Valentina Bevelacqua

Plasma levels and zymographic activities of matrix metalloproteinases 2 and 9 in type II diabetics with peripheral arterial disease

Deregulation of matrix metalloproteinases (MMPs) is an important factor contributing to the development of vascular lesions. Plasma levels and zymographic activities of MMP-2 and MMP-9 were investigated in type II diabetics with (n = 51) or without (n = 42) peripheral artery disease (PAD) and in normal volunteers (n = 23). Plasma MMP-2 levels were higher in type II diabetics with (p < 0.01) or without (p < 0.05) PAD in comparison with normal volunteers. Similarly, type II diabetics with (p < 0.0001) or without (p > 0.05) PAD had higher plasma MMP-9 levels than normal volunteers. Plasma zymographic activities of both MMP-2 and MMP-9 were positively correlated with their plasma levels. Plasma MMP-2 zymographic activity was higher in type II diabetics with PAD than type II diabetics without PAD (p > 0.05). Plasma MMP-9 zymographic activity was higher in type II diabetics with (p < 0.0001) or without (p < 0.0001) PAD in comparision with normal volunteers. Together, these results indicate that increased plasma levels and zymographic activities of MMP-2 and MMP-9 may contribute to PAD in type II diabetics. In particular, plasma MMP-9 may be a useful marker for the development of vascular disease in type II diabetics.

Analysis of BRAF mutation in primary and metastatic melanoma.

Mutation of BRAF has been proposed to contribute to melanoma development. However, it remains unclear whether or not BRAF mutation is associated with any particular stage of melanoma progression. Tumor biopsy specimens from patients with melanoma were analyzed to determine whether the frequency of BRAF mutation in metastatic melanoma differed from primary melanoma. BRAF mutation was present in 15 of 23 (61%) patients with primary melanoma and in 7 of 12 (58%) patients with metastatic melanoma. These results suggest that BRAF mutation in melanoma is most likely to occur prior to the development of metastatic disease.

Analysis of G(-174)C IL-6 polymorphism and plasma concentrations of inflammatory markers in patients with type 2 diabetes and peripheral arterial disease

Aims: To determine whether the G(−174)C interleukin 6 (IL-6) polymorphism influences the development of peripheral arterial disease (PAD) in individuals with type 2 diabetes. This was investigated by comparing the distribution of G(−174)C genotypes between patients with type 2 diabetes and PAD (PAD+) and those with type 2 diabetes but without PAD (PAD−). Plasma concentrations of IL-6, fibrinogen, C reactive protein (CRP), and vascular endothelial growth factor (VEGF) were also compared in PAD+ and PAD− patients. Methods: Blood samples were collected from 146 PAD+ and 144 PAD− patients. SfaNI was used to determine the G(−174)C genotype. Plasma concentrations of IL-6, fibrinogen, CRP, and VEGF were measured by an enzyme linked immunosorbent assay. Results: The GG genotype was more common in PAD+ patients than in PAD− patients. PAD+ patients also had increased mean plasma concentrations of IL-6, fibrinogen, CRP, and VEGF compared with PAD− patients. Mean plasma concentrations of IL-6, fibrinogen, and CRP in both PAD+ and PAD− patients were higher in those with the GG genotype than in those with the GC or CC genotypes. In contrast, mean plasma concentrations of VEGF in PAD+ and PAD− patients were not significantly different between those with different G(−174)C genotypes. Conclusions: These results support a model in which the GG genotype promotes PAD development among individuals with type 2 diabetes by inducing increased release of IL-6. Higher concentrations of IL-6 among those with the GG genotype is associated with increased plasma concentrations of fibrinogen and CRP.

Detection of BRAF Gene Mutation in Primary Choroidal Melanoma Tissue

Numerous BRAF mutations have been detected in melanoma biopsy specimens and cell lines. In contrast, several studies report lack of BRAF mutations in uveal melanoma including primary and metastatic choroidal and ciliary body melanomas. To our knowledge, for the first time, here we report a case of choroidal melanoma harboring the BRAF mutation (V600E). The activation of RAF/MEK/ERK pathway, although independent of BRAF mutation, was reported in uveal melanoma. The presence of V600E mutation indicates that the RAF/MEK/ERK pathway, in addition to cutaneous melanoma progression, may play a role in the choroidal melanoma development.

Absence of BRAF Gene Mutation in Non-Melanoma Skin Tumors

Basal cell carcinoma (BCC) is the most common skin cancer, and its incidence is increasing. It was proposed that the RAS oncogene significantly contributes to skin cancer development. Numerous BRAF mutations have been detected in melanoma biopsy specimens and cell lines. For the first time, in the present study, tumor biopsy specimens from 78 patients with BCC were screened for BRAF mutation within exons 11 and 15. Our results indicate that the BRAF gene does not appear to be frequently mutated in non-melanoma skin tumors such as BCC. These data suggest that other gene alterations may cause tumor development.

Analysis of the B-RafV600E mutation in cutaneous melanoma patients with occupational sun exposure.

Sun-exposure is one of the risk factors associated with the development of a cutaneous neoplasm. In melanoma, the Ras-Raf-MEK-ERK (MAPK) signaling pathway is constitutively activated through multiple mechanisms, including B-RAF mutation. It has been hypothesized that B-RAF mutations in melanocytic lesions arise from DNA damage induced by ultraviolet (UV) radiation. However, it is still discussed if B-RAF mutations are associated with melanoma patients exposed to the sun. Therefore, in the present study, the known B-RAFV600E mutation was analysed in melanoma samples from 30 indoor and 38 outdoor workers. B-RAFV600E mutation was detected in 52 and 73% of outdoor workers and indoor workers, respectively. Of note, this mutation was identified in 12 of 14 (85%) melanoma of the trunk diagnosed in indoor workers and in 9 of 19 (47%) samples from outdoor workers (p=0.03). By analyzing melanomas of other body sites, no statistical difference in the frequency of B-RAFV600E mutation was identified between the groups of workers. It appears that the mutation detected among indoor workers may be associated with a recreational or intermittent exposure to the sun, as usually the trunk is a sun-protected body site. Overall, these data indicate that the B-RAFV600E mutation detected in melanoma is not associated with a chronic exposure to the sun. Mutations detected in other genes may also contribute to melanoma development in the subset of patients exposed to UV radiation.

IL-6-174 G > C and MMP-9-1562 C > T polymorphisms are associated with increased risk of deep vein thrombosis in cancer patients.

BACKGROUND A growing body of evidence shows an increased risk of deep vein thrombosis (DVT) among cancer patients. Novel markers are needed to identify patients prone to develop DVT. The aim of the present study was to determine whether IL-6-174 G > C and MMP-9-1562 C > T polymorphisms may influence the development of DVT in cancer patients. METHODS Polymorphisms of IL-6 and MMP-9 were analyzed in 320 DNA samples from cancer patients (DVT+ and DVT-) and in 215 healthy donors. IL-6 and MMP-9 plasma levels were also measured by ELISA. RESULTS Distribution of -174 IL-6 genotype and -1562 MMP-9 were similar between healthy controls and DVT- cancer cases (OR = 0.98 and 1.04, respectively). Different results were obtained by compared healthy controls with DVT+ cancer patients. -174 IL-6 GG polymorphism was associated to DVT (OR = 2.07; 95% CI: 1.30-3.30), as well as -1562 MMP-9 CC polymorphism (OR = 2.60; 95% CI: 1.48-4.57). CONCLUSION The results of the present study support a model in which the GG and CC genotypes, respectively for IL-6-174 G > C and MMP-9-1562 C > T polymorphisms, are associated with a risk of DVT in cancer patients by inducing the release of IL-6 with subsequent increment of MMP-9. Overall, these findings may contribute to the management of DVT in cancer patients.

Soluble Adhesion Molecules and Cytokines in Children Affected by Recurrent Infections of the Upper Respiratory Tract

The objective of this study was to compare plasma levels of soluble adhesion molecules and Th1-Th2 type cytokines in 44 children with frequently recurrent respiratory infections (FRRI) of upper airways, defined as having nine or more episodes per year, and in 34 children without recurrence; all subjects were followed-up for 12 mo. The viral etiology was determined by cultures from nasal, pharyngeal, and ear secretions, using PCR and immunofluorescence. Plasma levels of five soluble adhesion molecules (E-selectin, P-selectin, L-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1) and interferon (IFN)-γ, IL-12, IL-4, and IL-10 were measured in patients and in 15 healthy controls using sandwich ELISA. During acute phase, all patients showed significant increase in plasma levels of soluble adhesion molecules; during the follow-up, the levels were greater in children with FRRI. A difference of cytokine profile was demonstrated between the patients with and without FRRI: an increased IL-4 and IL-10 release with decreased levels of IFN-γ and IL-12 suggested a skewing into Th2-type response, in patients with FRRI. This pattern persisted during the follow-up. In patients without recurrence, an increased IFN-γ and IL-12 release, together with decreased levels of IL-4 and IL-10, showed a skewing into Th1-type responses; in the follow-up these cytokines reached normal values. In conclusion, the abnormal levels of all examined parameters in children with FRRI may reflect the persistence of an inflammatory microenvironment in the airways and an activation of the immune system that may contribute to the frequently recurrence of the respiratory disease.

The unwelcome trio: HIV plus cutaneous and visceral leishmaniasis

Leishmania/Human Immunodeficiency Virus (HIV) coinfection has emerged as an extremely serious and increasingly frequent health problem in the last decades. Considering the insidious and not typical clinical picture in presence of immunosuppressive conditions, the increasing number of people travelling in endemic zones, the ability to survive, within both human and vector bodies, of the parasite, clinicians and dermatologists as the first line should be aware of these kind of “pathologic alliances,” to avoid delayed diagnosis and treatment. In this setting, the occurrence of cutaneous lesions can, paradoxically, aid the physician in recognition and approaching the correct staging and management of the two (or three) diseases. Treatment of these unwelcome synergies is a challenge: apart from the recommended anti‐retroviral protocols, different anti‐leishmanial drugs have been widely used, according with the standard guidelines for visceral leishmaniasis (VL), with no successful treatment regimen still been established.

Increased Levels of NF-kB-Dependent Markers in Cancer-Associated Deep Venous Thrombosis

Several studies highlight the role of inflammatory markers in thrombosis as well as in cancer. However, their combined role in cancer-associated deep vein thrombosis (DVT) and the molecular mechanisms, involved in its pathophysiology, needs further investigations. In the present study, C-reactive protein, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1β), matrix metalloproteases-9 (MMP-9), vascular endothelial growth factor (VEGF), tissue factor (TF), fibrinogen and soluble P-selectin, were analyzed in plasma and in monocyte samples from 385 cancer patients, of whom 64 were concomitantly affected by DVT (+). All these markers were higher in cancer patients DVT+ than in those DVT-. Accordingly, significantly higher NF-kB activity was observed in cancer patients DVT+ than DVT-. Significant correlation between data obtained in plasma and monocyte samples was observed. NF-kB inhibition was associated with decreased levels of all molecules in both cancer DVT+ and DVT-. To further demonstrate the involvement of NF-kB activation by the above mentioned molecules, we treated monocyte derived from healthy donors with a pool of sera from cancer patients with and without DVT. These set of experiments further suggest the significant role played by some molecules, regulated by NF-kB, and detected in cancer patients with DVT. Our data support the notion that NF-kB may be considered as a therapeutic target for cancer patients, especially those complicated by DVT. Treatment with NF-kB inhibitors may represent a possible strategy to prevent or reduce the risk of DVT in cancer patients.