Giovanni Maria Gaeta

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.

An Investigation on Micro-Raman Spectra and Wavelet Data Analysis for Pemphigus Vulgaris Follow-up Monitoring.

A wavelet multi-component decomposition algorithm has been used for data analysis of micro-Raman spectra of blood serum samples from patients affected by pemphigus vulgaris at different stages. Pemphigus is a chronic, autoimmune, blistering disease of the skin and mucous membranes with a potentially fatal outcome. Spectra were measured by means of a Raman confocal microspectrometer apparatus using the 632.8 nm line of a He-Ne laser source. A discrete wavelet transform decomposition method has been applied to the recorded Raman spectra in order to overcome problems related to low-level signals and the presence of noise and background components due to light scattering and fluorescence. This numerical data treatment can automatically extract quantitative information from the Raman spectra and makes more reliable the data comparison. Even if an exhaustive investigation has not been done in this work, the feasibility of the follow-up monitoring of pemphigus vulgaris pathology has been clearly proved with useful implications for the clinical applications.

Data analysis in Raman measurements of biological tissues using wavelet techniques.

Raman spectroscopy of oral tissues is a promising tool for in vivo diagnosis of oral pathologies, due to the high chemical and structural information content of Raman spectra. However, measurements on biological tissues are usually hindered by low level signals and by the presence of interfering noise and background components due to light diffusion or fluorescence processes. Numerical methods can be used in data analysis, in order to overcome these problems. In this work the wavelet multicomponent decomposition approach has been tested in a series of micro-Raman measurements performed on “in vitro” animal tissue samples. The experimental set-up was mainly composed by a He-Ne laser and a monochromator equipped with a liquid nitrogen cooled CCD equipped with a grating of 1800 grooves/mm. The laser light was focused on the sample surface by means of a 50 X optical objective. The resulting spectra were analysed using a wavelet software package and the contribution of different vibration modes have been singled out. In particular, the C=C stretching mode, and the CH2 bending mode of amide I and amide III and tyrosine contributions were present. The validity of wavelet approach in the data treatment has been also successfully tested on aspirin.

Oral pathology diagnosis by means of micro-Raman spectroscopy on biopsies and blood serum

Pemphigus vulgaris is a chronic, autoimmune, blistering disease of the skin and mucous membranes with a potentially fatal outcome. In this case micro-Raman spectroscopy (&mgr;-RS) can provide a powerful tool for a not invasive analysis of biological tissue for biopsy and in vivo investigation. Based on the evaluation of molecular vibration frequencies, the &mgr;-RS is able to detect the main molecular bonds of protein constituents, as the C-H and C-C ones. Changes in frequency or in the relative intensity of the vibration modes revealed by &mgr;-RS can be related to changes of chemical bond and of protein structure induced by pathology. Quantitative information on the intensity variation of specific Raman lines can be extracted by Partial Least Square (PLS) analysis. &mgr;-RS was performed on some samples of oral tissue and blood serum from informed patients affected by pemphigus vulgaris (an oral pathology) at different pathology stages. The spectra were measured by means of a Raman confocal microspectrometer apparatus using the 633 nm line of a He- Ne laser source. The main protein bonds are clearly detectable in the considered samples giving important information on the integrity and on the state of tissue and blood serum components (lipids and proteins), and consequently on the occurrence of pathology.

Static and dynamic light scattering properties of intralipid aqueous suspension for tissue phantom preparation and calibration

Intralipid is a material widely employed for the preparation of phantoms simulating optical properties of tissues in the field of optical imaging. Two main assumptions underlie the theoretical predictions of their scattering properties: the occurrence of single scattering for any concentrations of Intralipid, thus enabling the use of Mie theory, and a highly anisotropic g-factor giving a forward preferential direction of photon propagation. The importance of precisely estimating the optical properties of such phantoms requires that the accuracy of these assumptions and their range of applicability are very well-assessed. In this paper, we report the first step of an experimental investigation on the scattering properties of Intralipid suspensions at different concentrations. The study is carried out by the joined use of Time-Resolved Transmittance and Dynamic and Static Light Scattering techniques. By the analysis of the angular and temporal dependence of light scattered by Intralipid suspensions, a more reliable description of the scattering process occurring in the samples could be obtained. The results allow us to better elucidate the dependence of scattering properties of suspensions on Intralipid concentrations and composition. This will help in the design and realization of tissue phantoms with more and more reliable optical properties.

Wavelet data analysis of micro-Raman spectra for follow-up monitoring in oral pathologies

A wavelet multi-component decomposition algorithm has been used for data analysis of micro-Raman spectra from human biological samples. In particular, measurements have been performed on some samples of oral tissue and blood serum from patients affected by pemphigus vulgaris at different stages. Pemphigus is a chronic, autoimmune, blistering disease of the skin and mucous membranes with a potentially fatal outcome. The disease is characterized histologically by intradermal blisters and immunopathologically by the finding of tissue bound and circulating immunoglobulin G (IgG) antibody directed against the cell surface of keratinocytes. More than 150 spectra were measured by means of a Raman confocal microspectrometer apparatus using the 632.8 nm line of a He-Ne laser source. A discrete wavelet transform decomposition method has been applied to the recorded Raman spectra in order to overcome related to low-level signals and the presence of noise and background components due to light scattering and fluorescence. The results indicate that appropriate data processing can contribute to enlarge the medical applications of micro-Raman spectroscopy.

A Case of Pemphigus Vulgaris with Serum Autoantibodies Binding Blood Cell Antigen(s) on Immunofluorescence: A Case Report

Patients with pemphigus vulgaris (PV) may develop autoimmunity against an array of non-desmoglein antigens, including keratinocyte acetylcholine receptors. The latter, along with yet unknown autoantigens, could participate to PV pathogenesis. Here, we report a case of PV developing autoimmunity against peripheral blood mononuclear cell (PBMC) antigens. The patient was poorly responsive to steroidal and immunosuppressive drugs. Although the role for this PBMC antigen has not been established, it could affect the behaviour of immune cells that regulate autoantibody production in PV. Deregulation of PBMC activity may therefore influence patients response to pharmacological therapy.

Micro-Raman Spectroscopy and E-SEM analysis of hybrid layer at the dentine/resin interface of three different composite restorative resins

The formation of a hybrid layer at the dentin/resin interface is a critical point in the curative processes. New restorative materials are currently under investigation in order to improve the characteristics of this interface layer. In this paper micro-Raman spectroscopy and Environmental Scanning Electron Microscope (E-SEM) analysis have been performed to investigate the interface properties of three different restorative materials. The experimental investigation has been performed on tooth cavities prepared both by Er:YAG laser ablation and by conventional diamond bur. Laser prepared cavities were realized using 100 ms pulsed light beam with energy of 350 mJ, at a frequency of 20 Hz. The cavities were filled with three different composites: True-Vitality (Den-Mat), Enamel Plus (Micerium), Supreme (3M). The treated samples were sectioned perpendicularly to the exposed dentin surface and the morphological characterization of the samples was performed by means of E-SEM operating in wet mode. This equipment permits to obtain a high-resolution image of surface without conductive coating process. The same samples were then examined by micro-Raman spectroscopy that has already shown its validity in the study of dentin/resin interfaces.

Raman spectroscopy on dentin/resin interface of lased and unlased dental samples

Cavities in human teeth were fabricated by means of Er:YAG laser and compared with samples processed by using conventional drill. The cavities of lased and unlased samples were cured with two different composite resins ( Bis-GMA vs PEX) to investigate differences in their adhesion properties. Morphological characterization of the sample surfaces by SEM analysis was performed. The same samples were then examined by micro-Raman spectroscopy by using He-Ne laser visible light excitation, in order to characterize the dentin/resin interfaces. The Raman spectra exhibited typical peaks of dentine. Resin contributions to the Raman spectra were also detected and have been used to monitor the interface properties of the boundary region. The dependence of the Raman spectrum on spatial position has been monitored. In both lased and unlased samples the PEX diffusion in dentine is limited in a region of order of 10 μm depth. A larger interdiffusion region is observed at the boundary of dentine and Bis-GMA resin.