Impact of allogeneic stem cell transplantation on thyroid function.

PURPOSE: Primary hypothyroidism is a main endocrine complication after allogeneic stem cells transplantation (allo-SCT) in children, but in adults data on post-SCT hypothyroidism are limited. The aims of this observational, cross-sectional study were to assess the prevalence of hypothyroidism in adult allo-SCT recipients according to time from transplantation, and to identify risk factors. METHODS: One hundred and eighty-six patients (M 104; F 82; median age 53.4 years) who underwent allo-SCT between January 2010 and December 2017 were enrolled and divided into three groups, according to time from allo-SCT (1-3 years; 3-5 years; > 5 years). Pre-transplant TSH and fT4 levels were available for all patients. After transplantation, TSH, fT4 and anti-thyroperoxidase antibodies (TPO-Ab) were evaluated. RESULTS: After a follow-up of 3.7 years, 34 (18.3%) patients developed hypothyroidism, with higher prevalence in females (p < 0.001) and in patients who received matched unrelated donor grafts (p < 0.05). No difference in prevalence was found at different time points. Patients who developed hypothyroidism showed higher rate of TPO-Ab positivity (p < 0.05) and higher pre-transplant TSH levels (median 2.34 µU/ml) compared to those with preserved thyroid function (median 1.53 µU/ml; p < 0.001). Multivariable analysis identified higher pre-transplant TSH levels as a positive predictor of hypothyroidism (p < 0.005). The ROC curve analysis identified a pre-SCT TSH cutoff of 1.84 µU/ml, which can predict hypothyroidism with sensitivity 74.1% and specificity 67.2%. CONCLUSIONS: About one out of four patients developed hypothyroidism after allo-SCT, with a greater incidence in females. Pre-transplant TSH levels seem to predict the onset of post-SCT hypothyroidism.

The balance between rRNA and ribosomal protein synthesis up- and downregulates the tumour suppressor p53 in mammalian cells.

Data on the relationship between ribosome biogenesis and p53 function indicate that the tumour suppressor can be activated by either nucleolar disruption or ribosomal protein defects. However, there is increasing evidence that the induction of p53 does not always require these severe cellular changes, and data are still lacking on a possible role of ribosome biogenesis in the downregulation of p53. Here, we studied the effect of the up- and downregulation of the rRNA transcription rate on p53 induction in mammalian cells. We found that a downregulation of rRNA synthesis, induced by silencing the POLR1A gene coding for the RNA polymerase I catalytic subunit, stabilised p53 without altering the nucleolar integrity in human cancer cells. p53 stabilisation was due to the inactivation of the MDM2-mediated p53 degradation by the binding of ribosomal proteins no longer used for ribosome building. p53 stabilisation did not occur when rRNA synthesis downregulation was associated with a contemporary reduction of protein synthesis. Furthermore, we demonstrated that in three different experimental models characterised by an upregulation of rRNA synthesis, cancer cells treated with insulin or exposed to the insulin-like growth factor 1, rat liver stimulated by cortisol and regenerating rat liver after partial hepatectomy, the p53 protein level was reduced due to a lowered ribosomal protein availability for MDM2 binding. It is worth noting that the upregulation of rRNA synthesis was responsible for a decreased p53-mediated response to cytotoxic stresses. These findings demonstrated that the balance between rRNA and ribosomal protein synthesis controls the function of p53 in mammalian cells, that p53 can be induced without the occurrence of severe changes of the cellular components controlling ribosome biogenesis, and that conditions characterised by an upregulated rRNA synthesis are associated with a reduced p53 response.

Endoscopic appearance of GERD: putative role of cell proliferation.

BACKGROUND: Erosive esophagitis is a frequent endoscopic feature in patients with gastro-oesophageal reflux disease. However, most of patients with heartburn/regurgitation have a non-erosive reflux disease. The reason for this heterogeneous impact of gastro-oesophageal reflux disease on oesophageal mucosa is unknown to date. AIM: To evaluate the cell proliferation status of oesophageal epithelium in both healthy normal subjects and patients with gastro-oesophageal reflux disease with or without erosions. MATERIALS AND METHODS: All the subjects underwent endoscopy and biopsies were taken at 5 cm from the squamo-columnar junction. Specimens were analysed both at histology and at transmission electron microscopy. Cell proliferation was evaluated by MIB1 immunostaining. Of the 85 subjects were studied, 10 were healthy controls with normal pH-testing and macroscopical, histological and ultrastructural patterns; 37 were patients with erosive esophagitis, and 38 patients with non-erosive reflux disease. RESULTS: At histology, of the 37 patients affected by erosive esophagitis, 30 had normal mucosa and 7 showed mild oesophagitis. One patient with non-erosive reflux disease showed signs of oesophagitis at histology. At TEM, all patients with gastro-oesophageal reflux disease had ultrastructural patterns of damage i.e. dilations of intercellular spaces (DIS), and all controls had a normal ultrastructural pattern. The mean (+/-SD) MIB1-LI values of normal subjects and non-erosive reflux disease and erosive oesophagitis patients were 62.2% (+/-9.1), 29.7% (+/-7.2) and 16.2% (+/-5.2), respectively; there were significant differences among the three groups (p<0.001). CONCLUSIONS: Oesophageal mucosa of patients with reflux symptoms presents a decrease in MIB1 immunostaining of 50% and 25% in non-erosive reflux disease and erosive esophagitis patients with respect to normal subjects.

Different effects of ribosome biogenesis inhibition on cell proliferation in retinoblastoma protein- and p53-deficient and proficient human osteosarcoma cell lines.

OBJECTIVES: To evaluate the effects of rRNA synthesis inhibition on cell cycle progression and cell population growth according to the RB and p53 status. MATERIAL AND METHODS: RB- and p53-proficient U2OS cells and the RB- and p53-deficient SAOS-2 cells were used, rRNA transcription hindered by actinomycin D, and cell cycle analysed by flow cytometry. RESULTS: One hour of actinomycin D treatment induced in U2OS cells a block at the cell cycle checkpoints G(1)-S and G(2)-M, which was removed only after rRNA synthesis was resumed. rRNA synthesis inhibition did not influence cell cycle progression in SAOS-2 cells. No effect on cell cycle progression after actinomycin D-induced rRNA inhibition was also found in U2OS cells silenced for RB and p53 expression. A mild perturbation of cell cycle progression was observed in U2OS cells silenced for the expression of either RB or p53 alone. We also treated U2OS and SAOS-2 cells with actinomycin D for 1 h/day for 5 days. This treatment lightly reduced growth rate of the U2OS cell population, whereas cell population growth of SAOS-2 cells was completely inhibited. A marked reduction of ribosome content occurred in SAOS-2 cells after the long-term actinomycin D treatment, whereas no modification was observed in U2OS cells. CONCLUSIONS: These results demonstrate that inhibition of ribosome biogenesis does not hinder cell cycle progression in RB- and p53-deficient cells. A daily-repeated transitory inhibition of ribosome biogenesis leads to a progressive reduction of ribosome content with the consequent extinction of cancer cell population lacking RB and p53.

Controversial relationship between the expression of the RB pathway components and RB protein phosphorylation in human breast cancer.

Recent data challenge the relevance of the RB pathway to cancer based on RB inactivation, at least in breast tumors. To obtain information on the actual role of the components of the RB pathway in tumor progression we decided to investigate whether their quantitative changes were associated with variations in the level of RB phosphorylation in human breast cancer. A series of 68 human primary breast carcinomas was studied. Five cases were excluded from the study due to their lack of RB expression. In the remaining 63 cases the expression of cyclin D1, cdk4, cyclin E, and INK4a mRNA was assessed by real-time RT-PCR. The level of RB phosphorylated protein (ppRB) and p27 expression was immunohistochemically analyzed by measuring the percentage of stained cells (labeling index, LI). Cell proliferation rate was measured by Ki67 LI evaluation. The ppRB LI ranged from 5.2 to 73.8 and, as expected, was strongly related to the Ki67 LI (r=0.80; p<0.001). The expression of cyclin D1 mRNA, expressed in arbitrary units (a. u.), ranged from 1.15 to 123.0 and was inversely related to the ppRB LI (p=0.021) and Ki67 LI (p<0.001). Neither the cdk4 (range from 0.07 to 1.13 a. u.) nor the cyclin E (range from 0.13 to 9.27 a. u.) mRNA expression was significantly associated with the ppRB LI (p=0.962 and p=0.103, respectively). Cyclin E was related to Ki67 LI (p=0.022). Both INK4a mRNA (range from 0.01 to 0.60 a. u.) and p27 (LI from 0.0 to 73.1) values were inversely related to the ppRB LI (p=0.022 and p=0.014, respectively). Cyclin D1, cdk4, and cyclin E mRNA expressions were not significantly related to one another. In human primary breast cancers, the expression levels of the factors known to facilitate the cell cycle progression by RB protein phosphorylation were not positively related to ppRB-LI. Pathological increases of cyclin D, cdk4, and cyclin E are very likely associated with other biological functions other than their well-established action on cell cycle progression.

Relationship between the RB1 mRNA level and the expression of phosphorylated RB protein in human breast cancers: their relevance in cell proliferation activity and patient clinical outcome.

The aim of the present study was to ascertain the relationship between the level of RB1 mRNA and the expression of phosphorylated RB protein and the relevance of these two parameters in cancer cell proliferation and clinical outcome in human breast cancer. Sixty-eight primary human breast cancers were considered. The amount of RB1 mRNA was evaluated by quantitative RT-PCR analysis. The level of RB phosphorylation was immunohistochemical defined by measuring the phosphorylated (pp) RB labelling index (LI). Cell proliferation rate was measured by calculating the Ki67 LI. No relation was found between the RB1 mRNA level and the ppRB LI (p=0.565). Both RB1 mRNA value and ppRB LI were related (in an inverse and direct manner, respectively) to Ki67 LI. RB1 mRNA expression was more strictly associated with KI67 LI (p=0.001) than the ppRB LI (p=0.013). Regarding the patient clinical outcome, the separately considered RB parameters did not reach the prognostic significance. However, patients with low RB1 mRNA quantity and patients with high ppRB LI, taken together, had a significantly shorter disease free and overall survival than the group comprehending patients with high RB1 mRNA value and low ppRB LI, and this despite the low number of patients considered. Our results demonstrated that the ppRB LI was independent of the RB1 mRNA level; that both RB parameters are related to the cell proliferation rate and, if collectively considered, have a high informative value on breast tumour prognosis.

Detection of EWS chimeric transcripts by nested RT-PCR to allow reinfusion of uncontaminated peripheral blood stem cells in high-risk Ewing's tumor in childhood.

BACKGROUND AND OBJECTIVE: Ewing's tumors (ET) are primary malignancies of bone and soft tissues characterized in at least 96% of cases by specific fusion transcripts originating from recurrent chromosomal translocations. Clinical protocols for high-risk metastatic ETs include high-dose radiation/chemotherapy followed by autologous peripheral blood stem cell (PBSC) reinfusion. We used nested reverse transcriptase polymerase chain reaction (RT-PCR) to search for the presence of ET-specific transcripts in PBSC collections from patients with high-risk ET in order to collect harvests free from neoplastic cells but still sufficient to obtain early stable engraftment. DESIGN AND METHODS: Thirty-seven harvest samples from 15 ET patients treated with mobilizing chemotherapy were analyzed. Nested RT-PCR was performed to detect ET-specific transcripts in RNA extracted from the PBSC collections. RESULTS: A total of 30 harvests was performed. On average, 2 harvests (range 1-4) were sufficient to collect the minimum required number of mononuclear cells (2.5

Ischemia-reperfusion injury in rat fatty liver: role of nutritional status.

Fatty livers are more sensitive to the deleterious effects of ischemia-reperfusion than normal livers. Nutritional status greatly modulates this injury in normal livers, but its role in the specific setting of fatty liver is unknown. This study aimed to determine the effect of nutritional status on warm ischemia-reperfusion injury in rat fatty livers. Fed and fasted rats with normal or fatty liver induced by a choline deficient diet underwent 1 hour of lobar ischemia and reperfusion. Rat survival was determined for 7 days. Serum transaminases, liver histology and cell ultrastructure were assessed before and after ischemia, and at 30 minutes, 2 hours, 8 hours, and 24 hours after reperfusion. Survival was also determined in fatty fasted rats supplemented with glucose before surgery. The preischemic hepatic glycogen was measured in all groups. Whereas survival was similar in fasted and fed rats with normal liver (90% vs. 100%), fasting dramatically reduced survival in rats with fatty liver (14% vs. 64%, P <.01). Accordingly, fasting and fatty degeneration had a synergistic effect in exacerbating liver injury. Mitochondrial damage was a predominant feature of ultrastructural hepatocyte injury in fasted fatty livers. Glucose supplementation partially prevented the fasting-induced depletion of glycogen and improved the 7-day rat survival to 45%. These data indicate that rat fatty livers exposed to normothermic ischemia-reperfusion injury are much more sensitive to fasting than histologically normal livers. Because glucose supplementation improves both the hepatic glycogen stores and the rat survival, a nutritional repletion procedure may be part of a treatment strategy aimed to prevent ischemia-reperfusion injury in fatty livers.

Primary endocervical extraosseous Ewing's sarcoma/PNET.

A 36-year-old woman presented with intermenstrual spotting and was found to have a cystic mass involving the uterine cervix on a pelvic ultrasound examination. A necrotic and hemorrhagic tumor was excised by hysterectomy and processed for light and electron microscopic investigation and molecular analysis. Microscopic examination revealed a small round cell tumor that immunohistochemical studies (including staining for the highly restricted surface antigen p30/32MIC2) and ultrastructural studies indicated was an extraosseous Ewing's sarcoma (EES)/primitive neuroectodermal tumor (PNET). This diagnosis was established by detection of EWS/ERG fusion transcript through reverse transcription polymerase chain reaction (RT-PCR) with nested primers. Full body computed tomography failed to detect any extrauterine tumor, and the patient is clinically free of disease 18 months after hysterectomy. This case represents the first report of a primary EES/PNET arising in the uterine cervix.

Electron microscopic and immunocytochemical profiles of human subcutaneous fat tissue microvascular endothelial cells.

The ultrastructural and immunocytochemical characteristics of microvascular cells from human subcutaneous fat tissue were studied after the addition of collagenase and Percoll density gradient, respectively. Monoclonal and polyclonal antibodies directed against antigens specific for endothelial cells (factor VIII, Ulex europaeus, CD31, and CD34), pericytes (muscle-specific actin and desmin), adipocytes (S-100 protein), and monocytes-macrophages (MAC 387 and 150.95 protein) were demonstrated by alkaline phosphatase monoclonal anti-alkaline phosphatase and protein A-gold techniques. In addition, to determine whether the harvesting method interfered with microvascular cell function, DOT immunoassays of factor VIII and CD34 were conducted on solutions recovered at collagenase incubation as well as after nylon filtration and Percoll administration, respectively. After the collagenase step, the vast majority of microvascular cells had the typical ultrastructural and immunophenotypical features of endothelial cells. In sharp contrast, following the Percoll step, only 1% to 18% of microvascular cells stained with factor VIII, Ulex europeaus, and CD31, whereas 90% of them expressed the CD34 antigen. Surprisingly, DOT immunoassay revealed the presence of factor VIII in the washing buffer recovered after the Percoll step only. Consequently the decreased expression of common endothelial cell markers (factor VIII, Ulex europaeus, and CD31) observed at the end of the cell isolation procedure was related to the adverse effects of Percoll on endothelial cell function. The CD34 surface molecule, being highly resistant, is particularly well suited for unequivocal characterization of microvascular cells as true endothelium.

An immunohistochemical study of inflammatory abdominal aortic aneurysms.

Seven cases of inflammatory abdominal aortic aneurysms (IAs) were studied by light microscopy, transmission electron microscopy (TEM) and immunohistochemistry. Microscopically, atherosclerosis coexisted with adventitial fibrosis and inflammation. The inflammatory component showed a follicular and a diffuse pattern. Fibrous entrapment of fatty tissue, adventitial vasculitis, neuritis were also common findings. By TEM, sparse smooth muscle cells having dilated cisternae of rough endoplasmic reticulum, large bundles of collagen fibres and oedematous, amorphous fibrillary elastin were observed. By immunohistochemistry, the follicles mostly contained CD22+ B-cells. T4- (CD2+/CD4+/CD8-), T8-(CD2+/CD4-/CD8+) cells as well as macrophages (CD4+/CD11c+) and follicular dendritic reticulum cells (DRC1+) were also detected. The monoclonal antibody Ki-67 reacted with 2-48% of germinal center cells. In the fibrous extrafollicular adventitia, actively synthesizing plasma cells prevailed over T4-cells, and macrophages. Some of the macrophages were also activated (CD4+/CD11c+/CD25+/CD30-). IgM, IgG and C3c deposits were detected in the fibrous zone, in the germinal centers, within adventitial vessels and nerves. HLA-DR antigen was diffusely expressed in cells populating both the fibrous and the follicular zones as well as in endothelial and Schwann cells. These findings suggest that IAs could develop in some individuals affected by advanced atherosclerosis of the abdominal aorta through a pathogenic B-cell response to locally presented antigens.

Conventional and high resolution scanning electron microscopy of biological sectioned material.

Intracellular structures of embedded biological tissues (rat kidney, myocardium and small intestine) were observed by conventional-scanning electron microscopy (C-SEM) and high-resolution scanning electron microscopy (HR-SEM) after glass knife sectioning. C-SEM of semi-thin sections of material processed the same way as conventional transmission electron microscopy (TEM) provided strong backscattered electron (BSE)-dependent, two-dimensional secondary electron images (SEI(-)) which precisely integrated and further extended previous light microscopy (LM) observation of the same specimen. In addition, the three-dimensional (3-D) arrangement of intracellular organelles was appreciated using a mixture of acetone-soluble acrylic resin in place of epoxy resin embedding. Since the identification of such structures was hampered by the use of conventional fixations we introduced osmium maceration as a preliminary step to remove excess cytoplasmic matrix from the specimen. Consequently, semi-thin sections for LM and thin sections for TEM were obtained by sectioning of the tissue blocks. After resin removal, the sections were successfully observed in 3-D under a C-SEM. Finally, the deembedded, osmium treated sections proved to be smooth enough to facilitate deposition of continuous, ultra-thin (1 nm) chromium films and, therefore, HR-SEM studies of macromolecular cell membrane structures.

Electron microscopy of lipid deposits in human atherosclerosis.

The filipin probe associated with tannic acid stain was used to study intra- and extracellular lipids in surgically removed human atherosclerotic lesions (n = 20). In particular, intimal thickenings, fatty streaks and fibrolipidic plaques have been investigated by using mainly transmission and scanning electron microscopy. In the intimal thickenings, the lipid deposits were mainly localized in the subendothelial space as homogeneously sized particles (40-140 nm) and more heterogeneous uni-multilamellar vesicles (35-700 nm). Intermediate lipid forms were also observed. In the fatty streaks, the lipid deposits were intracellular and mainly observed in cells with a monocyte/macrophagic phenotype. Lipid inclusions, lipid lysosomal bodies and intracellular cholesterol crystals very similar to those observed in experimentally induced atherosclerosis were documented. In the fibrolipidic plaque the lipid deposits were found both in the intracellular and in the extracellular compartments. Lipids accumulated within arterial macrophages and smooth muscle cells, usually as lipid droplets. Clusters of lipoprotein-like particles (50 nm in diameter) as well as larger uni-multilamellar lipids (700 nm) with an occasional compound appearance were particularly observed bound to elastic tissue and collagen fibers. These morphological observations outline the complexity of lipid metabolism in the various histological aspects of human atherosclerosis.