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Perrone G.,Biomedical University of Rome | Gaeta L.M.,Biomedical University of Rome | Zagami M.,Biomedical University of Rome | Nasorri F.,Experimental Immunology Laboratory | And 9 more authors.
PLoS ONE | Year: 2012

Breast cancer cells with the CD44+/CD24- phenotype have been reported to be tumourigenic due to their enhanced capacity for cancer development and their self-renewal potential. The identification of human tumourigenic breast cancer cells in surgical samples has recently received increased attention due to the implications for prognosis and treatment, although limitations exist in the interpretation of these studies. To better identify the CD44+/CD24- cells in routine surgical specimens, 56 primary breast carcinoma cases were analysed by immunofluorescence and confocal microscopy, and the results were compared using flow cytometry analysis to correlate the amount and distribution of the CD44+/CD24- population with clinicopathological features. Using these methods, we showed that the breast carcinoma cells displayed four distinct sub-populations based on the expression pattern of CD44 and CD24. The CD44+/CD24- cells were found in 91% of breast tumours and constituted an average of 6.12% (range, 0.11%-21.23%) of the tumour. A strong correlation was found between the percentage of CD44+/CD24- cells in primary tumours and distant metastasis development (p = 0.0001); in addition, there was an inverse significant association with ER and PGR status (p = 0.002 and p = 0.001, respectively). No relationship was evident with tumour size (T) and regional lymph node (N) status, differentiation grade, proliferative index or HER2 status. In a multivariate analysis, the percentage of CD44+/CD24- cancer cells was an independent factor related to metastasis development (p = 0.004). Our results indicate that confocal analysis of fluorescence-labelled breast cancer samples obtained at surgery is a reliable method to identify the CD44+/CD24- tumourigenic cell population, allowing for the stratification of breast cancer patients into two groups with substantially different relapse rates on the basis of CD44+/CD24- cell percentage. © 2012 Perrone et al.


Maurelli R.,IDI Farmaceutici | Tinaburri L.,Vascular Pathology Laboratory | Gangi F.,Vascular Pathology Laboratory | Bondanza S.,Vascular Pathology Laboratory | And 7 more authors.
Journal of Cell Science | Year: 2016

The role of Ras in human skin tumorigenesis induction is still ambiguous. Overexpression of oncogenic Ras causes premature senescence in cultured human cells and hyperplasia in transgenic mice. Here, we investigated whether the oncogenic insult outcome might depend on the nature of the founding keratinocyte. We demonstrate that overexpression of the constitutively active Ras- V12 induces senescence in primary human keratinocyte cultures, but that some cells escape senescence and proliferate indefinitely. Ras overexpression in transient-amplifying- or stem-cell-enriched cultures shows that p16 (encoded by CDKN2A) levels are crucial for the final result. Indeed, transient-amplifying keratinocytes expressing high levels of p16 are sensitive to Ras-V12-induced senescence, whereas cells with high proliferative potential, but that do not display p16, are resistant. The subpopulation that sustains the indefinite culture growth exhibits stem cell features. Bypass of senescence correlates with inhibition of the pRb (also known as RB1) pathway and resumption of telomerase reverse transcriptase (TERT) activity. Immortalization is also sustained by activation of the ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1) and Akt pathways. Moreover, only transduced cultures originating from cultures bearing stem cells induce tumors in nude mice. Our findings demonstrate that the Ras overexpression outcome depends on the clonogenic potential of the recipient keratinocyte and that only the stem cell compartment is competent to initiate tumorigenesis. © 2016. Published by The Company of Biologists Ltd.


PubMed | Research Model and Services, IDI Farmaceutici, Molecular and Cellular Biology Laboratory, Experimental Immunology Laboratory and Vascular Pathology Laboratory
Type: Journal Article | Journal: Journal of cell science | Year: 2016

The role of Ras in human skin tumorigenesis induction is still ambiguous. Overexpression of oncogenic Ras causes premature senescence in cultured human cells and hyperplasia in transgenic mice. Here, we investigated whether the oncogenic insult outcome might depend on the nature of the founding keratinocyte. We demonstrate that overexpression of the constitutively active Ras-V12 induces senescence in primary human keratinocyte cultures, but that some cells escape senescence and proliferate indefinitely. Ras overexpression in transient-amplifying- or stem-cell-enriched cultures shows that p16 (encoded by CDKN2A) levels are crucial for the final result. Indeed, transient-amplifying keratinocytes expressing high levels of p16 are sensitive to Ras-V12-induced senescence, whereas cells with high proliferative potential, but that do not display p16, are resistant. The subpopulation that sustains the indefinite culture growth exhibits stem cell features. Bypass of senescence correlates with inhibition of the pRb (also known as RB1) pathway and resumption of telomerase reverse transcriptase (TERT) activity. Immortalization is also sustained by activation of the ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1) and Akt pathways. Moreover, only transduced cultures originating from cultures bearing stem cells induce tumors in nude mice. Our findings demonstrate that the Ras overexpression outcome depends on the clonogenic potential of the recipient keratinocyte and that only the stem cell compartment is competent to initiate tumorigenesis.


Orecchia A.,Molecular and Cell Biology Laboratory | Scarponi C.,Experimental Immunology Laboratory | Felice F.,Molecular and Cell Biology Laboratory | Cesarini E.,University of Rome La Sapienza | And 9 more authors.
PLoS ONE | Year: 2011

Histone deacetylases (HDAC) are key enzymes in the epigenetic control of gene expression. Recently, inhibitors of class I and class II HDAC have been successfully employed for the treatment of different inflammatory diseases such as rheumatoid arthritis, colitis, airway inflammation and asthma. So far, little is known so far about a similar therapeutic effect of inhibitors specifically directed against sirtuins, the class III HDAC. In this study, we investigated the expression and localization of endogenous sirtuins in primary human dermal microvascular endothelial cells (HDMEC), a cell type playing a key role in the development and maintenance of skin inflammation. We then examined the biological activity of sirtinol, a specific sirtuin inhibitor, in HDMEC response to pro-inflammatory cytokines. We found that, even though sirtinol treatment alone affected only long-term cell proliferation, it diminishes HDMEC inflammatory responses to tumor necrosis factor (TNF)α and interleukin (IL)-1β. In fact, sirtinol significantly reduced membrane expression of adhesion molecules in TNFã- or IL-1β-stimulated cells, as well as the amount of CXCL10 and CCL2 released by HDMEC following TNFα treatment. Notably, sirtinol drastically decreased monocyte adhesion on activated HDMEC. Using selective inhibitors for Sirt1 and Sirt2, we showed a predominant involvement of Sirt1 inhibition in the modulation of adhesion molecule expression and monocyte adhesion on activated HDMEC. Finally, we demonstrated the in vivo expression of Sirt1 in the dermal vessels of normal and psoriatic skin. Altogether, these findings indicated that sirtuins may represent a promising therapeutic target for the treatment of inflammatory skin diseases characterized by a prominent microvessel involvement. © 2011 Orecchia et al.


Scarponi C.,Experimental Immunology Laboratory | Butturini E.,University of Verona | Sestito R.,Experimental Immunology Laboratory | Sestito R.,Regina Elena Cancer Institute | And 4 more authors.
PLoS ONE | Year: 2014

The imbalance of the intracellular redox state and, in particular, of the glutathione (GSH)/GSH disulfide couple homeostasis, is involved in the pathogenesis of a number of diseases. In many skin diseases, including psoriasis, oxidative stress plays an important role, as demonstrated by the observation that treatments leading to increase of the local levels of oxidant species ameliorate the disease. Recently, dehydrocostuslactone (DCE) and costunolide (CS), two terpenes naturally occurring in many plants, have been found to exert various anti-inflammatory and pro-apoptotic effects on different human cell types. These compounds decrease the level of the intracellular GSH by direct interaction with it, and, therefore, can alter cellular redox state. DCE and CS can trigger S-glutathionylation of various substrates, including the transcription factor STAT3 and JAK1/2 proteins. In the present study, we investigated on the potential role of DCE and CS in regulating inflammatory and proliferative responses of human keratinocytes to cytokines. We demonstrated that DCE and CS decreased intracellular GSH levels in human keratinocytes, as well as inhibited STAT3 and STAT1 phosphorylation and activation triggered by IL-22 or IFN-γ, respectively. Consequently, DCE and CS decreased the IL-22- and IFN-γ-induced expression of inflammatory and regulatory genes in keratinocytes, including CCL2, CXCL10, ICAM-1 and SOCS3. DCE and CS also inhibited proliferation and cell-cycle progression-related gene expression, as well as they promoted cell cycle arrest and apoptosis. In parallel, DCE and CS activated the anti-inflammatory EGFR and ERK1/2 molecules in keratinocytes, and, thus, wound healing in an in vitro injury model. In light of our findings, we can hypothesize that the employment of DCE and CS in psoriasis could efficiently counteract the pro-inflammatory effects of IFN-γ and IL-22 on keratinocytes, revert the apoptosis-resistant phenotype, as well as inhibit hyperproliferation in the psoriatic epidermis. © PLOS ONE 2014.


Duryee M.J.,Experimental Immunology Laboratory | Duryee M.J.,University of Nebraska Medical Center | Willis M.S.,University of North Carolina at Chapel Hill | Schaffert C.S.,Experimental Immunology Laboratory | And 9 more authors.
American Journal of Physiology - Gastrointestinal and Liver Physiology | Year: 2014

Oxidative stress from fat accumulation in the liver has many deleterious effects. Many believe that there is a second hit that causes relatively benign fat accumulation to transform into liver failure. Therefore, we evaluated the effects of ethanol on ex vivo precision-cut liver slice cultures (PCLS) from rats fed a high-fat diet resulting in fatty liver. Agematched male Sprague-Dawley rats were fed either high-fat (obese) (45% calories from fat, 4.73 kcal/g) or control diet for 13 mo. PCLS were prepared, incubated with 25 mM ethanol for 24, 48, and 72 h, harvested, and evaluated for ethanol metabolism, triglyceride production, oxidative stress, and cytokine expression. Ethanol metabolism and acetaldehyde production decreased in PCLS from obese rats compared with age-matched controls (AMC). Increased triglyceride and smooth muscle actin production was observed in PCLS from obese rats compared with AMC, which further increased following ethanol incubation. Lipid peroxidation, measured by thiobarbituric acid reactive substances assay, increased in response to ethanol, whereas GSH and heme oxygenase I levels were decreased. TNF-α and IL-6 levels were increased in the PCLS from obese rats and increased further with ethanol incubation. Diet-induced fatty liver increases the susceptibility of the liver to toxins such as ethanol, possibly by the increased oxidative stress and cytokine production. These findings support the concept that the development of fatty liver sensitizes the liver to the effects of ethanol and leads to the start of liver failure, necrosis, and eventually cirrhosis. © 2014 the American Physiological Society.


Rongioletti F.,University of Cagliari | Failla M.C.,Experimental Immunology Laboratory | Atzori L.,University of Cagliari | Ferreli C.,University of Cagliari
Journal of Cutaneous Pathology | Year: 2016

POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein and skin signs) and AESOP (adenopathy and extensive skin patch overlying a plasmacytoma) syndromes are rare paraneoplastic conditions due to an underlying plasma cell dyscrasia. We report a 70-year-old patient with the rare coexistence of POEMS and AESOP syndromes and in whom skin signs, that differ both clinically and histologically, were the clues to the diagnosis of a plasma cell disorder. Vascular endothelial growth factor-A overexpression seems to be the common pathogenetic link of the different clinicopathological presentations of the skin lesions. © 2016 John Wiley & Sons A/S.


Carson J.S.,University of Nebraska Medical Center | Xiong W.,University of Nebraska Medical Center | Dale M.,University of Nebraska Medical Center | Yu F.,University of Nebraska Medical Center | And 6 more authors.
Journal of Vascular Surgery | Year: 2016

Objective Abdominal aortic aneurysm (AAA) is a pathologic dilation of the aorta. Inflammation of the aortic wall has been shown to be involved in AAA formation. Malondialdehyde-acetaldehyde (MAA) adducts are MAA/protein hybrids with immunogenic, proinflammatory, and profibrotic properties. Levels of MAA adducts are elevated in patients with coronary artery disease; however, the role of MAA adducts in AAA is unclear. We hypothesize that levels of circulating antibodies against MAA adducts are increased in patients with AAA. Methods Plasma samples were collected from mice and patients with AAA and control patients with atherosclerosis but not AAA. AAA was induced in mice by a standard CaCl2 protocol, with matching sham mice. Plasma levels of anti-MAA antibodies were quantified by enzyme-linked immunosorbent assay. Results Patients with AAA exhibited higher levels of immunoglobulin G and immunoglobulin A anti-MAA antibody subtypes (P =.049 and.026, respectively) compared with control patients. Conversely, immunoglobulin M anti-MAA antibodies in AAA patients were lower compared with control patients (P =.018). In CaCl2-treated mice, immunoglobulin G anti-MAA antibodies were elevated after AAA formation (P =.006). Conclusions The pattern of anti-MAA antibodies is able to distinguish between patients with AAA and patients with atherosclerosis but no AAA. These results demonstrate that MAA adducts are associated with AAA and suggest that they may play a role in either initiating or propagating chronic inflammation in AAA.


PubMed | Experimental Immunology Laboratory
Type: Journal Article | Journal: American journal of physiology. Gastrointestinal and liver physiology | Year: 2014

Oxidative stress from fat accumulation in the liver has many deleterious effects. Many believe that there is a second hit that causes relatively benign fat accumulation to transform into liver failure. Therefore, we evaluated the effects of ethanol on ex vivo precision-cut liver slice cultures (PCLS) from rats fed a high-fat diet resulting in fatty liver. Age-matched male Sprague-Dawley rats were fed either high-fat (obese) (45% calories from fat, 4.73 kcal/g) or control diet for 13 mo. PCLS were prepared, incubated with 25 mM ethanol for 24, 48, and 72 h, harvested, and evaluated for ethanol metabolism, triglyceride production, oxidative stress, and cytokine expression. Ethanol metabolism and acetaldehyde production decreased in PCLS from obese rats compared with age-matched controls (AMC). Increased triglyceride and smooth muscle actin production was observed in PCLS from obese rats compared with AMC, which further increased following ethanol incubation. Lipid peroxidation, measured by thiobarbituric acid reactive substances assay, increased in response to ethanol, whereas GSH and heme oxygenase I levels were decreased. TNF- and IL-6 levels were increased in the PCLS from obese rats and increased further with ethanol incubation. Diet-induced fatty liver increases the susceptibility of the liver to toxins such as ethanol, possibly by the increased oxidative stress and cytokine production. These findings support the concept that the development of fatty liver sensitizes the liver to the effects of ethanol and leads to the start of liver failure, necrosis, and eventually cirrhosis.

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