in the formation of hemidesmosomes and play a key role in the focal adhesion of epithelial cells to the basement membranes. The K3/K12 keratin pair is a major constituent of differentiated and stratified corneal epithelium. We have recently cloned the cDNA encoding the rabbit K12 keratin. In the present study we examined the expression of K12 keratin during the healing of alkali-burned rabbit corneas by slot-blot and in situ hybridization. Our results indicate that in normal cornea K12 keratin is equally expressed in all cell layers of stratified corneal epithelium and suprabasal layers of limbal epithelium, but not in bulbar conjunctival and other epithelia, i.e., lens, iris, and retinal pigment epithelium. The basal cells of the detached regenerating epithelium of the injured cornea express a very low level of K12 keratin. These observations are consistent with the notion that defective expression of K3/K12 keratins may play a role in the abnormal attachment of the regenerating epithelium to the basement membrane.

Topics: Animals; Autoradiography; Burns, Chemical; Cells, Cultured; Cornea; Corneal Injuries; Disease Models, Animal; DNA Probes; Epithelium; Eye Burns; Female; Gene Expression; In Situ Hybridization; Keratins; Male; Rabbits; RNA, Messenger; Sodium Hydroxide; Wound Healing

In alkali burned rabbit corneas activities of beta-glucuronidase, N-acetyl-beta-D-glucosaminidase and acid beta-galactosidase were studied histochemically in various time intervals after the traumatization. The technic with semipermeable membranes was employed. Within four days after the injury enzyme activities in the traumatized area were almost lacking. The corresponding activities in the unaffected part of the cornea were within the norm. On the 7th day enzyme activities were on an increase (but still subnormal) in the traumatized area. This area was surrounded by a zone of keratocytes with high levels of enzyme activities. This was particularly remarkable in keratocytes subjacent to the epithelium. The activation of all enzymes studied was present in the basal layer of the epithelium and in the endothelium as well. On the 14th day enzyme activities in the traumatized area were nearly restored and on the 32nd day they could not be distinguished from the normal cornea. Beta-galactosidase displayed a relatively maximal increase in the activity of all enzymes investigated.

Topics: Acetylglucosaminidase; Animals; Burns, Chemical; Cornea; Corneal Injuries; Endothelium; Epithelial Cells; Epithelium; Eye Burns; Galactosidases; Glucuronidase; Glycoside Hydrolases; Keratins; Lysosomes; Rabbits; Sodium Hydroxide; Time Factors; Wound Healing

Topics: Administration, Topical; Animals; Anti-Inflammatory Agents; Burns, Chemical; Chlorobutanol; Connective Tissue; Cornea; Drug Combinations; Fibroblasts; Glucocorticoids; Glycosaminoglycans; Histocytochemistry; Keratins; Methylprednisolone; Microscopy, Polarization; Neomycin; Rabbits; Regeneration; Staining and Labeling