A recurrent mutation in the BMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder of skeletal malformations and progressive extraskeletal ossification. We mapped FOP to chromosome 2q23-24 by linkage analysis and identified an identical heterozygous mutation (617G→A; R206H) in the glycine-serine (GS) activation domain of ACVR1, a BMP type I receptor, in all affected individuals examined. Protein modeling predicts destabilization of the GS domain, consistent with constitutive activation of ACVR1 as the underlying cause of the ectopic chondrogenesis, osteogenesis and joint fusions seen in FOP.

Genetic disorders of the LRP5-Wnt signalling pathway affecting the skeleton

Osteoporosis is a common, increasingly prevalent and potentially debilitating condition of men and women. Genetic factors are major determinants of bone mass and the risk of fracture, but few genes have been definitively demonstrated to be involved. The identification of these factors will provide novel insights into the processes of bone formation and loss and thus the pathogenesis of osteoporosis, enabling the rational development of novel therapies. In this article, we present the extensive genetic and functional data indicating that the LRP5 gene and the Wnt signalling pathway are key players in bone formation and the risk of osteoporosis, and that LRP5 signalling is essential for normal morphology, developmental processes and bone health.

Osteoclast formation from circulating precursors in osteoporosis

Objective: An imbalance between bone formation and bone resorption is thought to underlie the pathogenesis of reduced bone mass in osteoporosis. Bone resorption is carried out by osteoclasts, which are formed from marrow-derived cells that circulate in the monocyte fraction. Ihe aim of this study was to determine the role of osteoclast formation in the pathogenesis of bone loss in osteoporosis. Methods: The proportion of circulating osteoclast precursors and their relative sensitivity to the osteoclastogenic effects of M-CSF, 1,25(OH)2D3 and RANKL were assessed in primary osteoporosis patients and normal controls. Results: Although there was no difference in the number of circulating osteoclast precursors in osteoporosis patients and normal controls, osteoclasts formed from osteoporosis patients exhibited substantially increased resorptive activity relative to normal controls. Although no increased sensitivity to the osteoclastogenic effects of 1,25(OH)2D3 or M-CSF was noted, increased bone resorption was found in osteoporosis peripheral blood mononuclear cell (PBMC) cultures to which these factors were added. Conclusion: Our findings suggest that osteoclast functional activity rather than formation is increased in primary involutional osteoporosis and that dexamethasone acts to increase osteoclast formation.

Genetic studies of disorders of calcium crystal deposition

In summary, although many factors are likely to be involved in regulating calcification and ossification processes, studies of the causation of articular chondrocalcinosis and disorders of spinal ossification, such as DISH and OPLL, implicate control over inorganic pyrophosphate levels as being one of the most important factors in their aetiopathogenesis. The findings of these studies may prove relevant to other rheumatic diseases in which ectopic ossification occurs, such as AS.

Groucho homologue Grg5 interacts with the transcription factor Runx2-Cbfa1 and modulates its activity during postnatal growth in mice

Runx2-Cbfal, a Runt transcription factor, plays important roles during skeletal development. It is required for differentiation and function of osteoblasts. In its absence, chondrocyte hypertrophy is severely impaired and there is no vascularization of cartilage templates during skeletal development. These tissue-specific functions of Runx2 are likely to be dependent on its interaction with other proteins. We have therefore searched for proteins that may modulate the activity of Runx2. The yeast two-hybrid system was used to identify a groucho homologue, Grg5, as a Runx2-interacting protein. Grg5 enhances Runx2 activity in a cell culture-based assay and by analyses of postnatal growth in mice we demonstrate that Grg5 and Runx2 interact genetically. We also show that Runx2 haploinsufficiency in the absence of Grg5 results in a more severe delay in ossification of cranial sutures and fontanels than occurs with Runx2 haploinsufficiency on a wild-type background. Finally, we find shortening of the proliferative and hypertrophic zones, and expansion of the resting zone in the growth plates of Runx2(+/-)Grg5(-/-) mice that are associated with reduced Ihh expression and Indian hedgehog (Ihh) signaling. We therefore conclude that Grg5 enhances Runx2 activity in vivo.

Vascular growth factors and progenitor cells in cardiac allograft arteriosclerosis

Heart transplantation is the only therapeutic modality for many end-stage heart diseases but poor long-term survival remains a challenging problem. This is mainly due to the development of cardiac allograft arteriosclerosis (TxCAD) that is an accelerated form of coronary artery disease. Both traditional cardiovascular and transplantation-related risk factors for TxCAD have been identified but options for therapy are limited. TxCAD involves dysfunction of cardiac allograft vascular cells. Activated endothelial cells (EC) regulate allograft inflammation and secrete smooth muscle cell (SMC) growth factors. In turn, SMC and their progenitors invade the intima of the injured vessels and occlude the affected coronary arteries. Different vascular growth factors have to be delicately regulated in normal vascular development. In the present study, experimental heterotopic transplantation models were used to study the role of angiogenic and pro-inflammatory vascular endothelial growth factor (VEGF), EC growth factor angiopoietin (Ang), and SMC mitogen platelet-derived growth factor (PDGF) in the development of TxCAD. Pharmacological and gene transfer approaches were used to target these growth factors and to assess their therapeutic potential. This study shows that alloimmune response in heart transplants upregulates VEGF expression, and induces allograft angiogenesis that involves donor-derived primitive EC. Intracoronary adenoviral VEGF gene transfer increased macrophage infiltration, intimal angiogenesis and TxCAD. VEGF inhibition with PTK787 decreased allograft inflammation and TxCAD, and simultaneous PDGF inhibition with imatinib further decreased TxCAD. Specific inhibition of two VEGF-receptors (VEGFR) decreased allograft inflammation and TxCAD, and VEGFR-2 inhibition normalized the density of primitive and mature capillaries in the allografts. Adenovirus-mediated transient Ang1 expression in the allograft had anti-inflammatory and anti-arteriosclerotic effects. Adeno-associated virus (AAV)-mediated prolonged Ang1 or Ang2 expression had similar anti-inflammatory effects. However, AAV-Ang1 activated allograft SMC whereas AAV-Ang2 had no effects on SMC activation and decreased the development of TxCAD. These studies indicate an interplay of inflammation, angiogenesis and arteriosclerosis in cardiac allografts, and show that vascular growth factors are important regulators in the process. Also, VEGF inhibition, PDGF inhibition and angiopoietin therapy with clinically-relevant pharmacological agents or novel gene therapy approaches may counteract vascular dysfunction in cardiac allografts, and have beneficial effects on the survival of heart transplant patients in the future.

The genetics of osteoporosis

Introduction: Osteoporosis is the commonest metabolic bone disease worldwide. The clinical hallmark of osteoporosis is low trauma fracture, with the most devastating being hip fracture, resulting in significant effects on both morbidity and mortality. Sources of data: Data for this review have been gathered from the published literature and from a range of web resources. Areas of agreement: Genome-wide association studies in the field of osteoporosis have led to the identification of a number of loci associated with both bone mineral density and fracture risk and further increased our understanding of disease. Areas of controversy: The early strategies for mapping osteoporosis disease genes reported only isolated associations, with replication in independent cohorts proving difficult. Neither candidate gene or linkage studies showed association at genome-wide level of significance. Growing points: The advent of massive parallel sequencing technologies has proved extremely successful in mapping monogenic diseases and thus leading to the utilization of this new technology in complex disease genetics. Areas timely for developing research: The identification of novel genes and pathways will potentially lead to the identification of novel therapeutic options for patients with osteoporosis. © 2014 The Author.

Applications of coralline hydroxyapatite with bioabsorbable containment and reinforcement as bone graft substitute : An experimental study

The aim of the present experimental study was to find out if the applications of coralline hydroxyapatite (HA) can be improved by using bioabsorbable containment or binding substance with particulate HA in mandibular contour augmentation and by using bioabsorbable fibre-reinforced HA blocks in filling bone defects and in anterior lumbar interbody fusion. The use of a separate curved polyglycolide (PGA) containment alone or together with a fast resorbing polyglycolide/polylactide (PGA/PLA) binding substance were compared to the conventional non-contained method in ridge augmentation in sheep. The contained methods decreased HA migration, but the augmentations did not differ significantly. The use of the containment caused a risk for wound dehiscence and infection. Histologically there was a rapid connective tissue ingrowth into the HA graft and it was more abundant with the PGA containment compared to the non-contained augmentation and even additionally rich when the HA particles were bound with PGA/PLA copolymer. However, the bone ingrowth was best in the non-contained augmentation exceeding 10-12 % of the total graft area at 24 weeks. Negligible or no bone ingrowth was seen in the cases where the polymer composite was added to the HA particles and, related to that, foreign-body type cells were seen at the interface between the HA and host bone. The PGA and poly-dl/l-lactide (PDLLA) fibre-reinforced coralline HA blocks were studied in the metaphyseal and in the diaphyseal defects in rabbits. A rapid bone ingrowth was seen inside the both types of implants. Both PGA and PDLLA fibres induced an inflammatory fibrous reaction around themselves but it did not hinder the bone ingrowth. The bone ingrowth pattern was directed according to the loading conditions so that the load-carrying cortical ends of the implants as well as the implants sited in the diaphyseal defects were the most ossified. The fibre-reinforced coralline HA implants were further studied as stand-alone grafts in the lumbar anterior interbody implantation in pigs. The strength of the HA implants proved not to be adequate, the implants fractured in six weeks and the disc space was gradually lost similarly to that of the discectomized spaces. Histologically, small quantities of bone ingrowth was seen in some of the PGA and PDLLA reinforced coralline implants while no bone formation was identified in any of the PDLLA reinforced synthetic porous HA implants. While fragmented, the inner structure of the implants was lost, the bone ingrowth was minimal, and the disc was replaced by the fibrous connective tissue. When evaluated radiologically the grade of ossification was assessed as better than histologically, and, when related to the histologic findings, CT was more dependable than the plain films to show ossification of the implanted disc space. Local kyphosis was a frequent finding along with anterior bone bridging and ligament ossification as a consequence of instability of the implanted segment.

Heterotopias menores: delirando a vida como obra de arte

Esta tese se propõe a explorar as relações entre arte, loucura e polis a partir da aposta de que a vida possa ser permanentemente criada como obra de arte aberta às variações e diferenciações inerentes ao viver. Tal procedimento estético demanda a constituição de espaços-tempo heterotópicos, capazes de simultaneamente engendrar e abrigar contraposicionamentos às políticas de sujeição e controle contemporâneos. Para tanto, problematizamos as instituições arte, loucura e polis, extraindo, dessas formas estratificadas, virtualidades que possibilitem a emergência de modos menores, fazendo-as variar. Partimos da polis, dimensão imaterial e coletiva das cidades, ao mesmo tempo máquina abstrata de engendramento das formas que a habitam, a deslocam, a produzem e forma que a vida assume nesses territórios, para visibilizar como se engendram loucuras e artes de viver. Partimos de fragmentos históricos, literários e biográficos para pensar outros modos de habitar e tecer trajetórias na cidade, instando a tessitura de relações de criação de si e do mundo, por meio da invenção de espaços outros entre a loucura e a arte. Da loucura pensada por Foucault como positividade domesticada pelo saber médico, instauramos séries da experiência trágica ao fora e, deste, ao fluxo esquizo, proposto por Deleuze e Guattari para finalmente chegar a uma loucura menor, loucura que é antes dissolução das formas identitárias e estabilizadas e que materializa o delírio como dispositivo estético de criação de mundos. Finalmente, chegamos à arte, deslocando-a da dimensão de criação de objetos estéticos, para pensá-la como dimensão imanente ao próprio viver. Neste ponto nos valemos de Nietzsche, Deleuze e Foucault para pensar a vida como experimentação que resiste às armadilhas que a aprisionam em modelos préestabelecidos, vida que se cria a cada instante como obra de arte. Ativando, portanto, uma arte menor, arte de viver, damos corpo à proposição foucaultiana de uma estética da existência, procedimento por meio do qual se cria a si mesmo ao criar-se outro nas relações com o mundo. Procedimento, portanto, estético, ético e político. Instabilizadas e problematizadas as formas iniciais polis, loucura e arte , tomamos os casos Bispo do Rosário e Moacir para investigar, em suas trajetórias, como é possível do entrelaçamento entre elas, se constituírem estilizações da existência, de forma a fazer derivar o real e ficcionar modos de viver. Por fim, recorremos à poesia, delírio das palavras para ensaiar possibilidades de criação de espaços-tempo singulares, heterotopias menores, espécies de utopias efetivamente realizadas, como afirma Foucault, que nos permitam fazer da vida obra de arte.

Avaliação da idade e maturação óssea em crianças cardiopatas

O estudo do crescimento e desenvolvimento é essencial para a Ortodontia, pois cada criança possui um padrão único. Na presença de doenças sistêmicas como as cardiopatias, um exame mais detalhado deve ser feito, uma vez que estas podem alterar o crescimento e desenvolvimento. Um dos métodos mais utilizados nesta avaliação é a análise da calcificação dos ossos da mão e punho. Porém, as modificações no tamanho e forma das vértebras cervicais vêm sendo muito utilizadas nas últimas décadas pelo fato de ser realizada em radiografias cefalométricas laterais, rotineiramente utilizadas no diagnóstico ortodôntico. Inicialmente, os objetivos deste trabalho foram verificar a correlação entre os métodos de obtenção da idade óssea e dos estágios de maturação óssea que utilizam os indicadores presentes na região de mão e punho e os presentes nas vértebras cervicais em um grupo de crianças cardiopatas e não cardiopatas. A partir da correlação positiva e significativa, utilizou-se o método das vértebras cervicais para comparar a idade óssea, a diferença entre idade óssea e cronológica e os estágios de maturação óssea entre crianças cardiopatas e não cardiopatas. A amostra foi formada por 120 crianças com idades entre 4,83 a 14,66 anos, atendidas no Ambulatório de Pediatria do Hospital Universitário Pedro Ernesto. Entre estas, 73 eram cardiopatas, todas portadoras de cardiopatias congênitas cianóticas e 47 não cardiopatas, que faziam apenas acompanhamento de rotina, com idades médias de 9,3 e 8,9 anos respectivamente. A idade e maturação óssea foram verificadas através de radiografias cefalométricas laterais e carpais. A determinação da idade óssea foi realizada pelo método de Mito et al. nas radiografias cefalométricas laterais e pelo método de Greulich e Pyle nas radiografias carpais. E, os estágios de maturação óssea foram obtidos pelo método de Hassel e Farman nas radiografias cefalométricas laterais e pelo método de Singer nas radiografias carpais. A correlação entre os métodos de obtenção da idade óssea e dos estágios de maturação óssea apresentou valores positivos e significativos; tanto para o grupo cardiopata, com r = 0,478 (p<0,001) para idade óssea e r = 0,616 (p<0,001) para os estágios de maturação óssea, quanto para o grupo não cardiopata, com r = 0,366 (p=0,024) para idade óssea e r = 0,613 (p<0,001) para os estágios de maturação óssea. As idades ósseas não apresentaram diferença significativa entre os grupos (p=0,394). As diferenças entre as idades cronológicas e ósseas não apresentaram diferença significativa tanto no grupo cardiopata (p=0,418), quanto para o grupo não cardiopata (p=0,143). Também não foram encontradas diferenças significativas entre os grupos quando avaliada a quantidade de crianças que apresentavam idade óssea atrasada em relação à idade cronológica (p=0,395). O mesmo ocorreu quando avaliados os gêneros masculino (p = 0,060) e feminino (p = 0,313). A distribuição da amostra pelos estágios de maturação óssea não apresentou diferenças significativas entre os grupos (p=0,447). Os resultados do presente trabalho sugerem que a cardiopatia congênita, nesta faixa etária avaliada, não altera o padrão de maturação óssea analisado pelas vértebras cervicais.

Improved suppression of circulating complement does not block acute vascular rejection of pig-to-rhesus monkey cardiac transplants

At present, acute vascular rejection (AVR) remains a primary obstacle inhibiting long-term graft survival in the pig-to-non-human primate transplant model. The present study was undertaken to determine whether repetitive injection of low dose Yunnan-cobra venom factor (Y-CVF), a potent complement inhibitor derived from the venom of Naja kaouthia can completely abrogate hemolytic complement activity and subsequently improve the results in a pig-to-rhesus monkey heterotopic heart transplant model. Nine adult rhesus monkeys received a heterotopic heart transplant from wild-type pigs and the recipients were allocated into two groups: group 1 (n = 4) received repetitive injection of low dose Y-CVF until the end of the study and group 2 (n = 5) did not receive Y-CVF. All recipients were treated with cyclosporine A (CsA), cyclophosphamide (CyP) and steroids. Repetitive Y-CVF treatment led to very dramatic fall in CH50 and serum C3 levels (CH50 < 3 units/C3 remained undetectable throughout the experiment) and successfully prevented hyperacute rejection (HAR), while three of five animals in group 2 underwent HAR. However, the continuous suppression of circulating complement did not prevent AVR and the grafts in group 1 survived from 8 to 13 days. Despite undetectable C3 in circulating blood, C3 deposition was present in these grafts. The venular thrombosis was the predominant histopathologic feature of AVR. We conclude that repetitive injection of low dose Y-CVF can be used to continuously suppress circulating complement in a very potent manner and successfully prevent HAR. However, this therapy did not inhibit complement deposition in the graft and failed to prevent AVR. These data suggest that using alternative pig donors [i.e. human decay accelerating factor (hDAF)-transgenic] in combination with the systemic use of complement inhibitors may be necessary to further control complement activation and improve survival in pig-to-non-human primate xenotransplant model.

TMVA, a novel GPIb-binding protein, significantly prevents platelet microthrombi formation and prolongs discordant cardiac xenograft survival

In xenotransplantation, donor endothelium is the first target of immunological attack. Activation of the endothelial cell by preformed natural antibodies leads to platelet binding via the interaction of the glycoprotein (GP) Ib and von Willebrand factor (vWF). TMVA is a novel GPIb-binding protein purified from the venom of Trimeresurus mucrosquamatus. In this study, the inhibitory effect of TMVA on platelet aggregation in rats and the effect on discordant guinea pig-to-rat cardiac xenograft survival were investigated. Three doses (8, 20 or 40 mug/kg) of TMVA were infused intravenously to 30 rats respectively. Platelet aggregation rate was assayed 0.5, 12, and 24 h after TMVA administration. Wister rats underwent guinea pig cardiac cervical heterotopic transplantation using single dosing of TMVA (20 mug/kg, i.v., 0.5 h before reperfusion). Additionally, levels of TXB2 and 6-keto-PGF(1alpha) within rejected graft tissues were determined by radioimmunoassay. Treatment with TMVA at a dose of 20 or 40 mug/kg resulted in complete inhibition of platelet aggregation 0.5 h after TMVA administration. Rats receiving guinea pig cardiac xenografts after TMVA therapy had significantly prolonged xenograft survival. Histologic and immunopathologic analysis of cardiac xenografts in TMVA treatment group showed no intragraft platelet microthrombi formation and fibrin deposition. Additionally, the ratio of 6-keto-PGF(1alpha) to TXB2 in TMVA treatment group was significantly higher than those in control group. We conclude that the use of this novel GPIb-binding protein was very effective in preventing platelet microthrombi formation and fibrin deposition in a guinea pig-to-rat model and resulted in prolongation of xenograft survival. The increased ratio of PGI(2)/TXA(2) in TMVA treatment group may protect xenografts from the endothelial cell activation and contribute to the prolongation of xenograft survival.

O papel das BMPs na regeneração óssea

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária

Fertility preservation: Successful transplantation of cryopreserved ovarian tissue in a young patient previously treated for Hodgkin's disease

Cryopreservation of ovarian tissue is now offered as an experimental procedure to preserve the fertility of young patients with a high risk for premature ovarian failure resulting from cancer therapy. This is the only available option to preserve the fertility of prepubertal patients treated with gonadotoxic chemotherapy. At present, thousands of patients all over the world have undergone this procedure with the hope of later restoring their fertility. Although the efficiency of the transplantation of cryopreserved ovarian tissue to restore ovarian function has been established, reports of pregnancy are still very scarce. Here, we describe the second published full-term spontaneous pregnancy after an orthotopic and heterotopic transplantation of cryopreserved ovarian tissue in a 31-year-old woman previously treated by conditioning therapy for bone marrow transplantation for Hodgkin's disease. This birth gives compelling evidence for the graft origin of the gamete and confirms the efficacy of ovarian tissue transplantation in restoring human natural fertility after oncological treatment. This case report stresses the importance of proposing the ovarian tissue cryopreservation procedure to all young patients who require potentially sterilizing treatment, with all alternative options to preserve fertility being duly taken into consideration.

Developmental origins of precocial forelimbs in marsupial neonates.

Marsupial mammals are born in an embryonic state, as compared with their eutherian counterparts, yet certain features are accelerated. The most conspicuous of these features are the precocial forelimbs, which the newborns use to climb unaided from the opening of the birth canal to the teat. The developmental mechanisms that produce this acceleration are unknown. Here we show that heterochronic and heterotopic changes early in limb development contribute to forelimb acceleration. Using Tbx5 and Tbx4 as fore- and hindlimb field markers, respectively, we have found that, compared with mouse, both limb fields arise notably early during opossum development. Patterning of the forelimb buds is also accelerated, as Shh expression appears early relative to the outgrowth of the bud itself. In addition, the forelimb fields and forelimb myocyte allocation are increased in size and number, respectively, and migration of the spinal nerves into the forelimb bud has been modified. This shift in the extent of the forelimb field is accompanied by shifts in Hox gene expression along the anterior-posterior axis. Furthermore, we found that both fore- and hindlimb fields arise gradually during gastrulation and extension of the embryonic axis, in contrast to the appearance of the limb fields in their entirety in all other known cases. Our results show a surprising evolutionary flexibility in the early limb development program of amniotes and rule out the induction of the limb fields by mature structures such as the somites or mesonephros.