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The microstructural impact of diagenetic or post mortem alteration has been assessed in predominately human skeletal tissues. The method of assessment selected was microscopical analysis, mainly using backscattered electron imaging in a scanning electron microscope and, to a lesser extent, confocal reflection microscopy. The microstructural morphologies of post mortem alteration were investigated in archaeological material, both normal and pathological, from terrestrial and marine contexts. Further studies were undertaken on a case-by-case basis on skeletal material which offered some unique pathology, environmental context, spatial relationship, time variable, or mortuary practice. Additionally, the effect of diagenetic change on mitochondrial DNA (mtDNA) recovery and the potential location of DNA within the skeletal tissues were investigated. Two quantitative studies were undertaken to validate and measure the observed mineral density changes. The investigations showed that post mortem alteration or diagenetic change to skeletal material can be extensive, and can occur shortly after death. Diagenesis did not represent a post burial phenomenon as the term diagenesis suggests, but was found to have begun above ground in a range of exposural contexts. The implication of gut bacteria in the promotion of early bacterially-related microstructural change was strong, and it is proposed that body status at the point of, or soon after, death is important. Post mortem alteration to skeletal microstucture can provide environmental information, since terrestrial and marine contexts exhibited distinct morphologies. It may also provide localized environmental information within a stratigraphic matrix. Characterizing the post mortem microstructural and density changes to bone has helped to elucidate the preservational status of mtDNA in terms of its relative retrieval in archaeological specimens, and the potential location of mtDNA in bone. It is proposed that the shift in mineral density that was found in bacterially-remodelled specimens from terrestrial contexts, relative to the excellent preservation of marine specimens, may help to explain why marine vertebrates far outnumber terrestrial ones in the fossil record, since bacterially driven microstructural change is here considered to be a destructive form of fossilisation.

Orientador: Laurival Antonio De Luca / Resumo: O objetivo deste trabalho foi estudar através do exame Imunoistoquimico, a correlação entre alguns fatores Anatomopatológicos (tipo histológico e grau nuclear) e alguns fatores biológicos (receptores hormonais de estrógeno e de progesterona, proteína c-erbB-2 e proteína p53, no carcinoma de Paget da mama, doença rara e especial. No período entre Janeiro de 1980 a Dezembro de 1998 foram tratados no Instituto Brasileiro de Controle do Câncer, 6.303 casos de câncer de mama, deste total 98 casos foram diagnosticados como carcinoma de Paget, cuja incidência foi de 1,55%. Estudamos retrospectivamente 60 casos, sendo que 38 deles foram excluídos da análise, devido a limitação e escassez da amostra no material disponível, a idade das pacientes variou entre 26 e 84 anos, com média de 55,4 anos, as informações clínicas e terapêuticas foram obtidas dos prontuários das pacientes considerando, idade na época do diagnóstico, tamanho do tumor quando palpável, estadiamento clínico, e o tipo de cirurgia realizada. As amostras de tecido mamário foram recuperadas dos arquivos do Departamento de Anatomia Patológica do I.B.C.C. / MATTOSINHO. Após a revisão histológica, realizada por dois patologistas os casos foram classificados em quatro grupos: Grupo A- Doença de Paget (forma pura) n = 09 Grupo B- Doença de Paget + neoplasia ductal "in situ", n = 12 Grupo C- Doença de Paget, neoplasia ductal invasora, n = 30 Grupo D- Doença de Paget + neoplasia ductal "in situ"+ neoplasia ductal invasora, n = 09. Entre as variáveis anatomopatológicas, o grupo C prevaleceu com 30 casos (50%). O grau nuclear (GN-II) predominou com 45 casos (75%). Em relação as variáveis biológicas o receptor de estrógeno negativo predominou com 41 casos (68,3%), seguido pelo receptor de progesterona negativo com 40 casos (66,7%), podemos dizer que... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this paper was to study the immunohistochemical efects of Paget's disease, a rare and special carcinoma of the breast, by correlating anatomopathological (histological and nuclear grade) and biological (estrogen and progesterone hormonal receptors, c-erbB-2 protein and p53 protein) factors. Between January 1980 and December 1998, 6303 cases of breast cancer were treated at the Brazilian Institute of Cancer Control; 98 of these were diagnosed with Paget's carcinoma, an incidence of 1.55%. We retrospectively studied 60 of these cases; 38 were excluded due to lack of available sample material. Patient age varied between 26 and 84 years (mean 55.4), clinical and therapeutic information were obtained from patient's medical records considering age at time of diagnosis, tumor size when palpable, clinical stage, and type of surgery performed. Samples of breast tissue were retrieved the Anatomical Pathology Department, I.B.C.C. / MATTOSINHO. After histological review, by two different pathologists, they were classified into four groups: Group A- Paget's disease (pure form) n = 09 Group B- Paget's disease + "in situ" duct neoplasia n = 12 Group C- Paget's disease, invasive duct neoplasia n = 30 Group D- Paget's disease + "in situ" duct neoplasia + invasive duct neoplasia n = 09.Invasive duct neoplasia was the most prevalent anatomopathological variable (Group C, n = 30, 50%). Nuclear grade (GNII) was found in 45 cases (75%). In relation to the biological variables, the negative estrogen receptor was predominant with 41 cases (68.3%), followed by the negative progesterone receptor with 40 cases (66.7%); this correlation had good concordance by the Kappa test. The c-erbB-2 protein was positive in 53 cases (88.3%) and p53 was negative in 47 cases (78.3%). From these results, we concluded that there was no statistical... (Complete abstract click electronic address below) / Doutor

Introduction: Limited information is available about the clinical features of Paget's disease of bone among unselected patients in the community. We examined morbidity and mortality associated with this condition in a large inception cohort of Olmsted County, MN, residents with a new diagnosis of Paget's disease from 1950 through 1994. Materials and Methods: Survival was estimated using the Kaplan-Meier method. Cox proportional hazards models were used to assess the impact of various covariates on death. Results: Paget's disease of bone was diagnosed in 236 Olmsted County residents (mean age at diagnosis, 69.6 yr; 55% men). The majority were symptomatic at diagnosis (58%), and the proportion with symptoms did not change from the prescreening era (1950 to June 1974) to the postscreening era (July 1974-1994). Most patients had polyostotic disease (72%), and the pelvis (67%), vertebra (41%), and femur (31%) were the most common sites of involvement. Skeletal complications attributable to Paget's disease included bowing deformities (7.6%), fracture of pagetic bone (9.7%), and osteosarcoma (0.4%). Osteoarthritis was observed in 73% of patients, and 11% had a hip or knee replacement. Nonskeletal complications related to Paget's disease included cranial nerve (0.4%), peripheral nerve (1.7%), and nerve root (3.8%) compression, basilar invagination (2.1%), hypercalcemia (5.2%), and congestive heart failure (3.0%). Hearing loss, noted in 61%, was significantly higher than previously reported. Conclusions: Compared with white Minnesota residents, overall survival was slightly better than expected (p = 0.010). No clinical risk factors were identified that were associated with an increased risk of death.

Résumé : La maladie osseuse de Paget (MP) est un désordre squelettique caractérisé par une augmentation focale et désorganisée du remodelage osseux. Les ostéoclastes (OCs) de MP sont plus larges, actifs et nombreux, en plus d’être résistants à l’apoptose. Même si la cause précise de la MP demeure inconnue, des mutations du gène SQSTM1, codant pour la protéine p62, ont été décrites dans une proportion importante de patients avec MP. Parmi ces mutations, la substitution P392L est la plus fréquente, et la surexpression de p62P392L dans les OCs génère un phénotype pagétique partiel. La protéine p62 est impliquée dans de multiples processus, allant du contrôle de la signalisation NF-κB à l’autophagie. Dans les OCs humains, un complexe multiprotéique composé de p62 et des kinases PKCζ et PDK1 est formé en réponse à une stimulation par Receptor Activator of Nuclear factor Kappa-B Ligand (RANKL), principale cytokine impliquée dans la formation et l'activation des OCs. Nous avons démontré que PKCζ est impliquée dans l’activation de NF-κB induite par RANKL dans les OCs, et dans son activation constitutive en présence de p62P392L. Nous avons également observé une augmentation de phosphorylation de Ser536 de p65 par PKCζ, qui est indépendante d’IκB et qui pourrait représenter une voie alternative d'activation de NF-κB en présence de la mutation de p62. Nous avons démontré que les niveaux de phosphorylation des régulateurs de survie ERK et Akt sont augmentés dans les OCs MP, et réduits suite à l'inhibition de PDK1. La phosphorylation des substrats de mTOR, 4EBP1 et la protéine régulatrice Raptor, a été évaluée, et une augmentation des deux a été observée dans les OCs pagétiques, et est régulée par l'inhibition de PDK1. Également, l'augmentation des niveaux de base de LC3II (associée aux structures autophagiques) observée dans les OCs pagétiques a été associée à un défaut de dégradation des autophagosomes, indépendante de la mutation p62P392L. Il existe aussi une réduction de sensibilité à l’induction de l'autophagie dépendante de PDK1. De plus, l’inhibition de PDK1 induit l’apoptose autant dans les OCs contrôles que pagétiques, et mène à une réduction significative de la résorption osseuse. La signalisation PDK1/Akt pourrait donc représenter un point de contrôle important dans l’activation des OCs pagétiques. Ces résultats démontrent l’importance de plusieurs kinases associées à p62 dans la sur-activation des OCs pagétiques, dont la signalisation converge vers une augmentation de leur survie et de leur fonction de résorption, et affecte également le processus autophagique. / Abstract : Paget’s disease of bone (PDB) is a skeletal disorder characterized by focal and disorganized increases in bone turnover. In PDB, osteoclasts are larger, more active, more numerous, and resistant to apoptotic stimuli. While no single root cause has been identified, mutations to the gene encoding the p62 protein, SQSTM1, have been described in a significant population of patients with PDB. Among these mutations, the P392L substitution is the most prevalent, and overexpression of p62P392L in osteoclasts generates at least a partial pagetic phenotype in vitro. Normally this protein mediates a number of cell functions, from control of NF-κB signaling to autophagy. In human osteoclasts, a multiprotein complex containing p62 and protein kinases PKCζ and PDK1 (the principal kinase of Akt), form in response to stimulation by receptor activator of nuclear factor kappa-B ligand (RANKL), the principal osteoclastogenic-signaling cytokine. We found that PKCζ is involved in RANKL-induced activation of NF-κB, and that it contributed to a basal activation of NF-κB observed in p62P392L mutants. This may be regulated in part by a PKCζ dependent increase in p65 phosphorylation at Ser536 which we characterized, independent of IκB. This could represent one alternative pathway by which mutant p62 leads to increased NF-κB activation. We observed increased basal phosphorylation of survival regulators ERK and Akt in PDB that was reduced upon PDK1 inhibition. The activity of 4EBP1 and Raptor, associated with mTOR activity, were also altered in pagetic osteoclasts and regulated by PDK1 inhibition. We then identified autophagic defects common to pagetic osteoclasts; with higher basal levels of LC3II (associated with autophagic structures), regardless of p62 mutation, and reduced sensitivity to autophagy induction in PDB. These results suggest an accumulation of non-degradative autophagosomes. Inhibition of PDK1 not only induced apoptosis in PDB and controls, but significantly reduced resorption in PDB, and with regards to autophagy, PDK1 inhibition was more potent in PDB than in controls. Therefore PDK1/Akt signaling represents an important checkpoint to PDB osteoclast activation. In sum, these results demonstrate the importance of several p62-associated kinases in the over-activation of pagetic osteoclasts, through increased survival and altered signaling. As p62 mutations alone do not account for most cases of PDB, the characterization of these pathways may identify a common factor linking pagetic osteoclasts. Therefore these studies represent a novel approach to osteoclast apoptosis, activation, and autophagy associated with PDB.

Paget's disease of the bone is a skeletal disorder of unknown cause. This disease is characterised by excessive and abnormal bone remodelling brought about by increased bone resorption followed by disorganised bone formation. Increased bone turnover results in a disorganised mosaic of woven and lamellar bone at affected skeletal sites. This produces bone that is expanded in size, less compact, more vascular, and more susceptible to deformity or fracture than normal bone. Symptoms of Paget's disease may include bone pain, bone deformity, excessive warmth over bone from hypervascularity, secondary arthritis, and a variety of neurologic complications caused in most instances by compression of the neural tissues adjacent to pagetic bone. Genetic factors play a role in the pathogenesis of Paget's disease but the molecular basis remains largely unknown. The identification of the molecular basis of Paget's disease is fundamental for an understanding of the cause of the disease, for identifying subjects at risk at a preclinical stage, and for the development of more effective preventive and therapeutic strategies for the management of the condition. With this in mind, the aim of this project is to identify genetic loci, in a large pedigree, that may harbour genes responsible for Paget's disease of bone. A large Australian family with evidence of Paget's disease was recruited for these studies (Chapter 3). This pedigree has characterised over 250 individuals, with 49 informative individuals affected with Paget's disease of bone, 31 of whom are available for genotypic analysis. The pattern of disease in these individuals is polystotic, with sites of involvement including the spine, pelvis, skull and femur. Although the affected individuals have a severe early-onset form of the disease, the clinical features of the pedigree suggest that the affected family members have Paget's disease and not familial expansile osteolysis (a disease with some similarities to Paget's disease), as our patients have extensive skull and axial skeletal involvement. The disease is inherited as an autosomal dominant trait in the pedigree with high penetrance by the sixth decade. Due to the large size of this family and multiple affected members, this pedigree is a unique resource for the detection of the susceptibility gene in Paget's disease. The first susceptibility loci for Paget's disease of bone have been mapped by other investigators to chromosome 6p21 (PDB1) and 18q21.1-q22 (PDB2) in different pedigrees. Linkage analysis of the Australian pedigree in these studies was performed with markers at PDB1: these data showed significant exclusion of linkage, with LOD scores < - 2 in this region (Chapter 4). Linkage analysis of microsatellite markers from the PDB2 region excluded linkage with this region also, with a 30 cM exclusion region (LOD score < -2.0) centred on D18S42 (Chapter 4). This locus on chromosome 18q21.1-q22 contains a serine protease (serpin) cluster with similarities to chromosome 6p21. Linkage analysis of this region also failed to provide evidence of linkage to this locus (Chapter 4). These data are consistent with genetic heterogeneity of Paget's disease of bone. A gene essential for osteoclast formation encoding receptor activator of nuclear factor-kB (RANK), TNFRSF11A, has been previously mapped to the PDB2 region. Mutations in the TNFRSF11A gene have been identified segregating in pedigrees with Familial Expansile Osteolysis and early onset familial Paget's disease, however, linkage studies and mutation screening have excluded the involvement of RANK in the majority of Paget's disease patients. For the Australian pedigree, mutation screening at the TNFRSF11A locus revealed no mutations segregating with affected individuals with Paget's disease (Chapter 4). Based on these findings, our hypothesis is that a novel susceptibility gene relevant to the pathogenesis of Paget's disease of bone lies elsewhere in the genome in the affected members of this pedigree; this gene should be identifiable using a microsatellite genome-wide scan followed by positional cloning. A genome-wide scan of the Australian pedigree was carried out, followed by fine mapping and multipoint analysis in regions of interest (Chapter 5). The peak 2-point LOD scores from the genome-wide scan were LOD = 2.75 at D7S507 and LOD = 1.76 at D18S70. Two additional regions were also considered for fine mapping: chromosome 19p11-q13.1 with a LOD of 1.58 and chromosome 5q35-qter with a LOD of 1.57. Multipoint and haplotype analysis of markers flanking D7S507 did not support linkage to this region (Chapter 5). Similarly, fine mapping of chromosome 19p11-q13.1 failed to support linkage to this region (Chapter 5). Linkage analysis with additional markers in the region on chromosome 5q35-qter revealed a peak multipoint LOD score of 6.77 (Chapter 5). A distinct haplotype was shown to segregate with all members of the family, except the offspring of III-5 and III-6. Haplotype analysis of markers flanking D18S70 demonstrated a haplotype segregating with Paget's disease in a large sub-pedigree (descendants of III-3 and III-4) (Chapter 5). This sub-pedigree had a significantly lower age at diagnosis than the rest of the pedigree (51.2 + 8.5 vs. 64.2 + 9.7 years, p = 0.0012). Linkage analysis of this sub-pedigree demonstrated a peak two-point LOD score of 4.23 at marker D18S1390 (q = 0.00), and a peak multipoint LOD score of 4.71, at marker D18S70. An implication of these data is that 18q23 harbours a novel modifier gene for reducing the age of onset of Paget's disease of bone. A number of candidate Paget's genes have previously been identified on chromosome 18q23, including the nuclear factor of activated T cells (NFATc1), membrane-associated guanylated kinase (MAGUK) and a zinc finger protein. Candidate gene sequencing of these genes in these studies has failed to identify mutations segregating with affected family members in the sub-pedigree linked to chromosome 18q23 (Chapter 6). More recently, a mutation in the gene encoding the ubiquitin-binding protein sequestosome 1 (SQSTM/p62) has been shown to segregate with affected members of Paget's disease families of French-Canadian origin. In this study, a single base pair deletion (1215delC) was identified as segregating with the majority of affected members in the pedigree (Chapter 6). This deletion introduces a stop codon at amino acid position 392 which potentially results in early termination of the protein and loss of the ubiquitin binding domain. The three affected members of the family that do not share the affected haplotype do not carry a mutation in the coding region of SQSTM/p62. Screening of affected members from 10 further Paget's disease families identified the previously reported P392L mutation in 2 (20%) families. No SQSTM1/p62 coding mutations have been found in the remaining 8 families or in 113 aged matched controls. In conclusion, this project has identified genetic loci and mutations that segregate with individuals affected with Paget's disease. Further investigation of the functional significance of the genetic changes at these loci is expected to lead to a better understanding of the molecular basis of this disease.

Paget’s disease is characterised by focal regions of accelerated bone turnover. The aetiology is unknown, but genetic and environmental factors have been implicated. Pagetic lesions contain increased numbers of osteoclasts with abnormal morphology, so an osteoclast defect has been considered central to the pathogenesis. However, given osteoblasts regulate osteoclast differentiation and activity; osteoblast abnormalities may be important in the disease. This study aimed to identify features of pagetic osteoblasts that could clarify their role in Paget’s disease. Gene expression in osteoblasts and bone marrow cultured from pagetic lesions of 23 patients was compared to cells from unaffected tissue using both microarrays and real time RT-PCR. The results indicated global changes in gene expression in pagetic osteoblasts. A number of genes that can stimulate osteoclastogenesis, including interleukins 6 and 1β, and monocyte chemotactic factor 1 were up-regulated, but the RANKL/OPG ratio tended to be decreased. Genes involved in osteoblast differentiation were down-regulated, including the transcription factors RUNX2, DLX5 and SATB2, the osteogenic factor BMP2, and the matrix proteins osteocalcin and bone sialoprotein. Markers of less mature osteoblastic cells, alkaline phosphatase and matrix gla protein were up-regulated. The intermediate filament, keratin 18, was very significantly up-regulated in pagetic cells. Over-expression of this protein in osteoblasts using an adenoviral vector produced some changes in gene expression, but did not produce an overtly pagetic phenotype. Over-expression of SQSTM1 mutants found in some patients with Paget’s disease also produced only minor changes in osteoblast phenotype. The RNA from the primary cell cultures was also used to investigate the presence of measles virus and somatic mutations in SQSTM1 in the disease, but neither were identified in any of the patients. These results suggest that there are important changes in pagetic osteoblasts that are maintained when the cells are removed from the affected bone microenvironment. These include enhanced production of factors to stimulate osteoclastogenesis, while osteoblast differentiation and activity may be impaired. We were unable to identify genetic or environmental factors that could trigger these changes. The pagetic osteoblast is distinct from control cells, and is likely to contribute to the development of Paget’s disease.

Paget’s disease is characterised by focal regions of accelerated bone turnover. The aetiology is unknown, but genetic and environmental factors have been implicated. Pagetic lesions contain increased numbers of osteoclasts with abnormal morphology, so an osteoclast defect has been considered central to the pathogenesis. However, given osteoblasts regulate osteoclast differentiation and activity; osteoblast abnormalities may be important in the disease. This study aimed to identify features of pagetic osteoblasts that could clarify their role in Paget’s disease. Gene expression in osteoblasts and bone marrow cultured from pagetic lesions of 23 patients was compared to cells from unaffected tissue using both microarrays and real time RT-PCR. The results indicated global changes in gene expression in pagetic osteoblasts. A number of genes that can stimulate osteoclastogenesis, including interleukins 6 and 1β, and monocyte chemotactic factor 1 were up-regulated, but the RANKL/OPG ratio tended to be decreased. Genes involved in osteoblast differentiation were down-regulated, including the transcription factors RUNX2, DLX5 and SATB2, the osteogenic factor BMP2, and the matrix proteins osteocalcin and bone sialoprotein. Markers of less mature osteoblastic cells, alkaline phosphatase and matrix gla protein were up-regulated. The intermediate filament, keratin 18, was very significantly up-regulated in pagetic cells. Over-expression of this protein in osteoblasts using an adenoviral vector produced some changes in gene expression, but did not produce an overtly pagetic phenotype. Over-expression of SQSTM1 mutants found in some patients with Paget’s disease also produced only minor changes in osteoblast phenotype. The RNA from the primary cell cultures was also used to investigate the presence of measles virus and somatic mutations in SQSTM1 in the disease, but neither were identified in any of the patients. These results suggest that there are important changes in pagetic osteoblasts that are maintained when the cells are removed from the affected bone microenvironment. These include enhanced production of factors to stimulate osteoclastogenesis, while osteoblast differentiation and activity may be impaired. We were unable to identify genetic or environmental factors that could trigger these changes. The pagetic osteoblast is distinct from control cells, and is likely to contribute to the development of Paget’s disease.