Michele Lanza

The N-terminal fraction of desmoglein 3 encompassing its immunodominant domain is present in human serum: implications for pemphigus vulgaris autoimmunity.

Pemphigus vulgaris (PV) is considered as an autoimmune disease against a tissue-restricted antigen, desmoglein 3, a 130 kDa glycoprotein expressed by keratinocytes of skin and mucous membranes. Therefore, a breakdown of peripheral tolerance is generally invoked to explain this horror autotoxicus. The availability of a self-antigen and the strength of antigenic stimulation represent critical points in the regulation of immune system homeostasis. Our study shows for the first time that the immunodominant fraction of the PV self-antigen is present in sera of healthy individuals and patients as a circulating 30 kDa fragment (sDsg3). These findings provide a good explanation for the N-terminal specificity of antibody production and peptide recognition in PV patients by B and T cell, respectively. Moreover, the presence of the sDsg3 in human sera could allow to reconsider pemphigus as a disease against a circulating antigen; once produced, PV-autoantibodies also recognize the 130 kDa epidermal antigen desmoglein 3 on keratinocyte surface (kDsg3), thus triggering the acantholysis and the clinical manifestations of pemphigus.

The most widespread desmosomal cadherin, desmoglein 2, is a novel target of caspase 3-mediated apoptotic machinery

Apoptotic cells are known to regulate the ordered dismantling of intercellular contacts through caspase activity. Despite the important role of desmoglein (Dsg) 2 in epithelial cell–cell adhesion, the fate of this widespread desmosomal cadherin during apoptosis is yet poorly understood. Here, by means of pharmacological approaches, we investigated whether Dsg2 was targeted by caspases in HaCaT and HT‐29 cell lines undergoing staurosporine (STS)‐induced apoptosis. Results showed that STS induced a caspase‐dependent form of cell‐death in both keratinocytes (HaCaT) and enterocytes (HT‐29), that associated with progressive depletion of Dsg2 from cell lysates. The proteolytic processing of full‐length Dsg2 resulted in the appearance of a 70‐kDa fragment which was released into the cytosol. Consistently, immunofluorescence studies revealed that Dsg2 staining was abolished from cell surface whereas the cytoplasmic region of Dsg2 did localize intracellularly. Plakoglobin (Pg) also underwent cleavage and detached from Dsg2. Apoptotic changes paralleled with progressive loss of intercellular adhesion strength. All these biochemical, morphological, and functional changes were regulated by caspase 3. Indeed, in the presence of the caspase 3‐inhibitor z‐DEVD‐fmk, full‐length Dsg2 protein levels were preserved, whereas the amount of the 70‐kDa fragment was maintained on control levels. Furthermore, cells pretreated with z‐DEVD‐fmk retained the membrane labeling of Dsg2. Taken together, our data demonstrate that the apoptotic processing of Dsg2 is mediated by caspase 3 in epithelial cells. J. Cell. Biochem. 103: 598–606, 2008.

Long-term results of corneal collagen crosslinking for progressive keratoconus

PURPOSE To evaluate long-term keratoconus stability after corneal crosslinking (CXL) with riboflavin. METHODS In this prospective study, 57 eyes of 55 patients with progressive keratoconus, consecutively treated with ultraviolet A (UVA) - riboflavin CXL, were examined with the corneal topographer Pentacam, the biometer IOLMaster and the analyzer of corneal biomechanics Ocular Response Analyzer before and during a 24 months follow-up after CXL. RESULTS Twenty-four months after CXL, there was a significant improvement in best corrected visual acuity (BCVA) (P<0.01), a significant decrease in corneal thinnest point (CTP), keratometry readings at the keratoconus apex (K max), and corneal volume (CV) (P<0.01), and a significant increase in axial eye length (AL) (P=0.01). No significant changes in anterior chamber volume (ACV) and depth (ACD), (P=0.8), corneal hysteresis (CH) (P=0.16) and corneal resistance factor (CRF) (P=0.06) were found. However, in the subgroup of patients with decreased K max readings 24 months after treatment, both CH and CRF showed a significant reduction (P<0.01). CONCLUSION In the first month after the procedure, CXL induces a reduction in corneal volume. During the 24 months follow-up the cornea tends to recover its original volume with a persistence of the CXL efficacy.

Intraocular Pressure and Corneal Biomechanical Properties in Patients with Myotonic Dystrophy

PURPOSE To compare intraocular pressure (IOP) between patients with myotonic dystrophy (DM1) and normal subjects, taking into account corneal characteristics. To determine whether lower IOP measurements in patients with DM1 are due to thinner corneas. DESIGN Comparative case series. PARTICIPANTS Fifty-three eyes of patients with DM1 and 53 eyes of normal age- and sex-matched subjects. METHODS Corneal biomechanical properties and corneal compensated intraocular pressure (IOPcc) measured with the Ocular Response Analyzer (Reichert Inc., Depew, NY), central corneal thickness measured with the Oculus Pentacam (Oculus, Wetzlar, Germany), and IOP were evaluated in patients with DM1 and compared with age- and sex-matched healthy subjects. MAIN OUTCOME MEASUREMENTS Goldmann applanation tonometry, central corneal thickness, corneal hysteresis (CH), corneal resistance factor (CRF), and IOPcc. RESULTS Compared with the healthy subjects, patients with DM1 showed lower IOP (12.4+/-3.6 mm Hg vs. 14.9+/-3.4 mmHg) (P<0.01) and IOPcc (12.7+/-4.5 vs. 15.9+/-3.5) (P<0.01), and thicker cornea (575.9+/-35.02 mum vs. 556.3+/-33.2 microm) (P<0.01), but no significant changes in CH (P = 0.03) and CRF (P = 0.37). CONCLUSIONS Lower IOP in patients with DM1 is not related to differences in central corneal thickness or corneal biomechanical properties. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Corneal parameters and difference between goldmann applanation tonometry and dynamic contour tonometry in normal eyes.

PurposeTo compare the difference between 2 methods of measuring the intraocular pressure (IOP), namely, Goldmann applanation tonometry (GAT) and dynamic contour tonometry (DCT) and to study the relationship with the corneal parameters in patients with no glaucoma signs. MethodsOne hundred and eighteen eyes of 118 healthy subjects from 20 to 77 years of age underwent IOP measurements with DCT and GAT, central corneal thickness, and corneal volume measurements with Pentacam Scheimpflug camera. Pentacam examination has been performed first, then DCT and, after 10 minutes, GAT measurements. ResultsThe measurements with GAT ranged between 11 and 22 mm Hg (m=15.49±2.43 mm Hg), the measurements with DCT ranged between 10.5 and 25.1 mm Hg (m=17.59±2.9 mm Hg). DCT showed a statistically significant (P<0.0001) higher IOP measurement compared with GAT. A relation between differences in IOP with central corneal thickness and corneal volume was found, whereas no relation was found with corneal radius and age. ConclusionsOur results show a discrete correlation between GAT and DCT measurements, but DCT showed slightly higher values of IOP. If DCT should be considered the gold standard, higher values of IOP could still be considered normal. These 2 devices cannot to be used interchangeably.

Defining the involvement of proteinases in pemphigus vulgaris : Evidence of matrix metalloproteinase-9 overexpression in experimental models of disease

Pemphigus vulgaris (PV) acantholysis represents a complex phenomenon wherein a number of factors cooperates. PV serum is known to modulate important cellular events, including kinase activity, transcriptional regulation, and proteinase expression. Indeed, transduction of signals to the cell triggered by PV serum may induce proteinase up‐regulation potentially responsible for disruption of epidermal adhesion and, ultimately, blister formation. Here, we sought to investigate this hypothesis by using both in vivo and in vitro models of PV. Microarray analysis on mouse skin tissues suggested that the equilibrium between extracellular proteinases and their inhibitors moved towards enhanced proteolytic activity in PV neonatal mouse model, at least on the transcriptional level. Conversely, genes codifying cell adhesion proteins were dramatically down‐regulated. The effects of PV serum on the protein level were then studied in vitro both in keratinocyte monolayers and skin organ cultures focusing on matrix metalloproteinase (MMP) 9 expression and activity. By means of Western blotting, zymography, and living cell immunofluorescence studies, we showed that MMP‐9 was early overexpressed in keratinocytes exposed to PV serum, and subsequently secreted in the culture medium. However, we failed to demonstrate extracellular activation of MMP‐9, since it was found in its 92 kDa inactive form in serum‐free culture supernatants. Taken together, our data demonstrated that proteinase expression, particularly of MMP‐9, is modulated by PV serum and associated with PV acantholysis. J. Cell. Physiol. 212: 36–41, 2007.

If pemphigus vulgaris IgG are the cause of acantholysis, new IgG-independent mechanisms are the concause.

Pemphigus vulgaris (PV) is a disease of epidermal adhesion. Its pathogenesis is currently traced back to the action of autoantibodies against antigens located within the intercellular substance of keratinocytes, such as desmogleins and acetylcholine receptors. In the present paper, we sought to elucidate the non‐IgG‐mediated effects of PV sera on keratinocytes. Results showed that PV sera depleted of IgG were able to induce well‐defined changes on keratinocyte morphology and metabolic activity. Indeed, PV IgG‐free sera determined marked alterations on cell shape, accompanied by partial loss of keratinocyte–keratinocyte interactions within 48 h after treatment. Furthermore, PV IgG‐depleted sera caused a sharp reduction of cell viability along with a less sustained weakening of intercellular adhesion strength. In light of the above findings, loss of cell–cell adhesion in PV occurs as a result of the cooperating action of both IgG and non‐IgG‐mediated mechanisms. These data have remarkable consequences on experimental models of PV and might open new “biological” approaches to its therapy. Thus, researchers are well advised that PV pathophysiology cannot be faithfully reproduced by leaving non‐IgG serum factors out of consideration. J. Cell. Physiol. 212:563–567, 2007.

Reliability of the IOLMaster in measuring corneal power changes after photorefractive keratectomy

Purpose: To test the accuracy of the IOLMaster® (Carl Zeiss) in detecting corneal power changes after photorefractive keratectomy (PRK). Setting: Department of Ophthalmology, 2nd University of Naples, Naples, Italy. Methods: Two hundred twenty‐five consecutive eyes that had PRK (mean −5.13 diopters [D] ± 2.98 [SD] [range +0.25 to −16.25 D]) were analyzed. The data included preoperative and postoperative (1, 3, and 6 months) subjective refraction and computerized keratometry. Statistical analysis was performed to determine the correlation between the changes in the subjective refraction at the corneal plane and the changes in keratometry. Results: The mean difference between the changes in refraction and the measured corneal changes was 0.75 ± 1.13 D (range −3.84 to +7.68 D) at 1 month, 0.92 ± 1.10 D (range −0.87 to +7.93 D) at 3 months, and 0.75 ± 0.98 D (range −1.70 to +3.85 D) at 6 months. The difference was significant (P<.001). Conclusion: Automated keratometry provided by the IOLMaster did not accurately reflect the effective refractive changes after PRK, particularly in eyes that had a high dioptric treatment.

Trans epithelial corneal collagen crosslinking for progressive keratoconus: 6 months follow up.

PURPOSE To evaluate keratoconus biomechanical changes after transepithelial corneal collagen cross linking (TE CXL) using riboflavin and ultraviolet A (UVA). SETTING Second University of Naples, Naples, Italy. DESIGN Prospective non comparative case series study. METHODS Patients with progressive keratoconus were examined, before and during a 6 months follow up after TE CXL, with a Pentacam, an Ocular Response Analyzer and an IOLMaster. Best corrected visual acuity (BCVA), refraction, corneal thinnest point (CTP), keratometry readings at the keratoconus apex (Kmax), axial eye length (AL), corneal volume (CV) anterior chamber volume (ACV), anterior chamber depth (ACD), corneal hysteresis (CH) and corneal resistance factor (CRF) were evaluated. RESULTS Thirty-six eyes of 36 patients with progressive keratoconus were analyzed. Six months after treatment there was a significant improvement in BCVA (p<0.01), no significant changes in refraction (p=0.57), CTP (p=0.07), Kmax (p=0.88), AL (p=0.07), CV (p=0.38), ACV (p=0.07), ACD (p=0.7), CH (p=0.1) and CRF (p=0.3). CONCLUSIONS According to our results TE CXL stabilizes most of the patients with progressive keratoconus, without affecting in negative way the corneal elasticity.

Internalization of non-clustered desmoglein 1 without depletion of desmoglein 1 from adhesion complexes in an experimental model of the autoimmune disease pemphigus foliaceus.

Serum antibodies against desmoglein 1 (Dsg1) are known to induce the clinical and histological manifestations of pemphigus foliaceus (PF), autoimmune bullous disease targeting skin. The basic pathophysiological phenomenon of PF blistering is the disruption of epithelial integrity in the granular layer of the epidermis due to separation of keratinocytes from one another, or acantholysis. In this report we investigate the changes in subcellular distribution of Dsg1 in response to serum of patients with PF by using an in vitro model of PF. Immunofluorescence analysis on HaCaT cells indicates that non-clustered Dsg1 is markedly internalized after exposure to serum. However, binding of PF IgG to Dsg1-rich adhesion complexes (desmosomes) does not cause disruption of such structures nor depletion of clustered Dsg1, as revealed by colocalization of PF IgG and Dsg1 in a punctate staining on cell membrane 24 hours after treatment. Furthermore, morphological studies demonstrate that the dramatic alterations induced by PF sera are not the result of apoptotic programs. Taken together, our data strongly suggest that anti-Dsg1 antibodies from PF serum could cause the internalization of non-clustered Dsg1 and perturb the formation of new desmosomes but not directly disrupt Dsg1-containing junctions when stable contacts are already formed.