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author:("fraschini, A")
1.  IKKα/CHUK Regulates Extracellular Matrix Remodeling Independent of Its Kinase Activity to Facilitate Articular Chondrocyte Differentiation 
PLoS ONE  2013;8(9):e73024.
Background
The non-canonical NF-κB activating kinase IKKα, encoded by CHUK (conserved-helix-loop-helix-ubiquitous-kinase), has been reported to modulate pro- or anti- inflammatory responses, cellular survival and cellular differentiation. Here, we have investigated the mechanism of action of IKKα as a novel effector of human and murine chondrocyte extracellular matrix (ECM) homeostasis and differentiation towards hypertrophy.
Methodology/Principal Findings
IKKα expression was ablated in primary human osteoarthritic (OA) chondrocytes and in immature murine articular chondrocytes (iMACs) derived from IKKαf/f:CreERT2 mice by retroviral-mediated stable shRNA transduction and Cre recombinase-dependent Lox P site recombination, respectively. MMP-10 was identified as a major target of IKKα in chondrocytes by mRNA profiling, quantitative RT-PCR analysis, immunohistochemistry and immunoblotting. ECM integrity, as assessed by type II collagen (COL2) deposition and the lack of MMP-dependent COL2 degradation products, was enhanced by IKKα ablation in mice. MMP-13 and total collagenase activities were significantly reduced, while TIMP-3 (tissue inhibitor of metalloproteinase-3) protein levels were enhanced in IKKα-deficient chondrocytes. IKKα deficiency suppressed chondrocyte differentiation, as shown by the quantitative inhibition of.Alizarin red staining and the reduced expression of multiple chondrocyte differentiation effectors, including Runx2, Col10a1 and Vegfa,. Importantly, the differentiation of IKKα-deficient chondrocytes was rescued by a kinase-dead IKKα protein mutant.
Conclusions/Significance
IKKα acts independent of its kinase activity to help drive chondrocyte differentiation towards a hypertrophic-like state. IKKα positively modulates ECM remodeling via multiple downstream targets (including MMP-10 and TIMP-3 at the mRNA and post-transcriptional levels, respectively) to maintain maximal MMP-13 activity, which is required for ECM remodeling leading to chondrocyte differentiation. Chondrocytes are the unique cell component in articular cartilage, which are quiescent and maintain ECM integrity during tissue homeostasis. In OA, chondrocytes reacquire the capacity to proliferate and differentiate and their activation results in pronounced cartilage degeneration. Τηυσ, our findings are also of potential relevance for defining the onset and/or progression of OA disease.
doi:10.1371/journal.pone.0073024
PMCID: PMC3759388  PMID: 24023802
2.  MMP-13 loss associated with impaired ECM remodelling disrupts chondrocyte differentiation by concerted effects on multiple regulatory factors 
Arthritis and rheumatism  2010;62(8):2370-2381.
Purpose
To link MMP-13 activity and ECM remodeling to alterations in regulatory factors leading to a disruption in chondrocyte homeostasis.
Methods
Matrix-metalloproteinase-13 (MMP-13) expression was ablated in primary human chondrocytes by stable retrotransduction of short-hairpin RNAs. The effects of MMP-13 KD on key regulators of chondrocyte differentiation (Sox9, Runx2 and β-catenin), and angiogenesis (VEGF) were scored at the protein (immunohistochemistry or western blot) and RNA (real time PCR) levels in high density monolayer and micromass cultures under mineralizing conditions. Effects on cellular viability in conjunction with chondrocyte progression towards a hypertrophic-like state were assessed in micromass cultures. Alterations in Sox9 subcellular distribution were assessed by confocal microscopy in micromass cultures and also in OA cartilage.
Results
Differentiation of control chondrocyte micromasses progressed up to a terminal phase, with calcium deposition in conjunction with reduced cell viability and scant ECM. MMP-13 knock-down (KD) impaired ECM remodeling and suppressed differentiation in conjunction with reduced levels of Runx2, β-catenin and VEGF. MMP-13 levels in vitro and ECM remodeling in vitro and in vivo were linked to changes in Sox9 sub-cellular localization. Sox9 was largely excluded from the nuclei of chondrocytes with MMP-13 remodeled or degraded ECM, and exhibited an intranuclear staining pattern in chondrocytes with impaired MMP-13 activity in vitro or with more intact ECM in vivo.
Conclusions
MMP-13 loss leads to a break-down in primary human articular chondrocyte differentiation by altering the expression of multiple regulatory factors.
doi:10.1002/art.27512
PMCID: PMC2921033  PMID: 20506238
MMP-13; ECM remodeling; osteoarthritis; differentiation; shRNA retrovirus
3.  Differential requirements for IKKα and IKKβ in the differentiation of primary human osteoarthritic chondrocytes 
Arthritis and rheumatism  2008;58(1):227-239.
Objective
Osteoarthritic chondrocytes behave in an intrinsically de-regulated manner characterized by the chronic loss of healthy cartilage and inappropriate differentiation to a hypertrophic-like state. IKKβ and IKKα are essential kinases that activate NF-κB transcription factors, which regulate cellular differentiation and inflammation. This study reveals differential roles for each IKK in chondrocyte differentiation and hypertrophy.
Methods
Expression of IKKα or IKKβ were ablated in primary human chondrocytes by retro-transduction of specific shRNAs. Micromass cultures that faithfully undergo chondrogenesis to the terminal hypertrophic stage were established and ECM anabolism and remodelling were investigated by biochemical, immunohistochemical and ultrastructural techniques. Cellular parameters of hypertrophy (i.e. proliferation, viability and size) were also analyzed.
Results
Extracellular matrix remodelling and mineralization, processes characteristic of terminally differentiated hypertrophic cells, were defective upon IKKα or IKKβ loss. Silencing IKKβ markedly enhanced glycosaminoglycan accumulation, in conjunction with increased Sox9 expression. IKKα ablation dramatically enhanced Col2 deposition independent of Sox9 protein levels but instead in association with RUNX-2 suppression. Moreover IKKα deficient cells retained the phenotypes of pre-hypertrophic-like cells as evidenced by their smaller size and faster proliferation prior to micromass seeding along with the enhanced viability of their differentiated micromasses.
Conclusions
IKKα and IKKβ exert differential roles in ECM remodeling and endochondral ossification, events characteristic of hypertrophic chondrocytes and also factors often complicating osteoarthritis. Because IKKα's effects were more profound and pleotrophic in nature our observations suggest that exacerbated IKKα activity may be responsible for at least part of the characteristic abnormal phenotypes of osteoarthritic chondrocytes.
doi:10.1002/art.23211
PMCID: PMC2927097  PMID: 18163512
NF-κB; IKKs; osteoarthritis; chondrocytes; shRNA retroviruses
4.  Polyamine Depletion Inhibits NF-κB Binding to DNA and Interleukin-8 production in Human Chondrocytes Stimulated by Tumor Necrosis Factor-α 
Journal of cellular physiology  2005;204(3):956-963.
The activation of the NF-κB pathway by pro-inflammatory cytokines, such as tumor necrosis factor-α (TNFα), can be an important contributor for the re-programming of chondrocyte gene expression, thereby making it a therapeutic target in articular diseases. To search for new approaches to limit cartilage damage, we investigated the requirement of polyamines for NF-κB activation by TNFα in human C-28/I2 chondrocytes, using α-difluoromethylornithine (DFMO), a specific polyamine biosynthesis inhibitor. The NF-κB pathway was dissected by using pharmacological inhibitors or by expressing a transdominant IκBα super repressor. Treatment of C-28/I2 chondrocytes with TNFα resulted in a rapid enhancement of nuclear localization and DNA binding activity of the p65 NF-κB subunit. TNFα also increased the level and extracellular release of interleukin-8 (IL-8), a CXC chemokine that can have a role in arthritis, in an NF-κB-dependent manner. Pre-treatment of chondrocytes with DFMO, while causing polyamine depletion, significantly reduced NF-κB DNA binding activity. Moreover DFMO also decreased IL-8 production without affecting cellular viability. Restoration of polyamine levels by the co-addition of putrescine circumvented the inhibitory effects of DFMO. Our results show that the intracellular depletion of polyamines inhibits the response of chondrocytes to TNFα by interfering with the DNA binding activity of NF-κB. This suggests that a pharmacological and/or genetic approach to deplete the polyamine pool in chondrocytes may represent a useful way to reduce NF-κB activation by inflammatory cytokines in arthritis without provoking chondrocyte apoptosis.
doi:10.1002/jcp.20368
PMCID: PMC1226412  PMID: 15828019
Chondrocytes; Interleukin-8; NF-κB; Polyamines; Tumor necrosis factor-α
5.  Laparoscopic Fertility Sparing Management of Cervical Cancer 
Fertility can be preserved after conservative cervical surgery. We report on a 29-year-old woman who was obese, para 0, and diagnosed with cervical insufficiency at the first trimester of current pregnancy due to a previous trachelectomy. She underwent laparoscopic transabdominal cervical cerclage (LTCC) for cervical cancer. The surgery was successful and she was discharged two days later. The patient underwent a caesarean section at 38 weeks of gestation. Laparoscopic surgery is a minimally invasive approach associated with less pain and faster recovery, feasible even in obese women.
PMCID: PMC3973170
Laparoscopy; Cervical Cancer; Cerclage; Pregnancy; Trachelectomy
6.  The ACTonHEART study: rationale and design of a randomized controlled clinical trial comparing a brief intervention based on Acceptance and Commitment Therapy to usual secondary prevention care of coronary heart disease 
Background
Modifiable risk factors, including life-style habits and psychological variables, have been increasingly demonstrated to have an important role in influencing morbidity and mortality in cardiovascular patients, and to account for approximately 90% of the population risk for cardiac events.
Acceptance and Commitment Therapy (ACT) has shown effectiveness in promoting healthy behaviors, and improving psychological well-being in patients with chronic physical conditions. Moreover, a first application of an acceptance-based program in cardiac patients has revealed high treatment satisfaction and initial evidences of effectiveness in increasing heart-healthy behaviour. However, no clinical trial to date has evaluated the efficacy of an acceptance-based program for the modification of cardiovascular risk factors and the improvement of psychological well-being, compared to usual secondary prevention care.
Methods
Approximately 168 patients will be recruited from an outpatient cardiac rehabilitation unit and randomly assigned to receive usual care or usual care + a brief ACT-based intervention. The ACT group will be administered five group therapy sessions integrating educational topics on heart-healthy behaviours with acceptance and mindfulness skills. Participants will be assessed at baseline, six weeks later (post treatment for the ACT condition), at six and twelve months follow-up.
A partially-nested design will be used to balance effects due to clustering of participants into small therapy groups. Primary outcome measures will include biological indicators of cardiovascular risk and self-reported psychological well-being. Treatment effects will be tested via multilevel modeling after which the mediational role of psychological flexibility will be evaluated.
Discussion
The ACTonHEART study is the first randomized clinical trial designed to evaluate the efficacy of a brief group-administered, ACT-based program to promote health behavior change and psychological well-being among cardiac patients. Results will address the effectiveness of a brief treatment created to simultaneously impact multiple cardiovascular risk factors. Conducted in the context of clinical practice, this trial will potentially offer empirical support to alternative interventions to improve quality of life and reduce mortality and morbidity rates among cardiac patients.
Trial registration
clinicaltrials.gov/ (NCT01909102)
doi:10.1186/1477-7525-12-22
PMCID: PMC3936953  PMID: 24552555
Ischemic heart disease; Modifiable cardiovascular risk factors; Adherence; Psychological well-being; Quality of life; Acceptance and Commitment Therapy; Behavior modification; Randomized clinical trial
7.  Genital warts: comparing clinical findings to dermatoscopic aspects, in vivo reflectance confocal features and histopathologic exam*  
Anais Brasileiros de Dermatologia  2014;89(1):137-140.
Genital warts can be diagnosed through physical examination and confirmed by histopathology. Noninvasive methods are useful for ruling out other diagnoses with no harm to the patient. In this study the clinical findings were compared to dermoscopy, reflectance confocal microscopy (RCM), and to histopathology findings, in order to determine possible patterns that can aid diagnosis of the lesion. It was possible to identify structural changes on reflectance confocal microscopy that are already known by dermoscopy, in addition to cellular changes previously seen only by histopathological examination. This study shows the use of reflectance confocal microscopy in cases of genital warts, providing important information that can be used in further studies.
doi:10.1590/abd1806-4841.20141917
PMCID: PMC3938364  PMID: 24626658
Condylomata acuminata; Dermoscopy; Microscopy, confocal
8.  Stability of a Lipase Extracted from Seeds of Pachira aquatica in Commercial Detergents and Application Tests in Poultry Wastewater Pretreatment and Fat Particle Hydrolysis 
Enzyme Research  2013;2013:324061.
A protein extract containing a plant lipase from oleaginous seeds of Pachira aquatica was tested using soybean oil, wastewater from a poultry processing plant, and beef fat particles as substrate. The hydrolysis experiments were carried out at a temperature of 40°C, an incubation time of 90 minutes, and pH 8.0-9.0. The enzyme had the best stability at pH 9.0 and showed good stability in the alkaline range. It was found that P. aquatica lipase was stable in the presence of some commercial laundry detergent formulations, and it retained full activity up to 0.35% in hydrogen peroxide, despite losing activity at higher concentrations. Concerning wastewater, the lipase increased free fatty acids release by 7.4 times and promoted the hydrolysis of approximately 10% of the fats, suggesting that it could be included in a pretreatment stage, especially for vegetable oil degradation.
doi:10.1155/2013/324061
PMCID: PMC3884838  PMID: 24455209
9.  Human Adipose Stromal Cells (ASC) for the Regeneration of Injured Cartilage Display Genetic Stability after In Vitro Culture Expansion 
PLoS ONE  2013;8(10):e77895.
Mesenchymal stromal cells are emerging as an extremely promising therapeutic agent for tissue regeneration due to their multi-potency, immune-modulation and secretome activities, but safety remains one of the main concerns, particularly when in vitro manipulation, such as cell expansion, is performed before clinical application. Indeed, it is well documented that in vitro expansion reduces replicative potential and some multi-potency and promotes cell senescence. Furthermore, during in vitro aging there is a decrease in DNA synthesis and repair efficiency thus leading to DNA damage accumulation and possibly inducing genomic instability. The European Research Project ADIPOA aims at validating an innovative cell-based therapy where autologous adipose stromal cells (ASCs) are injected in the diseased articulation to activate regeneration of the cartilage. The primary objective of this paper was to assess the safety of cultured ASCs. The maintenance of genetic integrity was evaluated during in vitro culture by karyotype and microsatellite instability analysis. In addition, RT-PCR array-based evaluation of the expression of genes related to DNA damage signaling pathways was performed. Finally, the senescence and replicative potential of cultured cells was evaluated by telomere length and telomerase activity assessment, whereas anchorage-independent clone development was tested in vitro by soft agar growth. We found that cultured ASCs do not show genetic alterations and replicative senescence during the period of observation, nor anchorage-independent growth, supporting an argument for the safety of ASCs for clinical use.
doi:10.1371/journal.pone.0077895
PMCID: PMC3810264  PMID: 24205017
10.  Recurrence Methods for the Identification of Morphogenetic Patterns 
PLoS ONE  2013;8(9):e73686.
This paper addresses the problem of identifying the parameters involved in the formation of spatial patterns in nonlinear two dimensional systems. To this aim, we perform numerical experiments on a prototypical model generating morphogenetic Turing patterns, by changing both the spatial frequency and shape of the patterns. The features of the patterns and their relationship with the model parameters are characterized by means of the Generalized Recurrence Quantification measures. We show that the recurrence measures Determinism and Recurrence Entropy, as well as the distribution of the line lengths, allow for a full characterization of the patterns in terms of power law decay with respect to the parameters involved in the determination of their spatial frequency and shape. A comparison with the standard two dimensional Fourier transform is performed and the results show a better performance of the recurrence indicators in identifying a reliable connection with the spatial frequency of the patterns. Finally, in order to evaluate the robustness of the estimation of the power low decay, extensive simulations have been performed by adding different levels of noise to the patterns.
doi:10.1371/journal.pone.0073686
PMCID: PMC3774734  PMID: 24066062
11.  Cytosolic phosphorylated EGFR is predictive of recurrence in early stage penile cancer patients: a retropective study 
Background
Penile cancer (PC) is a rare tumor, and therapeutic options are limited for this disease, with an overall 5-year overall survival around 65-70%. Adjuvant therapy is not recommended for patients with N0-1 disease, despite up to 60% of these patients will die within 5 years from diagnosis.
Methods
Medical records of all patients who underwent radical surgery at University Federico II of Naples and at National Tumor Institute “Pascale” of Naples for early squamous cell carcinoma of the penis from January, 2000 to December, 2011 were retrieved. Paraffin wax embedded tissue specimens were retrieved from the pathology archives of the participating Institutions for all patients. Expression of p-EGFR, EGFR and positivity to HPV were evaluated along with other histological variables of interest. Demographic data of eligible patients were retrieved along with clinical characteristics such as type of surgical operation, time of follow up, time of recurrence, overall survival. A multivariable model was constructed using a forward stepwise selection procedure.
Results
Thirty eligible patients were identified. All patients were positive for EGFR by immunohistochemistry, while 13 and 16 were respectively positive for nuclear and cytosolic p-EGFR. No EGFR amplification was detected by FISH. Eight patients were positive for high-risk HPV by ISH. On univariable analysis, corpora cavernosa infiltration (OR 7.8; 95% CI = 0,8 to 75,6; P = 0,039) and positivity for cytosolic p-EGFR (OR 7.6; 95% CI =1.49 to 50; P = 0.009) were predictive for recurrence, while only positivity for cytosolic p-EGFR (HR =9.0; 95% CI 1.0-100; P = 0,0116) was prognostic for poor survival.
Conclusion
It is of primary importance to identify patients with N0-1 disease who are at increased risk of recurrence, as they do not normally receive any adjuvant therapy. Expression of p-EGFR was found in this series to be strongly related to increase risk of recurrence and shorter overall survival. This finding is consistent with the role of p-EGFR in other solid malignancies. Integration of p-EGFR with classic prognostic factors and other histology markers should be pursued to establish optimal adjuvant therapy for N0-1 PC patients.
doi:10.1186/1479-5876-11-161
PMCID: PMC3717121  PMID: 23819610
Epidermal growth factor receptor (EGFR) expression; Phospo-EGFR; Immunohistochemistry; Penile cancer; Tissue micro-array (TMA)
12.  New Insights into Osteogenic and Chondrogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells and Their Potential Clinical Applications for Bone Regeneration in Pediatric Orthopaedics 
Stem Cells International  2013;2013:312501.
Human mesenchymal stem cells (hMSCs) are pluripotent adult stem cells capable of being differentiated into osteoblasts, adipocytes, and chondrocytes. The osteogenic differentiation of hMSCs is regulated either by systemic hormones or by local growth factors able to induce specific intracellular signal pathways that modify the expression and activity of several transcription factors. Runt-related transcription factor 2 (Runx2) and Wnt signaling-related molecules are the major factors critically involved in the osteogenic differentiation process by hMSCs, and SRY-related high-mobility-group (HMG) box transcription factor 9 (SOX9) is involved in the chondrogenic one. hMSCs have generated a great interest in the field of regenerative medicine, particularly in bone regeneration. In this paper, we focused our attention on the molecular mechanisms involved in osteogenic and chondrogenic differentiation of hMSC, and the potential clinical use of hMSCs in osteoarticular pediatric disease characterized by fracture nonunion and pseudarthrosis.
doi:10.1155/2013/312501
PMCID: PMC3676919  PMID: 23766767
13.  Anatomical Correlates of Cognitive Functions in Early Parkinson's Disease Patients 
PLoS ONE  2013;8(5):e64222.
Background
Cognitive deficits may occur early in Parkinson's disease (PD) but the extent of cortical involvement associated with cognitive dysfunction needs additional investigations. The aim of our study is to identify the anatomical pattern of cortical thickness alterations in patients with early stage PD and its relationship with cognitive disability.
Methods
We recruited 29 PD patients and 21 healthy controls. All PD patients performed an extensive neuropsychological examination and 14 were diagnosed with mild cognitive impairment (PD-MCI). Surface-based cortical thickness analysis was applied to investigate the topographical distribution of cortical and subcortical alterations in early PD compared with controls and to assess the relationship between cognition and regional cortical changes in PD-MCI.
Results
Overall PD patients showed focal cortical (occipital-parietal areas, orbito-frontal and olfactory areas) and subcortical thinning when compared with controls. PD-MCI showed a wide spectrum of cognitive deficits and related significant regional thickening in the right parietal-frontal as well as in the left temporal-occipital areas.
Conclusion
Our results confirm the presence of changes in grey matter thickness at relatively early PD stage and support previous studies showing thinning and atrophy in the neocortex and subcortical regions. Relative cortical thickening in PD-MCI may instead express compensatory neuroplasticity. Brain reserve mechanisms might first modulate cognitive decline during the initial stages of PD.
doi:10.1371/journal.pone.0064222
PMCID: PMC3661458  PMID: 23717572
14.  Clinical Trials with Pegylated Liposomal Doxorubicin in the Treatment of Ovarian Cancer 
Journal of Drug Delivery  2013;2013:898146.
Among the pharmaceutical options available for treatment of ovarian cancer, increasing attention has been progressively focused on pegylated liposomal doxorubicin (PLD), whose unique formulation prolongs the persistence of the drug in the circulation and potentiates intratumor accumulation. Pegylated liposomal doxorubicin (PLD) has become a major component in the routine management of epithelial ovarian cancer. In 1999 it was first approved for platinum-refractory ovarian cancer and then received full approval for platinum-sensitive recurrent disease in 2005. PLD remains an important therapeutic tool in the management of recurrent ovarian cancer in 2012. Recent interest in PLD/carboplatin combination therapy has been the object of phase III trials in platinum-sensitive and chemonaïve ovarian cancer patients reporting response rates, progressive-free survival, and overall survival similar to other platinum-based combinations, but with a more favorable toxicity profile and convenient dosing schedule. This paper summarizes data clarifying the role of pegylated liposomal doxorubicin (PLD) in ovarian cancer, as well as researches focusing on adding novel targeted drugs to this cytotoxic agent.
doi:10.1155/2013/898146
PMCID: PMC3612436  PMID: 23577259
15.  Intra-articular delivery of adipose derived stromal cells attenuates osteoarthritis progression in an experimental rabbit model 
Introduction
Cell therapy is a rapidly growing area of research for the treatment of osteoarthritis (OA). This work is aimed to investigate the efficacy of intra-articular adipose-derived stromal cell (ASC) injection in the healing process on cartilage, synovial membrane and menisci in an experimental rabbit model.
Methods
The induction of OA was performed surgically through bilateral anterior cruciate ligament transection (ACLT) to achieve eight weeks from ACLT a mild grade of OA. A total of 2 × 106 and 6 × 106 autologous ASCs isolated from inguinal fat, expanded in vitro and suspended in 4% rabbit serum albumin (RSA) were delivered in the hind limbs; 4% RSA was used as the control. Local bio-distribution of the cells was verified by injecting chloro-methyl-benzamido-1,1'-dioctadecyl-3,3,3'3'-tetra-methyl-indo-carbocyanine per-chlorate (CM-Dil) labeled ASCs in the hind limbs. Cartilage and synovial histological sections were scored by Laverty's scoring system to assess the severity of the pathology. Protein expression of some extracellular matrix molecules (collagen I and II), catabolic (metalloproteinase-1 and -3) and inflammatory (tumor necrosis factor- α) markers were detected by immunohistochemistry. Assessments were carried out at 16 and 24 weeks.
Results
Labeled-ASCs were detected unexpectedly in the synovial membrane and medial meniscus but not in cartilage tissue at 3 and 20 days from ASC-treatment. Intra-articular ASC administration decreases OA progression and exerts a healing contribution in the treated animals in comparison to OA and 4% RSA groups.
Conclusions
Our data reveal a healing capacity of ASCs in promoting cartilage and menisci repair and attenuating inflammatory events in synovial membrane inhibiting OA progression. On the basis of the local bio-distribution findings, the benefits obtained by ASC treatment could be due to a trophic mechanism of action by the release of growth factors and cytokines.
doi:10.1186/ar4156
PMCID: PMC3672720  PMID: 23360790
16.  Co-Culture of Hematopoietic Stem/Progenitor Cells with Human Osteblasts Favours Mono/Macrophage Differentiation at the Expense of the Erythroid Lineage 
PLoS ONE  2013;8(1):e53496.
Hematopoietic stem cells (HSCs) are located in the bone marrow in a specific microenvironment referred as the hematopoietic stem cell niche, where HSCs interact with a variety of stromal cells. Though several components of the stem cell niche have been identified, the regulatory mechanisms through which such components regulate the stem cell fate are still unknown. In order to address this issue, we investigated how osteoblasts (OBs) can affect the molecular and functional phenotype of Hematopoietic Stem/Progenitor Cells (HSPCs) and vice versa. For this purpose, human CD34+ cells were cultured in direct contact with primary human OBs. Our data showed that CD34+ cells cultured with OBs give rise to higher total cell numbers, produce more CFUs and maintain a higher percentage of CD34+CD38- cells compared to control culture. Moreover, clonogenic assay and long-term culture results showed that co-culture with OBs induces a strong increase in mono/macrophage precursors coupled to a decrease in the erythroid ones. Finally, gene expression profiling (GEP) allowed us to study which signalling pathways were activated in the hematopoietic cell fraction and in the stromal cell compartment after coculture. Such analysis allowed us to identify several cytokine-receptor networks, such as WNT pathway, and transcription factors, as TWIST1 and FOXC1, that could be activated by co-culture with OBs and could be responsible for the biological effects reported above. Altogether our results indicate that OBs are able to affect HPSCs on 2 different levels: on one side, they increase the immature progenitor pool in vitro, on the other side, they favor the expansion of the mono/macrophage precursors at the expense of the erythroid lineage.
doi:10.1371/journal.pone.0053496
PMCID: PMC3551919  PMID: 23349713
17.  Role of the phloem in the biochemistry and ecophysiology of benzylisoquinoline alkaloid metabolism 
Benzylisoquinoline alkaloids (BIAs) are a diverse group of biologically active specialized metabolites produced mainly in four plant families. BIA metabolism is likely of monophyletic origin and involves multiple enzymes yielding structurally diverse compounds. Several BIAs possess defensive properties against pathogenic microorganisms and herbivores. Opium poppy (Papaver somniferum: Papaveraceae) has emerged as a model system to investigate the cellular localization of BIA biosynthesis. Although alkaloids accumulate in the laticifer cytoplasm (latex) of opium poppy, corresponding biosynthetic enzymes and gene transcripts are localized to proximal sieve elements and companion cells, respectively. In contrast, BIA metabolism in the non-laticiferous meadow rue (Thalictrum flavum; Ranunculaceae) occurs independent of the phloem. Evidence points toward the adoption of diverse strategies for the biosynthesis and accumulation of alkaloids as defensive compounds. Recruitment of cell types involved in BIA metabolism, both within and external to the phloem, was likely driven by selection pressures unique to different taxa. The biochemistry, cell biology, ecophysiology, and evolution of BIA metabolism are considered in this context.
doi:10.3389/fpls.2013.00182
PMCID: PMC3678098  PMID: 23781223
benzylisoquinoline alkaloid; phloem; laticifer; sieve element; plant defense
18.  Cognitive Impairment Affects Physical Recovery of Patients with Heart Failure Undergoing Intensive Cardiac Rehabilitation 
Purpose. To determine whether the presence of cognitive impairment (CI) affects physical recovery of patients with chronic heart failure (CHF) undergoing a cardiac rehabilitation program (CRP). Methods. We enrolled 80 CHF patients (M/F = 53/27). CI was evaluated by means of the Mini-Mental State Examination (MMSE), exercise tolerance was evaluated by six-minute walking test (6 mwt). All patients underwent a 6-week CRP program at 50–70% of maximal VO2. Patients were divided into two groups according to their MMSE (group 1: 16–23; group 2: 24–30). Results. MMSE resulted directly related to ejection fraction (r = 0.42; P = 0.03), and it was inversely related to creatinine (r = −0.36; P = 0.04). At 6 week group 1 had a lower increase in distance walked at 6 MWT than group 2 (P = 0.008). At multivariate logistic regression MMSE 16–23 predicted a reduced exercise recovery in the overall population (OR = 1.84; 95% CI = 1.50–2.18) and in women (OR = 1.42; 95% CI = 1.22–1.75), while it was not predicted in males. Conclusions. CI is a marker of advanced CHF and is an independent predictor of lower exercise recovery after CRP.
doi:10.1155/2012/218928
PMCID: PMC3544314  PMID: 23326665
19.  Nuclear damages and oxidative stress: new perspectives for laminopathies 
Mutations in genes encoding nuclear envelope proteins, particularly LMNA encoding the A-type lamins, cause a broad range of diverse diseases, referred to as laminopathies. The astonishing variety of diseased phenotypes suggests that different mechanisms could be involved in the pathogenesis of laminopathies. In this review we will focus mainly on two of these pathogenic mechanisms: the nuclear damages affecting the chromatin organization, and the oxidative stress causing un-repairable DNA damages. Alteration in the nuclear profile and in chromatin organization, which are particularly impressive in systemic laminopathies whose cells undergo premature senescence, are mainly due to accumulation of unprocessed prelamin A. The toxic effect of these molecular species, which interfere with chromatin-associated proteins, transcription factors, and signaling pathways, could be reduced by drugs which reduce their farnesylation and/or stability. In particular, inhibitors of farnesyl transferase (FTIs), have been proved to be active in rescuing the altered cellular phenotype, and statins, also in association with other drugs, have been included into pilot clinical trials. The identification of a mechanism that accounts for accumulation of unrepairable DNA damage due to reactive oxygen species (ROS) generation in laminopathic cells, similar to that found in other muscular dystrophies (MDs) caused by altered expression of extracellular matrix (ECM) components, suggests that anti-oxidant therapeutic strategies might prove beneficial to laminopathic patients.
doi:10.4081/ejh.2012.e45
PMCID: PMC3567764  PMID: 23361241
laminopathies; nuclear envelope; chromatin; muscular dystrophies; progeria; oxidative stress; DNA damage.
20.  Percutaneous vertebral augmentation for painful osteolytic vertebral metastasis: a case report 
Introduction
Vertebral metastases are associated with significant pain, disability, and morbidity. Open surgery for fracture stabilization is often inappropriate in this population due to a poor risk-benefit profile, particularly if life expectancy is short. Percutaneous vertebroplasty and kyphoplasty are appealing adjunctive procedures in patients with malignancy for alleviation of intractable pain. However, these patients have higher risk of serious complications, notably cement extravasation. Described in this report is a case of a painful osteolytic vertebral metastasis that was successfully treated by a novel percutaneous vertebral augmentation system.
Case presentation
A 42-year-old Caucasian female presented with a history of metastatic lung cancer unresponsive to radiation and chemotherapy with symptoms inadequately controlled by opiates over the previous 6 months. Magnetic resonance imaging and spiral computed tomography with two-dimensional reconstruction showed an osteolytic vertebral metastasis with complete involvement of the T10 vertebral body, extending to the cortical vertebral wall anteriorly and posteriorly. The patient was treated with percutaneous vertebral augmentation (Kiva® VCF Treatment System, Benvenue Medical, Inc, Santa Clara, CA) utilizing a novel coil-shaped polyetheretherketone implant designed to minimize the risk of cement extravasation. After the minimally invasive procedure, bone cement distribution within the vertebral body was ideal, with no observed cement extravasation. No complications were reported, pain completely resolved within 24 hours, and use of intravenous narcotics was progressively diminished within 1 week. Complete pain relief was maintained throughout 4 months of follow-up.
Conclusion
The Kiva System represents a novel and effective minimally invasive treatment option for patients suffering from severe pain due to osteolytic vertebral metastasis.
doi:10.2147/IMCRJ.S29569
PMCID: PMC3658248  PMID: 23754917
Kiva; minimally invasive; pain
21.  Antiapoptotic and Antiautophagic Effects of Eicosapentaenoic Acid in Cardiac Myoblasts Exposed to Palmitic Acid 
Nutrients  2012;4(2):78-90.
Apoptosis is a programmed cell death that plays a critical role in cell homeostasis. In particular, apoptosis in cardiomyocytes is involved in several cardiovascular diseases including heart failure. Recently autophagy has emerged as an important modulator of programmed cell death pathway. Recent evidence indicates that saturated fatty acids induce cell death through apoptosis and this effect is specific for palmitate. On the other hand, n-3 polyunsaturated fatty acids (PUFAs) have been implicated in the protection against cardiovascular diseases, cardiac ischemic damage and myocardial dysfunction. In the present study we show that n-3 PUFA eicosapentaenoic acid (EPA) treatment to culture medium of H9c2 rat cardiomyoblasts protects cells against palmitate-induced apoptosis, as well as counteracts palmitate-mediated increase of autophagy. Further investigation is required to establish whether the antiautophagic effect of EPA may be involved in its cytoprotective outcome and to explore the underlying biochemical mechanisms through which palmitate and EPA control the fate of cardiac cells.
doi:10.3390/nu4020078
PMCID: PMC3296992  PMID: 22413063
eicosapentaenoic acid; palmitic acid; apoptosis; autophagy; H9c2 cardiomyoblasts
22.  Does laparoscopic management of deep infiltrating endometriosis improve quality of life? A prospective study 
Background
Deep infiltrating endometriosis (DIE) can affect importantly patients' quality of life (QOL). The aim of this study is to evaluate the impact of the laparoscopic management of DIE on QOL after six months from treatment.
Methods
It is a prospective cohort study. In a tertiary care university hospital, between April 2008 and December 2009, 100 patients underwent laparoscopic management of DIE and completed preoperatively and 6-months postoperatively a QOL questionnaire, the short form 36 (SF-36).
Quality of life was measured through the SF-36 scores. Intra-operative details of disease site, number of lesions, type of intervention, period of hospital stay and peri-operative complications were noted.
Results
Six months postoperatively all the women had a significant improvement in every scale of the SF-36 (p < 0,0005). Among patients with intestinal DIE, significant differences in postoperative scores of SF-36 were not detected between patients submitted to nodule shaving and segmental resection (p > 0.05). There was no significant difference in the SF-36 scores at 6 months from surgery between patients who received postoperative medical treatment and patients who did not (p > 0.05).
Conclusions
Laparoscopic excision of DIE lesions significantly improves general health and psycho-emotional status at six months from surgery without differences between patients submitted to intestinal segmental resection or intestinal nodule shaving.
doi:10.1186/1477-7525-9-98
PMCID: PMC3247061  PMID: 22054310
23.  Expressed sequence tag analysis of khat (Catha edulis) provides a putative molecular biochemical basis for the biosynthesis of phenylpropylamino alkaloids 
Genetics and Molecular Biology  2011;34(4):640-646.
Khat (Catha edulis Forsk.) is a flowering perennial shrub cultivated for its neurostimulant properties resulting mainly from the occurrence of (S)-cathinone in young leaves. The biosynthesis of (S)-cathinone and the related phenylpropylamino alkaloids (1S,2S)-cathine and (1R,2S)-norephedrine is not well characterized in plants. We prepared a cDNA library from young khat leaves and sequenced 4,896 random clones, generating an expressed sequence tag (EST) library of 3,293 unigenes. Putative functions were assigned to > 98% of the ESTs, providing a key resource for gene discovery. Candidates potentially involved at various stages of phenylpropylamino alkaloid biosynthesis from L-phenylalanine to (1S,2S)-cathine were identified.
doi:10.1590/S1415-47572011000400017
PMCID: PMC3229120  PMID: 22215969
khat; (S)-cathinone; phenylpropylamino alkaloids biosynthesis; EST library; gene discovery
24.  Conceptual frameworks and empirical approaches used to assess the impact of health research: an overview of reviews 
Background
How to assess the impact of research is of growing interest to funders, policy makers and researchers mainly to understand the value of investments and to increase accountability. Broadly speaking the term "research impact" refers to the contribution of research activities to achieve desired societal outcomes. The aim of this overview is to identify the most common approaches to research impact assessment, categories of impact and their respective indicators.
Methods
We systematically searched the relevant literature (PubMed, The Cochrane Library (1990-2009)) and funding agency websites. We included systematic reviews, theoretical and methodological papers, and empirical case-studies on how to evaluate research impact. We qualitatively summarised the included reports, as well the conceptual frameworks.
Results
We identified twenty-two reports belonging to four systematic reviews and 14 primary studies. These publications reported several theoretical frameworks and methodological approaches (bibliometrics, econometrics, ad hoc case studies). The "payback model" emerged as the most frequently used. Five broad categories of impact were identified: a) advancing knowledge, b) capacity building, c) informing decision-making, d) health benefits, e) broad socio-economic benefits. For each proposed category of impact we summarized a set of indicators whose pros and cons are presented and briefly discussed.
Conclusions
This overview is a comprehensive, yet descriptive, contribution to summarize the conceptual framework and taxonomy of an heterogeneous and evolving area of research. A shared and comprehensive conceptual framework does not seem to be available yet and its single components (epidemiologic, economic, and social) are often valued differently in different models.
doi:10.1186/1478-4505-9-26
PMCID: PMC3141787  PMID: 21702930
Research governance; Research impact; Health research; Bibliometrics
25.  Cardiac Tamponade and Heart Failure Due to Myopericarditis as a Presentation of Infection with the Pandemic H1N1 2009 Influenza A Virus▿  
Journal of Clinical Microbiology  2010;48(6):2298-2300.
We describe a fatal case of myopericarditis presenting with cardiac tamponade in a previously healthy 11-year-old child. Pandemic H1N1 2009 influenza A virus sequences were identified in throat and myocardial tissues and pericardial fluid, suggesting damage of myocardial cells directly caused by the virus.
doi:10.1128/JCM.00418-10
PMCID: PMC2884467  PMID: 20392922

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