The role of autophagy in the pathogenesis of exposure keratitis

Incomplete tear film spreading and eyelid closure can cause defective renewal of the ocular surface and air exposure‐induced epithelial keratopathy (EK). In this study, we characterized the role of autophagy in mediating the ocular surface changes leading to EK. Human corneal epithelial cells (HCECs) and C57BL/6 mice were employed as EK models, respectively. Transmission electron microscopy (TEM) evaluated changes in HCECs after air exposure. Each of these models was treated with either an autophagy inhibitor [chloroquine (CQ) or 3‐methyladenine (3‐MA)] or activator [Rapamycin (Rapa)]. Immunohistochemistry assessed autophagy‐related proteins, LC3 and p62 expression levels. Western blotting confirmed the expression levels of the autophagy‐related proteins [Beclin1 and mammalian target of rapamycin (mTOR)], the endoplasmic reticulum (ER) stress‐related proteins (PERK, eIF2α and CHOP) and the PI3K/Akt/mTOR signalling pathway‐related proteins. Real‐time quantitative PCR (qRT‐PCR) determined IL‐1β, IL‐6 and MMP9 gene expression levels. The TUNEL assay detected apoptotic cells. TEM identified autophagic vacuoles in both EK models. Increased LC3 puncta formation and decreased p62 immunofluorescent staining and Western blotting confirmed autophagy induction. CQ treatment increased TUNEL positive staining in HCECs, while Rapa had an opposite effect. Similarly, CQ injection enhanced air exposure‐induced apoptosis and inflammation in the mouse corneal epithelium, which was inhibited by Rapa treatment. Furthermore, the phosphorylation status of PERK and eIF2α and CHOP expression increased in both EK models indicating that ER stress‐induced autophagy promoted cell survival. Taken together, air exposure‐induced autophagy is indispensable for the maintenance of corneal epithelial physiology and cell survival.     

Further evaluation of amniotic membrane banking for transplantation in ocular surface diseases

Objective: To define the best conditions foramniotic membrane preparation, storage and banking in its use for cornealreconstruction.Methods: Amniotic membrane pieces were prepared understerile conditions from placentas selected on the basis of donor medical andsocial history, serology, microbiological tests and histology. The pieces werekept at –140 °C but before grafting they werethawed and stored at 4 °C in RPMI medium, to have apreparation usable within 72 h. This procedure was validatedby testing its therapeutic effectiveness in 25 patients 13 of which had cornealulcers of various origin, 3 had sequelae of herpes simplex keratitis, 3 bandkeratopathy and 6 corneal stem cell deficiency due to chemical or thermalburns.Results: The preparation showed appreciableanti-inflammatory and analgesic effects. In the absence of corneal stem celldeficiency a stable re-epithelialisation was achieved in 15 out of 19 patients.When the limbus was lesioned, the amniotic membrane decreased vascularizationand increased the number of corneal epithelial cells only in 1 of the 6patients. No adverse reactions attributable to the tissue were recorded.Conclusions: A ready-to-use amniotic membrane preparationstored at 4 °C after cryopreservation has been tested incorneal reconstruction. Like the amniotic membrane thawed immediately beforegrafting, this preparation displayed full therapeutic effect in epithelialdefects with stromal ulceration but without severe limbal stem cell deficiency.In two years banking activity 463 pieces of the preparation were successfullydistributed to 90 Italian hospitals.     

Application of Preserved Human Amniotic Membrane for Corneal Surface Reconstruction

Objective: To evaluate the efficacy of preserved human amniotic membrane transplantation for reconstruction of the corneal surface diseases. Methods: Preserved human amniotic membrane transplantations were performed in 84 eyes of 78 patients for corneal surface reconstruction. The indications were limbal stem cell deficiency from Steven–Johnson syndrome, chemical burn and herpes keratitis (27 eyes), bullous keratopathy (26 eyes), persistent epithelial defect and dellen (17 eyes), band keratopathy (11 eyes), preparing for prosthesis (1 eye), corneal ulcer (1 eye) and acute chemical burn (1 eye). Results: Success was noted in 83.3% (70/84) eyes, partial success in 13.1% (11/84) eyes, and failure in 3.6% (3/84) eyes for an average follow-up of 10.5 months (3 – 29 months). No patient developed major immediate post-operative complications. Conclusion: Amniotic membrane transplantation can reduce inflammation, promote corneal epithelial healing, and decrease irritation in corneal surface problems.     

Isolation of a hemidesmosome-rich fraction from a human squamous cell carcinoma cell line

Hemidesmosomes are cell-to-matrix adhesion complexes anchoring keratinocytes to basement membranes. For the first time, we present a method to prepare a fraction from human cultured cells that are highly enriched in hemidesmosomal proteins. Using DJM-1 cells derived from human squamous cell carcinoma, accumulation of hemidesmosomes was observed when these cells were cultured for more than 10 days in a commercial serum-free medium without supplemental calcium. Electron microscopy demonstrated that numerous electron-dense adhesion structures were present along the basal cell membranes of DJM-1 cells cultured under the aforementioned conditions. After removing cellular materials using an ammonia solution, hemidesmosomal proteins and deposited extracellular matrix were collected and separated by electrophoresis. There were eight major polypeptides, which were determined to be plectin, BP230, BP180, integrin α6 and β4 subunits, and laminin-332 by immunoblotting and mass spectrometry. Therefore, we designated this preparation as a hemidesmosome-rich fraction. This fraction contained laminin-332 exclusively in its unprocessed form, which may account for the promotion of laminin deposition, and minimal amounts of Lutheran blood group protein, a nonhemidesmosomal transmembrane protein. This hemidesmosome-rich fraction would be useful not only for biological research on hemidesmosomes but also for developing a serum test for patients with blistering skin diseases.     

自身免疫性大疱病合并侵袭性肺曲霉病:3例报告

目的通过典型病例回顾探讨自身免疫性大疱病糖皮质激素治疗过程中合并肺曲霉病的诊断和治疗情况。方法报告3例自身免疫性大疱病合并侵袭性肺曲霉病病例。3例病例均行痰镜检、培养、抗原检测、胸部CT或者坏死组织病理检查。分离病原菌经形态学和分子生物学鉴定为烟曲霉、黄曲霉和刺孢裸胞壳。结果 3例病例证实为侵袭性肺曲霉病。进行以伏立康唑为主的综合治疗后均治愈。结论自身免疫性大疱病糖皮质激素治疗过程中应警惕肺曲霉病的发生。多种实验室检查可以帮助早期诊断,提高疗效。     

Crystal structure of a rigid four-spectrin-repeat fragment of the human desmoplakin plakin domain

The plakin protein family serves to connect cell-cell and cell-matrix adhesion molecules to the intermediate filament cytoskeleton. Desmoplakin (DP) is an integral part of desmosomes, where it links desmosomal cadherins to the intermediate filaments. The 1056-amino-acid N-terminal region of DP contains a plakin domain common to members of the plakin family. Plakin domains contain multiple copies of spectrin repeats (SRs). We determined the crystal structure of a fragment of DP, residues 175-630, consisting of four SRs and an inserted SH3 domain. The four repeats form an elongated, rigid structure. The SH3 domain is present in a loop between two helices of an SR and interacts extensively with the preceding SR in a manner that appears to limit inter-repeat flexibility. The intimate intramolecular association of the SH3 domain with the preceding SR is also observed in plectin, another plakin protein, but not in α-spectrin, suggesting that the SH3 domain of plakins contributes to the stability and rigidity of this subfamily of SR-containing proteins.     

Evidence for a role of autoantibodies to heat shock protein 60, 70, and 90 in patients with dermatitis herpetiformis

Heat shock proteins (Hsp) are highly conserved immunomodulatory molecules upregulated when cells are exposed to stressful stimuli, such as inflammation. Their involvement in various autoimmune diseases, including autoimmune bullous diseases and celiac disease, has been increasingly recognized. To further study the role of Hsp in autoimmune bullous diseases, we have investigated for the first time the humoral autoimmune response to Hsp40, Hsp60, Hsp70, and Hsp90 in patients with dermatitis herpetiformis (DH; n = 26), bullous pemphigoid (BP; n = 23), and pemphigus vulgaris (PV; n = 16), the first representing a cutaneous manifestation of celiac disease. While in patients with active BP and PV, serum levels of autoantibodies against these Hsp did not differ from the corresponding age- and gender-matched healthy controls (n = 9–14); circulating autoantibodies against Hsp60, Hsp70, and Hsp90 were found to be increased at the active disease stage of DH. Further analysis of this latter patient subgroup showed that these anti-Hsp autoantibodies decreased in parallel with serum autoantibodies against epidermal and tissue transglutaminase during remission of skin lesions following a gluten-free diet, revealing significantly positive correlations. Although further studies on larger groups of patients will be needed to confirm the present data, our results support the notion that autoantibodies against Hsp60, Hsp70, and Hsp90 deserve attention in the study of the mechanisms that promote the development and maintenance of DH and possibly also the underlying celiac disease as well as potential novel disease biomarkers.     

Bullous pemphigoid‐like skin blistering disease in a rhesus macaque (Macaca mulatta)

Autoimmune bullous disease is very uncommon in non‐human primates. We observed a bullous skin disease in a male rhesus monkey while conducting porcine islet xenotransplantation. Fifty days after the transplantation, multiple bullous skin lesions were observed. There was no mucosal involvement. Skin biopsy results demonstrated a subepidermal blister with no necrotic keratinocytes. Immunofluorescent staining showed linear IgG deposition at the roof of the blister. These skin lesions spontaneously disappeared. Considering these results, this monkey was diagnosed with bullous pemphigoid (BP). As far as we know, this is the first report of BP in non‐human primates.     

Introducing a null mutation in the mouse K6alpha and K6beta genes reveals their essential structural role in the oral mucosa

Mammalian genomes feature multiple genes encoding highly related keratin 6 (K6) isoforms. These type II keratins show a complex regulation with constitutive and inducible components in several stratified epithelia, including the oral mucosa and skin. Two functional genes, K6alpha and K6beta, exist in a head-to-tail tandem array in mouse genomes. We inactivated these two genes simultaneously via targeting and homologous recombination. K6 null mice are viable and initially indistinguishable from their littermates. Starting at two to three days after birth, they show a growth delay associated with reduced milk intake and the presence of white plaques in the posterior region of dorsal tongue and upper palate. These regions are subjected to greater mechanical stress during suckling. Morphological analyses implicate the filiform papillae as being particularly sensitive to trauma in K6alpha/K6beta null mice, and establish the complete absence of keratin filaments in their anterior compartment. All null mice die about a week after birth. These studies demonstrate an essential structural role for K6 isoforms in the oral mucosa, and implicate filiform papillae as being the major stress bearing structures in dorsal tongue epithelium.