Met protein and hepatocyte growth factor (HGF) in papillary carcinoma of the thyroid: evidence for a pathogenetic role in tumourigenesis.

In the last 10 years, evidence has accumulated that overexpression of Met protein is a distinguishing feature of almost every case of well-differentiated papillary carcinoma. Increased expression of the protein is probably due to enhanced transcription of the MET gene and/or to post-transcriptional mechanisms. So far, alterations of the MET gene have not been recognized, but evidence has been provided that activated RAS and RET can cause accumulation of MET RNA. Thus, the possibility exists that dysregulation of MET is the final result of different molecular pathways capable of inducing thyroid cell transformation; RET rearrangements might account for some of the cases, but the demonstration that the majority of papillary carcinomas do not have recognized alterations of the RET gene strongly suggests that MET gene dysregulation can also be achieved through other molecular pathways. Dysregulation of MET causes marked accumulation of Met protein in tumour cells that is promptly detected by immunohistochemistry. Thus, overexpression of Met protein might represent an immunohistochemical marker of papillary carcinoma, potentially helpful in problematic cases, but caution is required; moderate expression of Met protein is observed in non-neoplastic thyroid diseases, such as Graves' and Hashimoto's thyroiditis, and reagents active on paraffin sections may have a low affinity and/or low specificity for Met protein, leading to artifactual staining. Met protein-positive papillary carcinoma cells may produce hepatocyte growth factor (HGF) and may activate HGF through the urokinase-type plasminogen activator (uPA) bound to urokinase-type plasminogen activator receptor (uPA-R). Thus, papillary carcinoma cells possess the molecular machinery necessary for a productive HGF/Met interaction. In vitro studies have demonstrated that HGF enhances the motility and invasiveness of tumour cells and induces the synthesis and release of chemokines active in the recruitment of dendritic cells. These observations provide a rational basis for the understanding of two distinguishing features of papillary carcinoma. First, the tumour is often characterized by early metastatic spread to regional lymph nodes and by multifocal involvement of the gland, which suggests highly invasive behaviour. Second, a prominent peritumoural inflammatory reaction is often observed, which suggests cross-talk between tumour cells and the immune system.

Dendritic cells as a major source of macrophage-derived chemokine/CCL22 in vitro and in vivo.

Macrophage-derived chemokine (MDC)/CCL22 is a CC chemokine active on dendritic cells (DC), NK cells and Th2 lymphocytes. The present study was aimed at comprehensively investigating MDC production in vitro and in vivo. DC were the most potent producers of MDC among leukocytes tested. Endothelial cells did not produce MDC under a variety of conditions. Signals that induce maturation (lipopolysaccharide, IL-1, TNF, CD40 ligand, recognition of bacteria and yeast) dramatically augmented MDC production, and dexamethasone and vitamin D3 blocked it. Prostaglandin E(2), which blocked the acquisition of IL-12 production and the capacity to promote Th1 generation, did not affect MDC production. Using mass spectrometry-based techniques, DC supernatants were found to contain N-terminally truncated forms of MDC [MDC(3-69), MDC(5-69) and MD(C7-69)] as well as the full-length molecule. In vivo, CD1a(+), CD83(+), MDC(+) DC were found in reactive lymph nodes, and in Langerhans' cell histiocytosis. Skin lesions of atopic dermatitis patients showed that CD1a(+) or CD1b(+) DC, and DC with a CD83(+) phenotype were responsible for MDC production in this Th2-oriented disorder. Thus, DC are the predominant source of MDC in vitro and in vivo under a variety of experimental and clinical conditions. Processing of MDC to MDC(3-69) and shorter forms which do not recognize CCR4 is likely to represent a feedback mechanism of negative regulation.

Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells.

Members of the Toll-like receptor (TLR) family probably play a fundamental role in pathogen recognition and activation of innate immunity. The present study used a systematic approach to analyze how different human leukocyte populations express specific transcripts for the first five characterized TLR family members. TLR1 was expressed in all leukocytes examined, including monocytes, polymorphonuclear leukocytes, T and B cells, and NK cells. In contrast TLR2, TLR4, and TLR5 were expressed in myelomonocytic elements. Exposure to bacterial products, such as LPS or lipoarabinomannan, or to proinflammatory cytokines increased TLR4 expression in monocytes and polymorphonuclear leukocytes, whereas IL-10 blocked this effect. TLR3 was only expressed in human dendritic cells (DC) wherein maturation induced by bacterial products or cytokines was associated with reduced expression. TLR3 mRNA expression was detected by in situ hybridization in DC and lymph nodes. These results demonstrate that TLR1 through TLR5 mRNAs are differentially expressed and regulated in human leukocytes. In particular, expression of TLR3 transcripts is restricted to DC that are the only elements which express the full TLR repertoire. These data suggest that TLR can be classified based on expression pattern as ubiquitous (TLR1), restricted (TLR2, TLR4, and TLR5 in myelomonocytic cells), and specific (TLR3 in DC) molecules.

Cystic lymphoepithelial lesions of the parotid gland in HIV-1 infection.

The benign cystic lymphoepithelial lesion (BLL) of the parotid gland is a rare disorder affecting HIV-1-infected patients. Here we describe the clinical and histopathological features of 10 cases of BLL, who presented to our observation between November 1992 and December 1996, before the combination antiretroviral therapy was introduced.

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.

The benign cystic lymphoepithelial lesion of the parotid gland is a viral reservoir in HIV type 1-infected patients.

The presence of HIV-1 in cystic fluid aspirates from six cases of benign cystic lymphoepithelial lesion (BLL) of the parotid gland, a rare disorder affecting HIV-1-infected patients, has been investigated. HIV-1 p24 protein was present at a concentration ranging from 3 to 15 ng/ml, while it was undetectable in the peripheral blood of the same patients. The number of RNA copies of HIV-1 in the cystic fluids was high, ranging from 0.5 x 10(7) to 7.2 x 10(7) RNA copies/ml. BLL cystic fluid aspirates, despite the high level of HIV-1 RNA, were found to contain only a few infectious virions. The low infectivity correlated with the infrequent detection by electron microscopy of complete HIV-1 particles. The pathogenic mechanism leading to virus accumulation in the cystic fluid was studied by immunohistochemistry of tissue sections. p24 protein was associated with DRC-1+/S-100+ follicular dendritic reticulum cells, which were also present within the cystic cavities. Our findings are consistent with the possibility that the large amounts of virus present in the fluid derive from continuous shedding of HIV-1-infected cells from the surrounding lymphoid tissue.

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.

[A uterine pecoma: a case report]. / Pecoma uterino: descrizione di un caso.

The authors describe the first ever reported case of a malignant pecoma of the uterus infiltrating the ovary, the tube and two bowel loops. This extremely rare tumour usually shows a benign behaviour and seems to arise from the perivascular epithelioid cells (PEC).

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 mm(2). 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 [(3)H]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.

Intratumoral microvessel density and expression of ED-A/ED-B sequences of fibronectin in breast carcinoma.

The aim of this study was to examine the correlation between intratumoral microvessel density (iMVD) and the presence of cellular fibronectin isoforms, ED-A and ED-B, in order to identify those tumours with a prominent angiogenic phenotype. 91 cases of invasive ductal breast carcinoma were evaluated for TNM, histological grading, percentage of Ki-67+ cells and receptor hormonal status. iMVD was determined as a single microvessel count in a 200 x microscope field from the region of the tumour that appeared to be most densely vascular. When the mean values of iMVD of the various groups were compared, no significant difference was noted (Mann-Whitney test). When tumours were classified as high or low iMVD, based on a cut-off value (99 vessels/0.74 mm2), cases with high iMVD were significantly more numerous in poorly differentiated G3 tumours (P = 0.01, Chi-square test), and in tumours with lymph node metastasis (N0 versus N1 + N2; P = 0.002). The possibility that high iMVD was the expression of prominent vascular neoformation was explored using ED-A and ED-B isoforms of fibronectin as markers of neoformed vessels. ED-A + and/or ED-B + blood vessels were < 10% of total vessels, were detected in approximately 50% of cases independently of iMVD values, and were not more numerous in tumour areas with hot spot vascularisation. Our findings indicate that iMVD and expression of ED-A/ED-B reflect different aspects of tumour-associated angiogenesis.

Monocyte chemotactic protein-1 in the inflammatory pseudotumour of the lung.

Inflammatory pseudotumour of the lung is a lesion mainly composed of histiocytes. Histiocyte accumulation may arise from local proliferation of migratory cells, from cytokine induced recruitment of monocytes from the systemic circulation, or both. Cell proliferation was investigated with Ki-67 immunostaining and cytokine production with reverse transcriptase-polymerase chain reaction in two cases of inflammatory pseudotumour of the lung. It was found that the two lesions were composed mainly of non-proliferating (Ki-67 non-binding) macrophages that stained positive for CD68, CD14, CD4, and mannose receptor. Both cases contained mRNA transcripts for monocyte chemotactic protein-1 (MCP-1), a monocyte chemoattractant, and for interleukin 6 (IL-6), an inducer of plasma cell differentiation. One of the two cases also contained mRNA transcripts for IL-8, a neutrophil chemoattractant. These findings are consistent with the possibility that accumulation of non-proliferating histiocytes induced by MCP-1 is one of the pathogenic events occurring in inflammatory pseudotumour of the lung.

The monocyte chemotactic protein a (MCP-1) and interleukin 8 (IL-8) in Hodgkin's disease and in solid tumours.

AIMS: Monocyte chemotactic protein 1 (MCP-1) and interleukin 8 (IL-8) are small, inducible proteins with chemotactic activity for specific subsets of leucocytes. The possibility that MCP-1 and IL-8 are produced in tissues involved by Hodgkin's disease, thus contributing to the inflammatory-type background of the lesion, was investigated. METHODS: The presence of RNA transcripts for MCP-1 and IL-8 was investigated in biopsy samples of 24 cases of Hodgkin's disease, 17 non-Hodgkin's malignant lymphomas, 30 solid tumours, and 30 histologically normal tissues by means of reverse transcription-polymerase chain reaction (RT-PCR)/Southern blot analysis. RESULTS: MCP-1 expression was detected in 23 of 24 cases of Hodgkin's disease, in seven of 17 cases of B cell non-Hodgkin's lymphoma, and in seven of 14 cases of reactive lymphoid hyperplasia. IL-8 was present in six of 14 cases of Hodgkin's disease, and was seen only rarely in B cell non-Hodgkin's lymphoma and in reactive lymphoid tissues. MCP-1 and IL-8 RNA transcripts were detected in 13 of 25 carcinomas originating from the lung, breast, thyroid, and ovary. CONCLUSIONS: These findings are consistent with the possibility that MCP-1 and IL-8 are two additional cytokines involved in the pathogenesis of Hodgkin's disease.

Expression of Met protein and urokinase-type plasminogen activator receptor (uPA-R) in papillary carcinoma of the thyroid.

Met protein encoded by MET oncogene is the high affinity receptor for hepatocyte growth factor (HGF)/scatter factor (SF). HGF/SF has to be cleaved in its heterodimeric form by the urokinase-type plasminogen activator (uPA) to become active as a ligand for Met receptor. The expression of Met protein and of the high affinity receptor for uPA (uPA-R) was investigated in 39 samples of papillary carcinoma using immunohistochemistry. Reactivity for Met protein was present in 33 of 34 tumours, mostly with a diffuse pattern of staining. Reactivity for uPA-R was present in 78 per cent of papillary tumours and exhibited a pattern of staining similar to that of Met protein. Staining for uPA-R was present in 23 of 25 cases (92 per cent) of papillary carcinoma with prominent sclerosis, and in only 1 of 7 cases (14 per cent) without sclerosis. Peritumoural normal thyroid, follicular adenomas, and follicular carcinomas were negative for Met protein and for uPA-R. Hyperfunctioning tall thyroid cells showed weak membrane reactivity for uPA-R and for Met protein. The findings of immunohistochemistry were confirmed at the mRNA level using in situ hybridization, since the signal for uPA-R and Met RNAs was detected in most tumour cells of five cases of papillary carcinoma.

Basic fibroblast growth factor-induced angiogenic phenotype in mouse endothelium. A study of aortic and microvascular endothelial cell lines.

The mouse is the most commonly used species for in vivo studies on angiogenesis related to tumor development. Yet, to the best of our knowledge, very few reports on the in vitro interaction of the angiogenic basic fibroblast growth factor (bFGF) with mouse endothelial cells are available. Three mouse endothelial cell lines originated from aorta (MAECs), brain capillaries (MBECs), and heart capillaries (MHECs) were characterized for endothelial phenotypic markers, in vivo tumorigenic activity, and the capacity to respond in vitro to bFGF. These cells express angiotensin-converting enzyme, acetylated LDL receptor, constitutive endothelial nitric oxide synthase, and vascular cell adhesion molecule-1 and bind Griffonia simplicifolia-I lectin. When injected subcutaneously in nude mice, MAECs induced the appearance of slow-growing vascular lesions reminiscent of epithelioid hemangioendothelioma, whereas MBEC xenografts grew rapidly, showing Kaposi's sarcoma-like morphological features. No lesions were induced by injection of MHECs. MAECs, MBECs, and MHECs expressed both low-affinity heparan sulfate bFGF-binding sites and high-affinity tyrosine kinase receptors (FGFRs) on their surfaces. In particular, MAECs expressed FGFR-2/bek mRNA, whereas microvascular MBECs and MHECs expressed FGFR-1/flg mRNA. Accordingly, bFGF induced a mitogenic response and the phosphorylation of extracellular signal-regulated kinase-2 in all the cell lines. In contrast, upregulation of urokinase-type plasminogen activator expression was observed in bFGF-treated microvascular MBECs and MHECs but not in MAECs. Also, bFGF-treated MBECs and MHECs but not MAECs invaded a three-dimensional fibrin gel and formed hollow, capillary-like structures. The relevance of the modifications of the fibrinolytic balance of mouse microvascular endothelium in bFGF-induced angiogenesis was validated in vivo by a gelatin-sponge assay in which the plasmin inhibitors tranexamic acid and epsilon-aminocaproic acid given to mice in the drinking water inhibited neovascularization induced by the growth factor. In conclusion, differences in response to bFGF exist between large-vessel MAECs and microvascular MBECs and MHECs. Both in vitro and in vivo data point to a role of the profibrinolytic phenotype induced by bFGF in microvascular endothelial cells during mouse angiogenesis. Our observations make these endothelial cell lines suitable for further studies on mouse endothelium during angiogenesis and in angioproliferative diseases.

Kaposi's sarcoma cells express the macrophage-associated antigen mannose receptor and develop in peripheral blood cultures of Kaposi's sarcoma patients.

The mannose receptor (MR) is a surface 175-kd C-type lectin expressed by macrophages and dendritic cells. MR is involved in removal of effete cells, phagocytosis of mannose-coated particles, pinocytosis, and antigen presentation. Expression of MR was investigated in 17 biopsies of Kaposi's sarcoma (3 AIDS KS, 13 classical KS, and 1 transplant-associated KS) using three anti-MR monoclonal antibodies (3.29, D547, and PAM1). Immunostaining for MR was detected in 94 +/- 7% KS cells with spindle morphology. In normal tissues, MR was expressed by sinus-lining cells of spleen and lymph nodes, but it was not detected in endothelial cells lining normal hematic and lymphatic vessels, hemangioma, hemangioendothelioma, and lymphangioma. Expression of MR in KS cells prompted us to investigate the possibility that they derive from a circulating precursor cell. Peripheral blood mononuclear cells from 16 patients with KS (10 classical, 1 transplanted, and 5 AIDS) were cultured in PHA-conditioned medium for 10 to 14 days. Confluent monolayers of adherent spindle cells were detected in 8 of 11 classical KS, in 5 of 5 AIDS KS patients, and in 0 of 34 control patients. Peripheral-blood-derived KS-like cells were characterized by co-expression of macrophage and endothelial antigens being positive for CD45 (60%), CD68 (98%), MR (70%), CD14 (25%), VE-cadherin (70%), and von Willebrand factor (10%). When the immunophenotype of peripheral-blood-derived adherent cells was compared with that of KS spindle cells of tissue biopsies, it was found that both cell types are VE-cadherin+/MR+/CD68+, that peripheral-blood-derived spindle cells are CD34- and are less frequently stained for CD31 and von Willebrand factor, and that lesional KS cells do not express the leukocyte markers CD45 and CD18. Our findings are consistent with the possibility that KS lesions derive from tissue accumulation and local proliferation of a special subset of macrophages with endothelial features the normal counterpart of which are the sinus-lining cells of spleen and lymph nodes.

Expression of Met protein in thyroid tumours.

Met protein is a transmembrane 190 kD heterodimer with tyrosine kinase activity, encoded by c-MET oncogene. It serves as a high affinity receptor for hepatocyte growth factor (HGF)/scatter factor (SF), a cytokine which stimulates cell proliferation, motility, and invasion. Expression of Met protein was investigated in 116 thyroid tumours using an anti-Met mouse monoclonal antibody (DQ-13) active on paraffin-embedded material. Reactivity for DQ-13 was observed in 77 per cent of papillary carcinomas, in 70 per cent of Hürthle cell tumours, and rarely in other tumours. The staining was either uniformly present throughout the tumour or limited to nests of infiltrating tumour cells. In some Hürthle cell tumours, prominent accumulation of the protein was observed in the Golgi area. Reactivity for Met protein was decreased or absent in poorly differentiated tumours and was not influenced by tumour size, presence of lymph node metastases, or age of the patient. Immunostaining for Ki-67 revealed that cytoplasmic accumulation of Met protein was not associated with enhanced proliferation of tumour cells. Overexpression of Met protein in thyroid papillary carcinoma may result in increased motility of tumour cells, which in turn may account for intraglandular multifocal dissemination and early lymph node metastasis.