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author:("zappia, Marco")
1.  PKA regulatory subunit R2B is required for murine and human adipocyte differentiation 
Endocrine Connections  2013;2(4):196-207.
Adipogenesis is a complex process modulated by several factors, including cAMP signaling. The main cAMP target is protein kinase A (PKA), a tetrameric enzyme with four regulatory subunits showing tissue-specific expression and function: PRKAR2B is the main regulatory subunit in adipose tissue in mice and in adult humans. This study aimed to evaluate the expression of PKA regulatory subunits in human adipose tissue during fetal development and to investigate their role in the differentiation of 3T3-L1 and primary human preadipocytes. The expression of PKA regulatory subunits was evaluated in fetal adipose tissue (immunohistochemistry) and in cultured 3T3-L1 and primary human preadipocytes (western blot analysis). Cultured cells were transiently transfected with siRNA against PRKAR2B and induced to differentiate. Differentiation was evaluated by intracellular triglyceride staining (Oil Red O) and expression of molecular markers of adipocyte differentiation. In this study, we found that PRKAR2B is the main regulatory subunit in human adipose tissue during fetal development, from 12 weeks of gestation to the end of gestation, as well as in 3T3-L1 and primary human preadipocytes. The expression of PRKAR2B increases progressively during in vitro differentiation. The silencing of PRKAR2B abolishes the increase in the expression of peroxisome proliferator-activated receptor gamma (PPARγ (PPARG)), fatty acid synthase, aP2 (FABP4), and lipoprotein lipase, as well as intracellular triglyceride accumulation, resulting in impaired adipocyte differentiation in both mouse and human cell systems. In conclusion, PRKAR2B is the key PKA regulatory subunit involved in mouse and human adipose tissue development. The physiological increase in the expression of PRKAR2B is an essential event in adipogenesis in both mice and humans, and it might represent a possible target for future strategies for obesity treatment.
doi:10.1530/EC-13-0049
PMCID: PMC3847920  PMID: 24145613
PKA; cAMP; differentiation; adipocytes
2.  Screening for colorectal cancer with FOBT, virtual colonoscopy and optical colonoscopy: study protocol for a randomized controlled trial in the Florence district (SAVE study) 
Trials  2013;14:74.
Background
Colorectal cancer (CRC) is the most frequent cancer in Europe. Randomized clinical trials demonstrated that screening with fecal occult blood test (FOBT) reduces mortality from CRC. Accordingly, the European Community currently recommends population-based screening with FOBT. Other screening tests, such as computed tomography colonography (CTC) and optical colonoscopy (OC), are highly accurate for examining the entire colon for adenomas and CRC. Acceptability represents a critical determinant of the impact of a screening program. We designed a randomized controlled trial to compare participation rate and diagnostic yield of FOBT, CTC with computer-aided diagnosis, and OC as primary tests for population-based screening.
Methods/Design
A total of 14,000 subjects aged 55 to 64 years, living in the Florence district and never screened for CRC, will be randomized in three arms: group 1 (5,000 persons) invited to undergo CTC (divided into: subgroup 1A with reduced cathartic preparation and subgroup 1B with standard bowel preparation); group 2 (8,000 persons) invited to undergo a biannual FOBT for three rounds; and group 3 (1,000 persons) invited to undergo OC. Subjects of each group will be invited by mail to undergo the selected test. All subjects with a positive FOBT or CTC test (that is, mass or at least one polyp ≥6 mm) will be invited to undergo a second-level OC. Primary objectives of the study are to compare the participation rate to FOBT, CTC and OC; to compare the detection rate for cancer or advanced adenomas of CTC versus three rounds of biannual FOBT; to evaluate referral rate for OC induced by primary CTC versus three rounds of FOBT; and to estimate costs of the three screening strategies. A secondary objective of the study is to create a biological bank of blood and stool specimens from subjects undergoing CTC and OC.
Discussion
This study will provide information about participation/acceptability, diagnostic yield and costs of screening with CTC in comparison with the recommended test (FOBT) and OC.
Trial registration
ClinicalTrials.gov Identifier: NCT01651624.
doi:10.1186/1745-6215-14-74
PMCID: PMC3618219  PMID: 23497601
Colorectal cancer; Screening; FOBT; Colonoscopy; CT colonography; Virtual colonoscopy; CAD; Biological bank
3.  Breast cancer screening: are we seeing the benefit? 
BMC Medicine  2012;10:106.
A decline in breast cancer mortality has been observed in western European Countries since the middle of the 1990s.
Different methodological approaches, including case-control studies, incidence-based mortality studies, and trend studies, have been used to assess the effectiveness of mammography screening programmes in reducing breast cancer mortality. However, not all methods succeed in distinguishing the relative contributions of service screening and taking correctly into consideration the potential source of bias that might affect the estimate.
Recently, a review of six case-control studies confirmed a breast cancer mortality reduction ranging from 38% to 70% among screened women. This figure is in accordance with the estimate obtained from incidence-based mortality studies if screening compliance is taken into account. We will describe the methodological constraints of mortality trend studies in predicting the impact of screening on mortality and the necessary caution that must be applied when interpreting the results of such studies.
In conclusion, when appropriate methodological approaches are used, it is evident that mammographic screening programmes have contributed substantially to the observed decline in breast cancer mortality.
doi:10.1186/1741-7015-10-106
PMCID: PMC3447729  PMID: 22995098
Mammography screening; breast cancer mortality; case-control studies; incidence-based mortality studies; analysis of trends
4.  Balancing harms and benefits of service mammography screening programs: a cohort study 
Introduction
The use of screening mammography is still under debate within the medical community. The aim of this study is to define a balance sheet of benefits (breast cancer mortality reduction) and harms (overdiagnosis) for mammography screening programs.
Methods
We compared breast cancer incidence and mortality in two cohorts of women, defined as 'attenders' or 'non-attenders' on the basis of the individual attitudes towards screening, who were invited to the first round of the Florentine screening program. The effects of screening exposure on breast cancer incidence and mortality were evaluated by fitting Poisson regression models adjusted for age at entry, marital status and deprivation index. We performed a sensitivity analysis excluding 34 women not responding to the invitation with a breast cancer diagnosis in the following six months.
Results
In total, we included 51,096 women aged 50 to 69 years invited at the first screening round (1991 to 1993) and followed-up for breast cancer incidence and mortality until 31 December 2007 and 31 December 2008, respectively The estimate of mortality reduction varies from 45% among 50 to 59 year-old women up to 51% among 60 to 69 year-old women. The estimate of overdiagnosis, according to the cumulative-incidence method, is an additional 10% of all breast cancer cases among 60 to 69 year-old women screened.
Conclusions
Comparing the breast cancer mortality and breast cancer incidence between attenders and non-attenders, we have determined that the overall cost to save one life corresponds to no more than one over-diagnosed tumor (from 0.6 to 1 depending on the selection criteria of the cohort), even if a residual self-selection bias cannot be excluded.
doi:10.1186/bcr3090
PMCID: PMC3496124  PMID: 22230345
6.  Human papillomavirus infection and risk factors in a cohort of Tuscan women aged 18-24: results at recruitment 
BMC Infectious Diseases  2010;10:157.
Background
There is conclusive evidence that human papillomavirus (HPV) infections of the cervix are a necessary cause of cervical cancer. In Italy there are consistent data of HPV prevalence in women aged 25 - 64 years, but there is limited data for younger women. The objective of this on-going 3-year prospective cohort study is to investigate the prevalence, acquisition, clearance and persistence of HPV infections in young Tuscan women and the risk factors correlated with such events.
Methods
One thousand and sixty-six women aged between 18 and 24 years were enrolled and received an initial HPV test. They were asked to return to the clinic over the study period for further tests every 12 months, if their HPV HR result was negative, or every 6 months, if positive. Additionally, women with an HPV positive result were given a cytological examination and if the cytological diagnosis was ASC-US or more severe, only women with HPV HR, were referred for colposcopy.
Results
We present here data for the enrolment phase of the study. At baseline, within the study sample, just under 30% of women were infected by HPV and 19.3% of women were infected with oncogenic types. A relationship was highlighted between HPV infection, number of sexual partners (in particularly in the last 3 years) and the lifetime number of partner's partners. Condom use showed a slight protective effect in univariate analysis but these data were not statistically significant in multivariate analysis. The association between HPV infection and demographic and behavioural variables were tested by crude odds ratio (OR). Multivariate logistic regression was applied to compute the adjusted odds ratios.
Conclusions
The prevalence of oncogenic HPV types was high in young Tuscan women. The 3-year follow-up of this cohort may provide a better understanding of the processes of acquisition, clearance and persistence of infection and the correlated risk factors.
doi:10.1186/1471-2334-10-157
PMCID: PMC2898819  PMID: 20529280
7.  Protein Kinase A Regulatory Subunits in Human Adipose Tissue 
Diabetes  2009;58(3):620-626.
OBJECTIVE—In human adipocytes, the cAMP-dependent pathway mediates signals originating from β-adrenergic activation, thus playing a key role in the regulation of important metabolic processes, i.e., lipolysis and thermogenesis. Cyclic AMP effects are mainly mediated by protein kinase A (PKA), whose R2B regulatory isoform is the most expressed in mouse adipose tissue, where it protects against diet-induced obesity and fatty liver development. The aim of the study was to investigate possible differences in R2B expression, PKA activity, and lipolysis in adipose tissues from obese and nonobese subjects.
RESEARCH DESIGN AND METHODS—The expression of the different PKA regulatory subunits was evaluated by immunohistochemistry, Western blot, and real-time PCR in subcutaneous and visceral adipose tissue samples from 20 nonobese and 67 obese patients. PKA activity and glycerol release were evaluated in total protein extract and adipocytes isolated from fresh tissue samples, respectively.
RESULTS—Expression techniques showed that R2B was the most abundant regulatory protein, both at mRNA and protein level. Interestingly, R2B mRNA levels were significantly lower in both subcutaneous and visceral adipose tissues from obese than nonobese patients and negatively correlated with BMI, waist circumference, insulin levels, and homeostasis model assessment of insulin resistance. Moreover, both basal and stimulated PKA activity and glycerol release were significantly lower in visceral adipose tissue from obese patients then nonobese subjects.
CONCLUSIONS—Our results first indicate that, in human adipose tissue, there are important BMI-related differences in R2B expression and PKA activation, which might be included among the multiple determinants involved in the different lipolytic response to β-adrenergic activation in obesity.
doi:10.2337/db08-0585
PMCID: PMC2646060  PMID: 19095761
9.  Agreement between the AMPLICOR Human Papillomavirus Test and the Hybrid Capture 2 Assay in Detection of High-Risk Human Papillomavirus and Diagnosis of Biopsy-Confirmed High-Grade Cervical Disease▿  
Journal of Clinical Microbiology  2006;45(2):364-369.
The AMPLICOR HPV test (AMP) and the Hybrid Capture 2 assay (HC2) detect 13 high-risk human papillomavirus (HR-HPV) types. Evaluation of comparative performance with clinical samples is needed to allow informed implementation of AMP into clinical practice. AMP was used (i) to assess the prevalence of HR-HPV in 1,032 samples of known cytology, HC2 status, and/or confirmed histology; (ii) to determine agreement between AMP and HC2; (iii) to evaluate the clinical sensitivity and specificity for detecting HR-HPV; and (iv) to detect the presence of biopsy-confirmed high-grade cervical intraepithelial neoplasia. The prevalence of HR-HPV was 39.3% and 45.6% by AMP and HC2, respectively. Overall agreement was 89.2% (kappa value, 0.78). Of 509 HR-HPV-negative specimens by HC2, 488 (95.9%) were AMP negative. Of 427 HR-HPV-positive specimens by HC2, 347 (81.2%) were AMP positive. In comparing the ability to detect high-grade squamous intraepithelial lesions (HSIL), the two tests were positive for all HSIL samples. Both tests performed similarly on CIN2+ samples (clinical sensitivities were 96.7% and 97.8%, respectively, for AMP and HC2). The clinical specificities of AMP and HC2 were comparable (54.9% versus 51.6%; P = 0.18). Genotyping of 20 HC2-negative/AMP-positive cases using alternative technologies revealed target HR genotypes in 63.1% of cases and low-risk types in 15.7% of cases, while 21% of cases were negative. In conclusion, AMP provides a viable alternative to HC2, with good agreement for samples with high-grade cytology and similar sensitivity in detecting CIN2+ lesions.
doi:10.1128/JCM.00706-06
PMCID: PMC1828999  PMID: 17122008

Results 1-10 (10)