Multi-centre real-world validation of automated treatment planning for breast radiotherapy.

PURPOSE: To present the results of the first multi-centre real-world validation of autoplanning for whole breast irradiation after breast-sparing surgery, encompassing high complexity cases (e.g. with a boost or regional lymph nodes) and a wide range of clinical practices. METHODS: The 24 participating centers each included 10 IMRT/VMAT/Tomotherapy patients, previously treated with a manually generated plan ('manplan'). There were no restrictions regarding case complexity, planning aims, plan evaluation parameters and criteria, fractionation, treatment planning system or treatment machine/technique. In addition to dosimetric comparisons of autoplans with manplans, blinded plan scoring/ranking was conducted by a clinician from the treating center. Autoplanning was performed using a single configuration for all patients in all centres. Deliverability was verified through measurements at delivery units. RESULTS: Target dosimetry showed comparability, while reductions in OAR dose parameters were 21.4 % for heart Dmean, 16.7 % for ipsilateral lung Dmean, and 101.9 %, 45.5 %, and 35.7 % for contralateral breast D0.03cc, D5% and Dmean, respectively (all p < 0.001). Among the 240 patients included, the clinicians preferred the autoplan for 119 patients, with manplans preferred for 96 cases (p = 0.01). Per centre there were on average 5.0 ± 2.9 (1SD) patients with a preferred autoplan (range [0-10]), compared to 4.0 ± 2.7 with a preferred manplan ([0,9]). No differences were observed regarding deliverability. CONCLUSION: The automation significantly reduced the hands-on planning workload compared to manual planning, while also achieving an overall superiority. However, fine-tuning of the autoplanning configuration prior to clinical implementation may be necessary in some centres to enhance clinicians' satisfaction with the generated autoplans.

"A lactose-modified chitosan accelerates chondrogenic differentiation in mesenchymal stem cells spheroids".

Spheroids derived from human mesenchymal stem cells (hMSCs) are of limited use for cartilage regeneration, as the viability of the cells progressively decreases during the period required for chondrogenic differentiation (21 days). In this work, spheroids based on hMSCs and a lactose-modified chitosan (CTL) were formed by seeding cells onto an air-dried coating of CTL. The polymer coating can inhibit cell adhesion and it is simultaneously incorporated into spheroid structure. CTL-spheroids were characterized from a morphological and biological perspective, and their properties were compared with those of spheroids obtained by seeding the cells onto a non-adherent surface (agar gel). Compared to the latter, smaller and more viable spheroids form in the presence of CTL as early as 4 days of culture. At this time point, analysis of stem cells differentiation in spheroids showed a remarkable increase in collagen type-2 (COL2A1) gene expression (~700-fold compared to day 0), whereas only a 2-fold increase was observed in the control spheroids at day 21. These results were confirmed by histological and transmission electron microscopy (TEM) analyses, which showed that in CTL-spheroids an early deposition of collagen with a banding structure already occurred at day 7. Overall, these results support the use of CTL-spheroids as a novel system for cartilage regeneration, characterized by increased cell viability and differentiation capacity within a short time-frame. This will pave the way for approaches aimed at increasing the success rate of procedures and reducing the time required for tissue regeneration.

Active bone marrow segmentation based on computed tomography imaging in anal cancer patients: A machine-learning-based proof of concept.

PURPOSE: Different methods are available to identify haematopoietically active bone marrow (ActBM). However, their use can be challenging for radiotherapy routine treatments, since they require specific equipment and dedicated time. A machine learning (ML) approach, based on radiomic features as inputs to three different classifiers, was applied to computed tomography (CT) images to identify haematopoietically active bone marrow in anal cancer patients. METHODS: A total of 40 patients was assigned to the construction set (training set + test set). Fluorine-18-Fluorodeoxyglucose Positron Emission Tomography (18FDG-PET) images were used to detect the active part of the pelvic bone marrow (ActPBM) and stored as ground-truth for three subregions: iliac, lower pelvis and lumbosacral bone marrow (ActIBM, ActLPBM, ActLSBM). Three parameters were used for the correspondence analyses between 18FDG-PET and ML classifiers: DICE index, Precision and Recall. RESULTS: For the 40-patient cohort, median values [min; max] of the Dice index were 0.69 [0.20; 0.84], 0.76 [0.25; 0.89], and 0.36 [0.15; 0.67] for ActIBM, ActLSBM, and ActLPBM, respectively. The Precision/Recall (P/R) ratio median value for the ActLPBM structure was 0.59 [0.20; 1.84] (over segmentation), while for the other two subregions the P/R ratio median has values of 1.249 [0.43; 4.15] for ActIBM and 1.093 [0.24; 1.91] for ActLSBM (under segmentation). CONCLUSION: A satisfactory degree of overlap compared to 18FDG-PET was found for 2 out of the 3 subregions within pelvic bones. Further optimization and generalization of the process is required before clinical implementation.

Liposomes embedded with differentiating factors as a new strategy for enhancing DPSC osteogenic commitment.

Human dental pulp stem cell (DPSC) differentiation toward the osteoblastic phenotype is enhanced when culture media are supplemented with differentiating factors, i.e. ascorbic acid, ß-glycerophosphate and dexamethasone. Liposomes, spherical vesicles formed by a phospholipid bilayer, are frequently used as carriers for drugs, growth factors and hydrophobic molecules. The aim of this work was to speed up DPSC commitment to the osteogenic lineage by embedding differentiating factors within liposomes. Firstly, liposomes were prepared by rehydrating a phospholipidic thin film and characterised in terms of dimensions. Secondly, liposome-exposed DPSCs were characterised by their immunophenotypic profile. Levels of CD90 were significantly decreased in the presence of liposomes filled with ascorbic acid, ß-glycerophosphate and dexamethasone (Lipo-Mix) with respect to normal differentiation medium (DM), while CD73 and CD29 expression were enhanced, suggesting osteogenic commitment. Additionally, an appreciable extracellular matrix deposition is detected. Thirdly, the Lipo-Mix formulation better increases alkaline phosphatase activity and levels of Collagen I secretion with respect to DM. In parallel, the new liposome formulation is capable of decreasing the release of H2O2 and of triggering a precocious antioxidant cell response, redressing the redox balance required upon mesenchymal stem cell commitment to osteogenesis. It can be therefore hypothesised that Lipo-Mix could represent a suitable tool for clinical regenerative purposes in the field of tissue engineering by speeding up DPSC osteogenic commitment, mineralised matrix deposition and remodelling.

CD30 and ALK combination therapy has high therapeutic potency in RANBP2-ALK-rearranged epithelioid inflammatory myofibroblastic sarcoma.

BACKGROUND: Epithelioid inflammatory myofibroblastic sarcoma (eIMS) is characterised by perinuclear ALK localisation, CD30 expression and early relapse despite crizotinib treatment. We aimed to identify therapies to prevent and/or treat ALK inhibitor resistance. METHODS: Malignant ascites, from an eIMS patient at diagnosis and following multiple relapses, were used to generate matched diagnosis and relapse xenografts. RESULTS: Xenografts were validated by confirmation of RANBP2-ALK rearrangement, perinuclear ALK localisation and CD30 expression. Although brentuximab-vedotin (BV) demonstrated single-agent activity, tumours regrew during BV therapy. BV resistance was associated with reduced CD30 expression and induction of ABCB1. BV resistance was reversed in vitro by tariquidar, but combination BV and tariquidar treatment only briefly slowed xenograft growth compared with BV alone. Combining BV with either crizotinib or ceritinib resulted in marked tumour shrinkage in both xenograft models, and resulted in prolonged tumour-free survival in the diagnosis compared with the relapse xenograft. CONCLUSIONS: CD30 is a therapeutic target in eIMS. BV efficacy is limited by the rapid emergence of resistance. Prolonged survival with combination ALK and CD30-targeted-therapy in the diagnosis model provides the rationale to trial this combination in eIMS patients at diagnosis. This combination could also be considered for other CD30-positive, ALK-rearranged malignancies.

Predicting Quality of Life Changes in Hemodialysis Patients Using Machine Learning: Generation of an Early Warning System.

Objective To predict changes in the quality of life scores of hemodialysis patients for the coming month and the development of an early warning system using machine learning Methods It was a prospective cohort study (one-month duration) at the dialysis center of a tertiary care hospital in Pakistan. The study started on 1st October 2016. About 78 patients have been enrolled till now. Bachelor of Medicine and Bachelor of Surgery (MBBS) qualified doctors administered a proforma with demographics and the validated Urdu version of World Health Organization Quality Of Life-BREF (WHOQOL-BREF). It was to be repeated after one month to the same patient by the same investigator. Simple statistics were computed using SPSS version 24 (IBM Corp., Armonk, NY) while machine learning was performed using R (version 3.0) and Orange (version 3.1). Results Using machine learning algorithms, two models (classification tree and Naïve Bayes) were generated to predict an increase or decrease of 5% in a patient's WHOQOL-BREF score over one month. The classification tree was selected as the most accurate model with an area under curve (AUC) of 83.3% (accuracy: 81.9%) for the prediction of 5% increase in QOL and an AUC of 76.2% (accuracy: 81.8%) for the prediction of 5% decrease in QOL over the coming month. The factors associated with an increase of QOL by 5% or more over the next month included younger age (<19 years) and higher iron sucrose doses (>278mg/month). Drops in psychological, physical, and social domain scores lead to a decrease of 5% or more in QOL scores over the following month. Conclusion An early warning system, dialysis data interpretation for algorithmic-prediction on quality of life (DIAL) was built for the early detection of deteriorating QOL scores in the hemodialysis population using machine learning algorithms. The model pointed out that working on psychological and environmental domains, in particular, may prevent the drop in QOL scores from occurring. DIAL, if implemented on a larger scale, is expected to help patients in terms of ensuring a better QOL and in reducing the financial burden in the long term.

RANK/RANKL/OPG signaling pathways in necrotic jaw bone from bisphosphonate-treated subjects.

Osteonecrosis of the jaw (ONJ) is a chronic complication affecting long-term bisphosphonate-treated subjects, recognized by non-healing exposed bone in the maxillofacial region. The pathophysiological mechanism underlying ONJ has not been fully elucidated. The aim of the present study was to investigate the role of RANK/RANKL/OPG signaling pathway and, in parallel, to evaluate angiogenic and matrix mineralization processes in jaw bone necrotic samples obtained from bisphosphonate-treated subjects with established ONJ. Necrotic bone samples and native bone samples were processed for Light and Field Emission in Lens Scanning Electron Microscope (FEISEM) analyses, for Real-Time RT-PCR to evaluate the gene expression of TNFRSF11A (RANK), TNFSF11 (RANKL), and TNFSF11B (OPG) and for immunohistochemical analyses of VEGF and BSP expression. Morphological analyses performed by Light microscope and FEISEM show empty osteocytic lacunae and alteration of lamellar organization with degradation of the mineralized bone matrix in necrotic bone samples. A significant increase in TNFRSF11A, TNFSF11, TRAF6 and NFAT2 gene expression, and a reduction of TNFSF11B gene transcription level compared is also showed in necrotic bone compared to control samples. No significant difference of VEGF expression is evidenced, while lower BSP expression in necrotic bone compared to healthy samples is found. Even if the pathogenesis of bisphosphonate-associated ONJ remains unknown, a link between oral pathogens and its development seems to exist. We suppose lipopolysaccharide produced by bacteria colonizing and infecting necrotic bone and the surrounding viable area could trigger RANK/RANKL/OPG signaling pathway and, in this context, osteoclasts activation could be considered as a protective strategy carried out by the host bone tissue to delimitate the necrotic area and to counteract infection.

A dual role for ß1 integrin in an in vitro Streptococcus mitis/human gingival fibroblasts co-culture model in response to TEGDMA.

AIM: To evaluate the effect of TEGDMA on human gingival fibroblasts (HGFs) in vitro co-cultured with Streptococcus mitis, focusing on the signalling pathways underlying cell tissue remodelling and inflammatory response processes. METHODOLOGY: ß1 integrin expression was evaluated by means of imaging flow cytometry. The Western blot technique was used to investigate the expression of protein kinase C (PKC), extracellular signal-regulated kinase (ERK), matrix metalloproteinase 9 (MMP9) and 3 (MMP3). RT-PCR was performed to quantify nuclear factor-kb subunits (Nf-kb1, ReLa), IkB kinase ß (IkBkB), cyclooxygenase II (COX-2) and tumour necrosis factor-α (TNF-α) mRNA levels. Statistical analysis was performed using the analysis of variance (anova). RESULTS: When HGFs are co-cultured with S. mitis, ß1 integrin intensity, phosphorylated PKC (p-PKC), activated ERK (p-ERK), IkBkB mRNA level and MMP9 expression increased (for all molecules P < 0.05 HGFs versus HGFs co-cultured with S. mitis). A higher level of MMP3 in HGFs treated with TEGDMA was recorded (P < 0.05 HGFs versus HGFs exposed to TEGDMA). COX-2 inflammatory factor mRNA level appeared higher in HGFs exposed to 1 mmol L(-1) TEGDMA (P < 0.01 HGFs versus HGFs exposed to TEGDMA), whereas TNF-α gene expression was higher in HGFs co-cultured with S. mitis (P < 0.05 HGFs versus HGFs co-cultured with S. mitis). CONCLUSIONS: ß1 integrin triggered the signalling pathway, transduced by p-PKCα and involving ERK 1 and 2 and MMPs. This pathway resulted in an unbalanced equilibrium in tissue remodelling process, along with inflammatory response when HGFs are exposed to bacteria or biomaterial alone. On the contrary, the TEGDMA/S. mitis combination restored the balance between extracellular matrix deposition and degradation and prevented an inflammatory response.

pPKC α regulates through integrin ß 1 human gingival fibroblasts/Streptococcus mitis adhesion in response to HEMA.

AIM: To investigate in coculture of human gingival fibroblasts (HGFs) and Streptococcus mitis, the molecular mechanisms driving the response to 2-hydroxyethyl methacrylate (HEMA) in terms of eukaryotic/prokaryotic cell adhesion, signal transduction and apoptosis. METHODOLOGY: The clinical strain S. mitis DS12, cultured in Trypticase soy broth was added to HGFs, obtained from fragments of healthy marginal gingival tissue and cultured in DMEM, treated with 3 mmol L(-1) 2-hydroxyethyl methacrylate (HEMA) for 48 h and processed for microscopic, western blotting and flow cytometric analyses. RESULTS: 2-hydroxyethyl methacrylate (HEMA) treatment increased the adhesion between S. mitis and HGFs, which seemed to be mediated by the PKC α/integrin ß 1 signalling system, improved by the presence of saliva. It also reduced the viability and the adhesion of HGFs to polypropylene substrate in terms of procollagen I and MMP3 expression. The presence of saliva and S. mitis reduced the number of necrotic HGFs and upregulated the expression of both procollagen I and MMP3. CONCLUSIONS: These results shed more light on the biological and molecular events occurring in vitro in a coculture model that mimics the environment of the oral cavity with HEMA treatment. The key role played by oral bacteria and saliva in preventing inflammatory and toxic processes that occur in vivo in human gingival fibroblasts upon the release of dental material monomers is confirmed.

Maxillary sinus augmentation procedures through equine-derived biomaterial or calvaria autologous bone: immunohistochemical evaluation of OPG/RANKL in humans.

Autologous bone is considered the gold standard for bone regeneration, even if different heterologous bone substitutes have been proposed to overcome the limits related to its use. The aim of this study was to analyze and to compare the molecular events switched on by autologous or heterologous bone graft insertion, focusing on TGFß1 expression and OPG/RANKL ratio, to analyze resorption process, and estimating graft vascularization, new bone tissue deposition and its mineralization, through VEGF, BSP and SPARC expression evaluation, respectively. Patients needing pre-prosthetic rehabilitation of the posterior maxilla were treated using an equine-derived biomaterial (Group 1) or calvaria autologous bone (Group 2), according to the morphology of the bone defect. Bone graft integration was evaluated on bone samples obtained from the treated areas at the moment of dental implant insertion, by morphological and immunohistochemical analyses for TGFß1, OPG, RANKL, VEGF, BSP, and SPARC expression. Morphological analysis shows the presence of biomaterial residual granules in Group 1, in parallel to a good integration between graft and host tissue. Moderate TGFß1 expression is seen in both Group 1 and Group 2. OPG/RANKL ratio appear higher in Group 1; VEGF expression appears very strong in Group 1 and strong in Group 2, while BSP and SPARC expression results weak in Group 1 and moderate in Group 2. Results reveal the good integration between both types of graft and the host tissue, even though autologous graft seems to produce a faster regenerative process, as evidenced by the different expression of the investigated molecules. According to these observations, the clinical use of heterologous particulate equine-derived biomaterial may ensure long-term predictability of implant-prosthetic rehabilitation, comparable to that obtained with autologous bone graft.

Human carotid body neuroglobin, vascular endothelial growth factor and inducible nitric oxide synthase expression in heroin addiction.

AIMS: The carotid body (CB) represents the prime site for detecting and responding to hypoxia. Since the role of heroin in respiratory depression with consequent hypoxia is known, the authors were able to investigate morphological and molecular modifications occurring in the CB of heroin addicted subjects compared to subjects who died because of trauma. METHODS AND RESULTS: CB sampled from six 27 year old subjects, slides were treated with Mallory Trichrome staining or used for immunohistochemical analysis to detect Neuroglobin (NGB), Hypoxia Inducible Factor-1 (HIF-1α), Vascular Endothelial Growth Factor (VEGF), Inducible Nitric Oxide Synthase (i-NOS), Bax and cleaved Caspase-3 proteins. Mallory Trichrome staining shows an increase in the connective tissue in heroin subjects compared to controls and a parallel reduction in parenchymal area. Immunohistochemical analyses in heroin subjects found a decrease in NGB and an increase in HIF-1α and VEGF compared to controls; i-NOS expression was not statistically significant. Bax and cleaved caspase-3 were positive only in the heroin subjects. CONCLUSIONS: These results could confirm the typical hypoxic condition occurring in heroin addicts. Since NGB may function as a reactive oxygen or nitrogen species scavenger and as apoptotic cell death protector, the decrease in its expression may suggest a key role of this globin in human CB impairment due to heroin addiction.

Development and aging are oxygen-dependent and correlate with VEGF and NOS along life span.

During development and aging, vascular remodeling represents a critical adaptive response to modifications in oxygen supply to tissues. Hypoxia inducible factor (HIF) has a crucial role and is modulated by oxygen levels, with an age-dependent response in neonates, adult, and aged people. ROS are generated under hypoxic conditions and the accumulation of free radicals during life reduces the ability of tissues to their removal. In this immunohistochemical study we investigated the presence and localization of VEGF and iNOS in human carotid bodies (CB) sampled at autopsy from three children (mean age - 2 years), four adult young subjects (mean age - 44.3 years), and four old subjects (mean age - 67.3 years). VEGF immunoreactivity was significantly enhanced in CB tissues from the children (7.2 ± 1.2%) and aged subjects (4.7 ± 1.7%) compared with the young adults (1.4 ± 0.7%). On the other hand, iNOS immunoreactivity was enhanced in CB tissues from the children (0.4 ± 0.04%) and young adult subjects (0.3 ± 0.02%) compared with the old subjects (0.2 ± 0.02%). Prevention of oxygen desaturation, reducing all causes of hypoxemia from neonatal life to aging would decrease the incidence of diseases in the elderly population with lifespan extension.

2-Hydroxyethyl methacrylate inflammatory effects in human gingival fibroblasts.

AIM: To investigate the inflammatory response in human gingival fibroblasts (HGFs) treated with a relatively low 2-hydroxyethyl methacrylate (HEMA) concentration by studying reactive oxygen species (ROS) production, cyclooxygenase-2 (COX-2) and tumour necrosis factor-alpha (TNF-α) gene expression, and prostaglandin E2 (PGE2) release. METHODOLOGY: Cultured HGFs were exposed to 3 mmol L⁻¹ HEMA for 0, 24 or 96 h. ROS production was investigated by flow cytometry; TNF-α and COX-2 gene expression was determined by RT-PCR, and prostaglandin E2 production was detected by an enzyme immunoassay. RESULTS: After 24- or 96-h HEMA incubation, ROS levels were approximately eightfold and elevenfold higher than controls, whilst COX-2 gene expression was approximately twofold or fourfold higher than controls, respectively. Twenty-four-hour exposure enhanced TNF-α mRNA levels by approximately 66%, whilst after 96-h incubation, TNF-α gene expression was fivefold higher than controls. Ninety-six-hour HEMA treatment increased PGE2 concentration in the culture medium by around 17% compared with controls. CONCLUSIONS: 2-Hydroxyethyl methacrylate treatment (3 mmol L⁻¹) induced an inflammatory response in HGFs modulated by ROS production, as well as by the increase in TNF-α and COX-2 gene expression and by PGE2 release.

pPKCα mediated-HIF-1α activation related to the morphological modifications occurring in neonatal myocardial tissue in response to severe and mild hyperoxia.

In premature babies birth an high oxygen level exposure can occur and newborn hyperoxia exposure can be associated with free radical oxygen release with impairment of myocardial function, while in adult animal models short exposure to hyperoxia seems to protect heart against ischemic injury. Thus, the mechanisms and consequences which take place after hyperoxia exposure are different and related to animals age. The aim of our work has been to analyze the role played by HIF-1α in the occurrence of the morphological modifications upon hyperoxia exposure in neonatal rat heart. Hyperoxia exposure induces connective compartment increase which seems to allow enhanced blood vessels growth. An increased hypoxia inducible factor-1α (HIF-1α) translocation and vascular endothelial growth factor (VEGF) expression has been found upon 95% oxygen exposure to induce morphological modifications. Upstream pPKC-α expression increase in newborn rats exposed to 95% oxygen can suggest PKC involvement in HIF-1α activation. Since nitric oxide synthase (NOS) are involved in heart vascular regulation, endothelial NOS (e-NOS) and inducible NOS (i-NOS) expression has been investigated: a lower eNOS and an higher iNOS expression has been found in newborn rats exposed to 95% oxygen related to the evidence that hyperoxia provokes a systemic vasoconstriction and to the iNOS pro-apoptotic action, respectively. The occurrence of apoptotic events, evaluated by TUNEL and Bax expression analyses, seems more evident in sample exposed to severe hyperoxia. All in all such results suggest that in newborn rats hyperoxia can trigger oxygen free radical mediated membrane injury through a pPKCα mediated HIF-1α signalling system, even though specificity of such response could be obtained by in vivo administration to the rats of specific inhibitors of PKCα. This intracellular signalling can switch molecular events leading to blood vessels development in parallel to pro-apoptotic events due to an immature anti-oxidant defensive system in newborn rat hearts.

Inducible nitric oxide synthase-activated mitochondrial apoptotic pathway in hypoxic and aged rat hearts.

BACKGROUND: Hypoxia and aging determine on mammalian cells a stress response which implies modified production of oxidants, reactive oxygen species or reactive nitrogen species at the mitochondrial level, interfering with cell-signaling proteins and inducing mitochondrial damage, apoptosis occurrence and functional consequences. OBJECTIVE: Here we report the effects of hypoxia on the in vivo morphological and biochemical response of young and aged Wistar rat hearts. METHODS: Left ventricles were excised from each experimental point and processed. Investigations of vascular endothelial growth factor (VEGF) expression and apoptotic events, mitochondrial damage, were performed by light and electron microscopy, respectively; endothelial, inducible and neuronal NOS, PKCα, pPKCα, caspase-3 expression and Apaf-1/cytochrome c complex formation were assessed by Western blotting and co-immunoprecipitation analyses, respectively. RESULTS: Besides morphological modifications, which confirm mitochondrial suffering upon hypoxia exposure in both young and aged hearts, the role played by PKCα in controlling nitric oxide synthase (NOS) protein level was investigated. Downstream PKCα activation, a dramatic iNOS expression increase, concomitant to enhanced apoptotic cell percentage and Apaf-1/cytochrome c co-immunoprecipitation, is evident in the hypoxic young, suggesting iNOS-mediated activation of the mitochondrial apoptotic pathway. CONCLUSIONS: Moreover, overexpression of iNOS and VEGF in the hypoxic young rat hearts suggests that an increased VEGF level may allow coordinated development of the lymphatic and blood vasculature, necessary for fluid homeostasis and to counteract oxidative stress. Thus the inhibition of such growth factor proposes new therapeutic possibilities for diseases associated to vascular function and for solid tumors which show pathological angiogenesis and lymphoangiogenesis.

Vascular endothelial growth factor and e-nitric oxide synthase-mediated regenerative response occurring upon autologous and heterologous bone grafts.

Bone regeneration procedures allow oral rehabilitation with dental implants also in edentulous ridges with severe bone atrophy. The integration of grafted materials with the host tissue can initiate regenerative, inflammatory and apoptotic response. Since molecular mechanisms exist at the basis of such response, the aim of this work is to investigate, by immunohistochemical analyses, the expression of proteins involved in the graft integration process, in parallel to clinical and histological modifications, occurring on sites treated with extraoral autologous bone graft deriving from the parietal region of the calvaria (eAB), intraoral autologous bone graft deriving from mandibular ramus (iAB) and heterologous bone graft from swine (hB) in human patients. In our study, the immunohistochemical expression of BSP, VEGF, eNOS in eAB samples was significantly higher (p < 0.05) compared to values recorded in iAB and hB samples. The inflammatory response, investigated by iNOS expression, was found lower in all autologous samples (eAB and iAB) compared to hB, at statistically significant values. Moreover, the expression of the pro-apoptotic molecule, Bax, resulted significantly lower (p < 0.05) in eAB than in iAB and hB samples. These values, together with the low number of apoptotic cells detected in autologous samples, suggest a good regenerative response when extraoral autologous bone graft is used in comparison to the response from the other grafts, and also suggest the use of calvaria graft as a predictable therapeutic procedure for repairing severe bone defects in oral and maxillofacial surgery, not only by clinical and biomechanical criteria, but also from a biomolecular aspect.

p53 and telomerase control rat myocardial tissue response to hypoxia and ageing.

Cellular senescence implies loss of proliferative and tissue regenerative capability. Also hypoxia, producing Reactive Oxygen Species (ROS), can damage cellular components through the oxidation of DNA, proteins and lipids, thus influencing the shortening of telomeres.Since ribonucleoprotein Telomerase (TERT), catalyzing the replication of the ends of eukaryotic chromosomes, promotes cardiac muscle cell proliferation, hypertrophy and survival, here we investigated its role in the events regulating apoptosis occurrence and life span in hearts deriving from young and old rats exposed to hypoxia.TUNEL (terminal-deoxinucleotidyl -transferase- mediated dUTP nick end-labeling) analysis reveals an increased apoptotic cell number in both samples after hypoxia exposure, mainly in the young with respect to the old. TERT expression lowers either in the hypoxic young, either in the old in both experimental conditions, with respect to the normoxic young. These events are paralleled by p53 and HIF-1 α expression dramatic increase and by p53/ HIF-1 α co-immunoprecipitation in the hypoxic young, evidencing the young subject as the most stressed by such challenge. These effects could be explained by induction of damage to genomic DNA by ROS that accelerates cell senescence through p53 activation. Moreover, by preventing TERT enzyme down-regulation, cell cycle exit and apoptosis occurrence could be delayed and new possibilities for intervention against cell ageing and hypoxia could be opened.

Histone modifications associated with herpes simplex virus type 1 genomes during quiescence and following ICP0-mediated de-repression.

In the current study, it was shown that repressed virus genomes in quiescently infected MRC5 cells adopt a repressed histone-associated structure marked by the enrichment of deacetylated histones at a wide variety of herpes simplex virus type 1 (HSV-1) promoters. In addition, it was shown that genome de-repression, mediated by HSV-2 superinfection or delivery of ICP0 using a recombinant adenovirus vector, resulted in the enrichment of acetylated histones on HSV DNA. These data indicate that ICP0-mediated genome de-repression is intimately linked to enrichment of acetylated histones at virus promoters. The fold change in association of pan-acetylated histone H3 following Ad.TRE.ICP0-mediated de-repression consistently revealed promoter-specific variation, with the highest fold changes (>50-fold) being observed at the latency-associated transcript promoter and enhancer regions. Chromatin immunoprecipitation analyses using an antibody specific to the C terminus of histone H3 as a surrogate measure of nucleosome occupancy revealed little variability in the total loading of histone H3 at the various HSV promoters. This observation suggests that acetylation of histone H3 in response to ICP0 expression is not uniformly targeted across the HSV-1 genome during ICP0-mediated de-repression.