Multivariate linear regression analysis.

<p>Relationship between serum 25(OH) vitamin D₃ levels (dependent variable) and clinical-biochemical parameters in the study population. <i>Dependent Variable: 25(OH) vitamin D₃</i>.</p

IGFBP-3 inhibits tumour growth <i>in vivo</i>.

<p>(A) Effect of treatment with low (0.37 mg/Kg, green curve) and high (1.87 mg/kg, orange curve) IGFBP-3 doses on the growth over time of Me501 xenografts in SCID mice. Mean results are representative of two different experiments (each experiment on at least 8 mice). Tumor size was measured three times per week with calipers, and volume was calculated as described in the “Methods” section. (B) Immuno-histochemical staining of the cell proliferation marker Ki-67 in Me501 xenografts in tumours excised from SCID mice untreated (A–B) or treated with 1.87 mg/kg IGFBP-3 (C–D). (F) Graph reporting the mean values of Ki-67 positive cells in melanomas from control mice and mice treated with low and high doses of IGFBP-3. The proliferation index was determined as the percentage of proliferating cells (Ki-67 positive cells) in a population of about 800 cells. For this purpose two representative fields were randomly selected in each section and blind-counted by two independent observers. P value was calculated by Mann-Whitney U test.</p

IGFBP-3 up-regulates tyrosinase activity and increases melanin content in Me501 cells.

<p>(A) Tyrosinase activity measured by L-DOPA oxidation (left panel) and melanin content determined by measuring the absorbance at 405 nm (right panel) of lysates of Me501 cells grown in 2% serum, untreated or treated for 96 h with IGFBP-3 (2 µg/mL). (B) Tyrosinase activity of lysates of Me501 cell grown in the absence of serum for 24 and 48 h, treated and untreated with IGFBP-3 (C) Western blot analysis of tyrosinase content in Me501 cells after 0, 24 and 48 h of treatment with IGFBP-3 (2 µg/mL). (D) Phase-contrast microscopy of untreated and IGFBP-3-treated (2 µg/mL) Me501 cells, grown in the presence and in the absence of serum. Original magnification X100. (E, F) IGFBP-3 immuno-histochemical positivity in primary melanoma correlates positively with melanin content. IGFBP-3 is stained red, while melanin stains as dark-brown spots (E) X200 magnification (F) X400 magnification. The asterisks mark the IGFBP-3 immuno-positive melanocyte nests, the white and black arrows indicate the clusters of melanin and IGFBP–3 positive melanocytes, respectively. (G) Box plots showing the correlation between IGFBP-3 tumor score (calculated as described in the Methods) and sample pigmentation. P value was calculated by Mann-Whitney U test.</p

Low IGFBP-3 levels correlate with melanoma progression and patients' survival.

<p>(A) Kaplan-Meier survival curve of melanoma patients having seric IGFPB-3 concentration higher (red line) or lower (blue line) than 4.6 ng/mL. Cut-off value was chosen by ROC curve. P value was calculated by log rank test. (B) Kaplan-Meier survival curve of patients with IGF-1/IGFBP-3 ratio higher (red line) or lower (blue line) than 6.2. Cut-off value was chosen by ROC curve. P value was calculated by log rank test. (C) Mean IGFBP-3 serum levels measured twice (28 patients) during the follow-up. Orange boxes: Patients with stable disease. Blue boxes: patients with progressive disease. P value was calculated by ANOVA test. (D,G) IGFBP-3 immuno-staining in tissue samples from primary melanomas. (E, H) IGFBP-3 immuno-staining in tissue samples from metastatic melanomas. The asterisks indicate positive melanocytes nests in primary melanoma. The arrows indicate IGFBP-3 positive stromal cells. Original magnification, X100. (F,I) Graphic data processing for total (upper panel) and stromal (lower panel) IGFBP-3 immuno-staining score. 20 different samples were taken in each case and the number of IGFBP-3 positive cells was separately counted by two researchers. For each slide, at least 7–10 microscopic fields were randomly chosen. The immuno-histochemical score was evaluated as described in the Methods separately for melanocytic and stromal cells and expressed as tumour and stromal score respectively. A total score was derived for each sample as the sum of tumour and stromal score. Central boxes correspond to values from lower to upper quartile (25th–75th percentile). Middle lines show median. Vertical lines extend from minimum to maximum value. P value calculated by Mann-Whitney U test.</p

IGFBP-3 acts independently of IGF-1.

<p>A) Western blot analysis for the detection of IGF-1 in lysates or culture media (CM) of Me501 cells. IGF-1 rec represents the positive control with recombinant IGF-1 (5 ng). B) Western blot analysis for detection of Tyr 1135-phosphorylation on the IGF-1 receptor β. Me501 cells were grown for 24 h in the presence of 10% serum (left panels) or in the absence of serum (right panels). For each group, treatments with IGFBP-3 or with anti-IGF-1 antibodies were performed. The amount of phosphorylated IGF-1-β receptor was quantified by densitometric analysis using total receptor (which remained unchanged) as the internal standard. C) Scratch-repair capacity of Me501 cells in the presence of anti-IGF1-antibodies and of IGFBP-3. The images shown are representative of experiments that were repeated at least three times.</p

Correlation between IGFBP-3 serum level and patients' recorded variables.

<p>Abbreviations: IGFBP, insulin-like growth factor binding protein; SSM, superficial spreading melanoma; NM, nodular melanoma; ALM, acral lentiginous melanoma.</p><p>^*A receiver operating characteristic (ROC) curve analysis was used to find an average value of metastatic volume in order to dichotomize the patients into two subgroups. Each measurable metastases, measured according to RECIST guideline, was considered as a spherical formation and the volumes calculated using the following formula: volume  =  4/3 π r³.</p

Lysosomal Acid Lipase Activity Is Reduced Both in Cryptogenic Cirrhosis and in Cirrhosis of Known Etiology - Fig 1

<p>DBS-determined LAL activity in cryptogenic cirrhotics, cirrhotics of known etiology and healthy subjects (Panel A). Percentages of cirrhotic patients and healthy subjects with normal (≥0.8 nmol/spot/h), mildly reduced (range 0.4–0.8 nmol/spot/h), or severely reduced LAL activity (<0.4 nmol/spot/h) (Panel B).</p

Univariate and multivariate linear regression analysis to LAL activity in the entire study population.

<p>Univariate and multivariate linear regression analysis to LAL activity in the entire study population.</p

Univariate and multivariate linear regression analysis to LAL activity according to the three study subgroups and in the cirrhotic population as a whole.

<p>Univariate and multivariate linear regression analysis to LAL activity according to the three study subgroups and in the cirrhotic population as a whole.</p

Additional file 1: of No effects of oral vitamin D supplementation on non-alcoholic fatty liver disease in patients with type 2 diabetes: a randomized, double-blind, placebo-controlled trial

Study protocol. (PDF 134 kb