Mechanism of progression in cervical intraepithelial neoplasia

The cervical intraepithelial neoplasia (CIN) grade 1, 2 and 3 are the precursor lesions of cervical cancer. Due to the lack of useful study models, the molecular mechanisms involved in CIN progression are still largely unknown. The overall aim of my study was to investigate the molecular mechanisms occurring in CIN lesion progression. This goal was achieved by investigating gene expression profiles and methylation status of gene promoters in a novel study model of tumor progression, i.e. primary colonies of CIN2 and CIN3 keratinocytes derived from CIN2 and CIN3 lesions. To this purpose, the first aim of my study was to develop a rapid and simple cell culture protocol enables primary colonies of HPV16-CIN2 and HPV16-CIN3 keratinocytes to be derived from small tissue fragments of CIN2 and CIN3 lesions. The primary colonies of CIN2 and CIN3 keratinocytes were then investigated for presence epithelial and cervical markers showing cytokeratin -14, -17, -19 expression. In the second part of my study primary colonies of HPV16-CIN2 and HPV16-CIN3 keratinocytes were chosen to be investigated by microarray analysis. Differentially expressed genes were analyzed in normal cervical keratinocytes compared with HPV16-CIN2 keratinocytes and in HPV16-CIN2 keratinocytes compared with HPV16-CIN3 keratinocytes. Thirty-seven candidate genes with continuously decreasing or increasing expression during CIN progression were identified. Specifically, 23 down-expressed genes involved in antiviral immune response and differentiation and 14 over-expressed genes involved in proliferation and tumor invasiveness were identified. One of these genes, phosphoglycerate dehydrogenase, was chosen for further characterization. Quantitative reverse transcription-polymerase chain reaction and immunohistochemical analysis confirmed that expression of phosphoglycerate dehydrogenase consistently increases during progression of HPV16-CIN toward cancer. Phosphoglycerate dehydrogenase is likely to be associated with tumorigenesis and may be a potential prognostic marker for CIN progression. Finally, in the third part of my study, to verify whether down-expression of genes in CIN2 and CIN3 keratinocytes depended on DNA promoter methylation, I investigated the methylation status of RARB and IRF6 promoter, both tumor suppressor genes, in relationship with expression of their two pathway-correlated genes, p63 and c-JUN. The epigenetic analysis revealed a hypermethylation of RARB and IRF6 gene promoters in CIN2 and CIN3 keratinocytes compared to normal keratinocytes as well as a progressive hypermethylation of RARB promoter region from normal to CIN2 keratinocytes and from CIN2 to CIN3 keratinocytes. Consistently, a gradual up-regulation of p63 and c-JUN from CIN2 to CIN3 keratinocytes was detected. It is conceivable that the hypermethylation of RARB and IRF6 promoter region may cause a consequent RARB and IRF6 down-regulation and this reduction of expression could enhance c-JUN and p63 expression in CIN keratinocytes. In conclusion, in my study the molecular mechanisms occurring in CIN lesions progression were investigated. In particular, it was investigated gene expression profiles and methylation status of gene promoters in a novel study model, i.e. primary colonies of CIN2 and CIN3 keratinocytes derived from CIN2 and CIN3 lesions. Gene expression analysis revealed 37 down-expressed or over-expressed genes which may contribute to CIN progression. One of these genes, the phosphoglycerate dehydrogenase, which resulted over-expressed at both mRNA and protein level in CIN2 and CIN3 keratinocytes and in CIN2, CIN3 and cancer tissues, respectively, is likely to be associated with tumorigenesis and may be a potential prognostic marker for CIN progression. Aberrant promoter hypermethylation of RARB and IRF6 genes also may be a potential epigenetic prognostic marker for CIN progression

Stem Cell Fate and Immunomodulation Promote Bone Regeneration via Composite Bio-Oss®/AviteneTM Biomaterial

Bone defects in maxillofacial regions lead to noticeable deformity and dysfunctions. Therefore, the use of biomaterials/scaffolds for maxillofacial bone regrowth has been attracting great interest from many surgical specialties and experts. Many approaches have been devised in order to create an optimal bone scaffold capable of achieving desirable degrees of bone integration and osteogenesis. Osteogenesis represents a complex physiological process involving multiple cooperating systems. A tight relationship between the immune and skeletal systems has lately been established using the concept of “osteoimmunology,” since various molecules, particularly those regulating immunological and inflammatory processes, are shared. Inflammatory mediators are now being implicated in bone remodeling, according to new scientific data. In this study, a profiler PCR array was employed to evaluate the expression of cytokines and chemokines in human adipose derived-mesenchymal stem cells (hASCs) cultured on porous hydroxylapatite (HA)/Collagen derived Bio-Oss® /Avitene scaffolds, up to day 21. In hASCs grown on the Bio-Oss® /Avitene biomaterial, 12 differentially expressed genes (DEGs) were found to be up-regulated, together with 12 DEG downregulated. Chemokine CCL2, which affects bone metabolism, tested down-regulated. Interestingly, the Bio-Oss® /Avitene induced the down-regulation of pro-inflammatory interleukin IL-6. In conclusion, our investigation carried out on the Bio-Oss® /Avitene scaffold indicates that it could be successfully employed in maxillofacial surgery. Indeed, this composite material has the advantage of being customized on the basis of the individual patients favoring a novel personalized medicine approach

Genetics and Epigenetics of Bone Remodeling and Metabolic Bone Diseases

Bone metabolism consists of a balance between bone formation and bone resorption, which is mediated by osteoblast and osteoclast activity, respectively. In order to ensure bone plasticity, the bone remodeling process needs to function properly. Mesenchymal stem cells differentiate into the osteoblast lineage by activating different signaling pathways, including transforming growth factor β (TGF-β)/bone morphogenic protein (BMP) and the Wingless/Int-1 (Wnt)/β-catenin pathways. Recent data indicate that bone remodeling processes are also epigenetically regulated by DNA methylation, histone post-translational modifications, and non-coding RNA expressions, such as micro-RNAs, long non-coding RNAs, and circular RNAs. Mutations and dysfunctions in pathways regulating the osteoblast differentiation might influence the bone remodeling process, ultimately leading to a large variety of metabolic bone diseases. In this review, we aim to summarize and describe the genetics and epigenetics of the bone remodeling process. Moreover, the current findings behind the genetics of metabolic bone diseases are also reporte

Serum IgG antibodies from pregnant women reacting to mimotopes of simian virus 40 large T antigen, the viral oncoprotein

Simian virus 40 (SV40) large T antigen (LT) coding sequences were revealed in different human samples, whereas SV40 antibodies (Ab) were detected in human sera of cancer patients and healthy individuals, although with a lower prevalence. Previous studies carried out by the neutralization assay gave a SV40 seroprevalence, in the general population, up to 8%, although higher rates, 12%, were detected in kidney transplant children, in a group of HIV-positive patients, and in healthy females. In this study, serum samples from pregnant women, together with those from non-pregnant women, were analyzed to check the prevalence of IgG Ab reacting to SV40 LT antigens. Serum samples were collected from pregnant and non-pregnant women, with the same mean age. Women were in the range of 15-48 years old. Samples were assayed by an indirect ELISA employing specific SV40 LT mimotopes as antigens, whereas functional analysis was performed by neutralization of the viral infectivity in cell cultures. As a control, sera were analyzed for Ab against BK polyomavirus (BKPyV), which is a human polyomavirus homologous to SV40. Statistical analyses employed chi-square with Yates' correction, and Student's t tests. Indirect ELISAs indicated that pregnant women tested SV40 LT-positive with a prevalence of 17% (23/134), whereas non-pregnant women had a prevalence of 20% (36/180) (P > 0.05). Ab against BKPyV were detected with a prevalence of 80% in pregnant women and with a prevalence of 78% in non-pregnant women. These data indicate that SV40 infects at a low prevalence pregnant women. We may speculate that SV40, or a close human polyomavirus still undetected, could be transmitted from mother to fetus

Hydroxylapatite-collagen hybrid scaffold induces human adipose-derived mesenchymal stem cells to osteogenic differentiation in vitro and bone regrowth in patients

Tissue engineering-based bone graft is an emerging viable treatment modality to repair and regenerate tissues damaged as a result of diseases or injuries. The structure and composition of scaffolds should modulate the classical osteogenic pathways in human stem cells. The osteoinductivity properties of the hydroxylapatite-collagen hybrid scaffold named Coll/Pro Osteon 200 were investigated in an in vitro model of human adipose mesenchymal stem cells (hASCs), whereas the clinical evaluation was carried out in maxillofacial patients. Differentially expressed genes (DEGs) induced by the scaffold were analyzed using the Osteogenesis RT2 PCR Array. The osteoinductivity potential of the scaffold was also investigated by studying the alkaline phosphatase (ALP) activity, matrix mineralization, osteocalcin (OCN), and CLEC3B expression protein. Fifty patients who underwent zygomatic augmentation and bimaxillary osteotomy were evaluated clinically, radiologically, and histologically during a 3-year follow-up. Among DEGs, osteogenesis-related genes, including BMP1/2, ALP, BGLAP, SP7, RUNX2, SPP1, and EGFR, which play important roles in osteogenesis, were found to be upregulated. The genes to cartilage condensation SOX9, BMPR1B, and osteoclast cells TNFSF11 were detected upregulated at every time point of the investigation. This scaffold has a high osteoinductivity revealed by the matrix mineralization, ALP activity, OCN, and CLEC3B expression proteins. Clinical evaluation evidences that the biomaterial promotes bone regrowth. Histological results of biopsy specimens from patients showed prominent ossification. Experimental data using the Coll/Pro Osteon 200 indicate that clinical evaluation of bone regrowth in patients, after scaffold implantation, was supported by DEGs implicated in skeletal development as shown in "in vitro" experiments with hASCs

Significant low prevalence of antibodies reacting with simian virus 40 mimotopes in serum samples from patients affected by inflammatory neurologic diseases, including multiple sclerosis

Many investigations were carried out on the association between viruses and multiple sclerosis (MS). Indeed, early studies reported the detections of neurotropic virus footprints in the CNS of patients with MS. In this study, sera from patients affected by MS, other inflammatory (OIND) and non-inflammatory neurologic diseases (NIND) were analyzed for antibodies against the polyomavirus, Simian Virus 40 (SV40). An indirect enzyme-linked immunosorbent assay (ELISA), with two synthetic peptides, which mimic SV40 antigens, was employed to detect specific antibodies in sera from patients affected by MS, OIND, NIND and healthy subjects (HS). Immunologic data indicate that in sera from MS patients antibodies against SV40 mimotopes are detectable with a low prevalence, 6%, whereas in HS of the same mean age, 40 yrs, the prevalence was 22%. The difference is statistically significant (P = 0.001). Significant is also the difference between MS vs. NIND patients (6% vs. 17%; P = 0.0254), whereas no significant difference was detected between MS vs OIND (6% vs 10%; P>0.05). The prevalence of SV40 antibodies in MS patients is 70% lower than that revealed in HS

Hydroxylapatite-collagen hybrid scaffold induces human adipose-derived mesenchymal stem cells to osteogenic differentiation in vitro and bone regrowth in patients

Tissue engineering-based bone graft is an emerging viable treatment modality to repair and regenerate tissues damaged as a result of diseases or injuries. The structure and composition of scaffolds should modulate the classical osteogenic pathways in human stem cells. The osteoinductivity properties of the hydroxylapatite-collagen hybrid scaffold named Coll/Pro Osteon 200 were investigated in an in vitro model of human adipose mesenchymal stem cells (hASCs), whereas the clinical evaluation was carried out in maxillofacial patients. Differentially expressed genes (DEGs) induced by the scaffold were analyzed using the Osteogenesis RT2 PCR Array. The osteoinductivity potential of the scaffold was also investigated by studying the alkaline phosphatase (ALP) activity, matrix mineralization, osteocalcin (OCN), and CLEC3B expression protein. Fifty patients who underwent zygomatic augmentation and bimaxillary osteotomy were evaluated clinically, radiologically, and histologically during a 3-year follow-up. Among DEGs, osteogenesis-related genes, including BMP1/2, ALP, BGLAP, SP7, RUNX2, SPP1, and EGFR, which play important roles in osteogenesis, were found to be upregulated. The genes to cartilage condensation SOX9, BMPR1B, and osteoclast cells TNFSF11 were detected upregulated at every time point of the investigation. This scaffold has a high osteoinductivity revealed by the matrix mineralization, ALP activity, OCN, and CLEC3B expression proteins. Clinical evaluation evidences that the biomaterial promotes bone regrowth. Histological results of biopsy specimens from patients showed prominent ossification. Experimental data using the Coll/Pro Osteon 200 indicate that clinical evaluation of bone regrowth in patients, after scaffold implantation, was supported by DEGs implicated in skeletal development as shown in "in vitro" experiments with hASCs

High Human Papillomavirus DNA loads in Inflammatory Middle Ear Diseases

Background. Previous studies reported human papillomaviruses (HPVs) in middle ear tumors, whereas these viruses have been poorly investigated in chronic inflammatory middle ear diseases. The purpose of this study was to investigate HPVs in non-tumor middle ear diseases, including chronic otitis media (COM). Methods. COM specimens (n=52), including chronic suppurative otitis media (CSOM) (n=38) and cholesteatoma (COMC) (n=14), as well as normal middle ear specimens (NME) (n=56) were analyzed. HPV DNA sequences and DNA loads were analyzed by quantitative PCR. HPV genotyping was performed by direct sequencing of the amplimers. Results. HPV DNA was detected in 23% (12/52) of COM and in 30.4% (17/56) NME (p>0.05). Specifically, HPV DNA sequences were revealed in 26.3% (10/38) of CSOM and in 14.3% (2/14) COMC (p>.05). Interestingly, the HPV DNA load was higher in COMC (mean 7.47 copy/cell) than in CSOM (mean 1.02 copy/cell), and NME (mean 1.18 copy/cell) (P=.03 and P=.017 versus CSOM and NME, respectively). HPV16 and HPV18 were the main genotypes detected in COMC, CSOM and NME. Conclusions. This data indicates that HPV-positive CSOM and COMC are generally associated with higher viral DNA loads as compared to NME. In addition, for the first time, HPVs were detected in normal middle ear mucosa specimens. This result suggests that NME is an additional epithelial tissue that can be HPV infected

Measurement of Branching Fraction and Dalitz Distribution for B0->D(*)+/- K0 pi-/+ Decays

We present measurements of the branching fractions for the three-body decays B0 -> D(*)-/+ K0 pi^+/-$and their resonant submodes$B0 -> D(*)-/+ K*+/- using a sample of approximately 88 million BBbar pairs collected by the BABAR detector at the PEP-II asymmetric energy storage ring. We measure: B(B0->D-/+ K0 pi+/-)=(4.9 +/- 0.7(stat) +/- 0.5 (syst)) 10^{-4} B(B0->D*-/+ K0 pi+/-)=(3.0 +/- 0.7(stat) +/- 0.3 (syst)) 10^{-4} B(B0->D-/+ K*+/-)=(4.6 +/- 0.6(stat) +/- 0.5 (syst)) 10^{-4} B(B0->D*-/+ K*+/-)=(3.2 +/- 0.6(stat) +/- 0.3 (syst)) 10^{-4} From these measurements we determine the fractions of resonant events to be : f(B0-> D-/+ K*+/-) = 0.63 +/- 0.08(stat) +/- 0.04(syst) f(B0-> D*-/+ K*+/-) = 0.72 +/- 0.14(stat) +/- 0.05(syst)Comment: 7 pages, 3 figures submitted to Phys. Rev. Let