BRAF mutation and RASSF1A expression in thyroid carcinoma of southern Italy.

Aim of this work is to provide a detailed comparison of clinical-pathologic features between well-differentiated and poorly differentiated tumors according to their BRAF and RASSF1A status. We analyzed RASSF1A methylation by MSP and BRAF mutation by LCRT-PCR with LightMix® kit BRAF V600E in neoplastic thyroid tissues. Immunohistochemical evaluation of RASSF1A expression was also performed by standard automated LSAB-HRP technique. An overall higher degree of RASSF1A over-expression than normal thyroid parenchyma surrounding tumors (P < 0.05) has been found in all malignant well-differentiated lesions. Moreover, statistically significant higher levels of RASSF1A expression were observed in differentiated cancers associated to an inflammatory autoimmune background (P = 0.01). Amplifiable DNA for LC PCR with LightMix® kit BRAF V600E was obtained in nine PTCs, four FVPTCs, five ATCs, and one control. The V600E mutation was found in 13 of 18 (72%) tumors. BRAF was mutated in 6 of 9 (66%) classical PTC, in 2 of 4 (50%) follicular variant PTC and in all ACs (100%). The overall frequency of RASSF1A promoter methylation observed was 20.5% (9 cases out 44). Hypermethylation of RASSF1A in primary tumors was variable according to histotypes ranging from100% (5/5) in ACs to only 12.5% (4/32) in PTCs. We show a correlation between RASSF1A methylation status and RASSF1A protein expression. Finally, we conclude that BRAF V600E mutation and RASSF1A methylation were pathogenetic event restricted to a subgroup of PTC/FVPTCs in early stage and to clinically aggressive ATCs.

Beta HPV Type 15 Can Interfere With NF-κB Activity and Apoptosis in Human Keratinocytes.

E7 protein from cutaneous as well as mucosal HPV types can alter NF-κB activity. Conflicting literature data show a HPV-induced up- or down-regulation of the NF-κB pathway in different cell lines. In a previous study we detected the expression of E7 gene of HPV15 in a subungual tumor of a patient affected by incontinentia pigmenti (IP). IP is a rare X-linked genodermatosis in which the IKKγ gene is altered. From observations in transgenic IKKγ defective mice, it was suggested that IKK-deficient cells may undergo rapid hyper-proliferation and apoptosis/necrosis, leading to increased pro-inflammatory cytokine production in the neighboring IKK-positive cells. The objective of this study was to ascertain if beta HPV 15 can alter apoptosis and NF-κB pathway in normal and IKKγ-deficient keratinocytes. The human immortalized keratinocyte cell line (HaCaT), and human primary keratinocyte (HPK) cells were transduced with a retrovirus expressing E6-E7 proteins of HPV 15 and IKKγ was successful silenced mimicking the HPV15 infection and IP. HPV15 E6-E7 gene expression improved NF-κB activity in human keratinocytes even when IKKγ was silenced by siRNA. In IKKγ silenced keratinocyte cells, TNF-α-induced apoptosis was strongly reduced by the expression of HPV15 E6-E7 genes. Beta HPV15 exerted this anti-apoptotic activity by decreasing pro-apoptotic BAK and cleaved Caspase 3 proteins. In conclusion, we can speculate that presence of persistent infection by beta papillomavirus might influence the biological fate of IP by altering NF-κB activation and apoptosis in IKKγ mutated cells, favoring their survival and possibly the development of tumors in the late stage of disease. Taken together, our data reinforce the importance of host genetic background in the pathogenesis of HPV-associated skin lesions.

Role of ageing and coronary atherosclerosis in the development of cardiac fibrosis in the rabbit.

OBJECTIVE: Myocardial fibrosis contributes to the impairing of cardiac function and characterizes ageing, but is also a consequence of atherosclerotic ischemic disease. Since atherosclerosis is a slow progressive disease, which prevails in elderly populations, the aim of this study was to distinguish the contribution of ageing and atherosclerosis to cardiac fibrosis. METHODS: Coronary atherosclerosis was induced in 5-6-year-old rabbits by a hyperlipemic diet for 9 months. Left ventricular (LV) collagen was quantified by densitometric analysis after Sirius-Red staining; an immunohistochemical investigation of the interstitium was also performed. RESULTS: Atherosclerosis was associated to a marked increase of left ventricular interstitial collagen with the appearance of fibrotic foci and a decrease of coronary vessel endothelial nitric oxide synthase (eNOS) expression. In fibrotic foci, abundant macrophages co-localized with transforming growth factor beta-1 (TGFbeta-1)-positive myofibroblasts and vascular cell adhesion molecule-1 (VCAM-1) positive microvessels (52.3+/-3.9%). In normocholesterolemic rabbits, ageing resulted in a fourfold increase of myocardial interstitial collagen, with alpha-smooth muscle actin and TGFbeta-1 negative fibroblasts and VCAM-1 positive microvessels (19.4+/-1.2%) without macrophages, suggesting a role of endothelial dysfunction in age-related fibrosis. CONCLUSIONS: There is a distinct difference between ageing and coronary atherosclerosis-induced cardiac fibrosis, although the effects may be cumulative. In the cascade of events leading to myocardial remodeling, reparative fibrosis with TGFbeta-1-positive myofibroblasts and interstitial inflammation were the major findings in atherosclerotic old rabbits, whereas with ageing alone, interstitial fibrosis with TGFbeta-1 negative fibroblasts and VCAM-1 positive microvessels prevailed.

High levels of cellular retinol binding protein-1 expression in leiomyosarcoma: possible implications for diagnostic evaluation.

Retinoid bioavailability is regulated by the activity of specific cytoplasmic receptors. High levels of cellular retinol binding protein-1 (CRBP-1) have been documented during experimental arterial and wound-healing processes, but data concerning neoplastic smooth muscle tissues are scarce and/or controversial. This study reports that the expression of CRBP-1 is markedly higher in uterine and gastrointestinal leiomyosarcomas than in leiomyomas and normal myometrium; CRBP-1 was practically absent in normal gastrointestinal smooth muscle tissue. CRBP-1 positivity was particularly elevated in the epithelioid variant of leiomyosarcoma; it was associated with increased proliferative and apoptotic rates and inversely related to smooth muscle differentiation evaluated by alpha- and gamma-smooth muscle actin and desmin expression. Western blotting and reverse-transcription polymerase chain reaction confirmed the observations concerning CRBP-1 and actin isoform expression and revealed higher NF-kappa-Bp65 and RAR alpha and lower Bax protein levels in leiomyosarcoma than in the other conditions. These findings document that a high CRBP-1 expression is associated with smooth muscle malignancy and suggest that CRBP-1 expression represents a new useful marker for the classification of unusual variants of smooth muscle tumors.

Sortilin expression is essential for pro-nerve growth factor-induced apoptosis of rat vascular smooth muscle cells.

BACKGROUND: Sortilin, a member of the Vps10p-domain receptor family, has been demonstrated a key regulator in mediating cellular response to pro-neurotrophins. In the present study, we investigated the role of sortilin in the apoptotic pathway of vascular smooth muscle cells. METHODS AND PRINCIPAL FINDINGS: Immunohistochemistry revealed that sortilin was barely detectable in human and rat normal young vessels, while its expression was increased in human fibroatheromatous plaques. Sortilin immunodetection was also marked in the neointima of the rat aorta fifteen days after ballooning.In vitro, rat aortic intimal cells expressed higher sortilin levels than normal media SMCs; sortilin was distributed in the cytoplasm and in correspondence of the cell membrane. After 48 h, pro-nerve growth factor (proNGF) induced the strong dose-dependent increase of intimal cell apoptosis and the accumulation of sortilin protein. ProNGF was a more potent apoptotic inducer than equimolar or even higher concentration of NGF, whereas brain derived neutrotrophic factor was ineffective. Targeted interfering RNA-mediated sortilin reduction counteracted proNGF-induced apoptosis without affecting p75(NTR) expression. ProNGF-induced apoptosis was associated to NF-κB down-regulation and bax increase. Inhibition of NF-κB activity increased intimal cell apoptosis that did not further increase with the addition of proNGF. CONCLUSIONS: Our results indicate that sortilin expression characterizes human atheromatous lesions and rat aortic post-injury neointima, and suggest that sortilin represents an important regulator of proNGF-induced SMC apoptosis and arterial remodeling.

Kaposi's sarcoma in a patient treated with imatinib mesylate for chronic myeloid leukemia.

BACKGROUND: Chronic myeloid leukemia (CML) is a myeloproliferative disorder primarily characterized by the presence of the Philadelphia chromosome resulting from a reciprocal translocation between the long arms of chromosomes 9 and 22. This translocation determines a fusion gene, bcr-abl, which encodes a constitutively active protein, tyrosine kinase, resulting in decreased apoptosis, defective adhesion, and genomic instability in transformed cells. The tyrosine kinase activity and its effects represent a potential pharmacologic target of tyrosine kinase inhibitors, such as imatinib. Flare of Kaposi's sarcoma (KS) is well described in immunosuppressed patients treated with corticosteroids and rituximab, but has not yet been reported during treatment with imatinib. OBJECTIVE: We report a case of cutaneous KS lesions in a patient affected by CML treated with imatinib. CASE SUMMARY: A 61-year-old white male patient (weight, 90 kg) was diagnosed with CML in March 2006 at the Division of Hematology, University of Rome "Tor Vergata," Rome, Italy. He was treated with imatinib 400 mg/d, which improved his general condition with few adverse effects. After 12 months of treatment, molecular biology showed an important reduction in the peripheral blood of the fusion oncoprotein bcr-abl p210-b3a2. However, at the same time, multiple cutaneous violaceous papular-nodular lesions appeared on his left forearm. A punch biopsy showed the presence of KS, whereas a polymerase chain reaction assay documented the presence of human herpes virus type 8 (HHV8) DNA in the skin lesion. Serologic HIV was negative and HHV8 viremia was under the limit of quantitation of the assay. Total body computed tomography scan did not reveal any mucosal or visceral lesion. CONCLUSIONS: We present a case of a patient with CML who developed KS 12 months after starting treatment with imatinib 400 mg/d. The mechanism behind the development of the cutaneous lesions is unclear, and could have either a casual clinical association or be related to the study drug. According to the Naranjo adverse drug reaction probability scale, the development of KS in this case was probably associated with the imatinib treatment (score, 5-8).

Stem cell marker expression and proliferation and apoptosis of vascular smooth muscle cells.

Vascular endothelial Flt-1 and other stem cell markers are variably expressed in vascular smooth muscle cells (SMCs) during normal and pathological conditions, but their biological role remains uncertain. In normal rat aorta, rare flt-1+ and c-kit+ SMCs were detected. Fifteen days after injury, 61.8 +/- 3.8, 45.7 +/- 3% of the intimal cells resulted flt-1+ and c-kit+ and expressed low level of alpha-smooth muscle actin; CD133+ cells were 5.6 +/- 0.7%. BrDU+/flt-1+ largely predominated in the neointima, whereas BrDU+/CD133+ cells were rare. Forty-five and sixty days after injury, intimal proliferation such as BrDU+ cells was greatly reduced. After sixty days, intimal stem marker expression had almost disappeared whereas alpha-smooth muscle actin was restored. Flk-1 and Oct-4 SMC immunodection was consistently negative. In vitro, intimal cells obtained fifteen days after injury exhibited an epithelioid phenotype and increased flt-1 and c-kit protein and mRNA and low smooth muscle markers compared to spindle-shaped medial and intimal SMCs obtained after sixty days. Epithelioid clones, independently from layer of origin, were similar in stem cell marker expression. The anti-flt-1 blocking antibody added to epithelioid SMC cultures reduced serum-deprived apoptosis and migration but not PDGF-BB-induced proliferation, and increased cell-populated collagen lattice contraction. In conclusion, vascular SMC stem marker expression was variable, chronologically modulated and prevalent in epithelioid populations and clones; among stem markers, flt-1 expression critically regulates intimal SMC response to microenviromental changes.

Propionyl-L-carnitine reduces proliferation and potentiates Bax-related apoptosis of aortic intimal smooth muscle cells by modulating nuclear factor-kappaB activity.

Propionyl-l-carnitine (PLC) has been introduced among the therapeutic approaches of peripheral arterial disease, and more recently, an increase of intimal cell apoptosis has been demonstrated to contribute to its effectiveness in rabbit carotid postinjury myointimal hyperplasia prevention. How PLC mediates these effects on vascular smooth muscle cells (SMCs) remains poorly understood. We investigated the role of NF-kappaB in PLC-induced arterial remodeling. In vivo, daily PLC treatment 15 days after injury resulted in a reduction of relative rat aortic intimal volume, an increase of apoptosis, Bax up-regulation without changing the Bcl-2 level, and a reduction of NF-kappaB, vascular cell adhesion molecule-1, monocyte chemotactic protein-1, and survivin in myointimal thickening compared with controls. In the presence of 10% serum, a reduced G(1) --> S phase progression preceded PLC-induced intimal cell apoptosis; in 0.1% serum cultures, in a dose-dependent manner, PLC rapidly induced intimal cell apoptosis and reduced p65, p50, IAP-1, and IAP-2 expression. Inhibiting NF-kappaB activation through SN50 increased apoptotic rate and Bax expression in intimal but not in medial SMCs, and successive PLC treatment failed to induce a further increase in apoptotic rate. Bax antisense oligodeoxynucleotide reduced PLC-induced intimal cell apoptosis and cytochrome c release. The PLC-induced attenuation of NF-kappaB activity in intimal cells was also due to the increase of IkappaB-alpha bioavailability, as the result of a parallel induction of IkappaB-alpha synthesis and reduction of phosphorylation and degradation. Collectively, these findings document that NF-kappaB activity inhibition contributes to PLC-induced proliferative arrest and Bax-related apoptosis of intimal SMCs.

Propionyl-L-carnitine prevents age-related myocardial remodeling in the rabbit.

Age-related cardiac remodeling is characterized by changes in myocardial structure, which include fibrosis (ie, increased collagen concentration). The pathogenetic mechanisms of age-related cardiac changes and possible pharmacologic interventions are still a matter of investigation. A morphometric analysis of collagen accumulation was performed in Sirius Red-stained left ventricular sections of 3-month-old and 5-6-year-old animals after a 9-month period of propionyl-L-carnitine treatment (PLC; 120 mg Kg(-1) day(-1) per os); aged rabbits showed decreased interstitial collagen accumulation and no changes in cellularity and apoptotic rate compared to controls. Age-related expression of vascular cell adhesion molecule-1 (VCAM-1)-positive microvessels was also reduced in PLC-treated rabbits. In vitro, the 16-hour, 10-microM PLC treatment reduced collagen type 1 and VCAM-1 transcripts, which were investigated by reverse transcription-polymerase chain reaction, more markedly in cardiac fibroblasts from aged donors. In the latter, the anti-VCAM-1 antibody treatment was found to be associated with a reduction in collagen type I transcripts. Our results demonstrated that long-term PLC treatment partially prevents age-related interstitial remodeling and suggests that a more complex interstitial cell-to-cell signaling regulates senescent myocardium properties.

Prevalence and genotyping of human papillomavirus infection in women with vulvodynia.

BACKGROUND: Evidence of vulvar human papillomavirus infection varies and the frequency of the different genotypes has not been adequately assessed. METHODS: Fifty consecutive sexually active healthy patients with vulvodynia and suspected of human papillomavirus infection underwent a vulvoscopy and biopsy. Ten normal vulvar samples were also enrolled as control. Histological and vulvoscopic findings were compared in relation to human papillomavirus-DNA presence and genotyping by a broad-spectrum polymerase chain reaction and reverse hybridization line probe assay. RESULTS: Although the clinical and histological diagnoses did not always coincide, a good association was found (p<0.0001). Human papillomavirus-DNA was detected in 42% of all biopsies and in none of the controls, and less frequently in acetowhite-positive patients (33.3%, p<0.03). Squamous papillomatosis (74%) was the most frequent histological diagnosis, followed by condyloma (20%). Condyloma (90%) but not squamous papillomatosis (29.7%) was significantly associated with human papillomavirus-DNA presence. Out of the vulvoscopically normal patients, one (33%) was human papillomavirus-DNA positive. Out of the recorded microscopic features, only koilocytosis was associated with human papillomavirus-DNA presence. Eight different human papillomavirus genotypes were detected: high-risk 16 (43%), 31 (19%), 52 (14.3%), 68, and 59 (4.8% each), and low-risk types 6 (71.4%), 11, and 40 (4.8% each); 33.3% of infections were multiple, ranging from 2 to 4 genotypes. Out of the human papillomavirus-DNA positive squamous papillomatosis, 72.7% showed a high-risk type but the infection remained episomal. CONCLUSIONS: Our data confirm human papillomavirus as a frequent cause of vulvodynia and its frequent association with squamous papillomatosis or condyloma. The high-risk human papillomavirus in squamous papillomatosis suggests screening for possible undiagnosed cervical infection.

Cellular retinol binding protein-1 expression in endometrial hyperplasia and carcinoma: diagnostic and possible therapeutic implications.

Cellular retinol binding protein-1 (CRBP-1) contributes to the maintenance of the differentiative state of endometrial glandular cells through the regulation of bioavailability of retinol and derivatives, but its role in endometrial oncogenetic process remains unclear. Antibodies to CRBP-1, estrogen and progesterone receptors (ER and PR) were applied to paraffin sections of proliferative (n = 10) and secretory endometrium (n = 9), and to endometrial polyps (n = 6), simple (n = 7), complex (n = 3) and atypical endometrial hyperplasias (n = 9) as well as to 47 endometrioid carcinomas of different histological grade (G) (G1, n = 18; G2, n = 19; G3, n = 10). Four serous and two clear cell carcinomas were also examined. In glandular cells, CRBP-1 positivity was mainly cytoplasmic and rarely in the nuclei. CRBP-1 immunodetection was weakly positive in proliferative and low and focal in secretory endometrium and higher in atypical as compared to simple and complex hyperplasias. CRBP-1 expression in G1 endometrioid carcinomas was similar to that in atypical hyperplasias. In the latter, the highest CRBP-1 expression was observed in areas of squamous differentiation. Semiquantitative evaluation revealed a significant decrease of cytoplasmic CRBP-1 immunoreactivity with the increase of tumor grade. Among G3 endometrioid carcinomas, 60% were CRBP-1 negative, whereas the remaining cases showed a very low and focal positivity. Serous carcinomas were also CRBP-1 negative. When areas of different grading were present within the same tumor, less differentiated areas retained a lower CRBP-1 immunoreaction. The progressive decrease of CRBP-1 paralleled that of ER and PR immunodetection. RT-PCR in eight endometrioid carcinomas suggested a decrease of CRBP-1 with the increase of tumor grade also at transcriptional level. Our results indicate that CRBP-1 immunodetection may constitute an additional tool for histological grading of endometrial carcinoma. The CRBP-1 loss during the progression of endometrial cancer suggests a new potential target for pharmacological strategies aimed to counteract its progression by increased intracellular retinol bioavailability.

Alpha-actin isoform distribution in normal and failing human heart: a morphological, morphometric, and biochemical study.

We investigated the distribution of alpha-skeletal, alpha-cardiac, and alpha-smooth muscle actin isoforms in human heart during development, hypertrophy, and failure. At 20 weeks of fetal life, alpha-skeletal actin was localized in a small proportion of subendocardial and papillary muscle cardiomyocytes. At this gestation time, diffuse alpha-cardiac actin staining was observed, associated with focal expression of alpha-smooth muscle actin. In normal adult subjects, alpha-skeletal actin positive cardiomyocytes were distributed in a transmural gradient with the highest proportion located subendocardially. In myocardial hypertrophy and cardiomyopathies, the amount of alpha-skeletal actin was increased and diffuse staining was seen in all layers of ventricular myocardium, with the exception of idiopathic dilated cardiomyopathies. Cardiomyocytes were negative for alpha-smooth muscle actin in all pathological situations studied. As expected, fibroblasts in post-infarct scars expressed alpha-smooth muscle actin and transforming growth factor-beta1 but, surprisingly, were negative for these proteins in interstitial fibrosis. Our results demonstrate that increased expression of alpha-skeletal actin in the diseased human heart is associated with increased myocyte stretch, increased wall stress, and pressure overload, but not with idiopathic dilated cardiomyopathies. They also suggest that fibrotic changes develop with different mechanisms in scars versus interstitial fibrosis.