NT5E Genetic Mutation Is a Rare But Important Cause of Intermittent Claudication and Chronic Limb-Threatening Ischemia.

BACKGROUND: NT5Egenetic mutations are known to result in calcification of joints and arteries (CALJA), and worldwide, 14 patients from 7 families have been reported.Methods and Results:A total of 5 patients from 2 independent families with CALJA were found in Japan. Of them, 3 complained of intermittent claudication (IC), and 1 suffered from bilateral chronic limb-threatening ischemia (CLTI). Whole-exome sequencing analysis revealed an identical mutation pattern (c.G3C on the exon 1 start codon) that was unique compared withNT5Emutations reported in other countries. CONCLUSIONS: Vascular specialists need to recognize CALJA as a rare cause of ischemic IC and CLTI.

Chondrodysplasia and abnormal joint development associated with mutations in IMPAD1, encoding the Golgi-resident nucleotide phosphatase, gPAPP.

We used whole-exome sequencing to study three individuals with a distinct condition characterized by short stature, chondrodysplasia with brachydactyly, congenital joint dislocations, cleft palate, and facial dysmorphism. Affected individuals carried homozygous missense mutations in IMPAD1, the gene coding for gPAPP, a Golgi-resident nucleotide phosphatase that hydrolyzes phosphoadenosine phosphate (PAP), the byproduct of sulfotransferase reactions, to AMP. The mutations affected residues in or adjacent to the phosphatase active site and are predicted to impair enzyme activity. A fourth unrelated patient was subsequently found to be homozygous for a premature termination codon in IMPAD1. Impad1 inactivation in mice has previously been shown to produce chondrodysplasia with abnormal joint formation and impaired proteoglycan sulfation. The human chondrodysplasia associated with gPAPP deficiency joins a growing number of skeletoarticular conditions associated with defective synthesis of sulfated proteoglycans, highlighting the importance of proteoglycans in the development of skeletal elements and joints.

Development of an in vitro model to investigate joint ochronosis in alkaptonuria.

OBJECTIVES: Alkaptonuria (AKU) is a genetic disorder caused by lack of the enzyme responsible for breaking down homogentisic acid (HGA), an intermediate in tyrosine metabolism. HGA is deposited as a polymer, termed ochronotic pigment, in collagenous tissues. Pigmentation is progressive over many years, leading to CTDs including severe arthropathies. To investigate the mechanism of pigmentation and to determine how it leads to arthropathy, we aimed to develop an in vitro model of ochronosis. METHODS: Osteosarcoma cell lines MG63, SaOS-2 and TE85 were cultured in medium containing HGA from 0.1 µM to 1 mM. Cultures were examined by light microscopy and transmission electron microscopy, and Schmorl's stain was used to detect pigment deposits in vitro, following the observation that this stain identifies ochronotic pigment in AKU tissues. The effects of HGA on cell growth and collagen synthesis were also determined. RESULTS: There was a dose-related deposition of pigment in cells and associated matrix from 33 µM to 0.33 mM HGA. Pigmentation in vitro was much more rapid than in vivo, indicating that protective mechanisms exist in tissues in situ. Pigment deposition was dependent on the presence of cells and was observed at HGA concentrations that were not toxic. There was an inhibition of cell growth and a stimulation of type I collagen synthesis up to 0.33 mM HGA, but severe cell toxicity at 1 mM HGA. CONCLUSION: We have developed an in vitro model of ochronosis that should contribute to understanding joint destruction in AKU and to the aetiology of OA.

Apoptosis of ligamentous cells of the cranial cruciate ligament from stable stifle joints of dogs with partial cranial cruciate ligament rupture.

OBJECTIVE: To describe the presence and amount of apoptotic ligamentous cells in different areas of partially ruptured canine cranial cruciate ligaments (prCCLs) and to compare these findings with apoptosis of ligamentous cells in totally ruptured cranial cruciate ligaments (trCCLs). ANIMALS: 20 dogs with prCCLs and 14 dogs with trCCLs. PROCEDURES: Dogs with prCCLs or trCCLs were admitted to the veterinary hospital for stifle joint treatment. Biopsy specimens of the intact area of prCCLs (group A) and the ruptured area of prCCLs (group B) as well as specimens from trCCLs (group C) were harvested during arthroscopy. Caspase-3 and poly (ADP-ribose) polymerase (PARP) detection were used to detect apoptotic ligamentous cells by immunohistochemistry. RESULTS: No difference was found in the degree of synovitis or osteophytosis between prCCLs and trCCLs. No difference was found in degenerative changes in ligaments between groups A and B. A substantial amount of apoptotic cells could be found in > 90% of all stained slides. A correlation (r(s) = 0.71) was found between the number of caspase-3-and PARP-positive cells. No significant difference was found in the amount of apoptotic cells among the 3 groups. No significant correlation could be detected between the degree of synovitis and apoptotic cells or osteophyte production and apoptotic cells. CONCLUSIONS AND CLINICAL RELEVANCE: The lack of difference between the 3 groups indicates that apoptosis could be a factor in the internal disease process leading to CCL rupture and is not primarily a consequence of the acute rupture of the ligament.

Localization of cathepsin K and tartrate-resistant acid phosphatase in synovium and cranial cruciate ligament in dogs with cruciate disease.

OBJECTIVE: To localize cathepsin K and tartrate-resistant acid phosphatase (TRAP) in synovium and cranial cruciate ligament (CCL) of dogs with cruciate disease. ANIMALS: Dogs (n=15) with cruciate disease and ruptured CCL, and 12 dogs with intact CCL. METHODS: Synovium and CCL were examined histologically and cells containing cathepsin K or TRAP were identified immunohistochemically and histochemically, respectively. RESULTS: Increased cellular localization of cathepsin K and TRAP was detected in synovium and ruptured CCL in dogs with cruciate disease, when compared with tissues from dogs with intact CCL. Inflammation of synovium with TRAP+ macrophage-like cells was seen in 73% of dogs with CCL disease, but was not seen in dogs with intact CCL. The presence of cathepsin K and TRAP protein in synovium and CCL tissues was significantly correlated in dogs with CCL rupture. CONCLUSION: Inflammation of the epiligament of ruptured CCL with cathepsin K+ and TRAP+ macrophage-like cells forms part of a similar, more generalized chronic inflammatory change within the periarticular tissues of the stifle of a large proportion of dogs with CCL rupture. CLINICAL RELEVANCE: Production of matrix-degrading enzymes by the synovium may induce progressive pathologic rupture of the CCL. Therefore, these collagenolytic pathways may offer a novel target for medical therapy of joint inflammation in canine patients with cruciate disease.

Degenerative knee joint disease in mice lacking 3'-phosphoadenosine 5'-phosphosulfate synthetase 2 (Papss2) activity: a putative model of human PAPSS2 deficiency-associated arthrosis.

OBJECTIVE: Murine brachymorphism (bm) results from an autosomal recessive mutation of the Papss2 gene that encodes 3'-phosphoadenosine 5'-phosphosulfate synthetase 2, one of the principal enzymes required for the sulfation of extracellular matrix molecules in cartilage and other tissues. A spondyloepimetaphyseal dysplasia has been identified in Pakistani kindred having a mutation of PAPSS2. In addition to skeletal malformations that include short stature evident at birth due to limb shortening, brachydactyly, and kyphoscoliosis, affected individuals demonstrate premature onset degenerative joint disease. We investigated whether loss of Papss2 activity would similarly lead to degenerative joint disease in mice. METHODS: Mice carrying the bm mutation on a C57BL/6 background were obtained from the Jackson Laboratory. Limbs were analyzed by micro-computed tomography (microCT) and histology. RESULTS: At 12 months of age both male and female bm mice exhibited severe degenerative knee joint disease, with cartilage damage being primarily evident in the patello-femoral and medial compartments. Control 12-14-month-old C57BL/6 mice, in contrast, only occasionally demonstrated minimal cartilage damage. muCT imaging of bm limbs revealed shortened diaphyses associated with flared metaphyses in the proximal elements of both fore and hind limbs. Additionally, the bm hind limbs demonstrated extensive structural alterations, characterized by distortion of the patello-femoral groove, and prominent bowing of both tibia and fibula. CONCLUSIONS: The bm mutant, which develops severe articular cartilage lesions of the knee joint by approximately 12 months of age, represents a novel example of murine degenerative joint disease, possibly representing a model of human PAPSS2 deficiency-associated arthrosis.

The influence of repeated arthrocentesis and exercise on matrix metalloproteinase and tumour necrosis factor alpha activities in normal equine joints.

REASONS FOR PERFORMING STUDY: Matrix metalloproteinases (MMPs) and tumour necrosis factor alpha (TNF-alpha) may be useful as biomarkers of joint disease or inflammation. However, activity of both MMPs and TNF-alpha in synovial fluid (SF) may be influenced by nonpathological factors such as arthrocentesis or exercise. OBJECTIVE: To investigate the influence of repeated arthrocentesis and exercise on MMP and TNF-alpha activities in SF from normal equine joints. METHODS: SF was collected from the left metacarpophalangeal, radiocarpal and tarsocrural joints of 16 horses. Eight of these horses were subsequently subjected to an exercise programme on a treadmill and 8 were box-rested as controls. Arthrocentesis was repeated 14, 145, 17 and 24 days after the start of the exercise programme. General MMP and TNF-alpha activities were determined in SF. RESULTS: Repeated arthrocentesis caused a gradual increase but the exercise regimen no significant increase in MMP activity. There was a significant increase in TNF-alpha activity in SF collected from horses 2 h after cessation of the exercise programme. CONCLUSIONS AND POTENTIAL RELEVANCE: When using MMPs as biomarkers for joint disease, at least 14 days should elapse after previous arthrocentesis before subsequent SF collection. Moderate exercise does not increase MMP activity in SF from normal joints and it may be possible to ignore this as a source of error in evaluating MMP activity in diseased joints.

Synovial fluid gelatinase concentrations and matrix metalloproteinase and cytokine expression in naturally occurring joint disease in horses.

OBJECTIVES: To determine concentrations of matrix metalloproteinase (MMP)-2 and -9 in synovial fluid; and mRNA expression of MMP-1, -13, and -3; interleukin[IL]-1alpha and beta; and tumor necrosis factor (TNF)-alpha in synovial membrane and articular cartilage from horses with naturally occurring joint disease. SAMPLE POPULATION: Synovial fluid (n = 76), synovial membrane (59), and articular cartilage (45) from 5 clinically normal horses and 55 horses with joint disease categorized as traumatic (acute [AT] or chronic [CT]), osteochondritis dissecans (OCD), or septic (S). PROCEDURE: Synovial fluid gelatinase concentrations were analyzed, using zymography. Synovial membrane and articular cartilage mRNA expression for MMP-1, -3, and -13, IL-1alpha and beta, TNF-alpha, type-II collagen, and aggrecan were analyzed, using quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Synovial fluid pro-MMP-2 concentration was significantly higher in diseased joints than normal joints. Septic joints had significantly higher concentrations of pro and active MMP-9. Stromelysin-1 was expressed in > or = 80% of synovial membrane and articular cartilage samples and was strongly influenced by age. Collagenases were rarely expressed, with MMP-13 expressed only in diseased joints. Interleukin-1beta expression was significantly higher in all OCD samples and was influenced by age. Tumor necrosis factor-alpha expression was significantly higher in cartilage from joints with AT and OCD. There was no correlation between MMP or cytokines and type-II collagen or aggrecan expression. CONCLUSIONS AND CLINICAL RELEVANCE: Matrix metalloproteinase-2 and -3 are abundant in naturally occurring joint disease and normal joints. Interleukin-1beta and TNF-alpha may be important in the pathogenesis of OCD. Age affects MMP and IL-1beta concentrations.

Telomerase activity in the synovial tissues of chronic inflammatory and non-inflammatory rheumatic diseases.

Telomerase is a ribonucleoprotein complex which can compensate for telomeric loss originating from each cell division, and its activation plays a critical role in cellular immortality. We previously found that telomerase is activated not only in immortal cancer cells but also in activated lymphocytes. To assess the diagnostic significance of telomerase activity in RA synovial tissues, we quantitatively examined telomerase activity in synovial tissue samples obtained from 47 patients with RA, 31 with osteoarthritis (OA), and 23 with other joint diseases. Telomerase activity in synovial tissues was detected in 28 of 47 (59.6%) patients with RA, including monoarticular-type RA, but in none of those with other joint diseases except one case each of synovial chondromatosis and OA. Thus, the specificity of telomerase activity in synovial tissues for RA among joint diseases was 96.3% (52/54). In RA samples, the telomerase activity was detected in 14 of 27 (51. 9%) patients with total joint replacement, 7 of 12 (58.3%) open synovectomy cases, and 7 of 8 (87.5%) arthroscopic synovectomy cases. Detection of telomerase activity in synovial tissues is considered to be useful for diagnosis of RA, including monoarticular-type RA, or active inflammation with lymphocyte infiltration, and arthroscopy can be applied for this purpose.

[Superoxide dismutase and catalase levels in diseases of the aged]. / Niveles de superóxido dismutasa y catalasa en enfermedades del anciano.

UNLABELLED: Superoxide dismutase (SOD) and catalase (CAT) activities were measured in blood from 420 individuals: control population 126, males and females, age between 50 to 93 years of age without any relevant pathology. Pathological population: 294 patients, males and females, age between 50 to 93 years of age, with some disease in the cardiovascular system and in the osteoarticular system, myoma, prostatic pathologies, Chronic Obstructive Pulmonary Disease (EPOC), and Acute Cerebral Vascular Accident (ACVA). The method of Minami and Yoshikawa (SOD) and the method of Aebi (CAT) were judged the techniques of choice for a population study. STATISTICAL METHODS: ANOVA and Student's "t". 1) The results were that levels of activity for SOD and CAT were increased for women in control population, and 2) the level of activity for CAT decreases with aging. In the pathological population, we detected: 3) increased activity for SOD in cardiovascular diseases, myomas, EPOC and ACVA. 4) for CAT the level of activity decreases in cardiovascular and prostatic diseases, EPOC and ACVA. 5) while in osteoarticular diseases levels of activity for SOD and CAT were standard, but SOD level decreases with aging, for CAT in cardiovascular diseases and EPOC, too. Both enzymes work to balance the antioxidant system.

Niveles de superóxido dismutasa y catalasa en enfermedades del anciano / Dismutase and catalase superoxide levels in pathologies of the elderly

Se han determinado los niveles de superóxido dismutasa (SOD) y catalasa (CAT) en 420 individuos de uno y otro sexo y edades comprendidas entre 50 y 93 años. De ellos, 126 que no mostraban ninguna enfermedad relevante se utilizaron como grupo control. Los 294 restantes mostraban diferentes trastornos: alteraciones del sistema vascular (insuficiencias coronarias, hipertensión, infarto, etc.), alteraciones del sistema osteoarticular (artritis, polialtralgias, osteoporosis, etc.), miomas, afecciones, prostáticas, enfermedad pulmonar obstructiva crónica (EPOC) y accidente cerebral vascular agudo (ACVA). Para valorar SOD se utilizó el método de minami y Yoshikawa y el método de Aebi para valorar CAT. Métodos estadísticos: ANOVA y "t" de Student. En la población control se han obtenido: 1) niveles de SOD y CAT más elevados en mujeres que en varones. 2) la actividad de CAT disminuye con la edad. En la población con patologías: 3) la actividad de SOD está elevada en cardiovascular, miomas, EPOC y ACVA. 4) la actividad de CAT desciende en cardiovascular, próstata, EPOC y ACVA. 5) en osteoarticular actividad normal de SOD y CAT, aunque SOD desciende con la edad, CAT desciende con la edad en cardiovascular y EPOC. En general el comportamiento de ambos enzimas tiende a conseguir un equilibrio en el sistema antioxidante

[Overview of the current status of measurable parameters of cartilage metabolism in various body fluids]. / Uberblick über den aktuellen Stand der messbaren Parameter des Knorpelstoffwechsels in verschiedenen Körperflüssigkeiten.

The human cartilage and bone is characterized by a remodeling during the life, well balanced by neosynthesis and degradation of matrix components. In different joint diseases, it becomes imbalanced and the destruction of the cartilage supersedes the repair. In tissue processes in disease and in normal turnover of the matrix, these molecules are fragmented and released into surrounding fluids, in the synovial fluid, and then in the blood and the urine, where they can be detected. The quantitative measurement in the synovial fluid is more specific than in the other body fluids. The research process in recent years has suggested that these molecular markers of cartilage and bone matrix metabolism can be used to determine diagnosis, the disease severity rather than its presence or absence, the prognosis, and the response to therapy. They should help to identify the disease mechanism in different joint diseases not only on the tissue but also on the molecular level. The specific cartilage matrix markers promise to become useful tools in the future in clinical use. The research in this area is still in the early stages, with most results dated from the end of the 1980s and the 1990s.

Metalloproteinase inhibitors and the prevention of connective tissue breakdown.

The primary agents responsible for cartilage and bone destruction in joint diseases are active proteinases degrading collagen and proteoglycan. All four main classes of proteolytic enzymes are involved in either the normal turnover of connective tissue or its pathological destruction. These proteinases are made by different cells found within the joints. Both extracellular and intracellular pathways exist, and individual enzymes can be inhibited by specific proteinaceous inhibitors that block their activity. Recent research has implicated the matrix metalloproteinases in many of the processes involved in joint diseases. Conventional treatments do little to affect the underlying disease processes, and recently, the use of proteinase inhibitors has been suggested as a new therapeutic approach. A large variety of different synthetic approaches have been used and highly effective metalloproteinase inhibitors have been designed, synthesised and tested. These metalloproteinase inhibitors can prevent the destruction of animal cartilage in model systems and slow the progression of human tumours. Future patient trials will test the effectiveness of these compounds in vivo for the treatment of joint diseases.

Partial characterization of synovial fluid nucleotide pyrophosphohydrolase.

Synovial fluid adenosine triphosphate pyrophosphohydrolase, an enzyme which manifests increased activity in chondrocalcinosis and osteoarthritis, was partially characterized in synovial fluids from 41 patients who had a variety of arthropathies. Activity was found to be a soluble, heat labile, and divalent cation-dependent nonspecific nucleotide pyrophosphohydrolase with pH optimum 9.0-9.5.

Cytochemical investigation of cathepsin B in rheumatoid synovial membrane and fluid.

Cathepsin B was cytochemically investigated in the cells of synovial membranes and in the cell pellet of synovial fluids obtained from 50 patients with rheumatoid arthritis and eight patients with various nonrheumatoid arthropathies. The activity of Cathepsin B was estimated by using the substrate N-alpha-benzoyl-DL-arginine-naphthylamide HCl and diazoic dye Fast Corinth V in phosphate buffer pH 6.0 in the presence of EDTA and cysteine. A significant activity of cathepsin B was shown in lining mesothelial cells, in macrophages of the submesothelial infiltrations, as well as in fibroblasts prominent in the deep areas of rheumatoid synovial membranes. In the cell pellets of synovial fluids the highest activity of cathepsin B was found in the macrophages and polymorphonuclear leukocytes, accompanied by a variable activity in lymphocytes. The considerable activity of cathepsin B, an enzyme with degradative action upon collagen and proteoglycans, in the main cellular populations of rheumatoid synovial membranes and fluids, suggests its involvement in the genesis and maintenance of rheumatoid lesions.

I-Naphthyl acetate esterase isoenzymes in synovial fluids and radiography of temporomandibular and knee joints.

Esterase isoenzymes of synovial fluids were presented in cases of pain-dysfunction syndrome, osteoarthrosis, osteochondroma, malignant fibrous histiocytoma of temporomandibular joint, and hemarthrosis, rheumatoid arthritis of the knee joint. Radiographic features of them were also comparatively presented in several cases. The electrophoretogram of I-Naphthyl acetate esterase of pain-dysfunction syndrome showed the esterase-I, while when inflammatory process developed at joints, electrophoretic pattern of synovial fluids revealed a rather similar feature of sera with variable stainabilities of esterase-I, and -III, irrespective of any inflammation. Osteochondroma showed two components of esterase-I and -III, on the other hand malignant fibrous histiocytoma presented esterase-I and -III in the early stage but a more complicated pattern in recurrence. Among these conditions, the most similarity between synovial fluid and serum was demonstrated in the case of hemarthrosis on the electrophoretogram of I-Naphthyl acetate esterases.

Factors affecting the solubility of calcium pyrophosphate dihydrate crystals.

The solubility of triclinic calcium pyrophosphate dihydrate (CPPD) crystals was measured under varying conditions using 45Ca-labeled crystals, expressing solubility as micromoles per liter of 45Ca in solution. In a 0.1-M Tris-HC1 buffer pH 7.4, the solubility of accurately sized CPPD crystals (37-20mum) was 60muM with maximal solubility being attained after about 8 h incubation at 37degreeC. Reduction in crystal size, decrease in pH, increase in ionic strength, Mg++, citrate, and albumin all increased solubility. The most marked effects on solubility occurred when changing the calcium concentration or by enzymatic hydrolysis of inoganic pyrophosphate to orthophosphate. It was found that decreasing the ionized calcium level below 5 mg/100 ml resulted in a progressive enhancement of solubility. The observed solubility-enhancing effects of albumin could be explained solely on its calcium-binding ability and thereby, altered ionized calcium level. Diffusible calcium in synovial fluid was only 40% of the total calcium concentration, which means most joint fluids are normally near the critical concentration of 5 mg/100 ml of ionized calcium, below which solubility is enhanced. During surgery, especially parathyroidectomy, calcium levels fall, favoring dissolution of CPPD crystals. We speculate that the slight decrease in crystal size during dissolution frees them from their cartilaginous mold, resulting in a dose-dependent inflammatory reaction as they are "shed" into the joint space. Crystal shedding may be reinforced by the modest fall in joint fluid pH accompanying the inflammatory response.

An experimental study of surface injury to articular cartilage and enzyme responses within the joint.

An experimental model of degenerative joint disease on chondromalacia consists of a surgically-scarified articular surface of the adult dog knee joint. In 52 dogs, evaluated by histologic and enzymatic assays over a period of 1 to 110 weeks post-surgery, the levels of acid hydrolase activity varied on various areas of articular cartilage within the same joint. There was a transient rise in most of the acid hydrolases in the synovium as a response to arthrotomy of the knee joint. All of the acid hydrolases studied did not respond uniformly to surgically created trauma. There was evidence of repair of the cartilage lacerations even when the subchondral zone was not breached. Lacerations in the central portion of the patella rarely showed healing in contrast to those placed more to the periphery of the articular surface. There was no gross or histologic evidence of progressive degenerative joint disease up to 2 years post-surgery. Thus an injury inflicted to the surface of the articular cartilage may be in itself insufficient in severity to produce destructive changes in the joint. This should not be too surprising, since, clinically, all joint surface injury does not lead to degenerative arthritis. The joint seems to have an injury threshold whereby chondrocytes are capable of repairing surface injury if the damage is not massive or repetitive. Insofar as lacerations in the center of the patella rarely healed, while the peripheral ones showed consistent signs of healing, the site of injury, as well as the magnitude of injury, may be critical.