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1.
Int J Mol Sci ; 24(13)2023 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-37446173

RESUMO

Nitisinone has been approved for treatment of alkaptonuria (AKU). Non-invasive biomarkers of joint tissue remodelling could aid in understanding the molecular changes in AKU pathogenesis and how these can be affected by treatment. Serological and urinary biomarkers of type I collagen and II collagen in AKU were investigated in patients enrolled in the randomized SONIA 2 (NCT01916382) clinical study at baseline and yearly until the end of the study (Year 4). The trajectories of the biomarkers over time were observed. After treatment with nitisinone, the biomarkers of type I collagen remodelling increased at Year 1 (19% and 40% increase in CTX-I and PRO-C1, respectively), which was potentially reflected in the higher degree of mobility seen following treatment. The biomarkers of type II collagen remodelling decreased over time in the nitisinone group: C2M showed a 9.7% decline at Year 1, and levels then remained stable over the following visits; CTX-II showed a 26% decline at Year 3 and 4 in the nitisinone-treated patients. Nitisinone treatment induced changes in biomarkers of bone and cartilage remodelling. These biomarkers can aid patient management and deepen our knowledge of the molecular mechanisms of this rare disease.


Assuntos
Alcaptonúria , Humanos , Alcaptonúria/tratamento farmacológico , Biomarcadores , Cartilagem/patologia , Colágeno Tipo I
2.
Hum Mol Genet ; 28(23): 3928-3939, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31600782

RESUMO

Alkaptonuria is an inherited disease caused by homogentisate 1,2-dioxygenase (HGD) deficiency. Circulating homogentisic acid (HGA) is elevated and deposits in connective tissues as ochronotic pigment. In this study, we aimed to define developmental and adult HGD tissue expression and determine the location and amount of gene activity required to lower circulating HGA and rescue the alkaptonuria phenotype. We generated an alkaptonuria mouse model using a knockout-first design for the disruption of the HGD gene. Hgd tm1a -/- mice showed elevated HGA and ochronosis in adulthood. LacZ staining driven by the endogenous HGD promoter was localised to only liver parenchymal cells and kidney proximal tubules in adulthood, commencing at E12.5 and E15.5 respectively. Following removal of the gene trap cassette to obtain a normal mouse with a floxed 6th HGD exon, a double transgenic was then created with Mx1-Cre which conditionally deleted HGD in liver in a dose dependent manner. 20% of HGD mRNA remaining in liver did not rescue the disease, suggesting that we need more than 20% of liver HGD to correct the disease in gene therapy. Kidney HGD activity which remained intact reduced urinary HGA, most likely by increased absorption, but did not reduce plasma HGA nor did it prevent ochronosis. In addition, downstream metabolites of exogenous 13C6-HGA, were detected in heterozygous plasma, revealing that hepatocytes take up and metabolise HGA. This novel alkaptonuria mouse model demonstrated the importance of targeting liver for therapeutic intervention, supported by our observation that hepatocytes take up and metabolise HGA.


Assuntos
Alcaptonúria/enzimologia , Homogentisato 1,2-Dioxigenase/genética , Ácido Homogentísico/metabolismo , Fígado/enzimologia , Alcaptonúria/genética , Alcaptonúria/metabolismo , Animais , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Homogentisato 1,2-Dioxigenase/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas
3.
Calcif Tissue Int ; 109(3): 291-302, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34417863

RESUMO

Osteoarthritis (OA) is one of the most prevalent conditions in the world, particularly in the developed world with a significant increase in cases and their predicted impact as we move through the twenty-first century and this will be exacerbated by the covid pandemic. The degeneration of cartilage and bone as part of this condition is becoming better understood but there are still significant challenges in painting a complete picture to recognise all aspects of the condition and what treatment(s) are most appropriate in individual causes. OA encompasses many different types and this causes some of the challenges in fully understanding the condition. There have been examples through history where much has been learnt about common disease(s) from the study of rare or extreme phenotypes, particularly where Mendelian disorders are involved. The often early onset of symptoms combined with the rapid and aggressive pathogenesis of these diseases and their predictable outcomes give an often-under-explored resource. It is these "rarer forms of disease" that William Harvey referred to that offer novel insights into more common conditions through their more extreme presentations. In the case of OA, GWAS analyses demonstrate the multiple genes that are implicated in OA in the general population. In some of these rarer forms, single defective genes are responsible. The extreme phenotypes seen in conditions such as Camptodactyly Arthropathy-Coxa Vara-pericarditis Syndrome, Chondrodysplasias and Alkaptonuria all present potential opportunities for greater understanding of disease pathogenesis, novel therapeutic interventions and diagnostic imaging. This review examines some of the rarer presenting forms of OA and linked conditions, some of the novel discoveries made whilst studying them, and findings on imaging and treatment strategies.


Assuntos
COVID-19 , Coxa Vara , Osteoartrite , Sinovite , Humanos , Osteoartrite/genética , SARS-CoV-2
4.
Calcif Tissue Int ; 108(2): 207-218, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33057760

RESUMO

Alkaptonuria (AKU) is characterised by increased circulating homogentisic acid and deposition of ochronotic pigment in collagen-rich connective tissues (ochronosis), stiffening the tissue. This process over many years leads to a painful and severe osteoarthropathy, particularly affecting the cartilage of the spine and large weight bearing joints. Evidence in human AKU tissue suggests that pigment binds to collagen. The exposed collagen hypothesis suggests that collagen is initially protected from ochronosis, and that ageing and mechanical loading causes loss of protective molecules, allowing pigment binding. Schmorl's staining has previously demonstrated knee joint ochronosis in AKU mice. This study documents more comprehensively the anatomical distribution of ochronosis in two AKU mouse models (BALB/c Hgd-/-, Hgd tm1a-/-), using Schmorl's staining. Progression of knee joint pigmentation with age in the two AKU mouse models was comparable. Within the knee, hip, shoulder, elbow and wrist joints, pigmentation was associated with chondrons of calcified cartilage. Pigmented chondrons were identified in calcified endplates of intervertebral discs and the calcified knee joint meniscus, suggesting that calcified tissues are more susceptible to pigmentation. There were significantly more pigmented chondrons in lumbar versus tail intervertebral disc endplates (p = 0.002) and clusters of pigmented chondrons were observed at the insertions of ligaments and tendons. These observations suggest that loading/strain may be associated with increased pigmentation but needs further experimental investigation. The calcified cartilage may be the first joint tissue to acquire matrix damage, most likely to collagen, through normal ageing and physiological loading, as it is the first to become susceptible to pigmentation.


Assuntos
Alcaptonúria , Cartilagem/patologia , Condrócitos/patologia , Ocronose , Alcaptonúria/patologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Ocronose/patologia , Pigmentação
5.
J Inherit Metab Dis ; 44(3): 666-676, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33452825

RESUMO

A large alkaptonuria (AKU) cohort was studied to better characterize the poorly understood spondyloarthropathy of rare disease AKU. Eighty-seven patients attended the National Alkaptonuria Centre (NAC) between 2007 and 2020. Seven only attended once. Fifty-seven attended more than once and received nitisinone 2 mg daily. Twenty-three attended at least twice without receiving nitisinone. Assessments included questionnaire analysis, 18F Positron emission tomography computerised tomography (PETCT), as well as photographs of ochronotic pigment in eyes and ears at baseline when 2 mg nitisinone was commenced and yearly thereafter. Blood and urine samples were collected for chemical measurement. The prevalence of ochronosis, as well as pain, PETCT and combined pain and PETCT scores, was greatly increased at 90.5%, 85.7%, 100%, and 100%, respectively. Joint pain scores were greatest in proximal joints in upper and lower limbs. PETCT joint scores were higher in proximal joints in upper limb but higher in distal joints in the lower limb. Spine pain scores were highest in lumbar, followed by cervical, thoracic, and cervical regions at 77.4%, 59.5%, 46.4%, and 25%, respectively. PETCT spine scores were highest in thoracic followed by lumbar, cervical, and sacroiliac regions at 74.4%, 70.7%, 64.6%, and 47.8% respectively; ochronosis associated closely with spondyloarthropathy scores (R = .65; P < .0001). Nitisinone reversed ochronosis significantly, with a similar pattern of decreased joint and spine disease. Spondyloarthropathy is a highly prevalent feature in this NAC cohort. Ochronosis appears to be associated with spondyloarthropathy. Nitisinone decreases ochronosis and had a similar nonsignificant effect pattern on spondyloarthropathy.


Assuntos
Alcaptonúria/tratamento farmacológico , Cicloexanonas/administração & dosagem , Ácido Homogentísico/metabolismo , Articulações/patologia , Nitrobenzoatos/administração & dosagem , Ocronose/tratamento farmacológico , Coluna Vertebral/patologia , Idoso , Alcaptonúria/metabolismo , Estudos de Coortes , Feminino , Humanos , Articulações/diagnóstico por imagem , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Ocronose/metabolismo , Fenótipo , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Índice de Gravidade de Doença , Coluna Vertebral/diagnóstico por imagem , Reino Unido
6.
J Inherit Metab Dis ; 44(3): 656-665, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33314212

RESUMO

Arthroplasty in the spondyloarthropathy (SPOND) of alkaptonuria (AKU) in incompletely characterised. The aim was to improve the understanding of arthroplasty in AKU through a study of patients attending the National Alkaptonuria Centre (NAC). Eighty-seven patients attended the NAC between 2007 and 2020. Seven only attended once. Fifty-seven attended more than once and received nitisinone 2 mg daily. Twenty-three attended at least twice without receiving nitisinone. Assessments including questionnaire analysis eliciting details of arthroplasty and other surgical treatments for SPOND, 18 FPETCT and CT densitometry at the neck of hip and lumbar spine, as well as photographs of the eyes and ears were acquired from patients attending the National Alkaptonuria Centre (NAC) at baseline when 2 mg nitisinone was commenced, and yearly thereafter. Photographs were scored to derive ochronosis scores. Blood and urine samples were collected for chemical analyses. The prevalence of arthroplasty was 36.8%, similar in males and females, occurring especially in the knees, hips and shoulders. Multiple arthroplasties were found in 29 patients (33.3%) in this cohort. Incident arthroplasty was 6.5% in the nitisinone group and 7.1% in the no-nitisinone group. Incident arthroplasty was 11.3% in the group with baseline arthroplasty and 3.51% in the group without. A strong association of arthroplasty with SPOND (R = 0.5; P << .0001) and ochronosis (R = 0.54; P < .0001) was seen. Nitisinone had no significant effect on incident arthroplasty. Arthroplasty due to ochronosis and SPOND is common in AKU. Nitisinone decreased ochronosis but had no effect on arthroplasty in this cohort.


Assuntos
Alcaptonúria/complicações , Artroplastia/estatística & dados numéricos , Ocronose/complicações , Espondiloartropatias/diagnóstico por imagem , Espondiloartropatias/cirurgia , Idoso , Alcaptonúria/tratamento farmacológico , Estudos de Coortes , Cicloexanonas/administração & dosagem , Feminino , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Nitrobenzoatos/administração & dosagem , Ocronose/tratamento farmacológico , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Reino Unido
7.
BMC Musculoskelet Disord ; 22(1): 96, 2021 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-33478453

RESUMO

BACKGROUND: Hereditary multiple exostoses (HME) is a rare skeletal disorder characterised by a widespread. distribution of osteochondromas originating from the metaphyses of long bones. CASE PRESENTATION: This case study examines a 55-year-old male cadaver bequeathed to the University of Liverpool who suffered from HME, thus providing an exceptionally rare opportunity to examine the anatomical changes associated with this condition. CONCLUSIONS: Findings from imaging and dissection indicated that this was a severe case of HME in terms of the quantity and distribution of the osteochondromas and the number of synostoses present. In addition, the existence of enchondromas and the appearance of gaps within the trabeculae of affected bones make this a remarkable case. This study provides a comprehensive overview of the morbidity of the disease as well as adding to the growing evidence that diseases concerning benign cartilaginous tumours may be part of a spectrum rather than distinct entities.


Assuntos
Neoplasias Ósseas , Exostose Múltipla Hereditária , Osteocondroma , Neoplasias Ósseas/diagnóstico por imagem , Osso e Ossos , Diagnóstico por Imagem , Exostose Múltipla Hereditária/diagnóstico por imagem , Humanos , Masculino , Pessoa de Meia-Idade , Osteocondroma/diagnóstico por imagem
8.
J Inherit Metab Dis ; 43(2): 259-268, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31503358

RESUMO

Alkaptonuria (AKU) is caused by homogentisate 1,2-dioxygenase deficiency that leads to homogentisic acid (HGA) accumulation, ochronosis and severe osteoarthropathy. Recently, nitisinone treatment, which blocks HGA formation, has been effective in AKU patients. However, a consequence of nitisinone is elevated tyrosine that can cause keratopathy. The effect of tyrosine and phenylalanine dietary restriction was investigated in nitisinone-treated AKU mice, and in an observational study of dietary intervention in AKU patients. Nitisinone-treated AKU mice were fed tyrosine/phenylalanine-free and phenylalanine-free diets with phenylalanine supplementation in drinking water. Tyrosine metabolites were measured pre-nitisinone, post-nitisinone, and after dietary restriction. Subsequently an observational study was undertaken in 10 patients attending the National Alkaptonuria Centre (NAC), with tyrosine >700 µmol/L who had been advised to restrict dietary protein intake and where necessary, to use tyrosine/phenylalanine-free amino acid supplements. Elevated tyrosine (813 µmol/L) was significantly reduced in nitisinone-treated AKU mice fed a tyrosine/phenylalanine-free diet in a dose responsive manner. At 3 days of restriction, tyrosine was 389.3, 274.8, and 144.3 µmol/L with decreasing phenylalanine doses. In contrast, tyrosine was not effectively reduced in mice by a phenylalanine-free diet; at 3 days tyrosine was 757.3, 530.2, and 656.2 µmol/L, with no dose response to phenylalanine supplementation. In NAC patients, tyrosine was significantly reduced (P = .002) when restricting dietary protein alone, and when combined with tyrosine/phenylalanine-free amino acid supplementation; 4 out of 10 patients achieved tyrosine <700 µmol/L. Tyrosine/phenylalanine dietary restriction significantly reduced nitisinone-induced tyrosinemia in mice, with phenylalanine restriction alone proving ineffective. Similarly, protein restriction significantly reduced circulating tyrosine in AKU patients.


Assuntos
Alcaptonúria/dietoterapia , Alcaptonúria/tratamento farmacológico , Cicloexanonas/farmacologia , Dieta com Restrição de Proteínas , Nitrobenzoatos/farmacologia , Tirosinemias/dietoterapia , Alcaptonúria/metabolismo , Animais , Feminino , Humanos , Masculino , Camundongos , Fenilalanina/metabolismo , Tirosina/metabolismo , Tirosinemias/metabolismo
9.
J Inherit Metab Dis ; 43(5): 1014-1023, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32083330

RESUMO

For over two decades, nitisinone (NTBC) has been successfully used to manipulate the tyrosine degradation pathway and save the lives of many children with hereditary tyrosinaemia type 1. More recently, NTBC has been used to halt homogentisic acid accumulation in alkaptonuria (AKU) with evidence suggesting its efficacy as a disease modifying agent. NTBC-induced hypertyrosinaemia has been associated with cognitive impairment and potentially sight-threatening keratopathy. In the context of a non-lethal condition (ie, AKU), these serious risks call for an evaluation of the wider impact of NTBC on the tyrosine pathway. We hypothesised that NTBC increases the tyrosine pool size and concentrations in tissues. In AKU mice tyrosine concentrations of tissue homogenates were measured before and after treatment with NTBC. In humans, pulse injection with l-[13 C9 ]tyrosine and l-[d8 ]phenylalanine was used along with compartmental modelling to estimate the size of tyrosine pools before and after treatment with NTBC. We found that NTBC increased tyrosine concentrations in murine tissues by five to nine folds. It also significantly increased the tyrosine pool size in humans (P < .001), suggesting that NTBC increases tyrosine not just in serum but also in tissues (ie, acquired tyrosinosis). This study provides, for the first time, the experimental proof for the magnitude of NTBC-related acquired tyrosinosis which should be overcome to ensure the safe use of NTBC in AKU.


Assuntos
Alcaptonúria/tratamento farmacológico , Alcaptonúria/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/etiologia , Cicloexanonas/farmacologia , Nitrobenzoatos/farmacologia , Adulto , Idoso , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Fenilalanina/metabolismo , Tirosina/metabolismo , Adulto Jovem
10.
J Inherit Metab Dis ; 43(4): 737-747, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-31609457

RESUMO

The clinical effects of alkaptonuria (AKU) are delayed and ageing influences disease progression. Morbidity of AKU is secondary to high circulating homogentisic acid (HGA) and ochronosis. It is not known whether HGA is produced by or processed in the kidney in AKU. Data from AKU patients from four studies were merged to form a single AKU group. A control group of non-AKU subjects was generated by merging data from two non-AKU studies. Data were used to derive renal clearance and fractional excretion (FE) ratios for creatinine, HGA, phenylalanine (PHE) and tyrosine (TYR) using standard calculations, for comparison between the AKU and the control groups. There were 225 AKU patients in the AKU group and 52 in the non-AKU control group. Circulating HGA increased with age (P < 0.001), and was significantly associated with decreased HGA clearance (CLHGA ) (P < 0.001) and FEHGA (P < 0.001). CLHGA and FEHGA were increased beyond the theoretical maximum renal plasma flow, confirming renal production and emphasising the greater contribution of net tubular secretion than glomerular filtration to renal elimination of HGA. The kidneys are crucial to elimination of HGA. Elimination of HGA is impaired with age resulting in worsening disease over time. The kidney is an important site for production of HGA. Tubular secretion of HGA contributes more to elimination of HGA in AKU than glomerular filtration.


Assuntos
Alcaptonúria/metabolismo , Taxa de Filtração Glomerular , Ácido Homogentísico/metabolismo , Rim/metabolismo , Ocronose/etiologia , Adulto , Alcaptonúria/fisiopatologia , Estudos de Casos e Controles , Creatinina/metabolismo , Feminino , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Ocronose/fisiopatologia , Fenilalanina/metabolismo , Fatores Sexuais , Tirosina/metabolismo
11.
Angew Chem Int Ed Engl ; 59(29): 11937-11942, 2020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32219972

RESUMO

Alkaptonuria (AKU) is a rare disease characterized by high levels of homogentisic acid (HGA); patients suffer from tissue ochronosis: dark brown pigmentation, especially of joint cartilage, leading to severe early osteoarthropathy. No molecular mechanism links elevated HGA to ochronosis; the pigment's chemical identity is still not known, nor how it induces joint cartilage degradation. Here we give key insight on HGA-derived pigment composition and collagen disruption in AKU cartilage. Synthetic pigment and pigmented human cartilage tissue both showed hydroquinone-resembling NMR signals. EPR spectroscopy showed that the synthetic pigment contains radicals. Moreover, we observed intrastrand disruption of collagen triple helix in pigmented AKU human cartilage, and in cartilage from patients with osteoarthritis. We propose that collagen degradation can occur via transient glycyl radicals, the formation of which is enhanced in AKU due to the redox environment generated by pigmentation.


Assuntos
Alcaptonúria/metabolismo , Cartilagem Articular/metabolismo , Osteoartrite/metabolismo , Pigmentação , Espectroscopia de Ressonância de Spin Eletrônica , Ácido Homogentísico/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Oxirredução , Pigmentos Biológicos/química
12.
Clin Chem ; 65(4): 530-539, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30782595

RESUMO

BACKGROUND: Identification of unknown chemical entities is a major challenge in metabolomics. To address this challenge, we developed a comprehensive targeted profiling strategy, combining 3 complementary liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) techniques and in-house accurate mass retention time (AMRT) databases established from commercial standards. This strategy was used to evaluate the effect of nitisinone on the urinary metabolome of patients and mice with alkaptonuria (AKU). Because hypertyrosinemia is a known consequence of nitisinone therapy, we investigated the wider metabolic consequences beyond hypertyrosinemia. METHODS: A total of 619 standards (molecular weight, 45-1354 Da) covering a range of primary metabolic pathways were analyzed using 3 liquid chromatography methods-2 reversed phase and 1 normal phase-coupled to QTOF-MS. Separate AMRT databases were generated for the 3 methods, comprising chemical name, formula, theoretical accurate mass, and measured retention time. Databases were used to identify chemical entities acquired from nontargeted analysis of AKU urine: match window theoretical accurate mass ±10 ppm and retention time ±0.3 min. RESULTS: Application of the AMRT databases to data acquired from analysis of urine from 25 patients with AKU (pretreatment and after 3, 12, and 24 months on nitisinone) and 18 HGD -/- mice (pretreatment and after 1 week on nitisinone) revealed 31 previously unreported statistically significant changes in metabolite patterns and abundance, indicating alterations to tyrosine, tryptophan, and purine metabolism after nitisinone administration. CONCLUSIONS: The comprehensive targeted profiling strategy described here has the potential of enabling discovery of novel pathways associated with pathogenesis and management of AKU.


Assuntos
Alcaptonúria/metabolismo , Cicloexanonas/farmacologia , Metaboloma/efeitos dos fármacos , Nitrobenzoatos/farmacologia , Idoso , Alcaptonúria/tratamento farmacológico , Animais , Cromatografia Líquida/métodos , Cromatografia Líquida/estatística & dados numéricos , Bases de Dados de Compostos Químicos , Feminino , Técnicas de Silenciamento de Genes , Homogentisato 1,2-Dioxigenase/genética , Humanos , Masculino , Espectrometria de Massas/métodos , Espectrometria de Massas/estatística & dados numéricos , Metabolômica/métodos , Camundongos , Pessoa de Meia-Idade , Fenótipo
13.
J Inherit Metab Dis ; 42(5): 776-792, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31282009

RESUMO

Ochronosis is the process in alkaptonuria (AKU) that causes all the debilitating morbidity. The process involves selective deposition of homogentisic acid (HGA)-derived pigment in tissues altering the properties of these tissues, leading to their failure. Some tissues like cartilage are more easily affected by ochronosis while others such as the liver and brain are unaffected for reasons that are still not understood. In vitro and mouse models of ochronosis have confirmed the dose relationships between HGA and ochronosis and also their modulation by p-hydroxyphenylpyruvate dioxygenase inhibition. Ochronosis cannot be fully reversed and is a key factor in influencing treatment decisions. Earlier detection of ochronosis preferably by noninvasive means is desirable. A cause-effect relationship between HGA and ochronosis is discussed. The similarity in AKU and familial hypercholesterolaemia is explored and lessons learnt. More research is needed to more fully understand the crucial nature of ochronosis.


Assuntos
Alcaptonúria/patologia , Condrócitos/citologia , Ácido Homogentísico/metabolismo , Ocronose/patologia , Alcaptonúria/metabolismo , Animais , Cartilagem/metabolismo , Cartilagem/patologia , Condrócitos/metabolismo , Humanos , Camundongos , Oxirredução , Pigmentação
14.
Semin Cell Dev Biol ; 52: 53-7, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26891864

RESUMO

"Fundamental diseases" is a term introduced by the charity Findacure to describe rare genetic disorders that are gateways to understanding common conditions and human physiology. The concept that rare diseases have important lessons for biomedical science has been recognised by some of the great figures in the history of medical research, including Harvey, Bateson and Garrod. Here we describe some of the recently discovered lessons from the study of the iconic genetic disease alkaptonuria (AKU), which have shed new light on understanding the pathogenesis of osteoarthritis. In AKU, ochronotic pigment is deposited in cartilage when collagen fibrils become susceptible to attack by homogentisic acid (HGA). When HGA binds to collagen, cartilage matrix becomes stiffened, resulting in the aberrant transmission of loading to underlying subchondral bone. Aberrant loading leads to the formation of pathophysiological structures including trabecular excrescences and high density mineralised protrusions (HDMPs). These structures initially identified in AKU have subsequently been found in more common osteoarthritis and appear to play a role in joint destruction in both diseases.


Assuntos
Alcaptonúria/diagnóstico , Doenças Raras/diagnóstico , Alcaptonúria/genética , Alcaptonúria/fisiopatologia , Animais , Humanos , Camundongos , Ocronose/etiologia , Ocronose/fisiopatologia , Osteoartrite/etiologia , Osteoartrite/fisiopatologia , Doenças Raras/genética , Doenças Raras/fisiopatologia
15.
Rheumatology (Oxford) ; 56(1): 156-164, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28028161

RESUMO

OBJECTIVE: Alkaptonuria (AKU) is a rare autosomal recessive disease resulting from a single enzyme deficiency in tyrosine metabolism. As a result, homogentisic acid cannot be metabolized, causing systemic increases. Over time, homogentisic acid polymerizes and deposits in collagenous tissues, leading to ochronosis. Typically, this occurs in joint cartilages, leading to an early onset, rapidly progressing osteoarthropathy. The aim of this study was to examine tissue turnover in cartilage affected by ochronosis and its role in disease initiation and progression. METHODS: With informed patient consent, hip and knee cartilages were obtained at surgery for arthropathy due to AKU (n = 6; 2 knees/4 hips) and OA (n = 12; 5 knees/7 hips); healthy non-arthritic (non-OA n = 6; 1 knee/5 hips) cartilages were obtained as waste from trauma surgery. We measured cartilage concentrations (normalized to dry weight) of racemized aspartate, GAG, COMP and deamidated COMP (D-COMP). Unpaired AKU, OA and non-OA samples were compared by non-parametric Mann-Whitney U test. RESULTS: Despite more extractable total protein being obtained from AKU cartilage than from OA or non-OA cartilage, there was significantly less extractable GAG, COMP and D-COMP in AKU samples compared with OA and non-OA comparators. Racemized Asx (aspartate and asparagine) was significantly enriched in AKU cartilage compared with in OA cartilage. CONCLUSIONS: These novel data represent the first examination of cartilage matrix components in a sample of patients with AKU, representing almost 10% of the known UK alkaptonuric population. Compared with OA and non-OA, AKU cartilage demonstrates a very low turnover state and has low levels of extractable matrix proteins.


Assuntos
Envelhecimento/metabolismo , Alcaptonúria/metabolismo , Cartilagem Articular/metabolismo , Artropatias/metabolismo , Ocronose/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Ácido Aspártico/metabolismo , Biomarcadores/metabolismo , Proteína de Matriz Oligomérica de Cartilagem/metabolismo , Estudos de Casos e Controles , Feminino , Glicosaminoglicanos/metabolismo , Articulação do Quadril , Humanos , Articulação do Joelho , Masculino , Pessoa de Meia-Idade , Osteoartrite do Quadril/metabolismo , Osteoartrite do Joelho/metabolismo , Adulto Jovem
16.
Calcif Tissue Int ; 100(1): 95-106, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27796463

RESUMO

Accelerating the integration of a joint replacement or the healing of a bone fracture, particularly a complicated non-union fracture, would improve patient welfare and decrease healthcare costs. Currently, an autologous bone graft is the gold standard method for the treatment of complicated non-union fractures, but it is not always possible to harvest such a graft. A proactive highly inductive so-called smart material approach is pertinent in these cases. In this study, the surface chemistry of a previously approved material with desirable bulk material properties was modified to investigate its potential as an economical and effective alternative. The objective was to create stable synthetic chemical coatings that could guide cells along the osteogenic lineage required to generate mineralised tissue that would induce and accelerate bone healing. Primary human osteoblast-like cells were cultured in vitro for 7, 14 and 28 days on amine-terminated (chain length in the range 3-11) silane-modified glass surfaces with controlled nanotopography, to determine how surface chemistry and nanotopography change osteoblast function. The materials were characterised using atomic force microscopy (AFM), scanning electron microscopy (SEM), water contact angle (WCA) and a novel ninhydrin assay. The cells were analysed using qRT-PCR, von Kossa tinctural staining for mineralisation, and visualised using both transmitted white light and electron microscopy. Bone-like nodules, quantified using microscopy, only formed on the short-chain (chain length 3 and 4) amines after 7 days, as did the up-regulation of sclerostin, suggestive of a more mature osteoblast phenotype. In this paper, we report more rapid nodule formation than has previously been observed, without the addition of exogenous factors in the culture medium. This suggests that the coating would improve the integration of implants with bone or be the basis of a smart biomaterial that would accelerate the bone regeneration process.


Assuntos
Diferenciação Celular/fisiologia , Osteoblastos/citologia , Osteócitos/citologia , Regeneração Óssea/fisiologia , Osso e Ossos/citologia , Calcificação Fisiológica/fisiologia , Técnicas de Cultura de Células/métodos , Células Cultivadas , Humanos , Microscopia de Força Atômica/métodos , Osteogênese/fisiologia , Propriedades de Superfície
17.
Ann Rheum Dis ; 75(2): 362-7, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25475116

RESUMO

BACKGROUND: Alkaptonuria (AKU) is a serious genetic disease characterised by premature spondyloarthropathy. Homogentisate-lowering therapy is being investigated for AKU. Nitisinone decreases homogentisic acid (HGA) in AKU but the dose-response relationship has not been previously studied. METHODS: Suitability Of Nitisinone In Alkaptonuria 1 (SONIA 1) was an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study. The primary objective was to investigate the effect of different doses of nitisinone once daily on 24-h urinary HGA excretion (u-HGA24) in patients with AKU after 4 weeks of treatment. Forty patients were randomised into five groups of eight patients each, with groups receiving no treatment or 1 mg, 2 mg, 4 mg and 8 mg of nitisinone. FINDINGS: A clear dose-response relationship was observed between nitisinone and the urinary excretion of HGA. At 4 weeks, the adjusted geometric mean u-HGA24 was 31.53 mmol, 3.26 mmol, 1.44 mmol, 0.57 mmol and 0.15 mmol for the no treatment or 1 mg, 2 mg, 4 mg and 8 mg doses, respectively. For the most efficacious dose, 8 mg daily, this corresponds to a mean reduction of u-HGA24 of 98.8% compared with baseline. An increase in tyrosine levels was seen at all doses but the dose-response relationship was less clear than the effect on HGA. Despite tyrosinaemia, there were no safety concerns and no serious adverse events were reported over the 4 weeks of nitisinone therapy. CONCLUSIONS: In this study in patients with AKU, nitisinone therapy decreased urinary HGA excretion to low levels in a dose-dependent manner and was well tolerated within the studied dose range. TRIAL REGISTRATION NUMBER: EudraCT number: 2012-005340-24. Registered at ClinicalTrials.gov: NCTO1828463.


Assuntos
Alcaptonúria/tratamento farmacológico , Cicloexanonas/administração & dosagem , Inibidores Enzimáticos/administração & dosagem , Ácido Homogentísico/urina , Nitrobenzoatos/administração & dosagem , Adulto , Alcaptonúria/sangue , Alcaptonúria/urina , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Ácido Homogentísico/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Projetos de Pesquisa , Tirosina/sangue
18.
Purinergic Signal ; 12(4): 647-651, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27439698

RESUMO

It is widely accepted that the c-Fos gene has a role in proliferation and differentiation of bone cells. ATP-induced c-Fos activation is relevant to bone homeostasis, because nucleotides that are present in the environment of bone cells can contribute to autocrine/paracrine signalling. Gut hormones have previously been shown to have an effect on bone metabolism. In this study, we used the osteoblastic Saos-2 cell line transfected with a c-Fos-driven reporter stimulated with five gut hormones: glucose inhibitory peptide (GIP), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), ghrelin and obestatin, in the presence or absence of ATP. In addition, TE-85 cells were used to determine the time course of c-Fos transcript induction following stimulation with GLP-1, and GLP-2 with or without ATP, using reverse transcription qPCR. The significant results from the experiments are as follows: higher level of c-Fos induction in presence of GIP, obestatin (p = 0.019 and p = 0.011 respectively), and GIP combined with ATP (p < 0.001) using the luciferase assay; GLP-1 and GLP-2 combined with ATP (p = 0.034 and p = 0.002, respectively) and GLP-2 alone (p < 0.001) using qPCR. In conclusion, three of the gut peptides induced c-Fos, providing a potential mechanism underlying the actions of these hormones in bone which can be directed or enhanced by the presence of ATP.


Assuntos
Trifosfato de Adenosina/farmacologia , Grelina/farmacologia , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Peptídeo 2 Semelhante ao Glucagon/farmacologia , Osteoblastos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Linhagem Celular Tumoral , Humanos , Osteoblastos/metabolismo
19.
Purinergic Signal ; 12(1): 103-13, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26825304

RESUMO

Military recruits and elite athletes are susceptible to stress fracture injuries. Genetic predisposition has been postulated to have a role in their development. The P2X7 receptor (P2X7R) gene, a key regulator of bone remodelling, is a genetic candidate that may contribute to stress fracture predisposition. The aim of this study is to evaluate the putative contribution of P2X7R to stress fracture injury in two separate cohorts, military personnel and elite athletes. In 210 Israeli Defense Forces (IDF) military conscripts, stress fracture injury was diagnosed (n = 43) based on symptoms and a positive bone scan. In a separate cohort of 518 elite athletes, self-reported medical imaging scan-certified stress fracture injuries were recorded (n = 125). Non-stress fracture controls were identified from these cohorts who had a normal bone scan or no history or symptoms of stress fracture injury. Study participants were genotyped for functional SNPs within the P2X7R gene using proprietary fluorescence-based competitive allele-specific PCR assay. Pearson's chi-squared (χ (2)) tests, corrected for multiple comparisons, were used to assess associations in genotype frequencies. The variant allele of P2X7R SNP rs3751143 (Glu496Ala-loss of function) was associated with stress fracture injury, whilst the variant allele of rs1718119 (Ala348Thr-gain of function) was associated with a reduced occurrence of stress fracture injury in military conscripts (P < 0.05). The association of the variant allele of rs3751143 with stress fractures was replicated in elite athletes (P < 0.05), whereas the variant allele of rs1718119 was also associated with reduced multiple stress fracture cases in elite athletes (P < 0.05). The association between independent P2X7R polymorphisms with stress fracture prevalence supports the role of a genetic predisposition in the development of stress fracture injury.


Assuntos
Fraturas de Estresse/genética , Receptores Purinérgicos P2X7/genética , Adulto , Alelos , Atletas , Remodelação Óssea/genética , Estudos de Coortes , DNA/genética , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Israel , Masculino , Militares , Polimorfismo de Nucleotídeo Único/genética , Adulto Jovem
20.
Proc Biol Sci ; 281(1788): 20140786, 2014 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-24966314

RESUMO

Muscular contraction plays a pivotal role in the mechanical environment of bone, but controlled muscular contractions are rarely used to study the response of bone to mechanical stimuli. Here, we use implantable stimulators to elicit programmed contractions of the rat tibialis anterior (TA) muscle. Miniature stimulators were implanted in Wistar rats (n = 9) to induce contraction of the left TA every 30 s for 28 days. The right limb was used as a contralateral control. Hindlimbs were imaged using microCT. Image data were used for bone measurements, and to construct a finite-element (FE) model simulation of TA forces propagating through the bone. This simulation was used to target subsequent bone histology and measurement of micromechanical properties to areas of high strain. FE mapping of simulated strains revealed peak values in the anterodistal region of the tibia (640 µÎµ ± 30.4 µÎµ). This region showed significant increases in cross-sectional area (28.61%, p < 0.05) and bone volume (30.29%, p < 0.05) in the stimulated limb. Histology revealed a large region of new bone, containing clusters of chondrocytes, indicative of endochondral ossification. The new bone region had a lower elastic modulus (8.8 ± 2.2 GPa) when compared with established bone (20 ± 1.4 GPa). Our study provides compelling new evidence of the interplay between muscle and bone.


Assuntos
Módulo de Elasticidade , Contração Muscular , Músculo Esquelético/fisiologia , Tíbia/anatomia & histologia , Animais , Fenômenos Biomecânicos , Simulação por Computador , Estimulação Elétrica , Masculino , Modelos Biológicos , Ratos , Ratos Wistar , Tíbia/diagnóstico por imagem , Microtomografia por Raio-X
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