Study of Methionine Choline Deficient Diet-Induced Steatosis in Mice Using Endogenous Fluorescence Spectroscopy.

Non-alcoholic fatty liver disease is a highly prevalent condition worldwide that increases the risk to develop liver fibrosis, cirrhosis, and hepatocellular carcinoma. Thus, it is imperative to develop novel diagnostic tools that together with liver biopsy help to differentiate mild and advanced degrees of steatosis. Ex-vivo liver samples were collected from mice fed a methionine-choline deficient diet for two or eight weeks, and from a control group. The degree of hepatic steatosis was histologically evaluated, and fat content was assessed by Oil-Red O staining. On the other hand, fluorescence spectroscopy was used for the assessment of the steatosis progression. Fluorescence spectra were recorded at excitation wavelengths of 330, 365, 385, 405, and 415 nm by establishing surface contact of the fiber optic probe with the liver specimens. A multi-variate statistical approach based on principal component analysis followed by quadratic discriminant analysis was applied to spectral data to obtain classifiers able to distinguish mild and moderate stages of steatosis at the different excitation wavelengths. Receiver Operating Characteristic (ROC) curves were computed to compare classifier's performances for each one of the five excitation wavelengths and steatosis stages. Optimal sensitivity and specificity were calculated from the corresponding ROC curves using the Youden index. Intensity in the endogenous fluorescence spectra at the given wavelengths progressively increased according to the time of exposure to diet. The area under the curve of the spectra was able to discriminate control liver samples from those with steatosis and differentiate among the time of exposure to the diet for most of the used excitation wavelengths. High specificities and sensitivities were obtained for every case; however, fluorescence spectra obtained by exciting with 405 nm yielded the best results distinguishing between the mentioned classes with a total classification error of 1.5% and optimal sensitivities and specificities better than 98.6% and 99.3%, respectively.

Theranostic-PDT with the antibody anti isoform 4 SOD mitocondrial labeled with PpIX in the lung cancer cell line A-549.

BACKGROUND: In this work, a drug product composed of an IgM antibody derived from a hybridoma subclone 4C1F6D5G7B8 was prepared and further labeled with PpIX to be used in cell lines A-549 and MRC-5. The aim of this work was to evaluate the potential theranostic activity of the obtained product together with photodynamic therapy (PDT). METHODS: The IgM antibody labeled with PpIX was used in different concentrations to perform theranostics with PDT in cell lines A-549 and MRC-5 in order to identify the specificity of IgM antibody in lung cancer cells by means of a LED-irradiation system set at 630 nm. The location of the conjugate was further determined by confocal microscopy. RESULTS: The theranostic with conjugate Ab-PpIX in the A-549 cell lines showed fluorescence by confocal microscopy, whereas the MRC-5 cell line showed no reactivity. The PDT with the conjugate in the cell line A-549 decreased its viability 70% compared to the control. On the contrary, with the MRC-5 cell line no viability diference was shown. The confocal microscopy applied to the cell line A-549 showed that the Ab-PpIX was majorly located at the cytoplasm. CONCLUSION: Ab-PpIX showed therapeutical potential in lung cancer cells A-549 and had no activity in non-cancerous lung cells (MCR-5).

Diffuse reflectance spectroscopy as a possible tool to complement liver biopsy for grading hepatic fibrosis in paraffin-preserved human liver specimens.

A diffuse reflectance spectroscopy-based method to score fibrosis in paraffin-preserved human liver specimens has been developed and is reported here. Paraffin blocks containing human liver tissue were collected from the General Hospital of Mexico and included in the study with the patients' written consent. The score of liver fibrosis was determined in each sample by two experienced pathologists in a single-blind fashion. Spectral measurements were acquired at 450-750 nm by establishing surface contact between the optical probe and the preserved tissue. According to the histological evaluation, four liver samples showed no evidence of fibrosis and were categorized as F0, four hepatic specimens exhibited an initial degree of fibrosis (F1-F2), five liver specimens showed a severe degree of fibrosis (F3), and six samples exhibited cirrhosis (F4). The human liver tissue showed a characteristic diffuse reflectance spectrum associated with the progressive stages of fibrosis. In the F0 liver samples, the diffuse reflection intensity gradually increased in the wavelength range of 450-750 nm. In contrast, the F1-F2, F3, and F4 specimens showed corresponding 1.5-, 2-, and 5.5-fold decreases in the intensity of diffuse reflectance compared to the F0 liver specimens. At 650 nm, all the stages of liver fibrosis were clearly distinguished from each other with high sensitivity and specificity (92-100%). To our knowledge, this is the first study reporting a distinctive diffuse reflectance spectrum for each stage of fibrosis in paraffin-preserved human liver specimens. These results suggest that diffuse reflectance spectroscopy may represent a complementary tool to liver biopsy for grading fibrosis.