Parotid gland cancer.

<p>(<b>A</b>) CCEM shows proteasome immunofluorescence (green) distributed inside cytoplasmic PaCSs while sparing the nucleus (<b>A</b>, 3,000x). The boxed area is enlarged in <b>B</b> (50,000x) to magnify parallel 20S immunogold reactivity despite loss of density of barrel-like particles. Note in <b>A</b> mitochondria and clear unreactive areas merging with PaCS areas. In <b>C</b> (3,000x) a ballooning tumour cell with remnant of a central picnotic nucleus (arrow) is almost entirely formed by a degenerating PaCS only partly reactive to proteasome fluorescence. The boxed area, taken in <b>C</b> at the transition border between its highly and poorly fluorescent parts, shows in <b>D</b> (50,000x) the disappearance of particles and 20S proteasome immunogold below the border, in parallel with immunofluorescence. Another boxed area taken from the top of the heavily fluorescent part in <i>C</i>, shows well-preserved particles and proteasome immunogold in <b>E</b> (50,000x). (<b>F</b> and <b>G</b>) TEM (5,000x) showing two neoplastic cells, one with well-preserved normal nucleus (left), the other (right) with intranuclear accumulation of PaCS-like particles and proteasome immunogold, part of which is boxed in <b>F</b> and enlarged in <b>G</b> (50,000x). Note in <b>F</b> abundant mitochondria. n, nucleolus. (<b>H</b>) Bulging neoplastic cell with a small nucleus surrounded by a rim of dense cytoplasm and immersed in a lake of PaCS-type partly proteasome-reactive, particulate material (4,000x); the boxed area is enlarged in <b>I</b> (20,000x) with islets of residual cytoplasm, one of which is further enlarged in <b>L</b> (75,000x) to show a qualitative autophagic vesicle with double membrane enclosing a collection of well-preserved immunoproteasome-reactive PaCS-type particles. Note in <b>H</b> and <b>I</b> (arrowheads) three other double membrane delimited bodies enclosing PaCS-type particles.</p

Ovarian serous papillary, cervical endometrioid, and thyroid papillary carcinomas.

<p>Juxtanuclear PaCSs in ovarian serous papillary (<b>A</b>, 10,000x), cervical endometrioid (<b>D</b>, 10,000x), and thyroid papillary carcinoma (<b>G</b>, 10,000x), enlarged in <b>B</b>, <b>E</b>, and <b>H</b> (all 25,000x) and even further in <b>C</b>, <b>F</b>, and <b>I</b> (all, 50,000x), respectively, to show their particulate ultrastructure (partially lost in <b>I</b> and, focally, in <b>F</b>) and 20S proteasome immunogold reactivity.</p

<i>H. pylori</i>-colonized non-neoplastic human gastric epithelium (A–C) and well differentiated, gland-forming, deeply invasive, gastric cancer (D–M).

<p>(<b>A</b>) Foveolar epithelium showing 20S proteasome immunofluorescence (green) of infranuclear PaCSs. Overlapping of confocal fluorescence microscopy and toluidine blue images of a semithin resin section from an aldehyde-osmium-fixed block (500x). (<b>B</b>) Juxtanuclear PaCS (asterisk) in a foveolar cell (5,000x) from an adjacent section to <b>A</b>. (<b>C</b>) High resolution (X 65,000) TEM of a boxed PaCS area in <b><i>B</i></b> to show its particulate ultrastructure and 20S proteasome immunogold reactivity. (<b>D</b>) Toluidine blue stained semithin resin section (500x); note pink stained PaCSs (arrowheads), many of which subluminal. (<b>E</b>) Correlative confocal and electron microscopy (CCEM), of a consecutive thin section of the boxed area in <i>D,</i> shows 20S proteasome immunofluorescence (green spots) on a TEM background (5,000x). The juxtaluminal PaCS (arrowhead) is enlarged in <b>F</b> (20,000x) whose boxed area is further enlarged in <b>G</b> (50,000x) to illustrate its particulate ultrastructure with 20S proteasome immunogold reactivity. The same proteasome antibody was used on each face of the gilder finder grid used for CCEM. Note several basally located PaCSs in <b>E</b>. (<b>H</b>) Another thin section (5,000x) from the same tumour block with several supranuclear PaCSs, one of which (arrowhead) is enlarged in the top link inset (18,000x) to show an inner bacterial body (for comparison, another intracellular bacterium positive for H. pylori OMPs immunogold (21,000x) is shown in the bottom right inset), while an juxtaluminal PaCS (boxed area) is enlarged in <b><i>I</i></b> (25,000x) to illustrate NOD1 immunogold reactivity. N, nucleus. (<b>L</b>) TEM (20.000x) of another PaCS in another section from the same block immunostained with <i>H. pylori</i> OMP antibodies; boxed area enlarged in M (50,000x) to better magnify the immunogold particles and barrel-like putative proteasome particles, one of which (boxed in <b>M</b>) is further enlarged in the <b>L</b> inset (200,000x) to illustrate the characteristic punctuate-aligned structure.</p

Lung large cell carcinoma, hepatocarcinoma and colon adenocarcinoma.

<p>(<b>A</b>–<b>C</b>) Juxtanuclear PaCS in a lung large cell carcinoma (<b>A</b>, 10,000x), boxed area enlarged in <b>B</b> (50,000x) whose boxed area is enlarged in <b>C</b> (75,000x) to show its particulate structure and proteasome immunogold reactivity. (<b>D</b> and <b>E</b>) Hepatocellular carcinoma showing both hyaline bodies (hb), reactive for p62 protein in <b>D</b> (30,000x) and unreactive for 19S proteasome in <b>E</b> (25,000x), and PaCS (asterisks) unreactive for p62 and reactive for 19S proteasome. r, ribosomes. Inset to <b>D</b> (450x): hyaline and Mallory bodies react for p62 protein immunoperoxidase in formalin-fixed paraffin sections. (<b>F</b>–<b>H</b>) Colon adenocarcinoma (<b>F</b>, 10,000x) with partly intracellular and partly interstitial bacteria, a cytoplasmic vacuole (v) and a PaCS (boxed) bordering the cell plasma membrane, enlarged in <b>G</b> (40,000x) and <b>H</b> (75,000x). Note partial lysis of proteasome particles, though retaining 19S proteasome reactivity, development of irregular dense bodies and an encircling peripheral membrane, suggesting transition from a PaCS to an autophagic vacuole, also enclosing ribosome-like particles (r) and a bacterium (top right in <b>G</b> and <b>H</b>) with capsulated wall typical of Gram-positive organisms.</p

Pancreatic serous microcystic adenoma.

<p>(<b>A</b>, <b>B</b>) Formalin-fixed paraffin sections; note the clear, apparently “empty” cytoplasm of most cells in <b>A</b> (hematoxylin-eosin, 1,000x) and the poor reactivity in <b>B</b> to proteasome immunofluorescence under confocal microscopy (blue: nuclei; green: proteasome; 800x). (<b>C</b>, <b>D</b>) Semithin aldehyde-osmium fixed resin sections from the same tumour as in <b>A</b> and <b>B</b>, show abundant cytoplasmic PaCSs metachromatically stained pink with toluidine blue (<b>C</b>, 1,000x) and extensively proteasome immunofluorescent under confocal microscopy (<b>D</b>, green: proteasome; 500x). (<b>E</b>) CCEM showing proteasome immunofluorescent PaCS (5,000x); the same specimen under TEM only (<b>F</b>, 5,000x), boxed area enlarged in <b>G</b> (50,000x) shows the PaCS particulate pattern with 20S proteasome immunogold reactivity. (<b>H, I</b>) TEM of another specimen of the same tumour in <b>H</b> (5,000x), boxed area enlarged in <b>I</b> (30,000x) and further enlarged in the inset (50,000x) to show PaCS particulate ultrastructure and 19S proteasome immunogold.</p

Oligonucleotide sequences of CB₁, CB₂ and β-actin used for detection by qRT-PCR analysis.

<p>Oligonucleotide sequences of CB₁, CB₂ and β-actin used for detection by qRT-PCR analysis.</p

CB₁ and CB₂ gene and protein expression levels.

<p>A) qRT-PCR analysis of CB₁ and CB₂ in the duodenal mucosa of untreated celiac disease (UCD) patients, treated celiac disease (TCD) patients and control subject (CS) (n = 6). ***p<0.0001 <i>vs</i> CS, ^§§p<0.001 <i>vs</i> UCD, for CB₁; ^###p<0.0001<i>vs</i> UCD, for CB₂. B) CB₁ and CB₂ levels measured by ELISA in the biopsies of untreated celiac disease (UCD) patients, treated celiac disease (TCD) patients and control subjects (CS) (n = 6). *p<0.05 <i>vs</i> CS, ^##p<0.01 <i>vs</i> UCD, for CB₁@@@p<0.0001 <i>vs</i> CS, ^###p<0.0001 <i>vs</i> UCD, for CB₂. C) CB₁ and CB₂ binding activity in the duodenal mucosa of untreated celiac disease (UCD) patients, treated celiac disease (TCD) patients and control subjects (CS) (n = 4).</p

<i>Ex vivo</i> effect of PT-gliadin on mucosal expression of CB₁ and CB₂.

<p>CB₁ (A) and CB₂ (B) protein levels were detected by immunoblotting in treated celiac disease (TCD) biopsies incubated for 24 h with or without 1 mg/ml peptic tryptic digest of gliadin (PT-gliadin). Each example is representative of experiments performed in 11 TCD patients. Panels C and D show densitometry of CB₁ (C) and CB₂ (D) expression normalised for β-actin. *p<0.001 <i>vs</i> medium. Results are mean±SD.</p

Supplementary Material for: The Clinicopathologic Heterogeneity of Grade 3 Gastroenteropancreatic Neuroendocrine Neoplasms: Morphological Differentiation and Proliferation Identify Different Prognostic Categories

<b><i>Background/Aims:</i></b> Gastroenteropancreatic (GEP) neuroendocrine carcinomas (NECs) are defined as neuroendocrine neoplasms (NENs) with a Ki-67 index >20% according to the 2010 WHO classification. Some reports suggest that this category is heterogeneous. We retrospectively studied a series of 136 patients affected by grade 3 GEP-NECs with the aim to clarify the prognostic role of tumor morphological differentiation, proliferation, defect in mismatch repair proteins (MMRd), CD117 expression, and site of origin. The primary endpoint was the correlation between these parameters and the overall survival (OS). <b><i>Methods:</i></b> Univariate and multivariable Cox proportional hazards regression analyses were used to assess the prognostic significance of various clinical and histopathologic features. <b><i>Results:</i></b> With a median follow-up of 81 months, the median OS was 12.9 months. At multivariate analysis, morphological differentiation, Ki-67 index, MMRd, stage, and CD117 expression were independent prognostic markers in NECs. Three different prognostic categories of NECs were identified according to the degree of morphologic differentiation (well vs. poorly differentiated) and Ki-67 index (<55% vs. ≥55%). On this basis, median OS was 43.6 months in well-differentiated neoplasms with a Ki-67 index 20-55% (named type A), 24.5 months in poorly differentiated neoplasms with a Ki-67 index 20-55% (type B), and 5.3 months (p < 0.0001) in poorly differentiated neoplasms with a Ki-67 index ≥55% (type C). <b><i>Conclusions:</i></b> The present study suggests that GEP-NECs represent a heterogeneous group of neoplasms which can be better classified in different prognostic categories using both tumor morphology and Ki-67 index