bromochloroacetic-acid and Stevens-Johnson-Syndrome

To study the cytokeratin profile and keratinization-related gene expression in keratinized lid margins of chronic Stevens-Johnson syndrome (SJS) patients.. Posterior eyelid margins from 24 chronic SJS patients undergoing mucous membrane grafting and six healthy margins (orbital exenteration, fresh body donors) were studied using immunofluorescence staining (CK10, CK1, filaggrin, transglutaminase 1 (TGM1), (CK19, MUC5AC)) and quantitative PCR (keratinization-related genes-HBEGF, KGF, EGF, TGFα, TGFβ, and TNFα). The staining and gene expression were studied separately in the lid margin epidermis (LME) and lid margin conjunctiva (LMC).. The expression of CK 1/10, filaggrin, and TGM1 in the LMC was similar to the LME in SJS patients. CK19 was expressed only in the basal epithelial layer of the LMC with loss of MUC5AC expression. Increased expression of KGF (p ≤ 0.056), TNFα (p ≤ 0.02), and TGFα (p ≤ 0.01) was observed in the LME of SJS patients compared to normal LME. LMC of SJS patients showed an increased expression of HBEGF (p ≤ 0.002), EGF (p ≤ 0.0002), KGF (p ≤ 0.02), TNFα (p ≤ 0.04), TGFα (p ≤ 0.003), and TGFβ (p ≤ 0.001) compared to normal LMC. Significant differences were observed in the expression of these genes between LME and LMC of SJS patients. These genes were validated using String analysis, which revealed the positive regulation of keratinization.. In lid margins of SJS, there is an increased expression of keratinization-related genes compared to the normal lid margin. Keratinized LMC shares similar cytokeratin profile and keratinization gene expression as seen in cutaneous epithelium of SJS patients, indicating the possibility of the cutaneous epithelium as a source for keratinized LMC.

Topics: Epidermal Growth Factor; Eyelids; Gene Expression; Humans; Keratinocytes; Keratins; Stevens-Johnson Syndrome; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

To compare the regenerative potential of cultured oral mucosal epithelial cells sheets (COMECs) from Stevens-Johnson syndrome (SJS) subjects with those from non-SJS subjects.. Human oral mucosal epithelial cells from SJS and non-SJS subjects were cultured, and colony-forming efficiency (CFE), proliferative and migration potential, expression of cytokines/growth factors and stem cells were compared. COMECs from SJS and non-SJS subjects were transplanted into 12 limbal stem cell-deficient rabbits, and their regenerative potential was analyzed at 1 week after transplantation.. CFE (p>0.05, student's t test), cell proliferation potential (p>0.05, two-way ANOVA) and expression of the cytokeratins (K3, K4, K13, K19) in the oral mucosal epithelial cells from SJS subjects were similar to those of the cells from non-SJS subjects. The initial migratory potential of SJS cells was delayed compared to that of non-SJS cells (p <0.05, RM two-way ANOVA). The SJS cells expressed lower levels of EGF and higher levels of VEGF compared to that of non-SJS cells (p<0.05, one-way ANOVA). In vivo transplanted SJS-COMECs showed similar expression of K3, K4, and K13, proliferation markers (Ki-67; p>0.05, Mann-Whitney U test), and stem cell markers (p63; p>0.05, Mann-Whitney U test) compared to non-SJS COMECs. The initial epithelial defects in vivo were larger in the eyes treated with SJS-COMECs on day 3 (p<0.01, RM two-way ANOVA), but no differences were observed by day 7 between SJS- and non-SJS-COMECs.. These results suggest that, aside from differences in migratory potential, oral mucosal epithelial cells from SJS and non-SJS subjects are comparable in their regeneration potential in treating limbal stem cell deficiency.

Topics: Adolescent; Adult; Cell Movement; Cell Proliferation; Cells, Cultured; Cytokines; Epithelium, Corneal; Female; Humans; Keratins; Male; Middle Aged; Mouth Mucosa; Stem Cells; Stevens-Johnson Syndrome; Young Adult

To clarify the relationship between clinical symptoms and histological status in patients with ocular cicatricial pemphigoid (OCP) and Stevens-Johnson syndrome (SJS).. Clinical symptoms of four OCP and eight SJS patients in the chronic phase were scored with our recently proposed grading system. The histological status of the pannus tissue removed from the corneal surface during surgery was investigated using immunohistological techniques.. All participants showed total loss of the palisades of Vogt and conjunctivalization of the entire corneal surface. All pannus tissues expressed the conjunctival epithelium marker CK4/13. The pannus tissue in clinically keratinized SJS expressed skin epidermal major cytokeratins, but the tissues of nonkeratinized SJS did not.. Clinical observation and the use of our recently proposed grading system agreed with the immunohistological status with respect to keratinization, cell proliferation, and corneal/conjunctival cell typing. These findings facilitate our understanding of the pathogenesis of OCP and SJS, and will hopefully contribute to the development of future treatment strategies and improve predictions of the postoperative prognosis of ocular surface reconstruction in patients with OCP and SJS.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antigens, CD; Biomarkers; Cell Proliferation; Chronic Disease; Conjunctiva; Conjunctival Diseases; Cornea; Corneal Diseases; Epithelial Cells; Female; Fluorescent Antibody Technique, Indirect; Humans; Keratins; Ki-67 Antigen; Male; Middle Aged; Pemphigoid, Benign Mucous Membrane; Preoperative Period; Stem Cells; Stevens-Johnson Syndrome; Visual Acuity; Young Adult

To evaluate the use of autologous serum (AS) from patients with severe ocular surface disease (OSD) in the development of transplantable corneal and oral epithelial tissue equivalents and to compare it with the use of conventional culture methods by using fetal bovine serum (FBS).. AS was obtained from patients with severe OSD secondary to Stevens-Johnson syndrome. Corneal and oral epithelial cells were cultivated in medium supplemented with either AS or FBS. Corneal and oral epithelial equivalents were constructed on denuded amniotic membranes. The bromodeoxyuridine (BrdU) ELISA cell proliferation assay and colony-forming efficiency (CFE) of cells cultivated in AS- or FBS-supplemented media were compared. The morphologic characteristics and the basement membrane assembly of cultivated epithelial equivalents were analyzed by light and electron microscopy, as well as by immunohistochemistry.. BrdU proliferation assay and CFE analysis showed that human corneal and oral epithelial cells cultivated in AS-supplemented media had comparable proliferative capacities compared with FBS-supplemented media. The corneal and oral epithelial equivalents cultivated in AS- and FBS-supplemented media were morphologically similar and demonstrated the normal expression of tissue-specific keratins and basement membrane assembly. The presence of a well-formed stratified epithelium, a basement membrane, and hemidesmosomal attachments was confirmed by electron microscopy.. AS-supplemented cultures were effective in supporting the proliferation of human corneal and oral epithelial cells, as well as the development of transplantable epithelial equivalents. The use of AS is of clinical importance in the development of autologous xenobiotic-free bioengineered ocular surface equivalents for clinical transplantation.

Topics: Adult; Aged; Basement Membrane; Cell Culture Techniques; Cell Differentiation; Cell Proliferation; Cell Transplantation; Colony-Forming Units Assay; Conjunctival Diseases; Cornea; Culture Media; DNA; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Female; Fluorescent Antibody Technique, Indirect; Humans; Keratins; Male; Microscopy, Electron, Scanning; Middle Aged; Mouth Mucosa; Serum; Stevens-Johnson Syndrome

To report an assessment of the two-step surgical combination of cultivated autologous oral mucosal epithelial transplantation (COMET) and penetrating keratoplasty (PKP) used to treat patients with severe limbal deficiency disorders, and to investigate the keratin expression patterns of transplanted surviving oral mucosal epithelium.. Observational case series.. Two patients with Stevens-Johnson syndrome and chemical eye injury were treated by COMET followed, approximately six months later, by a PKP triple procedure. In the course of a mean follow-up period of 22.5 months, their clinical outcomes and the efficacy of this two-step surgical procedure were assessed. In addition, the keratin expression in corneal buttons excised during PKP were immunohistochemically examined to characterize the oral mucosal epithelium that survived ectopically on the cornea. In vivo laser confocal microscopy was used to investigate the structure of the epithelium on the corneal grafts.. The ocular surfaces were successfully reconstructed with cultivated autologous oral mucosal epithelial sheets and PKP. No clinical complications, such as persistent epithelial defects, rejections, or recurrence of cicatrization, were encountered. Postoperative best-corrected visual acuity was 20/125 in one patient and 20/100 in the other. The surviving oral mucosal epithelium, distinguished by its fluorescence pattern, consisted of an irregular, nonkeratinized, stratified epithelium without goblet cells. Immunohistochemical study demonstrated that K3, but not K12, was expressed in the transplanted cultivated oral mucosal epithelium that was similar to oral mucosal tissue. In vivo, the epithelial structure and cell density in the basal cell layer of the corneal grafts were similar to normal cornea.. This study presents a two-step surgical approach to treat severely scarred ocular surfaces by means of a combination of COMET and PKP. Clinical outcomes suggest that this treatment may be beneficial for the maintenance of the reconstructed ocular surface by providing oral mucosal epithelium around the corneal graft.

Topics: Aged; Cataract Extraction; Cell Count; Cell Transplantation; Cells, Cultured; Corneal Diseases; Corneal Opacity; Epithelial Cells; Epithelium, Corneal; Fluorophotometry; Humans; Keratins; Keratoplasty, Penetrating; Lens Implantation, Intraocular; Male; Microscopy, Confocal; Mouth Mucosa; Stevens-Johnson Syndrome; Transplantation, Autologous; Visual Acuity

To evaluate the use of autologous serum (AS)-derived cultivated oral epithelial transplants for the treatment of severe ocular surface disease.. We used AS from 10 patients with severe ocular surface disease and total limbal stem cell deficiency to develop autologous cultivated oral epithelial equivalents. These were compared with epithelial equivalents derived from conventional fetal bovine serum-supplemented medium. Surgery involved removal of the corneal pannus and surrounding diseased tissue and transplantation of the AS-derived epithelial equivalents. The oral equivalents were analyzed by review of histologic and immunohistochemical findings.. Oral epithelial sheets cultivated in AS- and fetal bovine serum-supplemented media were similar in morphology, and both formed basement membrane assembly proteins important for maintaining graft integrity. Complete corneal epithelialization was achieved within 2 to 5 days postoperatively. The ocular surface remained stable without major complications in all eyes during a mean +/- SD follow-up of 12.6 +/- 3.9 months. The visual acuity improved by more than 2 lines in 9 of 10 eyes, with transplanted oral epithelium surviving up to 19 months.. The successful use of an AS-derived oral epithelial equivalent to treat severe ocular surface disease represents an important advance in the pursuit of completely autologous xenobiotic-free bioengineered ocular equivalents for clinical transplantation.

Topics: Adult; Aged; Biomarkers; Cell Culture Techniques; Cell Transplantation; Collagen; Corneal Diseases; Culture Media; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Fas Ligand Protein; Female; Fluorescent Antibody Technique, Indirect; Fluorophotometry; Humans; Keratins; Laminin; Male; Middle Aged; Mouth Mucosa; Serum; Stevens-Johnson Syndrome; Transplantation, Autologous; Treatment Outcome

To analyze immunoregulation of autoreactive T cells specific for epidermal skin antigens, we crossed transgenic mice expressing ovalbumin selectively in keratinocytes under the keratin 5 promoter (K5-mOVA) with mice expressing a K(b)-restricted OVA-specific T cell receptor transgene (OT-I). In athymic double-transgenic mice, OT-I cells developed extrathymically and, at 8-12 weeks of age, initiated severe epidermal damage mimicking toxic epidermal necrolysis (TEN). In contrast, euthymic double-transgenic mice showed thymic deletion of OT-I cells, had few of these cells in the periphery, and never developed skin changes mimicking TEN. Adoptive transfer of OT-I cells isolated from euthymic double-transgenic mice induced TEN in athymic K5-mOVA single-transgenic mice. This spontaneous disease in athymic double-transgenic mice was prevented by transferring lymph node cells from euthymic mice, but was not prevented when CD4(+) or CD25(+) cells were depleted from this population. Although purified CD4(+)CD25(+) cells scarcely prevented the skin disease induced by adoptive transfer of OT-I cells, they efficiently prevented the disease when co-transferred with CD11c(+) dendritic cells. These results suggested that thymus-derived regulatory T cells cooperate with CD11c(+) dendritic cells to prevent life-threatening skin damage such as TEN.

Topics: Adoptive Transfer; Animals; CD11c Antigen; Immunophenotyping; Keratin-15; Keratin-5; Keratins; Mice; Mice, Inbred C57BL; Mice, Nude; Mice, Transgenic; Ovalbumin; Receptors, Interleukin-2; Stevens-Johnson Syndrome; T-Lymphocytes, Regulatory

In severe ocular surface diseases, pathologic keratinization of the ordinarily nonkeratinized corneal and conjunctival mucosal epithelia results in severe visual loss. The expression in conjunctivalized corneas of various proteins known to play important roles in the physiological keratinization process in human epidermis was examined to better understand the mechanism of keratinization.. Conjunctiva covering the cornea was examined in 12 eyes with ocular surface disease in the chronic cicatricial phase. These comprised four Stevens-Johnson syndrome, four ocular cicatricial pemphigoid, and four chemical injuries. Normal conjunctivas from four age-matched individuals served as controls. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to investigate transglutaminase 1 gene expression and immunohistochemistry to study the expression of transglutaminase 1 protein along with other keratinization-related proteins (involucrin, loricrin, filaggrin, and cytokeratins 1 and 10) and cytokeratin pairs 4/13 and 3/12.. Semiquantitative RT-PCR showed that transglutaminase 1 mRNA expression was upregulated in keratinized conjunctiva compared with normal. Also, in this tissue, immunohistochemistry demonstrated elevated levels of transglutaminase 1, involucrin, filaggrin, and the cytokeratin pair 1/10. Levels of loricrin and cytokeratin pairs 4/13 and 3/12, however, remained the same.. Various keratinization-related proteins, transglutaminase 1 included, are most likely involved in the pathogenesis of cicatrizing ocular surface diseases.

Topics: Adult; Aged; Aged, 80 and over; Conjunctiva; Conjunctival Diseases; Female; Filaggrin Proteins; Fluorescent Antibody Technique, Indirect; Humans; Intermediate Filament Proteins; Keratins; Male; Membrane Proteins; Middle Aged; Pemphigoid, Benign Mucous Membrane; Protein Precursors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stevens-Johnson Syndrome; Transglutaminases; Up-Regulation

Cicatricial conjunctivitis may be a sequel to systemic disorders (eg, Stevens-Johnson syndrome, cicatricial pemphigoid) or local disorders such as chemical burns. The cicatrisation is often associated with corneal epithelial changes that cause visual loss. These have been attributed to encroachment of the conjunctival epithelium over the cornea. However, the epithelial anomalies are poorly understood. We investigated the corneal epithelial changes in cicatricial conjunctivitis with an immunohistochemical study of intermediate filaments in normal and pathological specimens. Our results show that the normal corneal epithelium is immunoreactive for cytokeratin 3 (CK 3) but not cytokeratin 19 (CK 19), whereas normal conjunctival epithelium is CK 3 negative and CK 19 positive. Conjunctiva artificially transposed over the cornea (after therapeutic conjunctival flap reconstruction) retained the normal pattern of conjunctival cytokeratin expression (CK 3 negative, CK 19 positive). Conversely, the entire corneal epithelium exhibited the normal cytokeratin pattern (CK 3 positive, CK 19 negative) in 82% of Stevens-Johnson, 80% of cicatricial pemphigoid, and 69% of chemical burns specimens. The findings suggest that conjunctival encroachment is not responsible for the changes at the corneal surface in cicatricial conjunctivitis and that the abnormal corneal epithelium is derived from native corneal cells in these diseases.

Topics: Adult; Aged; Biomarkers; Conjunctivitis; Cornea; Epithelium; Humans; Intermediate Filaments; Keratins; Middle Aged; Pemphigoid, Benign Mucous Membrane; Staining and Labeling; Stevens-Johnson Syndrome; Surgical Flaps

Keratinization of the tarsal conjunctiva in an eye with adequate tears occurs following a number of conditions, including irritation and sensitivity to topical medications, Stevens-Johnson syndrome, radiation to the lid, and occasionally from unknown causes. The keratinized cells produce an epithelial keratitis with subsequent vascularization of the cornea. The morphogenesis of this condition is discussed, along with therapy including mucous membrane grafts and freezing of the tissue.

Topics: Animals; Conjunctiva; Cornea; Epidermis; Eye Diseases; Eyelid Neoplasms; Humans; In Vitro Techniques; Keratins; Keratitis; Metaplasia; Stevens-Johnson Syndrome; Vitamin A Deficiency

Topics: Birth Injuries; Blister; Drug Eruptions; Epidermolysis Bullosa; Erythema; Female; Herpes Simplex; Humans; Infant; Infant, Newborn; Infant, Newborn, Diseases; Keratins; Listeria monocytogenes; Lupus Erythematosus, Systemic; Male; Maternal-Fetal Exchange; Miliaria; Nevus; Pregnancy; Pregnancy Complications; Rubella; Skin Diseases; Skin Diseases, Infectious; Stevens-Johnson Syndrome; Sucking Behavior; Syphilis, Congenital; Urticaria Pigmentosa