Clara Rizzardi

Correlation between vascular endothelial growth factor, angiogenesis, and tumor-associated macrophages in invasive ductal breast carcinoma

Abstract. Angiogenic and anti-angiogenic factors, secreted by tumor, inflammatory, and stromal cells play an important role in regulation of neovascularization. Among the most important of these is vascular endothelial growth factor (VEGF), a specific mitogen for endothelium, which increases vascular permeability and induces proteolytic enzymes necessary for vascular remodeling. Tumor-associated macrophages (TAMs) can express complex functions related to tumor biology, including growth, proliferative rate, stroma formation and dissolution, and neovascularization. The aim of this study was to define, using immunohistochemical analysis, the microvessel density (MVD), VEGF expression, and TAMs level in 97 human invasive ductal breast carcinomas not otherwise specified (NOS), investigate a possible relationship between them and then correlate their values with tumor grade, mitotic activity index (MAI), tumor size and lymph-node status. Statistical analysis showed a strong positive relationship between MVD and VEGF expression (P<0.001). Furthermore, both MVD and VEGF expression were significantly correlated with tumor grade and lymph-node status, and TAMs infiltration with MAI. TAM level showed a significant positive connection with VEGF expression and MVD. These in situ observations suggest that VEGF stimulates angiogenesis in human invasive ductal breast carcinoma NOS and attracts macrophages to the tumor locus, which then may be involved in angiogenesis promotion. The expression of this angiogenic molecule, and MVD and TAM level, can provide additional prognostic significance and help in the identification of patients who need postoperative adjuvant therapy.

The interaction of asbestos and iron in lung tissue revealed by synchrotron-based scanning X-ray microscopy

Asbestos is a potent carcinogen associated with malignant mesothelioma and lung cancer but its carcinogenic mechanisms are still poorly understood. Asbestos toxicity is ascribed to its particular physico-chemical characteristics, and one of them is the presence of and ability to adsorb iron, which may cause an alteration of iron homeostasis in the tissue. This observational study reports a combination of advanced synchrotron-based X-ray imaging and micro-spectroscopic methods that provide correlative morphological and chemical information for shedding light on iron mobilization features during asbestos permanence in lung tissue. The results show that the processes responsible for the unusual distribution of iron at different stages of interaction with the fibres also involve calcium, phosphorus and magnesium. It has been confirmed that the dominant iron form present in asbestos bodies is ferritin, while the concurrent presence of haematite suggests alteration of iron chemistry during asbestos body permanence.

Lymphoepithelial carcinoma of the parotid glands and its relationship with benign lymphoepithelial lesions.

The salivary glands, despite their relatively simple morphology, give rise to more than 30 histologically distinct benign and malignant tumors. Salivary gland neoplasms comprise less than 2% of all tumors in humans and 3% of all head and neck tumors. They arise in the parotid gland in 80% of cases, and approximately 80% are benign and 20% are malignant. Among them are lymphoepithelial lesions, rare lesions of the salivary glands and especially of the parotid gland that are characterized by lymphocytic infiltration associated with an epithelial proliferation. They are divided into benign, which is considered as a tumorlike condition, and malignant, which is a rare carcinoma of the salivary glands. This article provides a review of the current knowledge on lymphoepithelial carcinoma with a look at its association with benign lesions and on the importance of making the correct diagnosis for the appropriate treatment.

Clinical aspects and management of bisphosphonates-associated osteonecrosis of the jaws

Objective. An increasing incidence of osteonecrosis of the jaws (ONJ) in patients treated with intravenous bisphosphonates has been reported in the literature. The aim of this study was to evaluate the clinical aspects, diagnostic investigations, and management of ONJ associated with bisphosphonates in a series of 12 patients. Method. Our patients included 1 asymptomatic and 11 symptomatic subjects. For the symptomatic patients, the osteonecrosis was diagnosed through histological investigations of exposed bone that showed avascular and necrotic tissue with inflammatory infiltrate. The patients were complaining of swelling, fever, and bone exposure involving the jaws. The asymptomatic patient presented as an occasional finding during a routine dental examination and the necrosis was confirmed on the basis of imaging investigations. Radiographic, scintigraphic, and microbiological examinations were carried out for all patients. Treatment included antibiotics, minor surgical interventions, and hyperbaric oxygen therapy. Results. The radiological investigations revealed osteolytic areas and the scintigraphy demonstrated increased bone metabolism. The microbiological analysis showed pathogenic micro-organisms in the majority of patients. Therapy was useful in obtaining short-term symptomatic relief. Conclusions. Histological, radiological, nuclear medicine, and microbiological investigations are important diagnostic tools for patients with bisphosphonates-associated osteonecrosis of the jaws. However, a long-term follow-up is necessary if we are to better understand the treatment outcome.

A tumor with composite pilo-folliculosebaceous differentiation harboring a recently described new entity - Melanocytic matricoma

We report on a case of a peculiar tumor of the pilosebaceous unit showing a composite histologic appearance. The case presented as a pigmented crusted lesion on the back of the nose of a 62-year-old woman with markedly sun-damaged skin. Histologically, the superficial portion of the neoplasm was composed of buds and nests of basaloid epithelium with varying degrees of pilar and sebaceous differentiation. Adjacent to this component, lobules of squamous cells with cytoplasmic glycogenation suggesting the mature outer root sheath were seen. In the underlying dermis, there was a well-defined nodular proliferation composed of variably pigmented basaloid matrical cells forming clusters of “shadow” or “ghost cells” admixed with numerous melanized dendritic melanocytes; this last component of the neoplasm was identical to a recently described entity, melanocytic matricoma. The small size, circumscription, and absence of necrosis favored benignity, although the cytologic atypia of matrical cells did not exclude malignancy. The case is interesting not only because it is the third reported case of a peculiar neoplasm imitating the epithelial-melanocytic interaction in the embryonal hair follicle or bulb of the anagen follicle but because the part resembling melanocytic matricoma presented as a component of a complex lesion. We believe that sunlight may have played a role in the development of this peculiar neoplasm.

SV40 Multiple Tissue Infection and Asbestos Exposure in a Hyperendemic Area for Malignant Mesothelioma

To assess the presence of SV40 in malignant mesothelioma tissue, 19 formalin-fixed paraffin-embedded pleural cancer samples of patients from a hyperendemic area of northeastern Italy were analyzed retrospectively. A total of 48 other tissues from the malignant mesothelioma subjects were investigated. The SV40 load was determined by real-time quantitative PCR. Exposure to asbestos was evaluated through a careful review of the occupational history of patients, supplemented by histology and isolation of asbestos bodies. Three of 19 (15.8%) malignant mesothelioma tissues harbored SV40 genomic signals. Two patients with SV40-positive malignant mesothelioma had viral sequences in another tissue. Overall, 3 of 18 (16.7%) normal liver tissues tested positive for SV40, as did 1 of 8 (12.5%) kidney tissues. SV40 viral loads were higher in malignant mesothelioma than in normal cells (P = 0.045). This survey shows that SV40 sustains infections in multiple tissues in malignant mesothelioma patients from a geographic area affected with asbestos-related mesothelioma.

Synchrotron soft X-ray imaging and fluorescence microscopy reveal novel features of asbestos body morphology and composition in human lung tissues

BackgroundOccupational or environmental exposure to asbestos fibres is associated with pleural and parenchymal lung diseases. A histopathologic hallmark of exposure to asbestos is the presence in lung parenchyma of the so-called asbestos bodies. They are the final product of biomineralization processes resulting in deposition of endogenous iron and organic matter (mainly proteins) around the inhaled asbestos fibres. For shedding light on the formation mechanisms of asbestos bodies it is of fundamental importance to characterize at the same length scales not only their structural morphology and chemical composition but also to correlate them to the possible alterations in the local composition of the surrounding tissues. Here we report the first correlative morphological and chemical characterization of untreated paraffinated histological lung tissue samples with asbestos bodies by means of soft X-ray imaging and X-Ray Fluorescence (XRF) microscopy, which reveals new features in the elemental lateral distribution.ResultsThe X-ray absorption and phase contrast images and the simultaneously monitored XRF maps of tissue samples have revealed the location, distribution and elemental composition of asbestos bodies and associated nanometric structures. The observed specific morphology and differences in the local Si, Fe, O and Mg content provide distinct fingerprints characteristic for the core asbestos fibre and the ferruginous body. The highest Si content is found in the asbestos fibre, while the shell and ferruginous bodies are characterized by strongly increased content of Mg, Fe and O compared to the adjacent tissue. The XRF and SEM-EDX analyses of the extracted asbestos bodies confirmed an enhanced Mg deposition in the organic asbestos coating.ConclusionsThe present report demonstrates the potential of the advanced synchrotron-based X-ray imaging and microspectroscopy techniques for studying the response of the lung tissue to the presence of asbestos fibres. The new results obtained by simultaneous structural and chemical analysis of tissue specimen have provided clear evidence that Mg, in addition to Fe, is also involved in the formation mechanisms of asbestos bodies. This is the first important step to further thorough investigations that will shed light on the physiopathological role of Mg in tissue response to the asbestos toxicity.

Insulin down-regulates TRAIL expression in vascular smooth muscle cells both in vivo and in vitro

To dissect the effect of hyperinsulinemia versus hyperglycemia on TNF‐related apoptosis inducing ligand (TRAIL) expression in the macrovascular district, we measured TRAIL mRNA and protein in four groups of animals: streptozotocin (SZT)‐induced diabetic rats, vehicle‐treated control animals, diabetic rats treated with insulin and non‐diabetic rats treated with insulin. While the aortas of diabetic rats did not show significant differences in TRAIL expression with respect to vehicle‐treated control animals, the aortas of both diabetic and non‐diabetic rats treated in vivo for 16 days with insulin showed a significant decrease in TRAIL expression with respect to either diabetic and control rats. Moreover, in vitro treatment of both rat and human vascular smooth muscle cells (VSMC) with insulin induced the down‐regulation of TRAIL protein. While the addition of recombinant TRAIL to rat VSMC promoted the dose‐dependent release of bioactive nitric oxide (NO), this effect was significantly counteracted by pre‐exposure of VSMC to insulin. These findings suggest that TRAIL might act as an endogenous regulator of the vascular tone and that chronic elevation of insulin might contribute to the vascular abnormalities characterizing type‐2 diabetes mellitus by down‐regulating TRAIL expression and activity. J. Cell. Physiol. 212: 89–95, 2007.

Calcium micro-depositions in jugular truncular venous malformations revealed by Synchrotron-based XRF imaging.

It has been recently demonstrated that the internal jugular vein may exhibit abnormalities classified as truncular venous malformations (TVMs). The investigation of possible morphological and biochemical anomalies at jugular tissue level could help to better understand the link between brain venous drainage and neurodegenerative disorders, recently found associated with jugular TVMs. To this end we performed sequential X-ray Fluorescence (XRF) analyses on jugular tissue samples from two TVM patients and two control subjects, using complementary energies at three different synchrotrons. This investigation, coupled with conventional histological analyses, revealed anomalous micro-formations in the pathological tissues and allowed the determination of their elemental composition. Rapid XRF analyses on large tissue areas at 12.74 keV showed an increased Ca presence in the pathological samples, mainly localized in tunica adventitia microvessels. Investigations at lower energy demonstrated that the high Ca level corresponded to micro-calcifications, also containing P and Mg. We suggest that advanced synchrotron XRF micro-spectroscopy is an important analytical tool in revealing biochemical changes, which cannot be accessed by conventional investigations. Further research on a larger number of samples is needed to understand the pathogenic significance of Ca micro-depositions detected on the intramural vessels of vein walls affected by TVMs.

Detection of PLGA-based nanoparticles at a single-cell level by synchrotron radiation FTIR spectromicroscopy and correlation with X-ray fluorescence microscopy

Poly-lactide-co-glycolide (PLGA) is one of the few polymers approved by the US Food and Drug Administration as a carrier for drug administration in humans; therefore, it is one of the most used materials in the formulation of polymeric nanoparticles (NPs) for therapeutic purposes. Because the cellular uptake of polymeric NPs is a hot topic in the nanomedicine field, the development of techniques able to ensure incontrovertible evidence of the presence of NPs in the cells plays a key role in gaining understanding of their therapeutic potential. On the strength of this premise, this article aims to evaluate the application of synchrotron radiation-based Fourier transform infrared spectroscopy (SR-FTIR) spectromicroscopy and SR X-ray fluorescence (SR-XRF) microscopy in the study of the in vitro interaction of PLGA NPs with cells. To reach this goal, we used PLGA NPs, sized around 200 nm and loaded with superparamagnetic iron oxide NPs (PLGA-IO-NPs; Fe3O4; size, 10–15 nm). After exposing human mesothelial (MeT5A) cells to PLGA-IO-NPs (0.1 mg/mL), the cells were analyzed after fixation both by SR-FTIR spectromicroscopy and SR-XRF microscopy setups. SR-FTIR-SM enabled the detection of PLGA NPs at single-cell level, allowing polymer detection inside the biological matrix by the characteristic band in the 1,700–2,000 cm−1 region. The precise PLGA IR-signature (1,750 cm−1 centered pick) also was clearly evident within an area of high amide density. SR-XRF microscopy performed on the same cells investigated under SR-FTIR microscopy allowed us to put in evidence the Fe presence in the cells and to emphasize the intracellular localization of the PLGA-IO-NPs. These findings suggest that SR-FTIR and SR-XRF techniques could be two valuable tools to follow the PLGA NPs’ fate in in vitro studies on cell cultures.