Stefania Scarpino

Papillary carcinoma of the thyroid: hepatocyte growth factor (HGF) stimulates tumor cells to release chemokines active in recruiting dendritic cells.

Tissue distribution of dendritic cells was investigated in eight cases of papillary carcinoma of the thyroid using immunohistochemistry. Most dendritic cells had an immature phenotype (CD1a++, CD11c+, CD40+, CD86-, HLA-DR-) and were located at the invasion edge of the tumor. This pattern of distribution was profoundly different from that of CD68+ macrophages, which were evenly distributed throughout the tumor. The ability of tumor cells to release chemotactic factors active on dendritic cells was investigated in primary cultures of the same cases of papillary carcinoma, and was compared to that of the corresponding normal thyroid cells obtained from the tumor-free contralateral lobe. Chemotactic activity of culture supernatants was tested against dendritic cells in a chemotaxis chamber. It was found that papillary carcinoma cells were active in releasing chemotactic activity, that hepatocyte growth factor (HGF; 100 ng/ml) or interleukin (IL)-1beta (10(3) U/ml) induced a fourfold increase in the amount of chemotactic activity released, and that normal thyroid cells obtained from the same patients were as effective as tumor cells. Characterization of chemokines at RNA level revealed that unstimulated cells contain large amounts of IL-8 and monocyte chemotactic protein (MCP)-1 RNAs, and that stimulation with HGF or IL-1beta induced RNAs for regulated upon activation normal T expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-3alpha, interferon-gamma-inducible protein 10 (IP-10), and, to a lesser extent, MIP-1alpha and MIP-1beta. The possibility that HGF/Met interaction has a biological role in vivo was investigated in serial sections of six tumors immunostained for CD1a+, Met protein, and HGF. It was found that all six tumors were intensely and diffusely positive for Met protein, that HGF staining was present in tumor cells of the advancing edge, and that HGF+/Met+ tumor cell nests were infiltrated by CD1a+ dendritic cells. The foregoing observations are consistent with the possibility that HGF stimulation of Met+ tumor cells is one of the molecular mechanisms involved in the recruitment of dendritic cells.

Increased expression of Met protein is associated with up-regulation of hypoxia inducible factor-1 (HIF-1) in tumour cells in papillary carcinoma of the thyroid

Met protein, the high affinity receptor for hepatocyte growth factor (HGF), was highly expressed by the tumour cells of 64 well‐differentiated papillary carcinomas of the thyroid. The p145 mature form and the p170 precursor form of the protein were both isolated from the tumours. Enhanced expression of Met protein was associated with a 9.5 ± 5‐fold increase in MET RNA transcript levels, suggesting increased transcription of the gene. In the same tumours, the levels of RNA transcripts for hypoxia inducible factor‐1 (HIF‐1), a potent stimulator of met gene transcription, were 4.5 ± 3‐fold higher than those present in the surrounding normal thyroid tissues. HIF‐1 is generally induced by hypoxia. Histological features suggestive of a hypoxia were observed in 37 of 50 tumours and included coagulative necrosis, psammoma bodies, cystic changes, intratumoural haemorrhage, and hyalinization of the fibrous stroma. Immunostaining for Met protein was particularly intense in some cells located at the tumour periphery which were characterized by an invasive phenotype. Microdissection of tumour cell nests from the invading front revealed that the levels of RNA transcripts for MET/HIF were higher than in the centre of the tumour in four of nine cases. Taken together, the findings of this study suggest that HIF‐1, perhaps driven by hypoxia, may be one of the factors leading to the increased transcription of met gene in papillary carcinoma and that this event is often more pronounced at the tumour periphery. Copyright

Expression of autoimmune regulator gene (AIRE) and T regulatory cells in human thymomas

Expression of the autoimmune regulator gene (AIRE) and the presence of CD25+/forkhead box p3 (FoxP3)+ T regulatory (Treg) cells were investigated in histologically normal adult thymi and in thymomas using immunohistochemistry and quantitative real‐time polymerase chain reaction (PCR). In the normal thymus staining for AIRE was detected in the nucleus of some epithelial‐like cells located in the medulla; in thymomas AIRE‐positive cells were extremely rare and could be detected only in the areas of medullary differentiation of two B1 type, organoid thymomas. RNA was extracted from 36 cases of thymoma and 21 non‐neoplastic thymi obtained from 11 myasthenic (MG+) and 10 non‐myasthenic (MG–) patients. It was found that AIRE is 8·5‐fold more expressed in non‐neoplastic thymi than in thymomas (P = 0·01), and that the amount of AIRE transcripts present in the thymoma tissue are not influenced by the association with MG, nor by the histological type. A possible involvement of AIRE in the development of MG was suggested by the observation that medullary thymic epithelial cells isolated from AIRE‐deficient mice contain low levels of RNA transcripts for CHRNA 1, a gene coding for acetylcholine receptor. Expression of human CHRNA 1 RNA was investigated in 34 human thymomas obtained from 20 MG– patients and 14 MG+ patients. No significant difference was found in the two groups (thymoma MG+, CHRNA1 = 0·013 ± 0·03; thymoma MG‐, CHRNA1 = 0·01 ± 0·03). In normal and hyperplastic thymi CD25+/Foxp3+ cells were located mainly in the medulla, and their number was not influenced by the presence of MG. Foxp3+ and CD25+ cells were significantly less numerous in thymomas. A quantitative estimate of Treg cells revealed that the levels of Foxp3 RNA detected in non‐neoplastic thymi were significantly higher (P = 0·02) than those observed in 31 cases of thymomas. Our findings indicate that the tissue microenvironment of thymomas is defective in the expression of relevant functions that exert a crucial role in the negative selection of autoreactive lymphocytes.

A rapid method of Sertoli cell isolation by DSA lectin, allowing mitotic analyses

We have developed a rapid and convenient method of Sertoli cell preparation for studying the growth kinetics of these cells in in vitro culture. Datura Stramonium agglutinin (DSA)-coated dishes were used to rapidly purify single Sertoli cells from immature rat testis. We have monitored by immunohistochemical markers the degree of contamination of our Sertoli cell preparation by other cell types. The cell preparation is essentially free of germ cells and interstitial cells and contains a minimal percentage of myoid cells. Sertoli cells isolated with this method retain functional activities such as the FSH responsiveness in terms of cAMP production. In addition, we have studied the proliferative activity of Sertoli cells isolated by lectin binding from rats of different ages. Sertoli cells exhibited a characteristic pattern of proliferation which was a function of the donor animal age. The proliferative activity of isolated Sertoli cells decreased with age, being much higher in 3 day-old rats than in older animals. A similar pattern was observed when the mitotic activity of Sertoli cells in response to mitogens present in the testicular extracts from 5 day-old rats was evaluated. The method described here reduces or eliminates many of the drawbacks of the conventional procedures used to isolate Sertoli cells, thus providing a useful tool in studies of growth kinetics and regulation of cell proliferation in vitro.

Hepatocyte growth factor (HGF) stimulates tumour invasiveness in papillary carcinoma of the thyroid

The present study has investigated the functional role of the Met receptor in primary cultures of 20 papillary carcinomas and of normal thyroid cells obtained from the same patients. Normal and tumour cells grew as adherent cells, formed a confluent monolayer after 10–20 days, had epithelial morphology, and were immunoreactive for cytokeratin, vimentin, and thyroglobulin. The potential effect of hepatocyte growth factor (HGF) on cell invasiveness was investigated in Boyden chambers, using a nucleopore filter coated with Matrigel as the barrier and HGF as the chemoattractant. Tumour cells of five out of seven cases of papillary carcinoma were more responsive to HGF than the corresponding normal cells in terms of the number of migrated cells per mm2. Involvement of the Met receptor in the HGF‐induced migratory response was suggested by the observation that the agonistic anti‐Met monoclonal antibody (MAb) DO‐24 was equally effective. HGF did not affect the proliferative activity of thyroid cells. Under the same experimental conditions, 10 per cent fetal bovine serum (FBS) induced a two‐fold increase in [3H]thymidine incorporation into normal cells and tumour cells. These findings are consistent with the possibility that HGF plays a crucial role in determining the invasiveness of tumour cells in papillary carcinoma of the thyroid. Copyright

Papillary carcinoma of the thyroid : Low expression of NCAM (CD56) is associated with downregulation of VEGF-D production by tumour cells

The expression of NCAM was investigated in tissue sections of 61 cases of papillary carcinoma and in 14 lymph node metastases using immunohistochemistry. Tumour cells of 18 primary tumours were not stained, whereas in the remaining 43 cases, NCAM was expressed in less than 5% tumour cells. Similar results were obtained when NCAM expression was evaluated at the RNA level. Reduced expression of NCAM is an early event since 6/15 cases (40%) of micro‐carcinoma were NCAM‐negative. NCAM‐positive tumour cells were more often located at the invasion front of the tumour. It has been reported that NCAM expression may affect lymphangiogenesis. In tissue sections immunostained for podoplanin, it was found that lymphatic vessels were extremely rare inside the body of the tumour, and were mostly associated with foci of chronic inflammation and/or of reparative fibrosis. Lymphangiogenesis is sustained by VEGF‐C, VEGF‐D, and FGF2. Analysis of micro‐dissected samples of the tumour and of the paired normal thyroid tissue revealed that RNA transcripts for VEGF‐D were significantly less numerous in the tumour tissue (p = 0.001). The potential role of NCAM in tumour cell biology was investigated by silencing the NCAM gene in the TPC1 thyroid papillary carcinoma cell line. It was found that NCAM down‐regulation caused a significant reduction (p < 0.05) in the expression of both VEGF‐C and VEGF‐D mRNAs. In addition, NCAM‐silenced TPC‐1 cells were more adhesive to different extracellular matrix components, and were less efficient in cell migration (59% reduction; p < 0.05) and invasiveness (68% reduction). These latter results confirm that modifications of NCAM expression cause profound alterations in the adhesive and migratory properties of tumour cells, but are in apparent discrepancy with the observation that loss of NCAM is usually associated with increased tumour invasiveness in vivo. Copyright

Papillary carcinoma of the thyroid: evidence for a role for hepatocyte growth factor (HGF) in promoting tumour angiogenesis.

The pattern of vascularization of papillary carcinoma was investigated in tumour sections from 31 cases and in primary cultures from 12 cases. Tumour sections were immunostained for von Willebrand Factor (vWF) to visualize blood vessels; for endothelial‐specific nitric‐oxide‐synthase (EC‐NOS), as a marker of endothelial cell activation; and for Ki‐67 to evaluate endothelial cell proliferation. It was found that endothelial cells lining venous vessels located in peritumoural fibrous tissue were intensely EC‐NOS‐positive and occasionally Ki‐67‐positive. Capillary vessels of tumour papillae were not stained for Ki‐67 and were weakly EC‐NOS‐positive. Primary cultures of papillary carcinoma cells were used as a potential source of factors active on endothelial cells. It was found that thyroid tumour cells contain RNAs for angiopoietin, vascular endothelial growth factor (VEGF), and VEGF‐C; moreover, they release large amounts of VEGF into culture supernatants and exert chemotactic activity in vitro for the endothelial cell line SIEC. The ability of papillary carcinoma cells to release angiogenic factors could be stimulated in vitro. Hepatocyte growth factor (HGF; 25 ng/ml) induced a 1.2‐ to 5‐fold increase in the amount of VEGF released by tumour cells and a 1.2‐ to 4.2‐fold increase in the amount of chemotactic activity present in culture supernatants. Met protein, the high affinity HGF‐receptor, is overexpressed in a large proportion of cases of papillary carcinoma. These findings are consistent with the possibility that HGF–Met protein interaction is one of the molecular mechanisms promoting the vascularization of papillary carcinoma of the thyroid. Copyright

Expression of EDA/EDB isoforms of fibronectin in papillary carcinoma of the thyroid

Cellular fibronectins containing the extracellular domain A or B (EDA and EDB) are particularly abundant in fetal and neoplastic tissues. The presence of EDA and EDB was investigated in 28 cases of papillary carcinoma of the thyroid using IST‐9 and BC‐1 monoclonal antibodies. Immunostaining for EDA and EDB was detected in tumour stroma, in tumour basement membranes, and in tumour blood vessels. EDA was present in 27 of the 28 cases, in 20 of which more than 75 per cent of the tumour stroma was stained. Immunostaining for EDB was detected in 23 of the 28 cases and was less pronounced than that for EDA, being present in less than 25 per cent of the tumour stroma in most cases. Reactivity for EDA/EDB was not observed in the adjacent normal thyroid in any of the cases investigated. In a group of 20 non‐papillary tumours, immunostaining for EDA was present in the stroma of three follicular carcinomas (one minimally and two widely invasive), one medullary carcinoma, and 5 of 16 follicular adenomas; expression of EDB was more restricted, being present in only the two cases of widely invasive follicular carcinoma. The presence of EDA and EDB was not correlated with the extent of fibrosis or the degree of tumour cell differentiation. Immunoreactivity was already present in microcarcinomas. These observations raise the possibility that the production of oncofetal fibronectins is an important step in papillary carcinoma tumourigenesis, perhaps facilitating adhesion and spreading of tumour cells. Copyright

Differential Modulation of Uncoupling Protein 2 in Kidneys of Stroke-Prone Spontaneously Hypertensive Rats Under High-Salt/Low-Potassium Diet

The stroke-prone spontaneously hypertensive rat (SHRsp) represents an animal model of increased susceptibility to high-salt diet–induced cerebral and renal vascular injuries. High blood pressure and genetic factors are viewed as major contributing factors. In high-salt–loaded SHRsp and stroke-resistant SHR animals, we determined blood pressure levels, degree of kidney lesions, renal uncoupling protein 2 (UCP2) gene and protein expression levels along with rattus norvegicus (rno)-microRNA (miR) 24 and 34a gene expression, nuclear factor-&kgr;B protein levels, and oxidative stress. In vitro, UCP2 gene silencing was performed in renal mesangial cells. We found more severe degree of renal damage in SHRsp at the end of 4-week high-salt dietary treatment as compared with stroke-resistant SHR, despite comparable blood pressure levels, along with increased rate of inflammation and oxidative stress. Kidney UCP2 gene and protein expression levels were significantly downregulated under high-salt diet in SHRsp, but not in stroke-resistant SHR. Differential UCP2 regulation was paralleled by differential expression of kidney rno-miR 24 and 34a, known to target UCP2 gene, in the 2 strains. UCP2 gene silencing in renal mesangial cells led to increased rate of reactive oxygen species generation, increased inflammation and apoptosis, reduced cell vitality, and increased necrosis. In conclusion, high-salt diet downregulates the antioxidant UCP2-dependent mechanism in kidneys of SHRsp, but not of stroke-resistant SHR. A parallel differential kidney miR regulation under high-salt diet in the 2 strains may contribute to the differential UCP2 modulation. UCP2 is a critical protein to prevent oxidative stress damage in renal mesangial cells in vitro.

Ndufc2 Gene Inhibition Is Associated With Mitochondrial Dysfunction and Increased Stroke Susceptibility in an Animal Model of Complex Human Disease

Background The genetic basis of stroke susceptibility remains to be elucidated. STR1 quantitative trait locus (STR1/QTL) was identified on rat chromosome 1 of stroke‐prone spontaneously hypertensive rat (SHRSP) upon Japanese‐style stroke‐permissive diet (JD), and it contributes to 20% of the stroke phenotype variance. Methods and Results Nine hundred eighty‐six probe sets mapping on STR1 were selected from the Rat RAE230A array and screened through a microarray differential expression analysis in brains of SHRSP and stroke‐resistant SHR (SHRSR) fed with either regular diet or JD. The gene encoding Ndufc2 (NADH dehydrogenase [ubiquinone] 1 subunit), mapping 8 Mb apart from STR1/QTL Lod score peak, was found significantly down‐regulated under JD in SHRSP compared to SHRSR. Ndufc2 disruption altered complex I assembly and activity, reduced mitochondrial membrane potential and ATP levels, and increased reactive oxygen species production and inflammation both in vitro and in vivo. SHRSR carrying heterozygous Ndufc2 deletion showed renal abnormalities and stroke occurrence under JD, similarly to SHRSP. In humans, T allele variant at NDUFC2/rs11237379 was associated with significant reduction in gene expression and with increased occurrence of early‐onset ischemic stroke by recessive mode of transmission (odds ratio [OR], 1.39; CI, 1.07–1.80; P=0.012). Subjects carrying TT/rs11237379 and A allele variant at NDUFC2/rs641836 had further increased risk of stroke (OR=1.56; CI, 1.14–2.13; P=0.006). Conclusions A significant reduction of Ndufc2 expression causes complex I dysfunction and contributes to stroke susceptibility in SHRSP. Moreover, our current evidence may suggest that Ndufc2 can contribute to an increased occurrence of early‐onset ischemic stroke in humans.